Effect of Crude Extract and Fractions from Elephantopus scaber on Hyperglycemia in Streptozotocin-Diabetic Rats
The effect of the crude extract and acetone fractions from Elephantopus scaber (Asteraceae) on glycemia was investigated in streptozotocin (STZ) diabetic rats. Oral administration of crude extract significantly reduced serum glucose levels and increased the lowered insulin concentrations in diabetic animals at 150 mg kg-1. Nevertheless, the hypoglycemic effect of the acetone fractions (mg kg-1) of Elephantopus scaber in diabetic rats was evident at 2 h after treatment, where the sugar lowering effect of the most effective fraction was more than 69%. Phytochemical analysis revealed the compound to be a terpenoid. Administration of glibenclamide, a reference drug (0.6 mg kg-1 bw) also produced a significant (p<0.005) reduction in blood glucose concentration in STZ-induced diabetic rats. Thus, the results of this experimental study shows that Elephantopus scaber has an anti-hyperglycemic action, is able to ameliorate the diabetic state and probably, is a source of potent hypoglycemic compounds.
Diabetes mellitus is a chronic disorder of metabolism caused by an absolute or relative lack of insulin. It is characterized by hyperglycemia in the postprandial and/or fasting state and in its severe form is accompanied by ketosis and protein wasting (Bell, 1991; Dima et al., 2007; Rotheri, 2007). Besides drugs classically used for the treatment of diabetes (insulin, sulphonylureas, biguanides and thiazolidinediones), several species of plants have been described in the scientific and popular literature as having a hypoglycaemic activity (Verspohl, 2002; De Sousa et al., 2004; Colca, 2006). Because of their perceived effectiveness, minimal side effects in clinical experience and relatively low costs, herbal drugs are prescribed widely even when their biologically active compounds are unknown (Valiathan, 1998; Mahady, 2005). The present study investigated the acute effect of the oral administration of crude extract and fractions of acetone extract of Elephantopus scaber on serum glucose and insulin levels in diabetic rats. A number of investigators have shown that cumarins, flavonoids, terpenoids and a host of other secondary plant metabolites, including arginine and glutamic acid possess medicinal properties (Marles and Farnsworth, 1995; Ross, 2001).
Although, the medicinal properties of Elephantopus scaber have been well established (Nadkarni, 1954), yet there are no reports available for the hypoglycemic activity of Elephantopus scaber in STZ-induced diabetic rats and also on the compounds responsible for the activity. So the aim of the study was to investigate the hypoglycemic effects of the crude extract of Elephantopus scaber and its role on insulin concentrations and also evaluate the hypoglycemic effects of the fractions of the acetone extract of Elephantopus scaber (using a bioassay guided separation) in streptozotocin induced diabetic rats.
MATERIALS AND METHODS
Elephantopus scaber Linn. popularly known as Elephant's foot, (Asteraceae) is a scabrescent aromatic herb distributed in the moist deciduous forests of the central Western Ghats. As per the traditional claims, the roots were used as an antipyretic, cardiotonic and diuretic (Nadkarni, 1954). Decoction of the roots and leaves is used as emollient and given in dysuria, diarrhea, dysentery and in stomach pain (Kirtikar and Basu, 1991). The aqueous extract of leaves is applied externally to treat eczema and ulcers (Chopra et al., 1956). Phytochemically the plant has been reported to contain sesquiterpene lactones deoxyelephantopin, isodeoxy-elephantopin and scabertopin (Paul Pui-Hay But et al., 1997). It also contains epifriedelinol, lupeol and stigmasterol (De-Silva et al., 1982). The pharmacological properties of the leaf extracts have been evaluated for diuretic, (Poli et al., 1992) anti-inflammatory, (Tsai and Lin, 1999) and hepatoprotective properties (Lin et al., 1995). Elephantopus scaber plants were collected from Kerala, India. The species was identified and authenticated at the Department of Botany of the College and the voucher specimen had been deposited.
Isolation and Identification of the Active Compound
The shade-dried plants were powdered and 1 kg was extracted using acetone
in a soxhlet apparatus and was evaporated to dryness under reduced pressure
in rotary evaporator. The yield of the acetone extract was 12.1 g %. The dry
residue of the crude extract obtained was stored at 4°C for further use.
The acetone extract was further chromatographed on a silica gel column (Merck
70-230 mesh, 400 g, 3.5i.d.x60 cm) and successively eluted with stepwise gradient
of ethyl acetate and hexane, then with acetone and ethyl acetate, with acetone
and chloroform and finally with chloroform and hexane. Seventeen fractions were
collected and each fraction was spotted on a precoated Silica gel 60 F254, 0.25
mm thick TLC plate (Merck) and fractions with similar Rf values in TLC pattern
were pooled together.
Male albino rats (Wistar strain, weighing 150-220 g) bred in the Laboratory
of Animal Medicine, Centre for Animal Health Studies, Tamilnadu Veterinary and
Animal Sciences Studies, Madhavaram, Chennai, Tamil Nadu, India were used. All
the animals were kept and maintained under laboratory conditions of temperature
(22+2°C), humidity (45+5%) and 12 h day: 12 h night cycle and were allowed
free access to food (standard pellet diet) and water ad libitum.
Induction of Diabetes in Rats
Diabetes was induced by a single intraperitoneal injection of streptozotocin
(single dose of 60 mg kg-1 b.wt.) dissolved in freshly prepared 0.01
M citrate buffer (pH 4.5) in a volume of 1 mL kg-1 b.wt. After 7
days of STZ administration, rats with blood sugar levels of 280-350 mg dL-1
and above, were considered as diabetic and were employed in the study. Blood
was collected from the tail vein.
Experimental Design and Treatment Schedule
The rats were randomly divided into nine groups of five animals each. Group
I served as control Group II was the untreated diabetic group. Groups I and
II received 0.1% carboxy methyl cellulose (CMC) orally. Groups III received
acetone extracts of E. scaber, orally at a dose of 150 mg kg-1
by gastric intubation, while Groups IV and V served as positive controls and
received humulin (Schmidt et al., 1999) and glibenclamide (Dhanabal et
al., 2006). The treatment was continued for 60 days by administering the
various crude extracts suspended in 0.1% CMC once daily. The rats were sacrificed
at the end of 60 days for estimation of glucose and insulin levels. Groups VI-IX
received fractions (F1-F4; 2 mg/animal) and blood sugar was determined after
2 h of administration of the fractions. The experiment was carried out in the
college during 2005-2006.
Estimation of Glucose and Biochemical Parameters
Blood samples were collected from tail vein in Eppendorff tubes (1.5 mL)
at 0th, 15th, 30th and 60th days and serum was separated by centrifuging the
samples at 5000 rpm for 10 min and immediately analysed for glucose content.
Students t-test and a probability level of p<0.05 were chosen as the
criterion of statistical significance. Values reported are mean±SD.
Administration of STZ produced diabetes in rats after a week. The effects of
the various extracts of E. scaber on blood glucose, serum insulin are
depicted in Table 1 and 2.
Determination of the Serum Glucose Concentration and Serum Insulin
Blood samples from the tail vein of the anesthetized rat were collected,
centrifuged and the serum was used to determine the glycemia by the glucose
oxidase method (De Sousa et al., 2004). Serum insulin was assayed using
Radio imnunoassay kit purchased from Disasorin, Italy.
Effect of the Acetone Crude Extract of E. scaber on Serum Glucose
Levels in STZ-Induced Diabetic Rats
The blood glucose level of untreated rats was 84.4±0.55 mg dL-1.
In the STZ diabetic rats, the level increased to 534.6±2.07 mg dL-1.
In the diabetic rats treated with the acetone extract of the plant, the blood
glucose decreased significantly and was to a level closer to the untreated control
and within the normal range.
Effect of the Acetone Fractions of E. scaber on Serum Glucose Levels
in STZ-Induced Diabetic Rats
Oral administration of the various fractions of E.scaber with doses
of 20 mg kg-1 significantly reduced the hyperglycemia in STZ-induced
diabetic rats and maximum reduction of serum glucose levels was observed at
2 h after treatment. Fraction 3 brought about 69% decrease in the blood sugar
levels as compared to the diabetic control (Table 3).
|| Effect of the acetone extract of Elephantopus scaber
on blood sugar levels in STZ-diabetic rats
||Effect of the acetone extract of Elephantopus scaber
(after 60 days treatment) on serum insulin levels in STZ-diabetic rats
|| Effect of the two compounds on blood sugar levels in STZ-diabetic
|All values are means±SD of six rats
Serum Insulin by Radioimmuno Assay
The serum insulin level of untreated rats was 51.06±1.18 μU
mL-1). The insulin level decreased to 6.14±0.14 μU mL-1
in the STZ-diabetic rats. In the ESA-treated diabetic rats the serum insulin
levels increased to 38.26±1.15 μU mL-1, which was close
to the normal untreated rats (Table 2).
Single dose administration of STZ produces diabetogenic effects in the first 24 h in rats. β-cells underwent conspicuous regression after treatment with streptozotocin (Szkudelski, 2001).Insulin-dependent diabetes mellitus (IDDM) is a disease caused by progressive destruction of the insulin secreting β-cells. Despite meticulous insulin therapy, the appearance of micro- and macro-angiopathy complications after 15 to 20 years of the disease is difficult to prevent in some patients. Presently, the only option to achieve permanent normoglycemia in diabetic patients is renewal of the β-cells or stimulate insulin secretion by medicinal plant (Robertson, 1993). The lowering of blood sugar level in streptozotocin-induced hyperglycemic animals through the administration of Elephantopus scaber extract may be due to insulin release from regenerated β-cells of the pancreas or by stimulation of insulin secretion (Palanichamy et al., 1988). Similar reports are available on the oral administration of aqueous extract of leaves of G. sylvestre normalized blood sugar levels of diabetic animals through β-cell regeneration (Shanmugasundaram et al., 1981).
In this study we have found that E. scaber decreases blood glucose in STZ diabetic rats. The possible mechanism by which E. scaber brings about its hypoglycemic action may be potentiating the insulin effect of plasma by increasing either the pancreatic secretion of insulin from the β-cells of islets of Langerhans or its release from bound insulin. The present study was conducted to evaluate the hypoglycemic and antidiabetic activity of the compounds from E. scaber, a very new herbal drug first time identified by us to get a berth in the group of antidiabetic herbal drugs. E. scaber did not exhibit any sign of toxicity. Since the main purpose of the preliminary acute toxicity study is to get some idea on conspicuous behavioral changes and death, if any and the alcoholic extract of E. scaber did not exhibit any toxic symptoms in the limited toxicity evaluation in male rats. A perusal of literature shows that studies were not carried out on this plant with regard to other pharmacological properties and phytochemistry. Type 1 diabetes occurs in a genetically susceptible human population as a result of the loss of the insulin-producing pancreatic beta cells. This accounts for the drastic drop in the insulin level in the diabetic rats. The serum insulin level decreased in diabetic animals, whereas E. scaber acetone extract treatment brought about a marked increase in serum insulin in streptozotocin-induced diabetic rats. This increase may be a consequence of the stimulation of insulin synthesis and secretion and or inhibition of insulin degradation, since many compounds present in plants have been demonstrated to produce these effects (Venkateswaran and Pari, 2003). For instance, benzoic acid-related molecules inhibit insulinase and enhance insulin effects (Aybar et al., 2001). The increased levels of insulin in extract-treated diabetic rats indicated that M. charantia extract stimulates insulin secretion from regenerated β-cells (Kameswara Rao et al., 2003). In the present study also, serum insulin level of diabetic animals treated with the extracts of Elephantopus scaber increased when compared to the diabetic controls. The biochemical mechanism of action appears to be through regeneration of the damaged β-cells and thereby, stimulate the secretion of insulin in β-cells as revealed by insulin assay.
The significant and consistent hypoglycemic effect of crude extract of E. scaber, as well as the acetone fractions, in diabetic rats indicates that this effect can be mediated by stimulation of glucose utilization by peripheral tissues. However, phytochemical and pharmacological studies, performed indicated the most effective fraction to be a terpenoid. Terpenoids appear to involve stimulation of pancreatic β-cells and subsequent secretion of insulin (Marles and Farnsworth, 1995). Further studies to isolate and to characterize the terpenoid compound found in the acetone fraction are underway to further elucidate the mechanism involved in the hypoglycemic effect.
We conclude that the crude acetone extract of E. scaber was effective in decreasing the serum glucose level, also increasing serum insulin by stimulating the pancreatic β-cells in diabetic rats and also, the acetone fractions showed the hypoglycemic effect in severe STZ-diabetic rats and it was more potent than the crude extract and that the plant was more effective than the reference drug, Glibenclamide.
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