Potential mechanism of anti-diabetic activity of Picrorhiza kurroa

Abstract

Picrorhiza kurroa Royle ex Benth. (Scrophulariaceae) is a traditional Ayurvedic herb known as Kutki. It is used as a remedy for diabetes by tribes of North Eastern Himalayan region of India. Present study was conducted to explore the mechanism of antidiabetic activity of standardized aqueous extract of Picrorhiza kurroa (PkE). PkE (100 and 200 mg/kg/day) was orally administered to streptozotocin induced diabetic rats, for 14 consecutive days. Plasma insulin levels were measured and pancreas of rat was subjected to histopathological investigations. Glucose transporter type 4 (GLUT-4) protein content in the total membrane fractions of soleus muscle was estimated by Western blot analysis. Plasma insulin level was significantly increased along with concomitant increase in GLUT-4 content of total membrane fractions of soleus muscle of diabetic rats treated with extract. There was evidence of regeneration of β-cells of pancreatic islets of PkE treated group in histopathological examinations. PkE increased the insulin-mediated translocation of GLUT-4 from cytosol to plasma membrane or increased GLUT-4 expression, which in turn facilitated glucose uptake by skeletal muscles in diabetic rats.

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Original Article
Potential mechanism of anti-diabetic activity of Picrorhiza kurroa
Gulam Mohammed Husain1, Richa Rai2, Geeta Rai2, Harikesh Bahadur Singh3, Ajit Kumar Thakur1, Vikas Kumar1,*
1Neuropharmacology Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu
University), Varanasi, India; 2Department of Molecular and Human Genetics, Faculty of Science, Banaras Hindu University,
Varanasi, India; 3Department of Mycology & Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University,
Varanasi, India
ABSTRACT
Picrorhiza kurroa Royle ex Benth. (Scrophulariaceae) is a traditional Ayurvedic herb known as Kutki. It
is used as a remedy for diabetes by tribes of North Eastern Himalayan region of India. Present study was
conducted to explore the mechanism of antidiabetic activity of standardized aqueous extract of Picrorhiza
kurroa (PkE). PkE (100 and 200 mg/kg/day) was orally administered to streptozotocin induced diabetic
rats, for 14 consecutive days. Plasma insulin levels were measured and pancreas of rat was subjected to
histopathological investigations. Glucose transporter type 4 (GLUT-4) protein content in the total
membrane fractions of soleus muscle was estimated by Western blot analysis. Plasma insulin level was
significantly increased along with concomitant increase in GLUT-4 content of total membrane fractions
of soleus muscle of diabetic rats treated with extract. There was evidence of regeneration of β-cells of
pancreatic islets of PkE treated group in histopathological examinations. PkE increased the insulin-
mediated translocation of GLUT-4 from cytosol to plasma membrane or increased GLUT-4 expression,
which in turn facilitated glucose uptake by skeletal muscles in diabetic rats.
Keywords Picrorhiza kurroa, type 2 diabetes, GLUT-4, pancreas, histopathology
INTRODUCTION
Type 2 diabetes mellitus has become a significant health
problem in both developed and developing countries. The
International Diabetes Federation has estimated that the
worldwide prevalence of diabetes mellitus is expected to
increase from 382 million people in 2013 to 592 million by
2035. There were 72.1 million people with diabetes mellitus in
the South East Asia region in 2013 and this number is expected
to increase to 123 million by 2035. India alone has 65.1 million
people living with diabetes mellitus, this places India second to
China with 98.41 million diabetic people (International
Diabetes Federation, 2013). Therefore, effective and safe
therapeutic interventions are necessary to deal with the
emerging epidemic of diabetes.
Diabetes is characterized by reduced insulin-mediated
glucose uptake associated with reduced Glucose transporter
type 4 (GLUT-4) expression (Berger et al., 1989). Down-
regulation of GLUT-4 in muscle and adipose tissue has also
been reported in type 2 diabetes (Kellerer et al., 1999).
Streptozotocin (STZ)-induced diabetes has also shown
significant low level of expression of GLUT-4 in rat muscles
(Hardin et al., 1993). It has suggested that the level of GLUT-4
expression determines the maximal effect of insulin on glucose
transport. Anti-diabetic treatments like phenolic rich plant
extracts are reported to improve expression as well as
translocation of GLUT-4 from cytosol to the plasma membrane
of skeletal muscle in STZ-induced diabetic rats (Ong et al.,
2011).
Picrorhiza kurroa Royle ex Benth. (Family:
Scrophulariaceae) is a small perennial herb, indigenous to India
and is commonly found on the Alpine Himalayas from Kashmir
to Sikkim at the altitude ranging from 3,000 - 5,000 m. It is a
well-known herb in the Ayurvedic system of medicine and has
traditionally been used to treat disorders of the liver and upper
respiratory tract, fever, dyspepsia, and chronic diarrhea
(Dwivedi et al., 1992; Luper, 1998). Kutkin is the active
principle of Picrorhiza kurroa and is comprised of kutkoside
and the iridoid glycoside picrosides I, II, and III (Basu et al.,
1970). Other identified active constituents are apocynin, drosin,
and nine cucurbitacin glycosides. Apocynin is a catechol that
has been shown to inhibit neutrophil oxidative burst in addition
to being a powerful anti-inflammatory agent, while the
cucurbitacins have been shown to be highly cytotoxic and
possess antitumor effect (Luper, 1998; Stuppner and Wagner,
1989).
Picrorhiza kurroa is mentioned in the classical text books
of Ayurveda as ‘Kutki’ and being used as a remedy for diabetes
by tribes of North Eastern Himalayan region (Chhetri et al.,
2005). Picrorhiza kurroa in various herbal formulations like
Picroliv, Livokin, Picrolax, Livomap, Tefroliv, Katuki, Arogya,
Kutaki and other related formulations have been used as
complementary and alternative medicine to treat a wide variety
of ailments (Ansari et al., 1991; Kumar et al., 2013). Earlier
preliminary findings have confirmed the antidiabetic effect of
Picrorhiza kurroa in rats as significant reduction in elevated
fasting blood glucose and control over dyslipidemia (Husain et
al., 2009). In the present study, we have investigated the
mechanism of antidiabetic effect of extract of Picrorhiza
kurroa (PkE) in rats. Apart from measurement of insulin level,
*Correspondence: Vikas Kumar
E-mail:
vikas.phe@iitbhu.ac.in
Received
March 20, 2014; Accepted November 17, 2014;
Published
November
30, 2014
doi: http://dx.doi.org/10.5667/tang.201
4.0013
©201
4 by Association of Humanitas Medicine
This is
an open access article under the CC BY-NC license.
(http://creativecommons.org/licenses/by-nc/3.0/)

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histopathological investigations of pancreas were performed to
observe the effect of PkE on pancreatic islets of β-cell.
Moreover, GLUT-4 content of soleus muscle of rats have been
analyzed using Western blot in order to explore the molecular
targets of PkE responsible for antidiabetic activity.
MATERIALS AND METHODS
Plant material
Dried aqueous extract of rhizomes of Picrorhiza kurroa was
procured in the month of December, 2008 from Promed
Research Centre, Gurgaon, India. The extract was standardized
to contain 5% kutkin using HPTLC. The plant material was
authenticated by Dr. Sateesh Chauhan (Promed Research
Centre, Gurgaon, India) and a voucher specimen (No.
Nov/2008/12) was preserved.
Animals
Adult Charles Foster rats (180 ± 10 g) were obtained from the
Central Animal House of the Institute of Medical Sciences,
Banaras Hindu University, Varanasi, India. The animals were
housed at the ambient temperature of 25 ± 1°C and 45-55%
relative humidity, with a 12:12 h light/dark cycle. Principles of
laboratory animal care guidelines (NIH publication #85-23,
revised in 1985) were always followed. Protocol of the study
was approved by Animal Ethics Committee of Banaras Hindu
University, Varanasi, India (Letter No. Dean/2009-10/693).
Induction of type 2 diabetes
Non insulin dependent diabetes mellitus (NIDDM) was induced
in overnight fasted rats by a single intraperitoneal (i.p.)
injection of 65 mg/kg streptozotocin (STZ; Merck, Germany),
15 min after the i.p. administration of 120 mg/kg nicotinamide
(SD Fine Chem, Mumbai, India) (Masiello et al., 1998).
Fasting blood samples were collected from the retro-orbital
venous plexus under light ether anaesthesia using a glass
capillary tube. Plasma was separated and glucose and insulin
levels were analysed. Hyperglycemia was confirmed by the
elevated glucose level (higher than 200 mg/dl) in the blood,
determined at 72 h and then on day 7 after injection (Wu and
Huan, 2008).
Animal grouping and drugs treatment
Rats were randomly assigned into different treatment groups
(n=6) as follows: Group I: Diabetic Control (vehicle-treated),
Group II: Normal Control (non-diabetic; vehicle treated),
Group III: Diabetic + PkE 100 mg/kg, Group IV: Diabetic +
PkE 200 mg/kg, and Group V: Diabetic + Glibenclamide 10
mg/kg. The doses of PkE were determined based on earlier
study with this extract (Husain et al., 2009). All the treatments
were started on 7th day after induction of diabetes (day 1 of
treatment), in the form of oral suspension in 0.3%
carboxymethyl cellulose (CMC), once daily for 14 days.
Histopathological study and GLUT-4 contents were analyzed in
high dose treated PkE group (i.e., 200 mg/kg).
Fasting plasma insulin measurement
Plasma insulin was measured at 0 day (before treatment) and
14th day of treatment by ELISA method (DRG Diagnostics,
GmbH, Germany) by using a microplate reader (Bio-Rad, CA,
USA).
GLUT-4 assay using Western blot
Rats were sacrificed on 14th day one h after the last dose
administration. Soleus muscle samples were isolated and
pooled for GLUT-4 assay. Protein was extracted from soleus
skeletal muscle by lysis in 50 mM Tris-Cl, 150 mM NaCl, 5
mM EDTA, 1% Triton X-100 for 20 min at 4°C. Dithiothreitol
and phenylmethylsulfonylfluoride were added to the lysis
buffer at the final concentration of 1mM (Zhou et al., 1998).
Protein was further quantified by the Bradford assay.
Protein (75 μg) was separated on 10% SDS polyacrylamide
gel and transferred to polyvinylidene fluoride membrane
overnight at 4°C at 30V using Bio-Rad mini trans-blot system
(Bio-Rad, CA, USA). Membrane was blocked with 5% BSA in
TBST (Tris-Buffered Saline Tween-20) for 3 h, followed by
incubation with primary antibody anti-GLUT-4 (IF8; Santa
Cruz biotechnology, CA, USA; 1:500) and anti -GAPDH
(Imgenex, Bhubaneswar, India; 1:2000) overnight at 4°C.
Washing was done with TBST for 3 times for 5 min each. The
blot was incubated with secondary antibodies; anti-mouse for
GLUT-4 (Santa Cruz Biotechnology, CA, USA; 1:1000) and
anti-rabbit for GAPDH (Santa Cruz biotechnology; 1:3000)
conjugated with horseradish peroxidase (HRP) for 2 h at room
temperature. After washing, the blot was developed using the
substrate for HRP i.e. tetramethylbenzidine (TMB; Genei,
Bangalore, India). The whole assay was conducted in triplicate
and densitometry analysis was done by using AlphaImager®
software.
Histopathological study
The pancreas was isolated immediately from the rats after
sacrificing and rinsed in ice-cold saline. The tissue samples
were fixated with 10% formaldehyde, dehydrated in a graded
series of ethanol, embedded in paraffin wax before section
preparation (about 5µm thickness) by using a microtome (Ross
et al., 1989). The sections were then stained with Haematoxylin
and Eosin dye and studied using digital optical microscope
(Nikon E200- Trinocular Microscope, Japan) for
histopathological changes.
Statistical analysis
Data of all experiments were expressed as Mean ± SEM (n = 6).
Statistical analysis was performed by one way ANOVA
followed by Student-Newman-Keuls multiple comparison test.
RESULTS
Effect on plasma insulin level
Plasma insulin level of diabetic rats was significantly reduced
compared to normal control rats. Both the doses of PkE
Fig. 1. Effect of Picrorhiza kurroa and glibenclamide on fasting insulin
level in diabetic rats.
*p < 0.001 vs. normal control; †p <
0.01 vs.
diabetic control.

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significantly increased plasma insulin level (increased by
34.13%, 47.62%, and 47.49% in 100 mg/kg PkE, 200 mg/kg
PkE and 10 mg/kg glibenclamide treated diabetic rats,
respectively) compared to vehicle treated diabetic control rats
on day 14 (Fig. 1).
Effect on GLUT-4
GLUT-4 protein content in total membrane fractions of STZ-
induced diabetic rats were significantly reduced compared to
normal control. PkE significantly increased the GLUT-4 protein
level compared to diabetic control. Glibenclamide treatment
did not significantly increase GLUT-4 protein compared to
diabetic control (Fig. 2).
Histopathological study
Normal control rats showed normal exocrine pancreatic acinar
architecture and pancreatic islets showing predominantly
insulin-producing β-cells with granular basophilic cytoplasm
and lesser glucagon-producing alpha cells with eosinophilic
cytoplasm. Diabetic rats challenged with nicotinamide-
streptozotocin showed marked decrease population of insulin
producing β-cells with granular basophilic cytoplasm and
abundance of eosinophilic glucagon-producing α-cells.
Shrinkage and vacuolization of islets and growth of fibrous
tissue was also visible. There were areas of eosinophilic
amorphous deposits within islets, suggesting cellular necrosis.
Two weeks treatment with PkE showed sign of regeneration of
islet β-cell and reversal of histopathological changes induced
by streptozotocin challenge. Glibenclamide treated rats showed
normal acini and endocrine islet of Langerhans with increased
β-cell mass due to regeneration (Fig. 3).
DISCUSSION
Stimulation of insulin release from remnant β-cell (Proks et al.,
2002) is involved in the observed effects of PkE in diabetic rats.
This is consonant with increased plasma insulin level in the
PkE treated rats, which is further supported by regeneration o f
islet β-cell observed following histopathological examinations
of pancreas compared to diabetic control rats.
Blood glucose regulation by insulin is mediated by
increased glucose transport into insulin sensitive tissues like
skeletal muscle, cardiac muscle and adipose tissue, which
express GLUT-4 and mediates the hormonal effect of insulin
(Zhou et al., 1998). Under physiological conditions, GLUT-4 is
localized in cytoplasm as intracellular membrane structures and
is translocated to the plasma membrane in response to insulin.
Insulin-stimulated translocation of glucose transporter protein
within the cell membrane is the rate-limiting step in
carbohydrate metabolism of skeletal muscle (Ziel et al., 1988).
Moreover, modulation of GLUT-4 expression as well as
translocation of GLUT-4 from cytosol to the plasma membrane
of skeletal muscle determines the maximal effect of insulin on
glucose transport (Ong et al., 2011).
PkE increased GLUT-4 content in the total membrane
fractions of skeletal muscle of STZ-induced diabetic rats,
which could be due to increased insulin mediated translocation
of GLUT-4 from cytosol to membrane. Another possibility
might be increased expression of GLUT-4 in the soleus muscle
in the present study. Consequently, there is an increase in
GLUT-4 mediated glucose uptake by the skeletal muscles.
Therefore, translocation of GLUT-4 to the membrane from
cytosol appears to be the major mechanism for the observed
antidiabetic activity of PkE. Glibenclamide, used in the present
study as standard anti-diabetic agent, is reported to inhibit the
ATP-sensitive K+ channels in pancreatic β-cells, which results
in cell membrane depolarization and opening of voltage-
dependent calcium channel. The intracellular level of Ca++ in
the β-cell increases and results in stimulation of insulin release
(Luzi and Pozza, 1997; Rai et al., 2012). There are equivocal
reports regarding the effect of glibenclamide on GLUT-4
expression. Some studies reported increased GLUT-4 protein
content in plasma membrane by glibenclamide treatment
(Kosegawa et al., 1999; Nizamutdinova et al., 2009). However,
Kern et al. (1993) reported no significant effect of
glibenclamide on the GLUT-4 protein level in skeletal muscle.
In the present study we did not find statistically significant
alteration in GLUT-4 protein of glibenclamide treated group.
The lack of effect of glibenclamide on GLUT-4 in diabetic rats
is consonant with the findings of Kern et al. (1993) which
showed that glibenclamide ensue glucose uptake through some
mechanism other than alterations in the level of the GLUT-4
glucose transporter protein.
PkE is widely used in India with no reported adverse
effects. The LD50 of kutkin is more than 2600 mg/kg in rats
Fig. 2. Typical immunoblot of GLUT-4 in the total membrane
fraction of soleus muscle.
*p < 0.001 vs. normal control; †p <
0.001
vs. diabetic control.
Fig. 3.Photomicrographs of Pancreas (Haematoxylin & Eosin
staining; X100). (A) Normal control
(non-
diabetic), (B) Diabetic
control, (C) Diabetic + PkE (200 mg/kg), and (D) Diabetic +
Glibenclamide. Hollow arrows indicate pancreatic islets; Solid arrows
indicate vacuolization of pancreatic islets.

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(Luper, 1998). Therefore, the doses tested in the present
investigation do not warrant any concern with regard to the
safety of Picrorhiza kurroa extract. Administration of
streptozotocin and appropriate protective dose of nicotinamide
induce a diabetic syndrome with reduced pancreatic insulin
stores that mimics some features NIDDM not shared by other
established animal models of diabetes (Masiello et al., 1998;
Tahara et al., 2008). Co-administration of nicotinamide and
STZ in rats is an experimental model based on selective
toxicity of STZ in β-cells by mitochondrial damage and
oxidative stress (Friederich et al., 2009; Masiello et al., 1998).
Human diabetes, in contrast, is not induced by STZ and it is not
known how oxidative toxicity contributes to β-cell
degeneration. Therefore, finding of the present study do not
necessarily prove that PkE will also be active in human
diabetes. It is possible that polyphenols in the PkE interfere
with the specific toxicity and apoptogenic effect of STZ.
Further clinical studies are required to establish the efficacy of
PkE in diabetic patients.
The present study revealed that standardized PkE increased
the insulin-mediated translocation of GLUT-4 from cytosol to
plasma membrane which result better glucose uptake by
skeletal muscles and improved glycaemic control in diabetic
rats. It may be concluded that GLUT-4 is at least partly
involved in the observed antidiabetic activity of PkE in rats.
ACKNOWLEDGEMENTS
GM Husain is grateful to the University Grants Commission,
Government of India, New Delhi, for providing the financial
assistance. Authors are thankful to Promed Research Centre,
Gurgaon, India for providing standardized extract of Picrorhiza
kurroa.
CONFLICT OF INTEREST
The authors do not have any conflict of interest in the present
study.
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