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International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 33
Antidiabetic activity of polyherbomineral
formulation: Chandrakala rasa
Alok Kumar Singh1, Santosh Kumar Maurya1, Damiki Laloo2,
Narendra Kumar Singh1, Ankit Seth1
1Department of Ayurvedic Pharmacy, Ayurvedic Pharmacy Laboratory, Rajiv Gandhi South Campus, Banaras
Hindu University, Barkachha, Mirzapur, Uttar Pradesh, India, 2Department of Pharmaceutical Sciences,
Girijananda Institute of Pharmaceutical Sciences, Assam Science and Technology University, Azara, Guwahati,
Assam, India
Abstract
Objective: Chandrakala rasa (CKR), a herbomineral formulation is used to treat diabetes mellitus in ayurvedic
system of health care. The present study aims to evaluate the antihyperglycemic potential of CKR in normoglycemic
and streptozotocin (STZ)-nicotinamide (NAD)-induced Type 2 diabetic rats. Materials and Methods: Effects of
CKR (100, 200 and 400 mg/kg, p.o.) on hypoglycemia as well as on oral glucose tolerance test (OGTT) were
evaluated in normoglycemic rats by measuring the blood glucose concentrations. Similarly, blood glucose level
was measured after 7, 14 and 21 days in STZ-NAD-induced diabetic rats treated with CKR. Different biochemical
parameters such as total cholesterol, triglyceride, low density lipoprotein-cholesterol, and high density lipoprotein-
cholesterol were estimated in a blood sample. In vivo antioxidant potential of CKR was measured in isolated liver
sample of rats. Results: CKR (400 mg/kg, p.o.) did not show any hypoglycemic effect in normoglycemic rats.
In OGTT, it significantly reduced the hike in blood glucose levels within 30-60 min after glucose administration
without causing any hypoglycemic effect. Administration of CKR significantly reduced the fasting blood glucose
levels on 7th, 14th and 21st days in STZ-NAD-induced diabetic rats. Treatment of rats with CKR reversed plasma
lipid profile as well as increases liver glycogen level significantly in STZ-NAD-induced diabetic rats. Treatment
with CKR in diabetic rats significantly restored the levels of lipid per-oxidation, superoxide dismutase and catalase
as compared to negative control rats. Conclusion: The present study showed that CKR has antidiabetic activity
probably because of antioxidant potential.
Key words: Antioxidant, hypoglycemia, lipid per-oxidation, superoxide dismutase, Type 2 diabetes
Address for correspondence:
Ankit Seth, Department of Ayurvedic Pharmacy,
Ayurvedic Pharmacy Laboratory, Rajiv Gandhi South
Campus, Banaras Hindu University, Varanasi - 221 005,
Uttar Pradesh, India. E-mail: ankitsethitbhu@gmail.com
Received: 29-09-2015
Revised: 23-12-2015
Accepted: 24-12-2015
INTRODUCTION
Diabetes mellitus is a chronic metabolic
disease characterized by derangements
in carbohydrate, protein and fat
metabolism.[1] It leads to hyperglycemia
resulting from a defect in insulin secretion,
or insulin resistance in the peripheral tissues
or both.[2] This hyperglycemic state produces
classical symptoms viz. polyuria, polydipsia,
polyphagia and weight loss.[3] It is assumed that
by 2030, the number of diabetic patients will
increase to 439 million which was 285 million
in 2010.[4] India has been declared as “Diabetic
Capital of the World” by the International
Diabetes Federation because 20% of the total
diabetic patients in the world found in India.
However, among the two major types of
diabetes, i.e., Type 1 and Type 2, Type 2 diabetes
mellitus is the most common form of diabetes
constituting 90-95% of the diabetic population
caused by obesity and an unhealthy lifestyle.[5] Hyperglycemia
is also associated with the generation of reactive oxygen
species (ROS) and consequent oxidative stress which leads
to[6] several diabetic complications such as nephropathy,
neuropathy, cardiovascular diseases and retinopathy.[7] The
treatment and management of diabetes become a challenge
before the medical practitioner globally.[8] Oral hypoglycemic
agents (glyburide, glimepiride, glipizide, metformin, and
pioglitazone, etc.) and insulin are basic pharmacotherapies
for diabetes mellitus.[9] Synthetic hypoglycemic agents put
ORIGINAL ARTICLE
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 34
forth serious side effects especially metallic taste, gastro-
intestinal discomfort and nausea.[10]
Furthermore, diabetes has been treated with several types of
folklore medicines (herbal and herbomineral preparations)
since ancient times. Bhasmas are the oldest metallic
preparation of nanoparticle range which attracted enormous
scientific and technological interest. Chandrakala rasa
(CKR) is a well-known polyherbometallic preparation,
used for the treatment of diabetes in ayurvedic system of
medicine.[11,12] CKR is a combination of six herbs with Vanga,
Lauha, Abhraka Bhasma, and Rasa Sindoor [Table 1].
It has been reported that metals in the Bhasmas are in the
nanoparticles range and are taken along with milk, butter,
honey, or ghee to enhance the absorption, elimination and
to remove the harmful effects.[13] Moreover, nanoparticles of
metals are increasingly gaining attention in the therapeutic
area due to their ease of preparation, chemical stability, and
unique optical properties.[14] Hence, the present study was
undertaken to investigate the antidiabetic potential of an
ayurvedic classical polyherbomineral preparation CKR in
experimentally induced diabetes in rats.
MATERIALS AND METHODS
Materials
The CKR was prepared as per the method given in ancient
ayurvedic text Bhaishajya Ratnavali,[11] at the Ayurvedic
Pharmacy Research Laboratory, Rajiv Gandhi South Campus,
Banaras Hindu University, Barkachha, Mirzapur, Uttar
Pradesh, India. Ingredients of CKR were given in Table 1.
Preparation of Vanga Bhashma
Vanga (tin metal) was melted and poured in Churnodaka (lime
water) and Nirgundi swarasa (Vitex nigundo Linn. juice)
mixed with Haridra (Curcuma longa Linn.). The process
was called as Dhalana and performed in specific apparatus
known as Pithera Yantra. The purified Vanga then fried with
Apamarga panchanga churna (powder of the whole plant of
Achyranthes aspera Linn.) to make it in powder form. Then,
the Vanga powder was triturated with Kumari swarasa (Aloe
vera L. Burm. fresh juice) and made into pellet form. The
pellets were heated at 500°C for 1 h and then allowed to
cool. This process was repeated 5 times to obtained Vanga
Bhasma.[15]
Preparation of Abhraka Bhashma
Abhraka (mica) was heated to red hot (at 800-850°C) and
immediately quenched into Triphala kwatha (decoction of
three fruits powder in equal quantity viz. Terminalia chebula
Retz., Terminalia bellarica Roxb., and Emblica officinale
Gaertn.). This process was repeated for 7 times. The whole
process was called as Nirvapa. Fresh Triphala kwatha was
used every time. After Nirvapa, Abhraka became brittle
and made in the form of powder. The powdered Abhraka
was mixed with ¼ part Shali dhanya (Oryaza sativa Linn.)
to make Dhanyabhraka. The Dhanyabhraka was triturated
with Kasamarda swarasa (juice of Cassia occidentalis
Linn. leaves) and made in the form of pellets. The pellets
were heated at 800°C for 1 h. After cooling, the process was
repeated for 40 times to obtained Abhraka Bhasma.[16]
Preparation of Lauha Bhasma
Tikshna Lauha (iron powder) has been used as the raw material
for the preparation of Lauha Bhasma. Tikshna Lauha was
heated to red hot (at 800-850°C), and it was dipped separately
into sesame oil, Takra (buttermilk), Gomûtra (cow urine),
Kanji (sour gruel) and a decoction of horse gram (Dolichos
biflorus Linn.). The process was repeated for 7 times, taking
fresh liquid media every time. After the procedure, Lauha
became brittle, and it was made in the form of powder. The
powdered Lauha was subjected to Bhanu paka (sun drying)
with Triphala kwatha and Sthali paka (boiling) with Triphala
kwatha. The powdered material was triturated with Triphala
kwatha and made pellets. The pellets were heated at 650°C
for 1 h. After self-cooling, the process was repeated for
20 times to obtained Lauha Bhasma.[17,18]
Preparation of Rasa Sindoor
For the preparation of Rasa Sindoor, Kumari Bhavita Kajjali
(mercuric sulfide, triturated with fresh juice of A. vera) was
taken in a glass bottle wrapped with seven layers of cloth
impregnated with clay. It was successively subjected to
heating for 3 h at 250°C, 450°C, and 650°C temperature in
Table 1: Ingredients of CKR
Ingredients Scientific name Quantity
Vanga Bhasma Incinerated tin metal 1 Part
Rasa Sindoor Red sulphide of mercury 1 Part
Abhraka Bhasma Incinerated biotite/mica 1 Part
Lauha Bhasma Incinerated iron 1 Part
Amalaki E. officinalis Gaertn. 1 Part
Shilajit Asphaltum 1 Part
Ela E. cardamomum Maton. 1 Part
Karpoor C. camphora Nees & Eberm. 1 Part
Shalmali B. ceiba Linn. Q. S.
Guduchi juice Tinospora cordifolia (Willd.)
Miers ex Hook.f. & Thoms.
Q. S.
E. officinalis: Emblica officinalis Gaertn., E. cardemonum: Elettaria
cardemonum Maton., C. camphora: Cinnamonum camphora Nees
& Eberm, B. ceiba: Bombax ceiba Linn., T. cordifolia: Tinospora
cordifolia (Willd.) Miers ex Hook.f. & Thoms., CKR: Chandrakala
rasa
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 35
a muffle furnace. Rasa Sindoor was collected in the form of
sublimate at the neck of the bottle.[19]
Preparation of CKR
For the preparation of CKR, all the ingredients (Vanga
Bhasma, Rasa Sindoor, Abhraka Bhasma, Lauha Bhasma,
Shilajit, E. officinalis, Elettaria Cardemonum, and
Cinnamomum camphora) were taken in equal quantities,
triturated 8 times separately with Tinospora cordifolia juice
and Bombax ceiba juice until dry powder obtained. CKR was
stored in an airtight container for the further experiment.
Drugs and Chemicals
Glibenclamide (GC), streptozotocin (STZ), nicotinamide
(NAD) (Sigma–Aldrich Co. LLC., New Delhi, India)
adenine dinucleotide, nitro blue tetrazolium (NBT) (Sisco
Research Laboratories Pvt. Ltd., Mumbai) were used for the
experimental purpose. All other reagent and solvents used in
the experiment were of the analytical grade.
In Vivo Antidiabetic Activity
Experimental animals
Adult Charles foster albino rats (140 ± 20 g) of either sex
were procured from the Central Animal House, Institute of
Medical Sciences, Banaras Hindu University; Varanasi. The
animals were kept in the laboratory at controlled temperature
(22 ± 2°C) and humidity (55 ± 10%) and 12 h light/12 h dark
cycle. The animals were provided with standard pelleted feed
(Amrut Pvt. Ltd. Pune, India) and water ad libitum. Rats were
kept in a standard laboratory environment for at least 1 week
before the commencement of the experiment. The protocols
for the study have been approved by the Institutional Animal
Ethical Committee of Banaras Hindu University, Varanasi
(Ref. No. Dean/13-14/CAEC/212).
Acute oral toxicity study
The acute oral toxicity study of CKR was performed
according to the Organization for Economic Co-operation
and Development-425 guidelines.[20] A single dose of CKR
2000 mg/kg, p.o. was administered in 24 h fasted rats (n = 5)
and observed at 0, 30, 60, 120, 180, and 240 min and then
once a day for next 14 days for any signs or symptoms of
toxicity or abnormalities. The number of rats that survived at
the end of the study period was recorded.
Oral Glucose Tolerance Test (OGTT)
30 normoglycemic rats were used for the experiment. The
animals were allowed free access of water ad libidum for
18 h before the experiment. The animals were divided into
five groups viz. control group, reference drug GC (10 mg/kg,
p.o., in 0.5% carboxymethylcellulose [CMC]) treated group,
and three test drug (CKR 100, 200, and 400 mg/kg, p.o.)
treated groups. The control group received only vehicle
(0.5%, CMC) by the oral route. 10 min after treatment with
reference and test drugs, glucose (2 g/kg, 10% solution in
water) was administered orally to each rat in all the groups.
The glucose level in the blood samples collected from tail
vein was determined by glucometer (Accu-Chek Meters,
Roche Diagnostics India, Pvt. Ltd.) based on enzymatic
glucose-oxidase method at 0 (before glucose administration),
30, 60, 90, and 120 min after glucose administration.[21]
Induction of Diabetes in Rats
The rats were divided into six groups (Group I-VI)
with six rats in each group. Group I received only water
(10 ml/kg, p.o.). Remaining Groups (II-VI) were administered
with STZ (60 mg/kg, i.p., in 0.1 M cold citrate buffer, pH 4.5)
15 min after the administration of NAD 100 mg/kg, i.p.) to
induce diabetes.[22] Glucose solution (20% in water) was
given to the STZ-NAD-treated rats for 24 h to avoid initial
hypoglycemic mortality induced by STZ-NAD. After 96 h
of the administration of STZ-NAD, blood samples of all the
animals groups were taken from tail vein for the estimation
of blood glucose levels. Diabetes was confirmed when blood
glucose level found above 250 mg/dL. After the induction
of diabetes, Group III was treated with standard drug GC
(10 mg/kg, p.o., in 0.5% CMC) and Groups IV-VI were
treated with the test drug CKR 100, 200, and 400 mg/kg, p.o.,
respectively, for 21 days. Body weights and blood glucose
level estimation were done weekly in overnight fasted
animals. On the next day, blood samples were collected from
a retro-orbital vein from the overnight fasted animals for
the estimation of various biochemical estimations. After the
experiment, all the animals were sacrificed for removal of
the liver.
Biochemical Analysis
Biochemical estimation kits (Span Diagnostic, Surat, Gujarat,
India) were used for the estimation of total cholesterol
(TC), triglyceride (TG), low density lipoprotein-cholesterol
(LDL-C), and high density lipoprotein-cholesterol (HDL-C)
estimation.
Tissue Preparation
The liver was carefully removed, weighed, and washed
with ice-cold saline to remove the traces of blood. The liver
tissue was sliced into pieces and homogenized (Glass Teflon
homogenizer, Thomas Scientific, Swedesboro, USA) in Tris–
HCl buffer (0.025 M, pH 7.5). The liver homogenate was
centrifuged at 10,000 g for 10 min at 41°C. The supernatant
was separated and used for the estimations of various
antioxidant enzymes.
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 36
Assay of Antioxidant Activity
The level of lipid per-oxidation (LPO) was estimated and
expressed in terms of malondialdehyde as per the method of
Ohkawa et al., 1979.[23] The activity of superoxide dismutase
(SOD) was estimated by following the procedure of Kakkar
et al., 1984[24] based on the reduction of NBT to blue colored
formazan in the presence of phenazine methosulfate. The
level of catalase (CAT) was estimated as per the method of
Sinha, 1972.[25]
Statistical Analysis
All the values of the experimental results were expressed
as mean ± standard error of mean. Two-ways ANOVA
followed by Bonferroni post-test was used to access effect
on normoglycemic, OGTT, and STZ-NAD-induced diabetic
rats. One-way ANOVA followed by Tukey’s multiple
comparison tests was performed for the statistical analysis
of the rest of parameters. Both the statistical analysis were
performed using GraphPad Prism, Version 5.0.1., Software.
RESULTS
Acute Oral Toxicity Study
Acute oral toxicity study of CKR did not show any toxicity
or behavioral changes at a dose level of 2000 mg/kg. This
finding suggests that CKR was safe or non-toxic to rats up
to 2000 mg/kg. The doses of CKR 100, 200, and 400 mg/kg,
b.w. were selected on the basis of pilot study for the in vivo
antidiabetic study.
Antidiabetic Study of CKR
Effect on blood glucose levels in fasted normal
rats
Figure 1 illustrates the effect of CKR on overnight fasted
rats. Two-way ANOVA revealed that there was a significant
difference between control group and treatment groups. GC
at the dose of 10 mg/kg, p.o., significantly reduced the blood
glucose level in rats when compared to normal control (NC)
group. However, CKR in all the doses tested did not show
any hypoglycemic effect on normal rats.
Effect on OGTT
The effect of CKR (100, 200, and 400 mg/kg, p.o.) on OGTT
is depicted in Figure 2. Two-way ANOVA indicates that there
were significant differences between experimental groups
after treatment. Animals treated with CKR (400 mg/kg), and
GC showed a significant decrease in blood glucose level
when compared to NC animals. The administration of CKR
significantly prevented the increase in blood glucose levels
without causing any hypoglycemic effect. The maximum
effect of CKR was observed at 30 and 60 min after the oral
glucose administration.
Effect on Fasting Blood Glucose Level of STZ-
NAD-Induced Diabetic Rats
Figure 3 indicates the effect of CKR (100, 200,
and 400 mg/kg, p.o.) on the STZ-NAD-induced diabetic
rats. Two-way ANOVA reveals that there were significant
differences in the experimental groups. A significant
increase in the level of blood glucose was observed in STZ-
NAD treated rats when compared to NC rats (P < 0.05).
Administration of CKR significantly reduced the fasting
blood glucose levels on 7th, 14th, and 21st days as compared
to diabetic control (DC). Treatment of diabetic rats with GC
also significantly reduced the increased blood glucose level.
Effect on plasma lipid profile
Effect of CKR on plasma lipid profile, i.e., TC, TGs, and
lipoproteins are shown in Table 2. The levels of plasma TC,
TGs, and LDL-C were significantly increased (P < 0.05),
whereas level of HDL-C was significantly decreased (P < 0.05),
in diabetic rats as compared to NC rats. The treatment of
Figure 2: Effect Chandrakala rasa on oral glucose tolerance
test in normal rats. *P < 0.05, compared to normal control;
#P < 0.05, compared to glibenclamide. (Two-way ANOVA
followed by Bonferroni post-test) (NC: Normal control,
Glib: Glibenclamide, CKR: Chandrakala rasa)
Figure 1: Effect of Chandrakala rasa on normal rats. Two-way
ANOVA followed by Bonferroni post-test revealed that there
was a non-significant difference between control group and
treatment group *P < 0.05 compared to normal control (NC:
Normal control, Glib: Glibenclamide, CKR: Chandrakala rasa)
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 37
rats with CKR reversed plasma lipid profile near to normal
values. This showed that treatment with CKR significantly
improved the lipid profile in diabetic animals. The effect of
CKR (200 and 400 mg/kg, p.o.) was found comparable with
that of GC (10 mg/kg, p.o.).
Effect on body weight
Effect of CKR treatment in rats on body weight and liver
glycogen are shown in Table 3. Changes of body weight in
DC group were remarkable in comparison to NC groups. The
mean body weight of diabetic rats was higher than control
group animals on the 21th day when treated with CKR, but
it was statistically significant (P < 0.05) for only 200 and
400 mg/kg, p.o. of CKR. The increase in body weight by the
administration of highest dose CKR was found comparable
to that GC treated group. A significant decrease (P < 0.05)
in liver glycogen content was observed in DC group as
compared to NC group. Rats treated with CKR 100 mg/kg,
p.o. did not show a significant increase in liver glycogen level;
however, rats treated with 200 and 400 mg/kg, p.o. showed
pronounced increases in liver glycogen level. GC treatment
also significantly increased (P < 0.05) liver glycogen level as
compared to DC rats.
Effect on antioxidant enzyme activity
Table 4 represents the concentration of thiobarbituric acid
reactive substances (TBARS) in liver samples of normal
and experimental rats. There was a significant elevation in
tissue TBARS in animals during diabetes as compared to the
normal group. Administration of CKR (200 and 400 mg/kg,
p.o.) significantly decreased the LPO in diabetic rats. The
effect of CKR at the dose level of 400 mg/kg, p.o. was found
comparable to GC. Statistical analysis by one-way ANOVA
on the effect of CKR on the activity of SOD and CAT showed
a significant effect of treatment with CKR. The activity of
SOD and CAT were found significantly lower in diabetic rats
as compared with their values in NC rats. Treatment with
CKR in diabetic rats significantly restored the enzyme levels
as compared to untreated diabetes animals.
DISCUSSION
The present study was designed to investigate the potential
antihyperglycemic, hypolipidemic, and antioxidant activity
of CKR in normal, glucose-loaded hyperglycemic, and STZ-
NAD-induced diabetic rats. The study revealed that CKR in
normoglycemic rats does not exert any significant decline in
blood glucose level, signifying that the CKR does not have
any hypoglycemic activity. However, the capacity of CKR to
lower blood glucose level in the OGTT suggests that animals
treated with CKR have better glucose utilization capacity.
Oral administration of CKR 100 mg/kg for 21 days caused
a significant decrease in blood glucose levels in diabetic
rats. Diabetes mellitus is a chronic metabolic disorder
characterized by hyperglycemia, basically due to over-
production or decreased utilization of glucose by the tissues.2
STZ-induced hyperglycemic condition is a most wildly
used model for evaluating the antidiabetic drugs. It causes
selective pancreatic islet β-cell necrosis mediated through the
release of nitric oxide and brings an increase in blood glucose
levels.[26] NAD, a potent antioxidant is added with STZ for
induction of Type II diabetes. It prevents the β-cell necrotic
action of STZ by free radicals scavenging activity and causes
only minor damage to pancreatic β-cell mass producing
Figure 3: Effect of Chandrakala rasa on the blood glucose
level of streptozotocin-nicotinamide-induced diabetic rats.
aP < 0.05, compared to normal control; bP < 0.05, compared
to diabetic control. (Two-way ANOVA followed by Bonferroni
post-test) (NC: Normal control, DC: Diabetic control,
Glib: Glibenclamide, CKR: Chandrakala rasa)
Table 2: Effect of CKR on lipid profile of STZ-NAD-induced diabetic rats
Group (n=6) Treatment (dose in mg/kg) TG (mg/dl) TC (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)
I NC 68.14±3.47 76.58±6.95 49.95±1.78 23.2±5.86
II DC 160.91±9.37a188.77±25.84a28.36±1.77a133.52±4.93a
III Glib (10) 79.71±4.58b91.7±5.3b51.16±3.73b29.89±4.87b
IV CKR (100) 115.69±19.55a147.02±12.68a32.48±1.94a83.69±11.3b
V CKR (200) 85.88±10.3b108.62±12.8b51.36±4.62b35.38±8.51b
VI CKR (400) 78.96±7.6b89.15±5.06b54.5±6.55b24.16±7.8b
Values are mean±SEM of six animals in each group. aP<0.05 compared to NC; bP<0.05 compared to DC; (One-way ANOVA followed
by Tukey’s multiple comparison test). NC: Normal control, DC: Diabetic control, Glib: Glibenclamide, CKR: Chandrakala rasa,
STZ: Streptozotocin, NAD: Nicotinamide, SEM: Standard error of mean
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 38
Type II diabetes.[27] For evaluating new antihyperglycemic
compounds in STZ-NAD-induced diabetes, sulfonylureas
such as GC are used as standard drug. Its action is mediated
through an increase in intracellular calcium in the β-cell,
which in turn stimulates insulin release.[28]
As the CKR did not cause the hypoglycemia in normoglycemic
rats but reduced blood glucose level in OGTT and STZ-NAD-
induced diabetic rats, CKR may act as antihyperglycemic,
rather than a hypoglycemic agent. Dyslipidemia is an
important factor in the determination of the course and status
of the disease. A reduction in insulin secretion causes a variety
of derangements in metabolic and regulatory mechanisms
leading to accumulation of lipids.[29] The findings of the
present study clearly show that CKR significantly reduced
the TG and TC in diabetic rats. Lipid-lowering effect of drugs
in diabetes reduces the risk of vascular complications.[30]
Changes in lipid profile (increase the level of TG, TC, LDL,
VLDL, and decrease HDL) increases the risk for coronary
heart diseases in diabetic patients.[31] HDL-C reduces the risk
of cardiovascular diseases through free radical scavenging
and anti-inflammatory actions and promotes the efflux of
cholesterol from the peripheral tissues to the liver.[32]
Altered carbohydrate metabolism promotes increased muscle
wasting, structural degradation of proteins or loss of muscle
proteins resulted in a decline in body weight.[33] An increase in
the body weight of diabetic rats treated with CKR (400 mg/kg
p.o.) suggesting a protective role of CKR on muscle wasting
might be due to the improvement in glycemic control and
increase synthesis of structural proteins.[34] Extracellular
glucose concentration and blood insulin level are two key
factors for conversion of glucose to glycogen in the liver cells.
The observed depletion of liver glycogen level in DC rat was
possibly due to the inactivation of the glycogen synthetase
systems or increased activity of glycogen phosphorylase,
reflecting of insulin deficiency.[35] The present study showed
the significant increase in the liver glycogen in diabetic rats
treated with CKR, may be due to the reactivation of glycogen
synthetase system which is responsible for the improvement
in the liver glycogen synthesis. Hence, CKR interferes
with glucose utilization and metabolism by storing excess
carbohydrates as glycogen.
Chronic hyperglycemia and impaired insulin secretion
may contribute to a reduction in levels of enzymatic (CAT)
and non-enzymatic antioxidants (total thiols) along with
increased free radicals regeneration, which can lead to
increased LPO.[36] This enhanced oxidative stress on the
β-cells was proposed as a key contributor to the development
of diabetes mellitus and its complications.[37] Enhanced levels
of TBARS in diabetic rats indicate the excessive formation
Table 3: Effect of CKR on body weight and liver glycogen in STZ-NAD-induced diabetic rats
Group
(n=6)
Treatment
(dose in mg/kg)
Body weight (g) Liver glycogen
(mg/g)
0th day 21th day
I NC 179.16±6.75 175.26±6.32 28.95±1.36
II DC 168.50±6.18 120.37±3.65a13.23±1.38a
III Glib (10) 174.66±5.72 169.87±5.3b23.93±1.62b
IV CKR (100) 162.33±3.71 139.28±4.78a15.92±0.32a
V CKR (200) 176.66±9.27 154.38±4.68ab 20.08±0.79ab
VI CKR (400) 174.16±5.97 164.36±2.89b24.72±1.42b
Values are mean±SEM of six animals in each group. aP<0.05 compared to NC; bP<0.05 compared to DC (one-way ANOVA followed
by Tukey’s multiple comparison test). NC: Normal control; DC: Diabetic control, Glib: Glibenclamide, CKR: Chandrakala rasa,
STZ: Streptozotocin, NAD: Nicotinamide, SEM: Standard error of mean
Table 4: Effect of CKR on TBARS, SOD, and CAT in STZ-NAD-induced diabetic rats
Group
(n=6)
Treatment
(dose in mg/kg)
TBARS
(nmol/mg protein)
SOD
(U/mg protein)
CAT (µ mol. H2O2
consumed/min/mg protein)
I NC 24.88±1.62 0.81±0.10 241.50±8.78
II DC 49.91±5.55a0.42±0.03a160.85±9.45a
III Glib (10) 29.79±2.08b0.78±0.12b239.86±20.13b
IV CKR (100) 40.25±2.85a0.54±0.05 213.90±8.30
V CKR (200) 33.89±3.96b0.76±0.05b235.46±15.95b
VI CKR (400) 26.42±3.11b0.86±0.05b238.69±12.59b
Values are mean±SEM of six animals in each group. aP<0.05 compared to normal control; bP<0.05 compared to diabetic control. (One-way
ANOVA followed by Tukey’s multiple comparison test). NC: Normal control, DC: Diabetic control, Glib: Glibenclamide, CKR: Chandrakala
rasa, TBARS: Thiobarbituric acid reactive substances, SOD: Superoxide dismutase, CAT: Catalase, STZ: Streptozotocin,
NAD: Nicotinamide, SEM: Standard error of mean
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 39
of free radicals and activation of LPO system which leads
to damage of membrane through LPO of unsaturated fatty
acids.[38] SOD and CAT remove free radicals and play a vital
role in maintaining the cell integrity. A decrease in the activity
of SOD and CAT can lead to an excessive accumulation of
free radicals (superoxide and hydrogen peroxide), which in
turn generate ROS, resulting in initiation and propagation of
LPO.[39] Therefore, decreased LPO and improved antioxidant
status by the CKR may be one of the mechanisms by
which CKR could contribute to the prevention of diabetic
complications.[40]
The result of the present study clearly shows that CKR
has free radical scavenging and anti-LPO potential. All the
herbal ingredients of CKR have antioxidant activity.[41-45]
The phytochemical screening of CKR revels the presence
of wide range of phytoconstituents such as alkaloids,
terpenoid, phenolics, glycosides, steroids, polysaccharides,
etc. Triterpenoids of B. ceiba were previously reported for
antidiabetic activity.[46] Furthermore, three alkaloids viz.,
palmatine, jatrorrhizine, and magnoflorine obtained from
T. cordifolia[47,48] have been reported for their antidiabetic
effect. Emblica officinalis also has antidiabetic activity.[49]
Shilajit, a herbomineral preparation used in the long-term
management of diabetes mellitus because of its multifaceted
action. It produces a better glycemic control along with
improvement in the lipid profile in animals.[50]
Antidiabetic activity of CKR may be due to the presence
of more than one antihyperglycemic principle and their
synergistic properties. Certain classes of compounds viz.
flavonoids, triterpenoids/sterols, alkaloids, and phenolics
are known to be bioactive antidiabetic principles. Phenolics
are found to be effective antihyperglycemic agents.[51] The
literature reveals that antioxidant activity of plant extract is
mainly due to the presence of phenolic compounds, which
may exert antioxidant effects as free radical scavengers, as
hydrogen donating sources or as singlet oxygen quenchers
and metal ion chelators.[51]
CONCLUSION
In the present investigation, we demonstrated that CKR
restored the altered serum glucose level, body weight; lipid
profile level near normal in STZ-induced diabetic rats. CKR
significantly enhanced the levels of endogenous antioxidant
enzymes (CAT, and SOD). On the basis of the findings
of the present study, it is clear that CKR has significant
hypoglycemic, hypolipidemic, and antioxidant potential.
REFERENCES
1. De Silva DD, Rapior S, Hyde K, Bahkali AH. Medicinal
mushrooms in prevention and control of diabetes
mellitus. Fungal Divers 2012;56:1-29.
2. Ansarullah, Bharucha B, Dwivedi M, Laddha NC,
Begum R, Hardikar AA, et al. Antioxidant rich
flavonoids from Oreocnide integrifolia enhance glucose
uptake and insulin secretion and protects pancreatic
ß-cells from streptozotocin insult. BMC Complement
Altern Med 2011;11:126.
3. Chan JC, Malik V, Jia W, Kadowaki T, Yajnik CS,
Yoon KH, et al. Diabetes in Asia: Epidemiology, risk
factors, and pathophysiology. JAMA 2009;301:2129-40.
4. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the
prevalence of diabetes for 2010 and 2030. Diabetes Res
Clin Pract 2010;87:4-14.
5. Mamtani M, Kulkarni H, Dyer TD, Almasy L,
Mahaney MC, Duggirala R, et al. Waist circumference
independently associates with the risk of insulin
resistance and type 2 diabetes in Mexican American
families. PLoS One 2013;8:e59153.
6. Sabitha V, Ramachandran S, Naveen KR,
Panneerselvam K. Antidiabetic and antihyperlipidemic
potential of Abelmoschus esculentus (L.) Moench. in
streptozotocin-induced diabetic rats. J Pharm Bioallied
Sci 2011;3:397-402.
7. Modak M, Dixit P, Londhe J, Ghaskadbi S,
Devasagayam TP. Indian herbs and herbal drugs used
for the treatment of diabetes. J Clin Biochem Nutr
2007;40:163-73.
8. Dewanjee S, Das AK, Sahu R, Gangopadhyay M.
Antidiabetic activity of Diospyros peregrina fruit: Effect
on hyperglycemia, hyperlipidemia and augmented
oxidative stress in experimental type 2 diabetes. Food
Chem Toxicol 2009;47:2679-85.
9. Bandawane D, Juvekar A, Juvekar M. Antidiabetic and
antihyperlipidemic effect of Alstonia scholaris Linn.
bark in streptozotocin induced diabetic rats. Indian J
Pharm Educ Res 2011;45:114-20.
10. Vishwakarma SL, Sonawane RD, Rajani M, Goyal RK.
Evaluation of effect of aqueous extract of Enicostemma
littorale Blume in streptozotocin-induced type 1 diabetic
rats. Indian J Exp Biol 2010;48:26-30.
11. Shastri AD. Bhaishajyaratnavali. 19th ed. Varanasi:
Chaukhambha Prakashan; 2008.
12. Das B, Mitra A, Hazra J. Management of Madhumeha
(diabetes mellitus) with current evidence and intervention
with Ayurvedic rasausadhies. Indian J Tradit Knowl
2011;10:624-8.
13. Rasheed A, Naik M, Mohammed-Haneefa KP, Arun-
Kumar RP, Azeem AK. Formulation, characterization
and comparative evaluation of x: A herbo-mineral Indian
traditional medicine. Pak J Pharm Sci 2014;27:793-800.
14. Alkilany AM, Murphy CJ. Toxicity and cellular uptake
of gold nanoparticles: What we have learned so far? J
Nanopart Res 2010;12:2313-2333.
15. Nagaraju V, Joshi D, Aryya NC. Toxicity studies on
Vanga Bhasma (Part I - with special reference to G. I. T.
liver and pancreas). Anc Sci Life 1984;4:32-5.
16. Bhatia B, Kale PG. Analytical evaluation of an Ayurvedic
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 40
formulation - Abhraka Bhasma. Int J Pharm Sci Rev Res
2013;23:17-23.
17. Gupta VK, Patgiri BJ. Standard manufacturing
procedure of Lauha Bhasma using Triphala media and
by employing electric muffle furnace heating. Ann Ayu
Med 2012;1:87-94.
18. Singh N, Reddy KR. Pharmaceutical study of Lauha
Bhasma. Ayu 2010;31:387-90.
19. Kupati VB, Jadar PG. Namburi phased spot test analysis
(NPST) of Rasasindura (Herbomineral preparation). Int
J Sci Res 2014;3:19-20.
20. OECD. Guidelines for Testing of Chemicals. Guidance
No. 425. Acute Oral Toxicity, Up and Down Procedure
(UDP); 2008. Available from: http://www.ntp.niehs.
nih.gov/iccvam/suppdocs/feddocs/OECD/OECDtg425.
pdf. [Last Accssed on 2013 Jan 03].
21. Hashim MA, Yam MF, Hor SY, Lim CP, Asmawi MZ,
Sadikun A. Anti-hyperglycaemic activity of swietenia
macrophylla king (meliaceae) seed extracts in
normoglycaemic rats undergoing glucose tolerance tests.
Chin Med 2013;8:11.
22. Bagri P, Ali M, Aeri V, Bhowmik M, Sultana S.
Antidiabetic effect of Punica granatum flowers: Effect
on hyperlipidemia, pancreatic cells lipid peroxidation
and antioxidant enzymes in experimental diabetes. Food
Chem Toxicol 2009;47:50-4.
23. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides
in animal tissues by thiobarbituric acid reaction. Anal
Biochem 1979;95:351-8.
24. Kakkar P, Das B, Viswanathan PN. A modified
spectrophotometric assay of superoxide dismutase.
Indian J Biochem Biophys 1984;21:130-2.
25. Sinha AK. Colorimetric assay of catalase. Anal Biochem
1972;47:389-94.
26. Szkudelski T. The mechanism of alloxan and
streptozotocin action in B cells of the rat pancreas.
Physiol Res 2001;50:537-46.
27. Fukaya M, Tamura Y, Chiba Y, Tanioka T, Mao J, Inoue Y,
et al. Protective effects of a nicotinamide derivative,
isonicotinamide, against streptozotocin-induced ß-cell
damage and diabetes in mice. Biochem Biophys Res
Commun 2013;442:92-8.
28. Rabbani SI, Devi K, Khanam S. Protective role of
glibenclamide against nicotinamide-streptozotocin
induced nuclear damage in diabetic Wistar rats.
J Pharmacol Pharmacother 2010;1:18-23.
29. Shukla K, Dikshit P, Tyagi MK, Shukla R, Gambhir JK.
Ameliorative effect of Withania coagulans on
dyslipidemia and oxidative stress in nicotinamide-
streptozotocin induced diabetes mellitus. Food Chem
Toxicol 2012;50:3595-9.
30. Movahedian A, Zolfaghari B, Sajjadi SE, Moknatjou R.
Antihyperlipidemic effect of Peucedanum
pastinacifolium extract in streptozotocin-induced
diabetic rats. Clinics (Sao Paulo) 2010;65:629-33.
31. Anwar M, Shousha WG, El-Mezayen HA, Raafat AW,
El-Wassef M, Naglaa MN, et al. Antiatherogenic effect
of almond oil in streptozotocin induced diabetic rats.
J Appl Pharm Sci 2013;3:59-65.
32. Eren E, Yilmaz N, Aydin O. High density lipoprotein and
it’s dysfunction. Open Biochem J 2012;6:78-93.
33. Bhardwaj M, Soni A, Mishra S, Tripathi S. Protective
effect of Commiphora wightii in metabolic activity of
streptozotocin (STZ) induced diabetes in rats. J Diabetes
Endocrinol 2014;5:19-28.
34. Eliza J, Daisy P, Ignacimuthu S, Duraipandiyan V.
Normoglycemic and hypolipidemic effects of costunolide
isolated from Costus speciosus (Koen ex. Retz.) Sm. in
streptozotocin-induced diabetic rats. Chem Biol Interact
2009;179:329-34.
35. Renjith RS, Rajamohan T. Protective and curative effects
of Cocos nucifera inflorescence on alloxan-induced
pancreatic cytotoxicity in rats. Indian J Pharmacol
2012;44:555-9.
36. Rahman K. Studies on free radicals, antioxidants, and
co-factors. Clin Interv Aging 2007;2:219-36.
37. Tiwari BK, Pandey KB, Abidi AB, Rizvi SI. Markers
of oxidative stress during diabetes mellitus. J Biomark
2013;2013:378790.
38. Subash-Babu P, Alshatwi AA, Ignacimuthu S.
Beneficial antioxidative and antiperoxidative effect
of cinnamaldehyde protect streptozotocin-induced
pancreatic β-cells damage in wistar rats. Biomol Ther
(Seoul) 2014;22:47-54.
39. Sellamuthu PS, Arulselvan P, Kamalraj S, Fakurazi S,
Kandasamy M. Protective nature of mangiferin on
oxidative stress and antioxidant status in tissues of
streptozotocin-induced diabetic rats. ISRN Pharmacol
2013;2013:750109.
40. Matough FA, Budin SB, Hamid ZA, Alwahaibi N,
Mohamed J. The role of oxidative stress and antioxidants
in diabetic complications. Sultan Qaboos Univ Med J
2012;12:5-18.
41. Poltanov EA, Shikov AN, Dorman HJ,
Pozharitskaya ON, Makarov VG, Tikhonov VP,
et al. Chemical and antioxidant evaluation of
Indian gooseberry (Emblica officinalis Gaertn. syn.
Phyllanthus emblica L.) supplements. Phytother Res
2009;23:1309-15.
42. Vishwakarma S, Chandan K, Jeba RC, Khushbu S.
Comparative study of qualitative phytochemical
screening and antioxidant activity of Mentha arvensis,
Elettaria cardamomum and Allium porrum. Indo Am J
Pharm Res 2014;4:2538-56.
43. Lee HJ, Hyun EA, Yoon WJ, Kim BH, Rhee MH,
Kang HK, et al. In vitro anti-inflammatory and anti-
oxidative effects of Cinnamomum camphora extracts.
J Ethnopharmacol 2006;103:208-16.
44. Sivakumar V, Rajan MS. Antioxidant effect of Tinospora
cordifolia extract in alloxan-induced diabetic rats. Indian
J Pharm Sci 2010;72:795-8.
45. Gandhare B, Soni N, Hemant JZ. In-vitro antioxidant
activity of Bombax ceiba. Int J Biomed Res 2012;1:31-6.
46. Bhavsar CJ, Talele GS. Potential anti-diabetic activity of
Singh, et al.: Antidiabetic activity of Chandrakala rasa
International Journal of Green Pharmacy • Jan-Mar 2016 • 10 (1) | 41
Bombax ceiba. Bangladesh J Pharmacol 2013;8:102-6.
47. Patel MB, Mishra S. Hypoglycemic activity of alkaloidal
fraction of Tinospora cordifolia. Phytomedicine
2011;18:1045-52.
48. Patel MB, Mishra SM. Magnoflorine from Tinospora
cordifolia stem inhibits α-glucosidase and is antiglycemic
in rats. J Funct Foods 2012;4:79-86.
49. Ansari A, Shahriar MS, Hassan MM, Das SR, Rokeya B,
Haque MA, et al. Emblica officinalis improves glycemic
status and oxidative stress in STZ induced type 2 diabetic
model rats. Asian Pac J Trop Med 2014;7:21-5.
50. Trivedi NA, Mazumdar B, Bhatt JD, Hemavathi KG.
Effect of Shilajit on blood glucose and lipid profile
in alloxan-induced diabetic rats. Indian J Pharmacol
2004;36:373-6.
51. Kumar R, Patel DK, Prasad SK, Laloo D,
Krishnamurthy S, Hemalatha S. Type 2 antidiabetic
activity of bergenin from the roots of Caesalpinia digyna
Rottler. Fitoterapia 2012;83:395-401.
Source of Support: Nil. Conflict of Interest: None declared.
