Research Paper - Effect of shilajit on blood glucose and lipid profile in alloxaninduced diabetic rats

Abstract

OBJECTIVE: To study the effect of shilajit (a herbomineral preparation) on blood glucose and lipid profile in euglycemic and alloxan-induced diabetic rats and its effects on the above parameters in combination with conventional antidiabetic drugs. MATERIAL AND METHODS: Diabetes was induced in albino rats by administration of a single dose of alloxan monohydrate 5% (125 mg/kg, i.p.). Effects of three different doses of shilajit (50, 100 and 200 mg/kg/day, orally), alone for 4 weeks and a combination of shilajit (100 mg/kg/day, orally) with either glibenclamide (5 mg/kg/day, orally) or metformin (0.5 g/kg/day, orally) for 4 weeks were studied on blood glucose and lipid profile. RESULTS: In the diabetic rats, all the three doses of shilajit produced a significant reduction in blood glucose levels and also produced beneficial effects on the lipid profile. The maximum effect was observed with the 100 mg/kg/day dose of shilajit. Combination of shilajit (100 mg/kg) with glibenclamide (5 mg/kg/day) or metformin (0.5 gm/kg/day) significantly enhanced the glucose-lowering ability and improvement in lipid profile than any of these drugs given alone. CONCLUSION: Shilajit is effective in controlling blood glucose levels and improves the lipid profile.

Full text

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Research
Paper
Effect of shilajit on blood glucose and lipid profile in alloxan-Effect of shilajit on blood glucose and lipid profile in alloxan-
Effect of shilajit on blood glucose and lipid profile in alloxan-Effect of shilajit on blood glucose and lipid profile in alloxan-
Effect of shilajit on blood glucose and lipid profile in alloxan-
induced diabetic ratsinduced diabetic rats
induced diabetic ratsinduced diabetic rats
induced diabetic rats
N. A. Trivedi, B. Mazumdar, J. D. Bhatt, K. G. Hemavathi
ABSTRACT
Objective: To study the effect of shilajit (a herbomineral preparation) on blood glucose and lipid
profile in euglycemic and alloxan-induced diabetic rats and its effects on the above parameters in
combination with conventional antidiabetic drugs.
Material and Methods: Diabetes was induced in albino rats by administration of a single dose of
alloxan monohydrate 5% (125 mg/kg, i.p.). Effects of three different doses of shilajit (50, 100 and
200 mg/kg/day, orally), alone for 4 weeks and a combination of shilajit (100 mg/kg/day, orally)
with either glibenclamide (5 mg/kg/day, orally) or metformin (0.5 g/kg/day, orally) for 4 weeks were
studied on blood glucose and lipid profile.
Results: In the diabetic rats, all the three doses of shilajit produced a significant reduction in blood
glucose levels and also produced beneficial effects on the lipid profile. The maximum effect was
observed with the 100 mg/kg/day dose of shilajit. Combination of shilajit (100 mg/kg) with
glibenclamide (5 mg/kg/day) or metformin (0.5 gm/kg/day) significantly enhanced the glucose-
lowering ability and improvement in lipid profile than any of these drugs given alone.
Conclusion: Shilajit is effective in controlling blood glucose levels and improves the lipid profile.
KEY WORDS: Biguanide, diabetes mellitus, herbomineral antidiabetic, sulfonylurea.
Department of
Pharmacology, Medical
College, Baroda -
390001, India.
Received: 10.1.2004
Revised: 21.4.2004
Accepted: 20.7.2004
Correspondence to:
N. A. Trivedi
E-mail:
natrivedi@yahoo.com
Introduction
Diabetes mellitus is a major public health problem in the
developed as well as developing countries. It is ranked sev-
enth among the leading causes of death, and third when all its
fatal complications are taken into account. Large-vessel athero-
sclerosis is the most common cause of death in diabetics. An
ideal oral treatment for diabetes would be a drug that not only
controls the glycemic level but also prevents the development
of atherosclerosis and other complications of diabetes. Unfor-
tunately, among the currently available drugs, the choice is
very limited. Alloxan is widely used to induce experimental
diabetes and is associated with marked reduction in islet cell
Super Oxide Dismutase (SOD) activity.1
Shilajit is a herbo-mineral drug, which oozes out from a
special type of mountain rocks in the peak summer months. It
is found at high altitudes ranging from 1000 to 5000 meters.
The active constituent of shilajit consists of dibenzo-alpha-
pyrones and related metabolites, small peptides (constituting
non-protein amino acids), some lipids and carrier molecules
(fulvic acids).2,3 Standard shilajit contains at least 5-7%
dibenzo-alpha-pyrones.2-4
Shilajit finds extensive use in Ayurveda, for diverse clini-
cal conditions. For centuries people living in the isolated vil-
lages in Himalaya and adjoining regions have used shilajit alone
or in combination with other plant remedies to prevent and
combat problems with diabetes.5 Medical researchers have
taken a more serious interest in determining if the claims re-
garding the antidiabetic effects of shilajit have scientific merit.
Studies done by Gupta6 and Bhattacharya7 have also reported
the antidiabetic actions of shilajit.
In the light of the above data, the objectives of the present
study were to evaluate (1) the effect of shilajit on blood glu-
cose and lipid profile in euglycemic and alloxan-induced dia-
betic rats and (2) to study its effect on the above parameters
in combination with conventional oral antidiabetic agents.
Material and Methods
Animals
Adult albino rats (250-300 gm) of either sex were used for
the study. They were housed at ambient temperature of 25±2
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Antidiabetic action of shilajit
oC and 45-55% humidity, with 12 h light dark cycle. Animals
were fed with standard laboratory diet and water was given
ad libitum. Animals described as fasted were deprived of food
for 18 h but had free access to water.
Study design
Each group consisting of six animals received the follow-
ing treatment. The first group of rats received normal saline
(vehicle of alloxan), which served as a euglycemic control, while
the second group received shilajit (100 mg/kg, p.o.). A single
dose (125 mg/kg, i.p.) of alloxan monohydrate 5% (dissolved
in normal saline) was used for induction of diabetes mellitus
in the rats. The induction of diabetes mellitus was confirmed
after the 5th day of alloxan treatment by estimation of elevated
fasting blood glucose (FBG) level. Only those rats with blood
glucose level ≥ 150 mg/dl were included in the study (Day 0).
These rats were further divided into various groups as fol-
lows: Group 3 served as diabetic control. While Groups 4, 5
and 6 were treated with three different doses of shilajit (50,
100 and 200 mg/kg/day/p.o. respectively). Groups 7 and 8
were treated with glibenclamide (5 mg/kg, p.o.) and a combi-
nation of glibenclamide (5 mg/kg) with shilajit (100 mg/kg)
respectively while Groups 9 and 10 received metformin (0.5
gm/kg, p.o.) and a combination of metformin (0.5 gm/kg) with
shilajit (100 mg/kg) respectively. Treatment with drugs was
started on the 6th day of the alloxan treatment (i.e. Day 1) and
was continued for 4 weeks. All the drugs were given orally as
a single dose in the morning. Blood glucose was measured
before starting the treatment (Day 0) and weekly thereafter
up to the end of the treatment period. Total cholesterol (TCh),
triglyceride (TG) and high-density lipoprotein (HDL) (i.e. lipid
profile) were measured on Day 0 and after the completion of
the treatment period (i.e. at the end of the 4th week).
Blood was collected by cardiac puncture just before drug
administration and 24 h after completion of the treatment.
Blood glucose and lipid profile were estimated by enzymatic
method using reagent kit (Span diagnostic Ltd., Surat, India).
Statistical analysis
The results are expressed as mean±SEM. Data on blood
glucose level were analyzed by one-way ANOVA followed by
Tukey’s post hoc test. While data on lipid profile were analyzed
by Student’s ‘t ’ test. Value of P less than 5% (P<0.05) was
considered statistically significant.
Results
A steady decrease in the body weight was observed in the
alloxan-treated rats which was significant after the 2nd week
of alloxan treatment. Shilajit per se had no effect on body weight
but attenuated the weight loss observed in alloxan-induced
diabetic rats (data not shown).
Effects of shilajit on euglycemic rats
A significant (P<0.001) reduction in the blood glucose level
was observed at the end of 2nd week of treatment with shilajit
(100 mg/kg) in the euglycemic rats, which remained persist-
ent up to 4 weeks of the treatment period (data not shown).
Moreover, a significant reduction in the level of TCh (P<0.001)
and TG (P<0.01) with significant increase (P<0.05) in the level
of HDL was noted at the end of the 4th week of treatment as
compared to the Day 0 value (Table 1).
Effects of shilajit on alloxan-induced diabetic rats
In alloxan-treated rats, the rise in blood glucose level
reached its peak value on the 5th day and then remained stable
throughout the study period (Figure 1). Treatment with all the
three doses of shilajit (50, 100 and 200 mg/kg) produced sig-
nificant reduction in the blood glucose level with maximum
reduction being achieved with the dose 100 mg/kg (P<0.001).
The peak reduction in blood glucose level with all the three
doses was observed at the end of the 2nd week of treatment,
Table 1
Effects of various treatments on lipid profile (Mean ± SEM) in euglycemic and alloxan-induced diabetic rats
Treatment (mg/kg) TCh (mg/dl) TG (mg/dl) HDL (mg/dl)
0 day 4 week 0 day 4 week 0 day 4 week
Control (normal saline) 098.6 ± 4.8 094.2 ± 4.0 78.6 ± 6.1 73.2 ± 5.6 34.6 ± 4.2 36.1 ± 3.4
Shi 100 092.7 ± 3.7 060.7 ± 6.3*** 75.9 ± 7.1 61.8 ± 6.3** 35.8 ± 2.8 41.8 ± 2.3*
Alloxan 094.2 ± 3.5 105.7 ± 2.8** 73.4 ± 2.3 85.2 ± 3.8** 34.4 ± 4.8 31.6 ± 5.6**
All + Shi (50) 108.1 ± 4.2 089.7 ± 3.9** 88.4 ± 3.8 69.6 ± 3.6** 31.5 ± 5.1 38.4 ± 3.8**
All + Shi (100) 102.4 ± 5.1 072.8 ± 4.8*** 89.1 ± 4.8 64.2 ± 3.9** 31.9 ± 2.4 43.4 ± 2.3***
All + Shi (200) 099.6 ± 2.9 073.8 ± 1.8*** 83.2 ± 3.2 53.7 ± 2.4*** 31.8 ± 4.1 42.4 ± 3.1***
All + Glib (5) 105.4 ± 3.4 090.1 ± 2.3** 88.9 ± 5.6 67.8 ± 5.2** 32.1 ± 4.8 35.1 ± 3.7*
All + Glib. + Shi (100) 100.8 ± 4.3 077.5 ± 4.3** a85.7 ± 4.8 59.8 ± 6.3** 30.7 ± 5.6 41.5 ± 6.1**a
All + Metformin(0.5) 108.6 ± 4.3 086.1 ± 3.9** 84.4 ± 3.1 60.0 ± 4.6** 30.2 ± 4.8 36.2 ± 3.7*
All + Met + Shi (100) 109.4 ± 6.2 062.7 ± 7.1***b,c 86.5 ± 4.3 67.2 ± 2.9**b31.5 ± 5.2 49.1 ± 6.9***b,c
n=6 rats in each group
*
P
<0.05, **
P
<0.01, ***
P
<0.001. *As compared to (initial) Day 0 treatment value
a
P
<0.05 as compared to glibenclamide (5 mg/kg)-treated animals in alloxan-induced diabetic rats.
b
P
<0.05 as compared to metformin (0.5 gm/kg)-treated animals in alloxan-induced diabetic rats.
c
P
<0.05 as compared to shilajit (100 mg/kg)-treated animals in alloxan-induced diabetic rats.
Shi - Shilajit, All - Alloxan, Glib - Glibenclamide, Met- Metformin
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375
which remained stable up to the 4th week (Figure 1).
Similar effects were also observed in the lipid profile. Treat-
ment with 50, 100 and 200 mg/kg of shilajit produced signifi-
cant reduction in TCh level, with maximum reduction caused
by 100 mg/kg (P<0.001). There was dose-dependent reduc-
tion in the TG level. All the three doses of shilajit also pro-
duced significant increase in the HDL level with the maximum
elevation being produced with the dose of 100 mg/kg (P<0.001)
(Table 1).
Effect of shilajit in combination with known antidiabetic
drugs
Combination of shilajit with glibenclamide significantly
(P<0.001) enhanced the glucose-lowering effect of shilajit (100
mg/kg) (P<0.05) or glibenclamide (P<0.01) per se (Figure 2).
Moreover, the effect of the combination treatment on the lipid
profile was significantly more than that of glibenclamide per
se (P<0.05), however, it was comparable to that produced by
shilajit (100 mg/kg) per se (Table 1).
Combination of shilajit (100 mg/kg) with metformin sig-
nificantly lowered the blood glucose level compared to that of
metformin per se (P<0.01). However, it was comparable to
that of shilajit (100 mg/kg). Moreover, the combination treat-
ment caused significant improvement in the lipid profile as
compared to that of shilajit (100 mg/kg) (P<0.05) or metformin
(P<0.05) per se.
Discussion
Although the precise mechanism of alloxan-induced dia-
betes remains unclear, there is increasing evidence that it in-
volves the degeneration of islet β-cells by accumulation of cy-
totoxic free radicals.1 Following its administration, alloxan is
concentrated in the islets and in the liver, where it is reduced
to dialuric acid. This acid is unstable in aqueous solutions and
undergoes oxidation back to alloxan, accompanied by genera-
tion of O2
-, hydrogen peroxide and hydroxyl radicals by Fenton
type reaction.1 The liver contains high super oxide dismutase
(SOD), catalase and glutathione peroxidase activities, which
can scavenge these free radicals. On the contrary, the islet
cells have low concentrations of these enzymes and are vul-
nerable to the cytotoxic effects of the free radicals. It is re-
ported that increase in islet cell SOD activity can prevent or
decrease alloxan toxicity.1
Experimental diabetes is suggested to result from initial
islet inflammation, followed by infiltration of activated
macrophages and lymphocytes in the inflammatory focus.
These cells might be the source of the cytotoxic oxygen radi-
cals. Shilajit has been reported to reduce macrophage and
lymphocyte activation and migration, as a part of its
immunomodulatory activity.7 Moreover, being an antioxidant
it will prevent damage to the pancreatic islet cell induced by
the cytotoxic oxygen radicals.7-9
In the present study, treatment with shilajit (100 mg/kg) in
euglycemic rats produced significant hypoglycemia. Gupta
et al6 suggested that long-term treatment with shilajit increases
the number of β-cells of pancreas, i.e. pancreatotrophic ac-
tion, which may result in better sensitivity of pancreatic β-
cells with prompt secretion of a large quantity of insulin in
response to hyperglycemia.
Combination of shilajit with glibenclamide produced a sig-
nificant decrease in the blood glucose level which is higher
than that produced by either drug alone. Thus it seems likely
that, apart from it’s pancreatic action, shilajit may also pos-
sess extrapancreatic action, which could have contributed to
its hypoglycemic action.
The hypoglycemic effect of shilajit (100 mg/kg) is signifi-
cantly higher than that of metformin (500 mg/kg). But the com-
bination of shilajit with metformin produced no further sig-
nificant reduction in the blood glucose level compared to that
produced by shilajit (100 mg/kg) per se.
Trivedi NA,
et al.
Figure 1: Effects of three different doses of shilajit (50, 100 and 200
mg/kg) on fasting blood glucose (FBG) level (mg/dl), in alloxan-
induced diabetic rats, on Day 0 and weekly up to 4 weeks of treatment.
(n=6). *
P
<0.001 as compared to Day 0 treatment value
All- Alloxan, Shi- Shilajit
Figure 2: Effects of glibenclamide (5 mg/kg) and metformin (0.5 gm/
kg) alone and in combination with shilajit (100 mg/kg) on fasting blood
glucose (FBG) levels (mg/dl), in alloxan-induced diabetic rats, on Day
0 and weekly up to 4 weeks of treatment (n=6).
*
P
<0.001 as compared to Day ‘0’ treatment value
a
P
<0.001 as compared to glibenclamide (5 mg/kg)-treated animals in
alloxan-induced diabetic rats.
b
P
<0.01 as compared to metformin (0.5 gm/kg)-treated animals in
alloxan-induced diabetic rats.
c
P
<0.001 as compared to shilajit (100 mg/kg)-treated animals in
alloxan-induced diabetic rats.
All- Alloxan, Shi- Shilajit, Glib- Glibenclamide, Met- Metformin
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376
All the three doses of shilajit also produced a significant
beneficial effect on the lipid profile in alloxan-induced diabetic
rats. It is reported that the derangement of glucose, fat and
protein metabolism during diabetes, results into the develop-
ment of hyperlipidemia.10-12 The beneficial effects on the lipid
profile by shilajit in alloxan-induced diabetic rats may be sec-
ondary to better glycemic control.
Moreover, shilajit produced significant beneficial effects in
the lipid profile in euglycemic rats also by reducing TCh and
TG and increasing HDL significantly. Therefore, it is likely that
shilajit-induced favorable changes in the lipid profile in dia-
betic rats may not only be due to better glycemic control (sec-
ondary), but could also be due to its direct action on lipid
metabolic pathways.
Combination of glibenclamide with shilajit failed to pro-
duce significant improvement in the lipid profile than that pro-
duced by shilajit (100 mg/kg) per se. This could be explained
on the basis that improvement in the lipid profile by
glibenclamide in diabetic rats may be due to better glycemic
control (i.e. secondary).13 Since glibenclamide acts by second-
ary mechanism, further improvement in the lipid profile was
not observed when used with shilajit. The effect on the lipid
profile produced by combination treatment is significantly (P<
0.01) more than that produced by glibenclamide per se.
Metformin produces beneficial effects on the lipid profile
mainly by correcting abnormal glucose metabolism.14 It also
produces moderate reduction in the triglyceride levels as a
result of decreased hepatic synthesis of very low-density lipo-
protein.13 A similar observation has been reported in our study.
Metformin has also been found to reduce the postprandial
hyperlipoproteinemia of intestinal origin significantly.13
Since the combination of shilajit with metformin produced
further improvement in the lipid profile except TG, than that
produced by metformin or Shilajit per se, it is suggested that
shilajit may be acting by some different mechanism than that
of metformin on lipid metabolic pathways.
Shilajit, a herbo-mineral preparation can offer a new and
promising approach in the long-term management of matu-
rity onset diabetes mellitus, because of its multifaceted ac-
tion. Since it can produce a better glycemic control along with
improvement in the lipid profile in animals, it is worthwhile to
try shilajit either as monotherapy or in combination with other
antidiabetic agents clinically.
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Antidiabetic action of shilajit
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Indian J Pharmacol | December 2004 | Vol 36 | Issue 6 | 373-376