European Journal of Pharmacology 667 (2011) 410–418

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European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Endocrine Pharmacology

Antidiabetic activity of γ-sitosterol isolated from Lippia nodiflora L. in streptozotocin

induced diabetic rats
Rangachari Balamurugan, Veeramuthu Duraipandiyan, Savarimuthu Ignacimuthu ⁎
Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai 600034, India

a r t i c l e i n f o a b s t r a c t

Article history: Lippia nodiflora L. (Verbenaceae) is a creeping perennial herb widely used in traditional system of medicine
Received 9 November 2010 to treat ulcers, bronchitis and heart diseases; it also possesses antidiabetic property. In the present study,
Received in revised form 26 April 2011 γ-sitosterol isolated from Lippia nodiflora was screened for its antidiabetic property in streptozotocin (STZ)
Accepted 11 May 2011
induced diabetic rats. Insulin secretion in response to glucose was evaluated in isolated rat islets. Oral
Available online 7 June 2011
administration of γ-sitosterol (20 mg/kg body weight) once daily for 21 days in STZ-induced diabetic rats
Keywords:
resulted in a significant decrease in blood glucose and glycosylated hemoglobin with a significant increase in
Lippia nodiflora plasma insulin level, body weight and food intake. Furthermore γ-sitosterol showed antihyperlipidemic
Streptozotocin activity as evidenced by significant decrease in serum total cholesterol, triglycerides and very low density
γ-Sitosterol lipoprotein-cholesterol levels coupled with elevation of high density lipoprotein-cholesterol levels in treated
Antidiabetic rats. A significant decrease in the activities of alanine aminotransaminase, aspartate aminotransaminase,
Insulin alkaline phosphatase and acid phosphatase in γ-sitosterol treated rats when compared to diabetic control rats
Hepatic marker enzyme indicated its protective role against liver damage. γ-Sitosterol increased insulin secretion in response to
glucose. Immunohistochemical study of pancreas also confirmed the biochemical findings. These results
indicated that γ-sitosterol, the compound isolated from L. nodiflora, possessed antihyperglycemic activity.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction Lippia nodiflora L. (Verbenaceae) is a creeping perennial herb with

Type 2 diabetes mellitus, also known as noninsulin dependent
diabetes mellitus, develops in middle or later life and affects 2–6% of
adults in most Western societies (Bailey, 2000). World Health
Organization (WHO) estimates that more than 220 million people
worldwide have diabetes and this number is likely to double by 2030
(Aragao et al., 2010). The pharmacological agents currently used for
the treatment of type 2 diabetes include sulfonylurea, biguanide,
thiazolidinedione and α-glycosidase inhibitors. These agents, how-
ever, have restricted usage due to several undesirable side effects and
fail to significantly alter the course of diabetic complications (Rang
and Dale, 1991). The high prevalence of diabetes as well as its long-
term complications has led to an ongoing search for hypoglycemic
agents from natural sources (Nicasio et al., 2005). Herbal remedies
have been used since ancient times for the treatment of diabetes
mellitus. About 90% of the world population in rural areas of
developing countries relies solely on traditional medicines for their
primary health care (Hassan et al. 2010).
⁎ Corresponding author at: Entomology Research Institute, Loyola College,
Nungambakkam, Chennai 600 034, Tamil Nadu, India. Tel.: + 91 44 2817 8348; fax:
+ 91 44 2817 5566.
E-mail address: entolc@hotmail.com (S. Ignacimuthu).
small white flowers; it is found in wet grounds and grassy pastures.
The plant is distributed throughout India, Ceylon, Baluchistan, and
Africa. In the Ayurveda system the plant is used as an aphrodisiac and
to treat ulcers, bronchitis and heart diseases. In Yunani medicine, the
plant is used as diuretic and to treat fever and cold. The herb possesses
cooling and diuretic properties and stops knee joint pain. The plant
made into a poultice is used as a maturant for boils (Durairaj et al.
2008).
Antimalarial activity has been reported from the herb (Mukherjee,
1991); leaves of the plant were reported to possess anti-inflammatory,
analgesic, and antipyretic activity (Forestieri et al., 1996). Gastro-
protective effect has also been described (Khalil et al., 2007).
Hypoglycemic property of a methanol extract of L. nodiflora has
been reported by us (Balamurugan et al., 2010). Resin, stigmasterol,
β-sitosterol, sugars (Pacual et al., 2001) as well as essential oil
constituents such as monoterpenes and sesquiterpenes (Terblanche
and Kornelius, 1996) have been isolated from the plant and
identified. Presence of Lippiflorin A and Lippiflorin B, flavonoids like
nepetin, jaceosidin, hispidulin, flavone monosulfates and flavone
disulfates has also been reported from aerial parts (Tomas-Barberan
et al., 1987). γ-Sitosterol, a steroidal compound, was isolated from
L. nodiflora by us. It has been previously isolated from soya and its
antihyperlipidemic activity has been described (Best et al., 1954). The
same compound has also been reported to occur in Abelmoschus manihot
(Jain et al., 2009) and Polygonum bistorta (Manoharan et al., 2005). There

0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.05.025
 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418
are no reports on the hypoglycemic effects of γ-sitosterol. This study
was carried out to assess the antidiabetic property of γ-sitosterol in
STZ-induced diabetic rats.
2. Materials and methods
2.1. Plants
L. nodiflora L. (Verbenaceae) was collected from Salem district,
Tamil nadu, India in January 2007. Dr. S. Somasundaram, National
Institute of Siddha, Chennai, identified the plant. The voucher
specimen (ERIP-4) was deposited in the herbarium at the Entomology
Research Institute, Loyola College, Chennai.
2.2. Isolation and identification of active compound
3 kg of L. nodiflora whole plant powder was soaked in 9 l of
methanol for 72 h with intermittent shaking. The extract was filtered
through Buchner funnel and concentrated using vacuum rotary
evaporator at 40 °C. Ninety-seven grams of crude methanol extract
was obtained and dissolved in methanol (200 ml) and water
(300 ml). The solution was partitioned with diethyl ether in a 1:1
ratio using a separating funnel. The diethyl ether part was concen-
trated to obtain the extract. Fifty four grams of this extract was
chromatographed on a silica gel column (Merck 10–200 mesh, 750 g
3.5 i.d. × 60 cm) and successively eluted with stepwise gradient of
hexane:ethyl acetate solvent system (5%, 25%, 50%, 75% and 100%).
184 fractions were collected and finally five fractions were obtained
based on TLC profiles. A light green color precipitate was obtained in
fraction 2 eluted with 75% ethyl acetate and 25% hexane solvent
system. This was finally washed with 100% ethyl acetate, spotted on a
precoated silica gel (60 F254) s 0.25 mm thick TLC plate (Merck) and
run in toluene:ethyl acetate (9:1) solvent system. Single spot was
obtained. It was subjected to gas chromatography–mass spectrometry
(GC–MS) analysis.
2.3. Gas chromatography–mass spectrometry (GC–MS)
Fraction 2 was quantified using gas chromatograph (GCMS-
Shimadzu) equipped with a CPB-capillary column (mm i.d. × 50 m
length) mass spectrometer (ion source 200 °C, RI 70 eV) programmed
at 40–280 °C with a rate of 4 °C/min. Injector temperature was 280 °C;
carrier gas was He (20 psi).
2.4. Experimental animal
Healthy adult Wistar male albino rats raised at Entomology
Research Institute, Loyola College were used for the study; their
body weight reached 170–235 g and they were 60–70 days old at the
moment of experiments. The rats were housed individually in poly
propylene cages and maintained under standard conditions (12 h
light and 12 h dark cycle, 25 ± 3 °C); they were fed with standard rat
pellet diet (Pranav agro industry Ltd, Maharastra) and water
ad libitum. The study was approved by the Animal Ethics Committee
of the Institute (IAEC-ERI-LC-10).
2.5. Experimental induction of non-insulin dependent diabetes mellitus
Non-insulin dependent diabetes mellitus was induced in overnight
fasted adult Wistar strain male albino rats weighing 170–235 g by
single intraperitoneal injection of freshly prepared streptozotocin,
(Sigma-Aldrich, Bangalore) (40 mg/kg body weight) in 0.1 M citrate
buffer (pH = 4.5). Seven days after STZ administration blood glucose
level of each rat was determined. Rats with blood glucose level above
200 mg/dl were considered diabetic and were included in the study.
411
2.6. Experimental design
The rats were divided into five groups with six rats in each group.
10% DMSO was used as vehicle. Single dose of γ-sitosterol (20 mg/kg
body weight) was suspended in vehicle (10% DMSO) and was
administered orally every day for 21 days.
Group 1 Served as normal control (10% DMSO alone)
Group 2 Normal rats + 20 mg/body weight of γ-sitosterol in 10%
DMSO
Group 3 Diabetic control (10% DMSO alone)
Group 4 Diabetic rats + 20 mg/kg body weight of γ-sitosterol in 10%
DMSO
Group 5 Diabetic rats + glibenclamide 600 μg/kg body weight in 10%
DMSO
Body weight and food intake were measured on the first and the
last day of the experiment. 21 days after administration animals were
decapitated, blood was collected, and serum was separated immedi-
ately. Liver, muscle and tissues were excised and stored at − 40 °C for
biochemical estimations. Pancreas was dissected out for immunohis-
tochemical work.
2.7. Biochemical analysis
2.7.1. Estimation of fasting plasma glucose and plasma insulin
Fasting glucose level was estimated by glucose oxidase–peroxidase
method (Triender, 1969). The assay of insulin in the serum of normal
and STZ-induced diabetic rats was performed by ELISA.
2.7.2. Estimation of liver and muscle glycogen level
The glycogen level of liver and skeletal muscles was measured by
anthrone method (Carrol et al. 1956).
2.7.3. Estimation of glycosylated hemoglobin
Glycosylated hemoglobin (HbA1c) was estimated by the method
of Nayak and Pattabiraman (1981).
2.7.4. Estimation of glucose 6-phosphatase and glucose 6-phosphate
dehydrogenase
Glucose 6-phosphatase was determined by the method of Koide
and Oda (1959). Glucose 6 phosphate dehydrogenase was estimated
by the method of Bergmeyer (1984).
2.7.5. Estimation of lipid profile
Lipid profile (total cholesterol, high-density lipoprotein-cholesterol
and triglyceride) levels in serum were determined according to the
instructions of the manufacturer (Merck, Mumbai, India). Very low-
density lipoprotein cholesterol was calculated by Friedewald's formula:
VLDL: Triglycerides/5 (Friedewald et al., 1972).
2.7.6. Estimation of plasma liver marker enzymes
Alanine aminotransaminase (ALT) and aspartate aminotransami-
nase (AST) activities were determined by the method of Reitman and
Frankel (1957). The activity of serum alkaline phosphatase (ALP) and
acid phosphatase (ACP) was measured by the method of King (1959).
2.7.7. Insulin secretion experiments
Non-fasting male Wistar rats (180–250 g bodyweight) were
euthanized by cervical dislocation and decapitation. Islets from rat
pancreas were isolated by collagenase digestion method (Moskalewski,
1965). In all the in vitro experiments islets were preincubated with
0.5 ml of incubation buffer containing 11.1 mM glucose alone or in
combination with various concentrations of γ-sitosterol (5, 10, 15 and
20 μg/kg body weight) at 37 °C for 30 min in a shaking water bath under
95% O2 and 5% CO2 atm. After the preincubation the buffer was removed,
412 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418
new additions were made in a similar fashion and incubated under
similar conditions. Aliquots of 50 μl were removed from static
incubation mixture at 0, 10 and 60 min and were frozen immediately
until insulin assay was performed using ELISA method. For inhibition
experiments, islets were preincubated with 0.25 mM diazoxide or
0.25 mM EGTA or 0.25 mM nimodipine along with and without 20 μg
γ-sitosterol and 11.1 mM glucose for 30 min. Insulin released after 10
and 60 min was measured by ELISA method as indicated above.
2.7.8. Immunohistochemical analysis
For immunohistochemical examination, the pancreas was re-
moved, washed in saline, kept in 10% formalin and fixed in paraffin.
The preparation was washed in running water overnight and then
sectioned (5 μm in thickness). Sections were used for immunohisto-
chemical analysis. The sections of control and treated rats were
dewaxed, then incubated for 1 h at room temperature in 0.3%
hydrogen peroxide in PBS, followed by 3 washings in PBS. Sections
were then incubated for 16 h, at 4 °C, in PBS containing 2% normal
goat serum (NGS) and 0.5% triton X-100 and washed in PBS. They
were then incubated for overnight at 4 °C with the primary
monoclonal antibody (Mouse anti-EMA, anti-insulin). Incubation
was followed by 3 washes in PBS-2% NGS. The primary antibodies
Fig. 1. GC–MS spectral analysis of γ-sitosterol isolated from L. nodiflora. A—GC–MS chromotograph of γ-sitosterol. B—mass spectra of γ-sitosterol.
were bounded by a biotinylated anti-mouse secondary antibody in
PBS for 1 h at room temperature. Sections were then incubated in
avidin–biotin complex linked to peroxidase. The peroxidase was
visualized with 0.03% diaminobenzidine hydrochloride and 0.005%
hydrogen peroxide in 0.1 M Tris buffer. Sections were counterstained
with hematoxylin (Hsu et al. 1981).
2.7.9. Statistical analysis
One way ANOVA and Student's t-test (SPSS program; version 11.5)
were carried out to compare the data with the level of significance set
at 0.05 against diabetic control.
3. Results
3.1. Identification of active compound
The purified fraction 2 was quantified using GC–MS chromatograph.
The GC–MS spectrum revealed the presence of compound γ-sitosterol
(100%). The molecular weight of the compound was m/z: 414, 43(100),
396, 381, 329, 303, 273 (Fig. 1). From the GC–MS analysis fraction 2 was
identified as γ-sitosterol (Fig. 2). The mass spectral values corresponded
to literature (Jain et al., 2009).
 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418 413

3.4. Effect of γ-sitosterol on plasma insulin and glycosylated hemoglobin

(HbA1c)

Table 3 presents the effect of γ-sitosterol on plasma insulin levels

and glycosylated hemoglobin levels in normal and STZ-induced
diabetic rats after 21 days of treatment. In diabetic rats, there was a
significant decrease in plasma insulin (− 78.83%) and an increase in
HbA1c (49.60%) when compared to normal control rats. Oral
administration of γ-sitosterol significantly increased plasma insulin
(69.05%), and decreased HbA1c (66.96%) when compared to diabetic
control rats.

3.5. Effect of γ-sitosterol on liver and muscle glycogen content

Table 4 shows the liver and muscle glycogen content in normal

and diabetic rats. There was a decrease in liver (78.18%) and muscle
Fig. 2. Structure of γ-sitosterol. (81.98%) glycogen content in STZ-induced diabetic rats when
compared to normal control rats. When γ-sitosterol (20 mg/kg body
weight) was administered to diabetic rats for 21 days it increased the
muscle (76.86%) and hepatic (77.15%) glycogen content significantly
3.2. Effect of γ-sitosterol on body weight when compared to diabetic control rats.

Tables 1 and 2 show the effect of γ-sitosterol on body weight and

food intake of normal and STZ-induced diabetic animals. The STZ-
induced animals showed decrease in body weight (− 36.13%) and an
increased in food intake compared to normal control animals. Oral
administration of γ-sitosterol (20 mg/kg body weight) for 21 days
significantly increased (7.6%) body weight and decreased food intake
(−54.41%) of diabetic animals compared to diabetic control animals.
3.3. Effect of γ-sitosterol on fasting blood glucose
Plasma glucose level was measured in normal and experimental
animals on days 0, 7, 15 and 21 of the treatment. STZ-induced diabetic
rats showed significant increase in the levels of blood glucose
when compared to normal rats. Oral administration of γ-sitosterol
(20 mg/kg body weight) resulted into a significant decrease in fasting
blood glucose level compared to diabetic control (Table 2).
3.6. Effect of γ-sitosterol on carbohydrate metabolism enzymes
Table 5 shows the activities of carbohydrate metabolism enzymes in
the liver of normal and STZ-induced diabetic rats. The activity of glucose
6 phosphatase enzyme increased in diabetic rats whereas the activity of
glucose 6 phosphate dehydrogenase decreased when compared with
normal control rats. Oral administration of γ-sitosterol (20 mg/kg body
weight) decreased the activity of glucose 6 phosphatase (40.07%) and
increased the activity of glucose 6 phosphate dehydrogenase (38.52%)
significantly when compared to diabetic control rats.
3.7. Effect of γ-sitosterol on lipid profile
Table 6 shows the activity of γ-sitosterol on total cholesterol, HDL-
cholesterol, triglycerides and VLDL-cholesterol in treated, normal control
and diabetic control rats. Administration of γ-sitosterol for 21 days

Table 1
Effect of γ-sitosterol on body weight and food intake in STZ-induced diabetic rats.

Group Body weight Food intake

0 day(g) 21st day(g) 0 day g/day 21st day g/day

Normal control 186.25 ± 8.98 203.75 ± 7.46 17.86 ± 1.40 19.68 ± 0.63
Normal + γ-sitosterol 20 mg/kg body weight 187.50 ± 7.77 203.75 ± 6.88 16.42 ± 1.15 16.30 ± 1.70
Diabetic control 202.50 ± 12.99 148.75 ± 3.14 34.83 ± 1.24 36.04 ± 1.78
Diabetic + γ-sitosterol 20 mg/kg body weight 196.25 ± 4.26 210.00 ± 5.40a 33.97 ± 0.92 16.45 ± 1.09a
Diabetic + glibenclamide 600 μg/kg bodyweight 186.25 ± 3.721 215.00 ± 6.45b 34.63 ± 1.39 21.25 ± 0.99b

All values are mean ± S.E.M for six animals.

a
Values deviate very significantly from diabetic control.
b
Values deviate significantly from diabetic control group (P ≤ 0.05).

Table 2
Effect of γ-sitosterol on fasting plasma glucose level in STZ-induced diabetic rats.

Groups Plasma glucose level

0 day 1st week 2nd week 3rd week

Normal control 90.10 ± 3.80 91.00 ± 2.16 89.13 ± 3.19 90.24 ± 2.54
Normal + γ-sitosterol 20 mg/kg body weight 92.15 ± 1.22 93.08 ± 1.21 92.69 ± 1.38 92.16 ± 1.47
Diabetic control 370.67 ± 7.15 412.49 ± 23.57 420.18 ± 36.14 445.23 ± 13.64
Diabetic + γ-sitosterol 20 mg/kg body weight 343.28 ± 5.13 322.11 ± 25.39a 267.38 ± 3.59b 191.14 ± 6.78b
Diabetic + glibenclamide 600 μg/kg body weight 356.86 ± 22.64 342.02 ± 16.75b 284.97 ± 8.45 181.63 ± 9.02b

All values are in (mg/dl) mean ± S.E.M for six animals.

a
Values deviate significantly from diabetic control group (P ≤ 0.05).
b
Values deviate very significantly from diabetic control groups.
414 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418

Table 3 at 10 and 60 min when compared with control group without the
Effect of γ-sitosterol on plasma insulin and glycosylated hemoglobin in STZ-induced addition of γ-sitosterol (Fig. 3).
diabetic rats.
Effects of diazoxide, EGTA and nimodipine on glucose and γ-
Groups Plasma insulin and glycosylated sitosterol-induced insulin release were studied. Diazoxide, a potent
hemoglobin K +-ATP channel opener, is a well known inhibitor of glucose-induced
Glycosylated hemoglobin Plasma insulin insulin release. Incubation of pancreatic islets with 11.1 mM glucose
(mg/g of Hb) (U/ml) and 20 μg γ-sitosterol in the presence of 0.25 mM diazoxide caused a
Normal control 0.47 ± 0.006 19.99 ± 1.27 40% inhibition in insulin release when compared with γ-sitosterol
Normal + γ-sitosterol 20 mg/kg 0.45 ± 0.004 19.95 ± 1.06 treated islets without diazoxide. When islets were incubated with
body weight 11.1 mM glucose, 20 μg γ-sitosterol and 0.25 mM EGTA or 0.25 mM
Diabetic control 0.94 ± 0.009 4.23 ± 0.44
Diabetic + γ-sitosterol 20 mg/kg 0.56 ± 0.011a 13.36 ± 1.08a
nimodipine, no change was observed in the release of insulin as
body weight compared with γ-sitosterol treated islets without EGTA or nimodipine
Diabetic + glibenclamide 600 μg/kg 0.51 ± 0.006a 14.52 ± 1.53b (Fig. 4).Decreased secretion of insulin was observed in the absence of
body weight γ-sitosterol.
All values are in (mg/dl) mean ± S.E.M for six animals.
a
Values deviate very significantly from diabetic control groups.
b
Values deviate significantly from diabetic control group (P ≤ 0.05). 3.10. Immunohistochemical study

The pancreatic islets in the normal control present numerous

Table 4
Effect of γ-sitosterol on liver and muscle glycogen content in STZ-induced diabetic rats. insulin-immunoreactive cells in both central and peripheral parts of
the islets of Langerhans (Fig. 5-A). The pancreatic islets administered
Groups Glycogen content
with γ-sitosterol (20 mg/kg body weight) display similar characters
Liver glycogen Muscle glycogen (Fig. 2-B). After the onset of diabetes only a few insulin-positive islets
(mg/100 mg wet weight) (mg/100 mg wet weight) were observed (Fig. 5-C). However administration of γ-sitosterol
Normal control 46.94 ± 0.25 9.99 ± 0.75 (20 mg/kg bodyweight) to diabetic rats enhanced the number of
Normal + γ-sitosterol 45.98 ± 1.26 10.54 ± 1.09 immunoreactive insulin-secreting cells in pancreatic islets (Fig. 5-D).
20 mg/kg body weight
Diabetic control 10.02 ± 0.55 1.80 ± 0.10
Diabetic + γ-sitosterol 43.86 ± 2.57a 7.78 ± 0.37a 4. Discussion
20 mg/kg body weight
a a
Diabetic + glibenclamide 46.377 ± 1.25 8.70 ± 0.39
600 μg/kg body weight The currently available drug regimens for management of diabetes
mellitus have certain drawbacks and therefore there is a need to find
All values are mean ± S.E.M for six animals.
a
Values deviate very significantly from diabetic control groups (P ≤ 0.05). safer and more effective antidiabetic drugs (Grover et al., 2000). The
aim of the present study was to evaluate the antidiabetic property of
γ-sitosterol isolated from L. nodiflora in STZ-induced diabetic rats. The
experimental diabetic model used in this study corresponds to type 2
decreased the total cholesterol, triglycerides and VLDL-cholesterol levels
diabetes, since low dose (35 mg/kg body weight) exerts partial beta-
and increased HDL-cholesterol significantly when compared to diabetic cytotoxic effect (Aybar et al., 2001) with residual beta cells secreting
control rats.
insufficient insulin and causing type 2 diabetes as a result (Gomes et
al., 2001).
3.8. Effect of γ-sitosterol on serum liver marker enzymes STZ-induced hyperglycemia has been described as a useful
experimental model to study the activity of hypoglycemic agents
Table 7 shows the activities of serum liver marker enzymes such as (Junod et al., 1969). The mechanism by which STZ brings about a
AST, ALT, ALP and ACP in normal control and diabetic control rats. The diabetic state includes selective destruction of pancreatic beta cells,
activities of AST, ALT, ALP and ACP increased significantly in diabetic leading to hypoinsulinemia which as a result decreased glucose
rats. Oral administration of γ-sitosterol or glibenclamide for 21 days uptake and hyperglycemia which is the characteristic feature of
improved the level of these parameters. diabetes mellitus (Marles and Farnsworth, 1995).
STZ-induced diabetes is characterized by a severe loss in body
3.9. Effect of γ-sitosterol on glucose-induced insulin release weight (Al-Shamaony et al., 1994) and increased food intake
(Szkudelski and Szkudeslka, 2002). Body weight loss might be the
When pancreatic islets were incubated with 11.1 mM glucose in result of protein wasting due to unavailability of carbohydrate as an
the presence of γ-sitosterol, a significant increase in insulin release energy source (Chen and Ianuzzo, 1982). Oral administration of γ-
was observed. There was a dose-dependent increase in insulin release sitosterol improved body weight and decreased food intake in diabetic
when islets were incubated with various concentrations of γ-sitosterol rats which might result from an improvement in glycemic control.

Table 5
Effect γ-sitosterol on carbohydrate metabolic enzymes in STZ-induced diabetic rats.

Groups Carbohydrate metabolic enzymes

Glucose 6-phosphatase (Ua/min/mg protein) Glucose 6 phosphate dehydrogenase(Ub/mg protein)

Normal control 0.17 ± 0.0009 4.48 ± 0 .412

Normal control + γ-sitosterol 20 mg/kg body weight 0.17 ± 0.0013 4.33 ± 0.203
Diabetic control 0.39 ± 0.0020 2.18 ± 0.459
Diabetic + γ-sitosterol 20 mg/kg body weight 0.23 ± 0.0008a 3.49 ± 0.239a
Diabetic + glibenclamide 600 μg/kg body weight 0.22 ± 0.0023b 3.68 ± 0.296a

All values are in mean ± S.E.M for six animals.

a
Values deviate significantly from diabetic control group (P ≤ 0.05); μmol of Pi liberated per hour.
b
Values deviate very significantly from diabetic control groups; nmol of NADPH formed per minute.
 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418 415

Table 6
Effect of γ-sitosterol on lipid profile in STZ-induced diabetic rats.

Groups Serum lipid profile

Total cholesterol HDL-Cholesterol VLDL-Cholesterol Triglycerides

Normal control 50.10 ± 2.00 51.38 ± 3.34 10.54 ± 0.34 51.66 ± 3.71
Normal + γ-sitosterol 20 mg/kg body weight 52.45 ± 2.12 53.63 ± 1.25 10.17 ± 0.23 50.85 ± 2.39
Diabetic control 231.03 ± 5.17 29.35 ± 2.14 40.90 ± 0.34 204.34 ± 2.61
Diabetic + γ-sitosterol 20 mg/kg body weight 117.66 ± 9.16 49.57 ± 2.67a 27.62 ± 0.18a 141.23 ± 1.21a
Diabetic + glibenclamide 600 μg/kg body weight 129.63 ± 3.94b 49.57 ± 2.54a 28.34 ± 0.23a 139.54 ± 1.25a

All values are (mg/dl) mean ± S.E.M for six animals.

a
Values deviate very significantly from diabetic control groups.
b
Values deviate significantly from diabetic control group (P ≤ 0.05).

Table 7
Effect of γ sitosterol on serum liver marker enzymes in STZ-induced diabetic rats.

Groups Serum liver markers enzymes

AST (U/dl) ALT (U/dl) ACP (U/dl) ALP (U/dl)

Normal control 31.71 ± 1.73 52.43 ± 2.52 54.36 ± 1.34 12.50 ± 0.452
Normal γ-sitosterol 20 mg/kg body weight 31.56 ± 1.82 53.83 ± 2.64 57.15 ± 1.17 12.41 ± 0.439
Diabetic control 67.06 ± 0.91 88.91 ± 1.27 83.32 ± 1.42 23.85 ± 1.35
Diabetic + γ-sitosterol 20 mg/kg body weight 41.81 ± 1.97a 54.91 ± 7.15a 63.82 ± 1.51a 16.60 ± 1.72a
Diabetic + glibenclamide 600 μg/kg body weight 42.82 ± 2.24a 56.30 ± 1.85b 66.48 ± 1.45a 17.57 ± 1.14a

All values are mean ± S.E.M for six animals.

a
Values deviate significantly from diabetic control group (P ≤ 0.05).
b
Values deviate very significantly from diabetic control groups.

In the present study, STZ-induced diabetic rats exhibit a significant
decrease in plasma glucose level when treated with γ-sitosterol.
Glucose lowering effect of γ-sitosterol might be due to stimulation of
surviving β-cells of islets of Langerhans leading to more insulin
release. This was confirmed by the increased levels of plasma insulin
in diabetic rats treated with γ-sitosterol and the increase in the
amount of immunoreactive insulin-secreting cells in many parts of
the pancreatic islets as shown by our immunohistochemical analysis.
Li et al. (2004) reported that the mechanism of action of terpenoids
and steroids might occur through stimulation of pancreatic islets
leading to an increase in insulin-induced glucose uptake.
Studies with isolated pancreatic islets bring evidence for a glucose-
dependent action of the γ-sitosterol. Insulin release was increased at
different concentrations and the insulinotropic effect was observed at
10 and 60 min. This suggested that γ-sitosterol causes release of
insulin from secretory pool as well as reserve pool. This has been
confirmed for several anti-diabetic compounds (Dickinson et al., 1997,
Ohta et al., 1999).
To understand the mechanism of insulin release by γ-sitosterol a
study was performed by incubating pancreatic islets with diazoxide,
a potent K +-ATP channel opener and a known inhibitor of insulin
release. γ-Sitosterol was able to partially reverse the inhibitory effect
of diazoxide on glucose-induced insulin release at 10 min and 60 min.
This suggested that γ-sitosterol afforded some protection to β-cells.
When islets were incubated with EGTA (chelator of exogenous Ca 2+)
and nimodipine (inhibitor of Ca 2+ influx) along with glucose there
was no change in insulin release. This implies that γ-sitosterol does
not require Ca 2+ influx (Hisatomi et al., 1996; Komatsu et al., 1998).
Since diazoxide is a potent K + ATP channel opener, it stimulates the
ROS production by opening of mitochondrial K + ATP channel (Nagy
et al., 2004). We think that γ-sitosterol works by the principle of
closing the K + ATP channel and releases insulin from the islets of the
pancreas. Hence γ-sitosterol may have an antioxidant property.
Moreover, Gupta et al. (2011) also reported the antioxidant property
of β-sitosterol.
In uncontrolled or poorly controlled diabetes, there is an increased
glycosylation of a number of proteins, including Hb (Alberti and Press,
1982). HbA1c was found to increase in diabetic patients up to 16%

Fig. 4. Effect of γ-sitosterol (alone and in combination with other compounds) on

Fig. 3. Concentration dependent effect of γ-sitosterol on glucose-induced insulin release glucose-induced insulin release from rat pancreatic islets. Values are mean ± S.E.M.
from rat pancreatic islets. Values are mean ± S.E.M. (n = 5). *, P b 0.05; **, P b 0.001 as (n = 4). *, P b 0.001 as compared with untreated control group. +, P b 0.01. ++, P ≤ 0.05
compared with untreated control group. as compared with compound treated control.
416 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418

Fig. 5. A—immunohistochemical expression of insulin secreting cells in pancreatic islets of a normal rat (X 400). B—normal rats treated with 20 mg/kg body weight of γ-sitosterol for
21 days, immunohistochemical expression of insulin secreting cells in pancreatic islets of a normal rat (X 400). C—diabetic control, negative staining for insulin secreting cell in a STZ-
diabetic rat. Arrow indicates a positively stained insulin-secreting cell (X 400). D—positive insulin-secreting cells in a diabetic rat after treatment with the γ-sitosterol 20 mg/kg body
weight (X 400) E—positive insulin secreting cells in a diabetic rats after treatment with glibenclamide 600 μg/kg body weight.

(Koeing et al., 1976). The level of HbA1c is a reliable index of glycemic
control in diabetes mellitus (Gabbay, 1976) which reflects the average
blood glucose concentration (Murray et al., 2000). In our study, HbA1c
increased in diabetic animals compared to normal rats. γ-Sitosterol
and glibenclamide treatments reduced the HbA1c values to near
normal levels due to improved glycemic control.
Glycogen is the primary intracellular storable form of glucose and
its level in various tissues is a direct reflection of insulin activity as
insulin promotes intracellular glycogen deposition by stimulating
glycogen synthase and inhibiting glycogen phosphorylase (Golden et
al., 1979). The glycogen content of skeletal muscle and liver markedly
decreased in diabetic animals (Welihinda and Karuanayake, 1986) in
proportion to insulin deficiency (Stalmans et al., 1997). The diabetic
rats treated with γ-sitosterol increased muscle and liver glycogen
contents significantly when compared to the diabetic control which
could be due to increased secretion of insulin.
A decrease in the activity of glucose 6-phosphate dehydrogenase
has been shown to slow down the pentose phosphate pathway in
diabetic conditions (Abdel-Rahim et al., 1992). Oral administration
of γ-sitosterol to diabetic animals significantly increased liver glucose
6-phosphate dehydrogenase activity via increased secretion of
insulin. This might have increased the influx of glucose into the
pentose monophosphate shunt resulting in an increased production
of the reducing agent NADPH with concomitant decrease in oxidative
stress (Ugochukwu and Babady, 2002).
Glucose 6 phosphatase is an important enzyme for the final step
of gluconeogenesis or glucogenolysis in which it catalyzes the
hydrolysis of glucose 6 phosphate to glucose and phosphate. Glucose
is transported out of the liver to increase blood glucose concentration.
Normally insulin inhibits the hepatic glucose production by suppres-
sing glucose 6 phosphatase and fructose 1, 6-bisphosphatase activity
(Chen et al., 2000; Wiernsperger and Bailey, 1999). Administration
of γ-sitosterol to diabetic rats decreased the activity of glucose 6
phosphatase when compared to diabetic control rats, thereby
decreasing gluconeogenesis and glycogenesis.
The marked increase in serum triglycerides, total cholesterol,
VLDL-cholesterol and decreased HDL-cholesterol in diabetic rats is in
agreement with the findings of Nikkila and Kekki (1973). The most
common lipid abnormalities in diabetes are hypertriglyceridemia
and hypercholesterolemia (Khan et al., 1995; Mitra et al., 1995). In
the present study, administration of γ-sitosterol to STZ-induced
diabetic rats significantly improved these parameters in a positive
way. β-Sitosterol isolated from soya has been shown to lower the
serum cholesterol (Best et al., 1954). Hwang et al. (2008) reported
that the antihyperglycemic and antihypercholesterolemic properties
of β-sitosterol were mediated by AMPK activation via LKB1, increasing
 R. Balamurugan et al. / European Journal of Pharmacology 667 (2011) 410–418
glucose uptake and GLUt4 translocation to the plasma membrane. A
similar mechanism could account for γ-sitosterol effects.
The liver is necrotized in STZ-induced diabetic rats (Ohaeri, 2001)
resulting in increased activities of serum enzymes including AST, ALT,
ALP, and ACP in plasma mainly due to the leakage of these enzymes
from the liver cytosol into the blood stream (Concepcion Navarro
et al., 1993). Treatment of the diabetic rats with γ-sitosterol caused
significant reduction in the activity of these enzymes in plasma when
compared to diabetic control and consequently alleviated liver
damage caused by STZ-induced diabetes.
In conclusion, results show that γ-sitosterol markedly reduced
hyperglycemia in STZ-induced diabetic rats due to increased insulin
secretion and inhibition of gluconeogenesis. This investigation
revealed that the active compound γ-sitosterol from L. nodiflora
could be used to treat diabetes mellitus.
Acknowledgment
The authors gratefully acknowledge the Indian Council of Medical
Research, New Delhi for financial assistance (no. 59/9/2005/BMS/TRM).
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