Antidiabetic activity of hydro-alcoholic stem
bark extract of Callicarpa arborea Roxb. with
antioxidant potential in diabetic rats
Julfikar Ali Junejo, Mithun Rudrapal, Lalit Mohan Nainwal, Kamaruz
Department of Pharmaceutical Sciences, Dibrugarh University,
Dibrugarh, Assam, India
Biomedicine & Pharmacotherapy Vol.95 , 2017
• Diabetes mellitus (or Diabetes) is a
metabolic disease characterized by
hyperglycemia with abnormal
carbohydrate, fat and protein
metabolism resulting from defects
in insulin secretion, insulin action/both.
• Oxidative stress (OS) is believed to be the underlying cause of cellular
injury, tissue damage or organ dysfunctions commonly associated
with diabetic complications.
• Callicarpa arborea Roxb., known as beautyberry (English), is a shrub
or small evergreen tree, which belongs to family Verbenaceae.
Ethnobotanical study indicates that paste of the stem bark and juice
of C. arborea has been used by the tribals of Mizoram state of
Northeast India to cure cuts and wounds.
The leaves infusion has also been used for the management of
Moreover, since a biochemical relationship exists between diabetic
hyperglycemia and cellular oxidative stress, the antidiabetic activity
evaluation of the hydro-alcoholic extract (HAE) of C. arborea stem
bark was carried out along with the antioxidant activity study.
2. Materials and Methods
All chemicals and reagents, Commercial reagent kits were procured
from Rankem, Mumbai and Himedia Laboratories Ltd., Mumbai, and
SPAN Diagnostics Ltd., Surat (India).
2.2 Plant material
Stem bark of Callicarpa arborea Roxb. were collected from forest areas
of Dibrugarh district, Assam during the month of December 2014.
The plant species was identiﬁed and authenticated.
2.3 Preparation of hydro-alcoholic extract (HAE)
100 g of powdered stem barks was used for the preparation of extract.
Powdered stem barks were extracted using sufﬁcient quantity (1000
ml) of ethanol:water mixture (7:3) by cold maceration for 24 h.
2.4 Test Animals
Healthy Wistar male albino rats (240–260 g)
2.5 Phytochemical screening
Preliminary phytochemical screening of the HAE was carried out for
detection of the presence of various phytoconstitutents.
2.6 Estimation of phenolic and ﬂavonoid contents
The total phenolic content of the HAE was evaluated following the
Folin-Ciocalteu colorimetric method
The total ﬂavonoid content was estimated using the aluminum chloride
2.7 In vitro antioxidant activity
The in vitro antioxidant activity of HAE was carried out by the following
three assay methods in accordance with previously reported
procedures with minor modiﬁcations. Lipid peroxidation scavenging
activity, Hydroxyl radical scavenging activity and Superoxide radical
2.8 Acute oral toxicity study
Over-night fasted rats were randomly divided into six groups of six
Rats of different groups were administered with increasing doses (250,
500, 1000, 2000 and 2000 mg/kg b.w.) of the HAE.
One group was maintained as normal control and was given vehicle
The animals were observed individually for ﬁrst one hour for any gross
behavioral changes if any, and then periodically for the next 24 h, and
then at every 24 h for any signs of acute toxicity over a period of 14
2.9 Oral glucose tolerane (OGT) test
Glucose 2 g/kg was fed orally 30 min after the administration of HAE
and metformin hydrochloride.
Blood was withdrawn from the tail vein at 0, 30, 60, 90 and 120 min
Group 1 (n=6) Normal control vehicle (0.5% CMC w/v in NS).
Group 2 (n=6) Test group HAE at 250 mg/kg b.w.
Group 3 (n=6) Test group HAE at 500 mg/kg b.w.
Group 4 (n=6) Treatment group metformin hydrochloride (5
2.10 Hypoglycemic activity in streptozotocin-induced diabetic rats
Diabetes was induced in overnight fasted animals by a single
intraperitoneal (i.p) injection of streptozotocin (STZ, 55 mg/ kg b.w. in
Blood was collected from the tail vein each time for the determination
of glucose levels on 0, 7, 14 and 21 day.
Group 1 (n=6) Normal control vehicle (0.5% CMC w/v in NS).
Group 2 (n=6) Diabetic control vehicle (0.5% CMC w/v in NS).
Group 3 (n=6) Test group 1 HAE at 250 mg/kg b.w.
Group 4 (n=6) Test group 2 HAE at 500 mg/kg b.w.
Group 5 (n=6) Standard group metformin hydrochloride (5 mg/kg
2.11 Liver and kidney function tests
Blood was collected , The serum was separated by centrifugation
(2000 rpm, 10 min) for estimation of various biochemical
Serum insulin levels were measured by the microplate ELISA method
using a commercial kit (SPAN Diagnostics Ltd.).
Serum lipid proﬁle was estimated using commercially available kits
(SPAN Diagnostics kit). Serum was used to estimate glutamate
oxaloacetate transaminase (GOT), glutamate pyruvate
transaminase (GPT) and alkaline phosphatase (ALP), total protein
(TPR) and creatinine (CRTN).
2.12 In vivo antioxidant activity
On 21st day, all the groups of animals were anaesthetized using
diethyl ether, liver was dissected out, washed with normal saline
and one part was preserved in 10% formalin for histopathological
The other part of liver was homogenized by ice chilled Tris- HCl
buffer and used for activities/levels of superoxide dismutase (SOD),
catalase, reduced Glutathione (GSH), Glutathione peroxidase
(GPx), and malondialdehyde (MDA).
2.13 Histopathological studies
At the end of 21th day of treatment, the animals were fasted for 12 h,
anaesthetized using diethyl ether and sacriﬁced by cervical dislocation.
Pancreas and liver were instantly dissected out, excised and rinsed in
ice-cold saline solution. Tissues were processed and after ﬁxation,
tissues were dehydrated in ethanol (70–95%), cleared in xylene, and
embedded in parafﬁn, solid transverse sections of 4–5 mm thickness
were obtained by using a rotary microtome.
The section were stained with haematoxin-eosin and histopathological
observations were carried out under a light microscope.
2.14 Statistical analysis
one-way ANOVA followed by Student’s t-test.
3.1 Phytochemical screening
The percent yield of crude dried extract was found to be 68.78%, w/w
per dry weight of powdered stem barks. The results of preliminary
phytochemical screening revealed the presence of alkaloids,
glycosides, saponins, tannins, ﬂavonoids and phenolic compounds.
3.2 Acute toxicity study
No sign and symptoms of acute toxicity and mortality up to 2000
mg/kg body weight dose were observed during the whole
For further studies, the doses were ﬁxed as 250 and 500 mg/kg body
3.3 In vitro antioxidant activity
Fig. 3.3.1. Superoxide radical scavenging activity. Values are mean ± SEM
of three replicate experiments. HAE, quercetin and gallic acid exhibited 78.47 ±
0.20%, 82.12 ± 0.44% and 85.33 ± 0.34% of scavenging effect respectively at the
highest tested concentration of 250 mg/ml.
Fig.3.3.2. Hydroxyl radical scavenging activity. Values are mean ± SEM
of three replicate experiments. The percent inhibition of hydroxyl radicals
were 83.45 ± 0.18%, 87.37 ± 0.30% and 86.14 ± 0.49 for HAE, quercetin and gallic
acid respectively at the concentration of 250 mg/ml.
Fig. 3. Lipid peroxidation scavenging activity.
Values are mean ± SEM of three replicate experiments.
%Scavenging activity of HAE is statistically signiﬁcant at p < 0.05,
compared to quercetin & gallic acid (standards). 64.11 ± 0.27%, while quercetin and gallic
acid inhibited by 74.27 ± 0.65%, and 71.09 ± 0.34%, respectively at 250 mg/ml
Fig. 4. OGT test. Values are mean ± SEM of three replicate
experiments. Activities of HAE and metformin are statistically
signiﬁcant at p < 0.05, compared to normal control.
In OGT, HAE (250 and 500 mg/kg) showed signiﬁcant (p < 0.05) reduction of glucose load (plasma glucose level) as compared to normal
3.8. Effect of HAE on lipid proﬁle in diabetic rats
In diabetic rats, the levels of triglycerides (TG), total cholesterol (TC),
and low density lipoprotein (LDL) were signiﬁcantly increased and
high density lipoprotein (HDL) level was signiﬁcantly decreased. In
HAE (250 and 500 mg/kg) treated groups the TG, TC and LDL levels
activities were signiﬁcantly (p < 0.05) reduced and the HDL level was
signiﬁcantly (p < 0.05) increased as compared to diabetic control rats.
Increased levels of MDA, an indicator of LPO, in diabetic rats were signiﬁcantly (p < 0.05)
reduced after treatment with HAE (250 & 500 mg/kg) as compared to the normal rats.
Fig. 5. Histology of pancreas of experimental rats after treatment with HAE, 500
(A) Normal control, (B) Diabetic control, (C) Diabetic treated with HAE (500
mg/kg), (D) Diabetic treated with metformin.
Fig. 6. Histology of liver of experimental rats after treatment with HAE 500
mg/kg. (A) Normal control, (B) Diabetic control, (C) Diabetic treated with
HAE (500 mg/kg), (D) Diabetic treated with metformin.
The intraperitoneal administration of STZ damages partially the
insulin secreting b-cells of the pancreas by breaking DNA strands
leading to decreased endogenous insulin release which ultimately
results in diabetes mellitus.
Administration of HAE to diabetic rats showed a signiﬁcant reduction
in the levels of blood glucose and an increase in the levels of serum
insulin and it was further supported by histopathological
The liver glycogen content was markedly reduced in diabetic
animals, which was in proportion to insulin deﬁciency.
Diabetic rats treated with HAE increased signiﬁcantly the liver
glycogen content as compared to the diabetic control, which could
be due to increased insulin secretion.
Under normal circumstances insulin activates enzyme
lipoprotein lipase and hydrolyses triglycerides.
Diabetic rats treated with HAE signiﬁcantly improved
serum TG and TC.
The signiﬁcant control of the levels of serum lipids in the
HAE treated diabetic rats might be attributed to
improvements in insulin levels.
Signiﬁcant lowering of LDL cholesterol and raise in HDL
cholesterol were observed in treated diabetic rats.
Diabetic rats showed signiﬁcantly increased level of CRTN.
The signiﬁcant reduction in the level of CRTN in HAE
treated diabetic rats indicated that the HAE prevented the
progression of renal damage in diabetic rats.
In diabetes mellitus, high glucose level can inactivate
antioxidant enzymes SOD, CAT, GSH and GPx by glycating
these proteins thus producing induced oxidative stress,
which in turn, causes lipid peroxidation.
Furthermore, malonaldehyde (MDA) is one of the end
products in LPO process.
LPO in the tissue homogenate was determined by measuring the
amounts of MDA produced primarily.
SOD, CAT, GSH and GPx activities were increased to normal
indicating the efﬁcacy of HAE in attenuating the oxidative stress
(OS) and eventual inhibition of LPO in diabetic liver.
Decrease in MDA level indicated reduced rate of LPO in HAE
In this study, a marked increase in the concentration of TBARS and
MDA were observed in STZ induced diabetic rats indicating the LPO
of tissues under OS.
Since the HAE signiﬁcantly decreased TBARS levels as well as MDA
in liver of diabetic rats indicating strong lipid peroxidation
scavenging activity of the HAE as antioxidant agent. 30
Superoxide directly initiates LPO and plays an important role in the
formation of other ROS like hydroxyl radicals, which induce oxidative
damage in lipids, proteins and DNA.
Hydroxyl radical is directly involved in the LPO process. Hydroxyl
radicals are more potent than superoxides, and HAE could effectively
scavenge these radicals together with the inhibition of LPO, where in
it scavenged active oxygen species by preventing the propagation of
free radical chain reaction as antioxidant.
It is now assumed that the antioxidant activity is responsible for the
antidiabetic action of the HAE, and phenolic compounds and
ﬂavonoids present in the HAE may be involved in reducing underlying
cellular OS and eventual hypoglycemic reactions.
The present study concludes that HAE showed potent hypoglycemic
activity in diabetic rats compared to normal rats with signiﬁcant
improvement in body weight, levels of serum insulin, liver glycogen,
serum lipids, liver and kidney biochemical markers and liver
antioxidant enzymes levels of experimental animals.
The antioxidant activity of C. arborea stem bark reported herein
signiﬁes the potential of this plant species as herbal antioxidant with
possible role in the prevention of oxidative stress-induced diabetes
and associated disease complications.
The authors declare that there are no conﬂicts of interest.
1)Title – Clear, brief
Study design was not mentioned
2) Abstract - Provided an accurate summary of the
background, key methods, principal findings and
conclusions of the study
3) Background- Included sufficient scientific
4) Objectives - Yes, objectives were Clearly described.
5) Ethical statement – Yes, animal ethics committee approval
6) Study design - The number of experimental and control
groups was mentioned for each experiment; sampling
method was mentioned
7) Experimental procedures – were explained in detail,
method of euthanasisa also mentioned.
8) Experimental animals - Provided details of the animals
used, including species, strain, sex, developmental stage,
weight, source of animal
9) Housing and husbandary – type of facility, type of cage,
number of cage companions was mentioned.
10) Sample size - the number of animals in each group was
mentioned in each experiment.
Doesn’t mention How sample size was calculated?
11) Allocating animals to experimental groups – full details of
how animals were allocated, treated and assessed in different
experimental groups given.
12)Experimental outcomes -Yes, they were Clearly defined
13) Statistical methods – Yes, details of the statistical method
used for each analysis was mentioned
14) Baseline data- Yes, it was mentioned.
15) Numbers analysed- the mean of all the animals
6/6 present in each group was used in analysis
16) Outcomes and estimation – Yes, it was
mentioned. P < 0.05 was considered statistically
significant, all the outcomes were mentioned
graphically and in tabular form.
17) Adverse events- No, there were no adverse
18) Interpretation/ scientific implications- Yes,
interpretations of the study were mentioned
19) Generalisability/ translation- Yes, the findings of
the study were relevant to human biology
20 ) Funding – NO, source of funding was not
3.3.3. Lipid peroxidation scavenging activity
LP induced by Fe2+/ascorbate in rat liver homogenate was
found to be inhibited by the HAE in a concentration
dependant manner and a considerable amount of lipid
peroxidation inhibitory effect was observed by 64.11 ±
0.27%, while quercetin and gallic acid inhibited by 74.27 ±
0.65%, and 71.09 ± 0.34%, respectively at 250 mg/ml
Test results were considered statistically signiﬁcant when
compared to standard drugs (p < 0.05). 40
3.11. Histopathological observations
3.11.1. Effect of HAE on pancreatic section in normal and diabetic
Histopathological studies of pancreas (Fig. 5) of STZ-treated diabetic
rats exhibited reduction in the dimensions of islets, damaged b-cell
population and extensive necrotic changes followed by ﬁbrosis and
HAE (500 mg/kg) and metformin treated rats restored the necrotic
and ﬁbrotic changes and also increased the number and increased the
size of the islets (C).
In normal control group normal acini and normal cellular in the islets
of langerhans in the pancreas were observed (A). 41
3.11.2 Effect of HAE on liver section in normal and diabetic rats
Photomicrographs of liver (Fig. 6) showed normal hepatic cells with
well preserved cytoplasm, nucleus, nucleolus and central vein (A).
In case of diabetic rats, the normal lobular structure was preserved.
The central vein was prominent and prominently congested. Focal
areas of hemorrhage were also seen. Vacuoliza- tion and fatty change
were evident. The portal tracts appeared normal (B).
In diabetic treated group (HAE 500 mg/kg), the hepatocytes portal
tracts and central veins appeared normal (C).
HAE treated group is comparable with metformin treated group (D)
and changes in treated groups are signiﬁcant (p < 0.05) compared to
normal control group. 42
Hinweis der Redaktion
Hindi- kumhar, nepali – guren,
like drowsiness, restless- ness, writhing, convulsions and symptoms of toxicity and mortality
The acute toxicity study was done as per OECD guideline-425.
Carboxymethylcellulose (CMC) solution (0.5% w/v in normal saline) was used as vehicle.
The animals conﬁrmed as diabetic (after 72 h of STZ injection) by the elevated plasma glucose levels (200– 300 mg/dl) was used for the experiment.
The total phenolic content of the HAE, calculated from the calibration curve of gallic acid (R2 = 0.986), was 52.17 ± 2.48 GE/g, and
the total ﬂavonoid content (R2 = 0.988), calculated from the calibration curve of quercetin was 39.10 ± 2.15 QE/g.
3.6. Effect of HAE on blood glucose levels in diabetic rats
STZ-treated diabetic rats exhibited signiﬁcant increase in the levels of blood glucose in comparison to normal rats.
After treatment with HAE the blood glucose levels were signiﬁcantly (p < 0.05) reduced compared to the diabetic control rats at both the doses 250 & 500 mg/kg Table 2.
The activity of HAE was found less than that of metformin (5 mg/kg) treated group.
In diabetic rats the body weight, insulin level and glycogen content were signiﬁcantly decreased.
After 21 days of treatment with HAE at 250 and 500 mg/kg the body weight was signiﬁcantly (p < 0.05) increased, insulin level and glycogen content were also signiﬁcantly (p < 0.05) increased as compared to diabetic rats
Metormin (5 mg/kg) treated rats also showed signiﬁcant effects on blood levels of SGOT, SGPT, ALKP, TPR and CRTN in diabetic rats.
3.9. Effect of HAE on SGOT, SGPT, ALKP, TPR and CRTN in diabetic rats
There was a signiﬁcant increase in activities of SGOT, SGPT and ALKP in diabetic rats.
After treatment with HAE (250 & 500 mg/kg) the activities of SGOT, SGPT and ALKP activities were signiﬁcantly (p < 0.05) reduced as compared to diabetic control rats.
A signiﬁcant decrease in serum total protein (TPR) level and a signiﬁcant increase in creatinine (CRTN) level were observed in diabetic rats.
After treatment with HAE at 250 and 500 mg/kg doses for 21 days the TPR level was signiﬁcantly increased (p < 0.05) and CRTN level was signiﬁcantly (p < 0.05) decreased compared to diabetic control rats.
Metformin (5 mg/kg) also showed signiﬁcant (p < 0.05) reduction of these enzymes.
3.10. Effect of HAE on liver enzymes and MDA
In treated diabetic rats, the activities of SOD, CAT, GSH and GPx were signiﬁcantly increased.
There was a signiﬁcant (p < 0.05) reduction in the activities of these antioxidant enzymes in diabetic rats as compared to normal rats.
which clearly revealed that diabetic treated (HAE) animals showed increase in the number of islets, lesser degree of shrinkage and restoration of necrosis of b-cells of pancreas.
The decrease in liver glycogen content in diabetes is due to the lack of insulin which ultimately results in the inactivation of glycogen synthase enzyme.The signiﬁcant increase in the glycogen levels of the HAE treated diabetic animals might be because of the reactivation of glycogen synthase system.
In insulin deﬁcient diabetics, the plasma free fatty acid concentration is elevated as a result of increased free fatty acid outﬂow from fat depots, where the balance of the free fatty acid esteriﬁcation–triglyceride lipolysis cycle is displaced in favour of lipolysis.
SOD, CAT, GSH and GPx are enzymatic antioxidants that play a vital role in preventing oxidative damage to cells.
SOD reduces the superoxide radical into hydrogen peroxide (H2O2).
The other enzymatic antioxidant CAT catalyzes the reduction of hydrogen peroxides into water molecule and protects the tissues against reactive hydroxyl radicals.
When cell has increased levels of SOD without a proportional increase in GPx, cells face a peroxide overload challenge.
Attenuating – reducing
STZ produces oxygen radicals in the body, which cause pancreatic injury and could be responsible for increased blood sugar level in animals.
Moreover, abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to the development of insulin resistance.
The changes in pancreas morphology in metformin treated group (D) are similar to HAE treated rats, where changes are signiﬁcant (p < 0.05) compared to normal control group.