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Centre for Biotechnology, Muthayammal College of Arts and Sciences, Kakkaveri, Rasipuram, Namakkal 637408, Tamilnadu, India
Division of Industrial Toxicology and Pollution Control, Manonmaniam Sundaranar University, Sri Paramakalyani Centre for Environmental Sciences,
Alwarkurichi 627412, Tamilnadu, India
i n f o
Article history:
Received 2 May 2009
Accepted 17 June 2009
Keywords:
Garlip
Polyherbal formulation
Streptozotocin
Antihyperlipidemic activity
Antiperoxidation
1. Introduction
a b s t r a c t
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This study was undertaken to investigate the effect of Garlip, a polyherbal drug composed of aqueous
extract of six medicinal plants on blood glucose, plasma insulin, tissue lipid prole, and lipidperoxidation
in streptozotocin induced diabetic rats. Aqueous extract of Garlip a, polyherbal drug was administered
orally (200 mg/kg body weight) for 30 days. The different doses of Garlip on blood glucose and plasma
insulin in diabetic rats were studied and the levels of lipid peroxides (TBARS and Hydroperoxide) and tissue lipids (cholesterol, triglyceride, phospholipids and free fatty acids) were also estimated in streptozotocin induced diabetic rats. The effects were compared with tolbutamide. Treatment with Garlip and
tolbutamide resulted in a signicant reduction of blood glucose and increase in plasma insulin. Garlip
also resulted in a signicant decrease in tissue lipids and lipid peroxide formation. The effect produced
by Garlip was comparable with that of tolbutamide. The decreased lipid peroxides and tissue lipids
clearly showed the antihyperlipidemic and antiperoxidative effect of Garlip apart from its antidiabetic
effect.
2009 Elsevier Ltd. All rights reserved.
great strides that have been made in the understanding and management of diabetes, the disease and disease related complications
are increasing unabated (Tiwari and Madhusudana Rao, 2002). In
spite of the presence of known antidiabetic medicine in the pharmaceutical market, remedies from medicinal plants are used with
success to treat this disease (Bhattaram et al., 2002). Many traditional plant treatments for diabetes are used throughout the world.
Plant drugs (Bailey and Day, 1989) and herbal formulation (Mitra
et al., 1996; Annapurna et al., 2001; Bhattacharya et al., 1997)
are frequently considered to be less toxic and more free from side
effects than synthetic one.
Based on the WHO recommendations hypoglycemic agents of
plant origin used in traditional medicine are important (WHO,
1980). The attributed antihyperglycemic effects of these plants is
due to their ability to restore the function of pancreatic tissues
by causing an increase in insulin output or inhibit the intestinal
absorption of glucose or to the facilitation of metabolites in insulin
dependent processes. Hence treatment with herbal drugs has an
effect on protecting b-cells and smoothing out uctuation in glucose levels (Jia et al., 2003; Elder, 2004). In general, there is very little biological knowledge on the specic modes of action in the
treatment of diabetes, but most of the plants have been found to
contain substances like glycosides, alkaloids, terpenoids, avonoids etc., that are frequently implicated as having antidiabetic effects (Loew and Kaszkin, 2002).
In the experiment, a total of 42 rats (30 diabetic surviving rats, 12 normal rats)
were used. The rats were divided into seven groups of six rats each after the induction of streptozotocin diabetes.
Group1: Normal treated rats.
Group2: Normal rats given aqueous solution of Garlip (200 mg/kg body weight)
daily using an intragastric tube for 30 days.
Group 3: Diabetic control rats.
Group 4: Diabetic rats given aqueous solution of Garlip (50 mg/kg body weight)
daily using an intragastric tube for 30 days.
Group 5: Diabetic rats given aqueous solution of Garlip (100 mg/kg body
weight) daily using an intragastric tube for 30 days.
Group 6: Diabetic rats given aqueous solution of Garlip (200 mg /kg body
weight) daily using an intragastric tube for 30 days.
Group 7: Diabetic rats given aqueous solution of tolbutamide (250 lg/kg body
weight) daily using an intragastric tube for 30 days.
At the end of 30 days, all the rats were killed by decapitation under pentobarbitone sodium (60 mg/kg) anaesthesia. Blood was collected in tubes containing
potassium oxalate and sodium uoride solution for the estimation of blood glucose
and plasma was separated for the assay of insulin. Liver and kidney were immediately dissected out, washed in ice-cold saline to remove the blood. The tissues were
weighed and 10% tissue homogenate was prepared with 0.025 M TrisHCl buffer,
pH 7.5. After centrifugation at 3000 rpm for 10 min, the clear supernatant was used
to measure thiobarbituric acid reactive substances (TBARS) and hydroperoxides. For
the determinations of lipids the liver and kidney tissues were weighed and lipids
were extracted from tissues by the method of Folch et al. (1957).
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Male Wistar albino rats (weighing 160200 g) were procured from the Animal
house, Bharathidasan University, Tiruchirapalli under standard environmental conditions (12 h light/dark cycles at 2528 C, 6080% relative humidity) and kept in
clean and dry cages and maintained in well-ventilated animal house. Animals were
divided into 8 groups of ve each and were fed with standard diet and water ad libitum. All studies were conducted in accordance with the National Institute of Health
guide (1985).
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Garlip was suspended in distilled water and administered orally through intragastric tube at the following doses of 50, 100 and 200 mg/kg body weight.
2.5. Streptozotocin-induced diabetes
Rats were made diabetic by single administration of streptozotocin (60 mg/kg/
i.p) dissolved in 0.1 M-citrate buffer, pH 4.5. Forty-eight hours later, blood samples
were collected and glucose levels were determined to conrm the development of
diabetes. Only those animals which showed hyperglycemia (blood glucose levels > 300 mg/dl) were used in the experiment.
Table 1
Garlip (composition and concentration).
S.No.
Botanical name
Common name
Family
Part used
Concentration (mg/dl)
1
2
3
4
5
6
Allium sativum
Terminalia bellirica
Alpinia galanga
Glycyrrhize glabra
Aloe vera
Cynodon dactylon
Garlic
Belliric myrobalan
Greater galangal
Liquorice
Indian aloe
Dhub grass
Liliaceae
Combretaceae
Zingiberaceae
Fabaceae
Liliaceae
Poaceae
Bulb
Fruits
Rhizome
Roots
Pulp
Whole plant
50
30
25
25
40
50
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4. Discussion
Diabetes mellitus is one of the most common chronic disease
and is associated with hyperlipidemia and co-morbidities such as
obesity, hypertension. Hyperlipidemia is a metabolic complications of both clinical and experimental diabetes (Bierman et al.,
1975). Streptozotocin induces a wide variety of animal species by
damaging the insulin secreting pancreatic b-cell, resulting in a decrease in endogenous insulin release, which paves the ways for the
decreased utilization of glucose by the tissues (Gilman et al., 1990).
In our study, we have observed that Garlip decreases blood glucose in streptozotocin diabetic rats. The possible mechanism of action of extract could be correlated with the reminiscent effect of
the hypoglycemic sulphonylureas that promote insulin secretion
by closure of K+ATP channels, membrane depolarization and
stimulation of Ca2+ inux, an initial key step in insulin secretion.
In this context, number of other plants has also been reported to
have antihyperglycemic and insulin stimulatory effects (Mazunder
et al., 2005; Kumar et al., 2007a). Like the plant extract, tolbutamide also produced signicant reduction in blood glucose levels
of streptozotocin diabetic rats.
Since streptozotocin is known to destroy pancreatic b-cells, the
present ndings appear to be in consonance with the earlier suggestion of Kumar et al. (2006a) that sulphonylureas have extrapancreatic antihyperglycemic mechanism of action secondary to
their insulin secreting effect and the attendant glucose uptake into,
and utilization by, the tissues.
Apart from the regulation of carbohydrate metabolism, insulin
also plays an important role in the metabolism of lipids. Insulin
is potent inhibitor of lipolysis. Since it inhibits the activity of the
hormone sensitive lipases in adipose tissue and suppresses the release of free fatty acids (Loci et al., 1994). During diabetes, enhanced activity of this enzyme increases lipolysis and releases
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3. Results
of acetyl acetone reagent. After mixing, the tubes were kept in a water bath at 65 C
for 1 h, the contents were cooled and absorbance was read at 420 nm. The triglyceride content was expressed as mg/100 g tissue.
Phospholipid content was determined by the method of Zilversmit and Davis
(1950). To 0.1 ml of lipid extract, added 1 ml of 5 N H2SO4 and 1 ml of Conc.
HNO3 and digested to a colourless solution. The phosphorus content in the extract
was determined by the method of Fiske and Subbarow (1925). The values were expressed as g/100 g tissue.
Free fatty acids were estimated by the method of Falholt et al. (1973). 0.1 ml of
lipid extract was evaporated to dryness. one milliliter of phosphate buffer, 6 ml of
extraction solvent and 2.5 ml of copper reagent were added. All the tubes were shaken vigorously. Two hundred milligrams of activated silicic acid was added and left
aside for 30 min. The tubes were centrifuged and 3 ml of the copper layer was
transferred to another tube containing 0.5 ml of diphenyl carbazide and mixed
carefully. The absorbance was read at 550 nm immediately. The amount of free
fatty acids was expressed as mg/100 g tissue.
Groups
Table 2
Changes in blood glucose and plasma insulin levels of control and experimental animals.
Normal
Diabetic control
Diabetic + Garlip (50 mg/kg)
Diabetic + Garlip (100 mg/kg)
Diabetic + Garlip (200 mg/kg)
Diabetic + tolbutamide (250 mg/kg)
81.04 2.29
262.24 22.23b
209.58 12.46c
155.58 11.69d
104.16 6.56e
110.65 9.35e
11.26 0.96a
3.48 0.69c
5.59 0.34d
6.03 0.45d
7.15 0.45e
6.32 0.48de
Values are given as mean SD for six rats in each group. Values not sharing a common superscript letter differ signicantly at p < 0.05 (DMRT).
Table 3
Changes in levels of TBARS and hydroperoxides in liver and kidney of control and experimental animals (mM/100 g tissue).
Groups
Normal
Normal + Garlip (200 mg/kg)
Diabetic control
Diabetic + Garlip (200 mg/kg)
Diabetic + tolbutamide (250 mg/kg)
TBARS
Hydro peroxide
Liver
Kidney
Liver
Kidney
0.58 0.066a
0.56 0.079a
1.54 0.06b
0.64 0.053c
0.67 0.088c
0.68 0.05a
0.78 0.06a
1.65 0.12b
0.97 0.07c
1.02 0.08c
70.38 4.54a
68.59 5.14a
99.98 5.98b
80.55 5.69c
84.35 5.45c
55.34 4.55a
52.48 4.75a
78.29 4.58b
60.99 4.78c
62.45 5.56c
Values are given as mean SD for six rats in each group. Values not sharing a common superscript letter differ signicantly at p < 0.05 (DMRT).
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Table 4
Changes in levels of cholesterol, free fatty acids, triglycerides and phospholipids in liver of control and experimental animals (mg/100 g wet tissue).
Groups
Cholesterol
Normal
Normal + Garlip (200 mg/kg)
Diabetic control
Diabetic + Garlip (200 mg/kg)
Diabetic + tolbutamide (250 mg/kg)
335.44 18.90
328.38 5.74a
496.56 13.10b
398.65 17.54c
405.21 9.79c
606.10 1.76
601.93 9.00a
921.60 44.60b
769.16 3.30c
802.80 3.77c
Triglycerides
Phospholipids
a
344.50 23.20
341.10 21.00a
622.50 18.80b
456.25 17.30c
530.80 35.70d
1598.00 19.30a
1593.10 24.10a
1858.60 18.70b
1718.80 14.40c
1769.30 17.60c
Values are given as mean SD for six rats in each group. Values not sharing a common superscript letter differ signicantly at p < 0.05 (DMRT).
Table 5
Changes in levels of cholesterol, free fatty acids, triglycerides and phospholipids in kidney of control and experimental animals (mg/100 g wet tissue).
Cholesterol
Triglycerides
Phospholipids
Normal
Normal + Garlip (200 mg/kg)
Diabetic control
Diabetic + Garlip (200 mg/kg)
Diabetic + tolbutamide (250 mg/kg)
375.90 16.58a
371.08 8.28a
546.90 23.80b
435.20 12.18c
449.90 13.49c
434.01 1.50a
432.51 1.60a
743.00 5.70b
556.80 8.50c
600.30 3.40d
282.50 14.70a
278.75 14.60a
501.10 34.10b
382.90 9.28c
438.66 39.30d
1447.60 26.90a
1442.50 43.30a
2041.50 33.69b
1684.00 28.80c
1819.30 34.70d
Groups
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Values are given as mean SD for six rats in each group. Values not sharing a common superscript letter differ signicantly at p < 0.05 (DMRT).
more free fatty acids into the circulation (Agardh et al., 1999). Increased fatty acids concentration also increases the b-oxidation
of fatty acids, producing more acetyl CoA and cholesterol during
diabetes. In normal condition, insulin increases the receptor-mediated removal of LDL-cholesterol and decreased activity of insulin
during diabetes causes hypercholesterolemia. Hypercholesterolemia and hypertriglyceridemia have been reported to occur in diabetic rats (Bopanna et al., 1997). The increased concentration of
cholesterol could result in a relative molecular ordering of the
residual phospholipids resulting in a decrease in membrane uidity (Dario et al., 1996).
The increased concentration of free fatty acids in liver and kidney may be due to lipid breakdown and this may cause increased
generation of NADPH, which results in the activation of NADPH
dependent microsomal lipid peroxidation. Liver and kidney phospholipids were increased in diabetic control rats. Phospholipids is
present in cell membrane and make up vast majority of the surface
lipoprotein forming a lipid bilayer that acts as an interface with
both polar plasma environment and non-polar lipoprotein of lipoprotein core (Cohn and Roth, 1996).
Phospholipids are vital part of biomembrane rich in PUFA,
which are susceptible substrate for free radicals such as O2 - and
OH radicals (Ahmed et al., 2001). Increased phospholipids levels
in tissues were reported by Sharmila Banu et al. (2007) and Kumar
and Murugesan (2008) in streptozotocin diabetic rats. Administration of Garlip decreased the levels of tissue free fatty acids and
phospholipids.
Accumulation of triglycerides is one of the risk factors in Coronary Heart Disease (CHD). The signicant increase in the level of
triglycerides in liver and kidney of diabetic control rats may be
due to the lack of insulin. Since under normal condition, insulin
activates the enzyme lipoprotein lipase and hydrolysis triglycerides (Frayn, 1993). Garlip reduces triglycerides in tissues of streptozotocin-induced diabetic rats and may prevent the progression
of CHD. The results show increased lipid peroxidation in the tissues
(liver and kidney) of diabetic control group. Previous studies have
reported that there was an increased lipid peroxidation in liver,
heart, kidney and brain of diabetic rats (Kumar et al., 2006b,
2007b, 2008a,b). This may be because the tissues contain relatively
high concentration of easily peroxidizable fatty acids.
Liver during diabetes, showed a relatively severe impairment in
antioxidant capacity than kidney. The kidney exhibits a characteristic pattern of changes during diabetes (Aragno et al., 1999). The
increase in oxygen free radicals in diabetes could be primarily
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