Medical plants play an important role in the management of diabetes mellitus especially in developing countries where resources are meager. Plant-based drugs have been used against various diseases since a long time. The nature has provided abundant plant wealth for all the living creatures, which possess medicinal virtues. Therefore, there is a necessity to explore their uses and to conduct pharmacognostic and pharmacological studies to ascertain their therapeutic properties. In fact, nowadays, diabetes is a global problem. Hence, the present study aims to open new avenues for the improvement of medicinal uses of Verbesina encelioides Benth. roots for the selected area for diabetes.. Dried aqueous and alcoholic extracts were subjected for hypoglycaemic activity in swiss albino mice (30-40g). Blood sugar level was determined using digital glucometer. The oral administration of roots extracts at doses of 400 mg/ kg lead to a significant blood glucose reduction in normal and in Streptozotocin, alloxan diabetic mices significantly within 4 h. Continued, daily administration of the drug produced a sustained effect.
KEYWORDS: Verbesina encelioides , Alloxan-induced diabetes, Streptozotocin -induced diabetes, Hypoglycaemic activity.
From time immemorial plants are used as medicine around the world and plant based medicine has been the mainstay of traditional societies in dealing with health problems.1 The World Health Organization (WHO) has estimated that 80% of the earth’s (6 billion) inhabitants rely upon traditional medicine for their primary health care needs and major part of this therapy involves the use of plant extracts or their active principles.2 Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia, polyuria, polydypsia, polyphagia, glycosuria, hyperlipidemia, negative nitrogen balance, pruritis, unexpected weight loss and some times ketonemia.3-5 At present number of diabetics worldwide is 150 million and this is likely to increase to 300 million or more by the year 2025.6 This rise in number is due to sedimentary lifestyle, consumption of energy rich diet, obesity, higher life span etc.7 Regions with greatest potential are Asia and Africa, where diabetes mellitus rates could rise 2-3 folds than the present rates. Many medicine have been recommended for the treatment of diabetes.8-12 The two major types of diabetes mellitus are Type-I insulin dependent and Type-II non-insulin dependent . Type-I Insulin-dependent diabetes mellitus or juvenile-onset diabetes (IDDM). Type-II Non-insulin dependent diabetes mellitus (NIDDM) or maturity-onset diabetes:
Treatments of this disorder take three main factors:
(i) Diet and exercise
(ii) Insulin replacement therapy
(iii) Oral hypoglycemic agents 13
Before the advent of insulin and oral hypoglycemic drugs, the major form of treatment involved the use of plants. Recently, some medicinal plants have been reported to be useful worldwide and have been used empirically as antidiabetic and remedies.14
There is an increasing demand by patients to use the natural products with antidibetic activity due to side effects associated with the use of insulin and oral hypoglycemic agents such as sulfonylureas, metformin, α-glycosidase inhibitors, troglitazone.15-17 Antihyperglycemic effects of these plants are attributed 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. More than 400 plant species having hypoglycemic activity are known to have been recommended, and recent investigations have affirmed the potential value of these,18-20 however searching for new antidiabetic drugs from natural plants is still attractive because they contain substances which take alternative and safe effects on diabetes mellitus.21 Chemical studies directed at the isolation, purification and identification of substances responsible for the anti-diabetic activity are also being conducted.22 Most of plant contain glycosides, alkaloids, terpenoids, flavonoids, cartenoids, etc. that are frequently implicated as having antidiabetic effect.23
MATERIAL AND METHODS:
The dried root powder
(2.5kg) of Verbesina encelioides
Benth. was taken and
subjected to extraction with ethanol in soxhlet
apparatus so as to yield 4.54% w/w of extract and aqueous extract was prepared
by cold maceration yielded 2.05% w/w. Extracts were kept in a dessicator and stored in refrigerator for future biological
evaluation. Alloxan was purchased from LOBA CHEMIE,
Swiss albino mice of either sex (weighing 30-45 g) were used as per experimental protocols approved by Institutional committee, The experimental protocol has been approved by the Institutional Animal Ethics committee and by the Regulatory body of the government. The animals were housed under standard environmental condition (25 ± 2°C and relative humidity 50 ± 5%) and fed with standard diet and water, ad libitum. The animals were acclimatized to laboratory environment for a period of 14 days before performing the experiments.
The mice were divided into six groups of six animals each. The first group comprised the control. The second received the standard reference control drug. The remaining four groups were administered test dose. The test doses were prepared in Tween 20 (1%) as suspension in distilled water to get the desired concentration of the extracts. The dosages selected were as follows:
i) Solvent control
1% Tween 20 in distilled water (2 ml/kg).
ii) Reference control
Glibenclamide:2.5 mg/kg body weight in distilled water.
iii) Diabetogenic agent
Alloxan monohydrate: 120 mg/kg in normal saline.
Streptozotocin: 60 mg/kg in citrate buffer (pH 4.5)
iv) Test drugs
Ethanol extract- 200mg/kg
Ethanol extract- 400mg/kg
Aqueous extract-200 mg/kg
Aqueous extract-400 mg/kg
Swiss albino mice of either sex were used. The animals were fasted overnight, kept under laboratory conditions and allowed free access to water. The animals were divided in to different groups of six animals each. The ethanolic and aqueous extracts were administered orally at a dose of 5 mg/kg. The control group received a similar volume of 1% Tween 20 in distilled water. Mortality in each group was observed for 3 to 7 days. If no mortality was observed, the procedure was repeated for higher doses such as 50, 500, 1000, 1500 and 2000 mg/kg.24 The doses of 200 and 400 mg/kg were selected based on the results of preliminary toxicity testing.25
The animals were fasted for 18 h, but were
allowed free access to water before and throughout the duration of experiment.
At the end of the fasting period, taken as zero time (0 h), blood was withdrawn
(0.1 ml) from the tip of the tail of each mice under
mild ether anaesthesia. The blood glucose level was
measured with haemoglucostrips supplied by B. Braun
Medical Industries Sdn. Bhd.,
Figure 1: Percentage reduction in blood glucose level in normoglycaemic mice
The acclimatized animals were kept fasting for 24 h with water ad libitum and injected intraperitoneally a dose of 120 mg/kg of alloxan monohydrate in normal saline. After 1 h, the animals were provided feed ad libitum. The blood glucose level was checked before alloxanisation and 24 h after alloxanisation. Animals were considered diabetic when the blood glucose level was raised beyond 200 mg/100 ml of blood. This condition was observed at the end of 48 h after alloxanisation. The animals were then segregated in to six groups of six mice in each. Group-I served as solvent control which received vehicle (2 ml/kg) only through oral route. Group-II received glibenclamide (2.5 mg/kg) and served as reference control. Group-III and IV received the ethanolic extract at a dose of 200 and 400 mg/kg body weight respectively, through oral route. Group-V and VI received the aqueous extract at a dose of 200 and 400 mg/kg body weight respectively, in a similar manner. Blood glucose levels were examined after 1, 2, 4, 8 and 10 h of administration of single dose of test samples.22
Figure 2: Percentage reduction in blood glucose level in alloxan induced diabetic mice.
The 18 h fasted mice were divided in to six groups of six animals each. The animals were made diabetic by injection of a dose of streptozotocin 60 single mg/kg body weight dissolved in 0.1 ml of citrate buffer (pH 4.5) by intravenous injection in to the tail vein. Animals were considered diabetic when blood glucose level was raised beyond 200 mg/100 ml of blood. Group–I served as solvent control and received only vehicle (2 ml/kg) through oral route. Group–II received glibenclamide (2.5 mg/kg). Group-III and IV received the ethanolic extract at a dose of 200 and 400 mg/kg body weight respectively,
through oral route. Group-V and VI received the aqueous extract at a dose of 200 and 400 mg/kg body weight respectively. Blood glucose levels were examined after 1, 2, 4, 8 and 10 h of administration of single dose of test samples.5
The albino mice of either sex (20-30 g) maintained on a standard diet (water ad libitum) and fasted overnight. The animals were killed by decapitation and diaphragms were taken out quickly avoiding trauma and divided in to two halves. The hemidiaphrams were then rinsed in cold Tyrode solution (without glucose) to remove any blood clots and were then placed in small conical flasks containing 2ml Tyrode solution with 2 g% glucose and incubated for 30 min at 37 oC in an atmosphere of 95% O2-5% CO2 with shaking. Six sets of experiments were performed. The diaphragms were exposed to:
(a) Tyrode solution with 2g% glucose which served as control,
(b) Tyrode solution with 2g% glucose + insulin (0.25 IU/ml),
(c) Tyrode solution with 2g% glucose + ethanol extract (200 mg/ml)
(d) Tyrode solution with 2g% glucose + aqueous extract (200 mg/ml)
(e) Tyrode solution with 2g% glucose + insulin (0.25 IU/ml) + ethanol extract (200 mg/ml).
(f) Tyrode solution with 2g% glucose + insulin (0.25 IU/ml) + aqueous ethanol extract (200 mg/ml).
Following incubation, the hemidiaphrams were taken out and weighed. Glucose uptake was calculated as the difference between the initial and final glucose content in the incubation medium. The glucose estimation was carried out on autoanalizer.
Data expressed as mean± S.E.M. statistical comparison between different groups were done using one-way analysis of variance (ANOVA) followed by the Dunnett’s ‘t’ test, to judge the difference between various groups. Significance was accepted at P<0
In acute toxicity study, there were no behavioral changes up to 4 h and no mortality was observed up to 7 days even at the maximum tested dose level of 2000 mg/kg.
Figure 3: Percentage reduction in blood glucose level in streptozotocin induced diabetic mice.
The ethanolic and aqueous extracts of Verbesina encelioides Benth. roots produced significant decrease in the blood glucose level when compared with the control in the normoglycaemic mice [Table-1]. The reduction in blood glucose level was observed 2h after oral administration of single dose of the standard drug and extracts, the reduction in blood glucose level was reached maximum after 10 h for all the test doses and standard drug, it was more significant (P<0.01) for the 400 mg/kg ethanol extract and aqueous extract (200 and 400 mg/kg). While 200 mg/kg ethanol extract showed less significance (P<0.05) when compared with the control. The percentage reduction in blood glucose is shown in Figure-1. The maintenance of normoglycaemia depends on the result of the interwork of islets of β-cell secretion and insulin sensitivity in the periphery and in the liver. The results corroborated that the test extracts showed hypoglycaemic properties throughout the experimental period in the normoglycaemic mice. Normal animals are often used for potential oral hypoglycaemia in addition to diabetic animal model as information regarding mechanism of action.
Alloxan-induced diabetic mice:
The perusals of Table-2 reveals that the ethanolic and aqueous extracts in alloxan induced diabetic mice produced significant (p<0.01) decrease in blood glucose level after 4h of administration of extracts when compared with the control. The percentage decrease in blood glucose level for ethanolic and aqueous extract at the dose level of 400 mg/kg is 64.21% and 63.48 % respectively, which is comparable to standard drug glibenclamide (68.14%) as shown in Figure-2. Alloxan is a urea derivative which induces “chemical diabetes” in a wide variety of animal species by damaging the insulin secreting pancreatic β-cells, resulting in a decrease in endogenous insulin release.26 Numerous studies demonstrated that a variety of plant extracts effectively lowered the glucose level in alloxan-induced diabetic animals.27,28 In the present study, ethanolic and aqueous extracts of V. encelioides roots effectively decrease the blood glucose in alloxan- induced diabetic mice, which is almost equal to that of glibenclamide.
The ethanolic and aqueous extracts exhibited significant antidiabetic effect in streptozotocin induced diabetic mice [Table-3]. The standard drug, ethanol extract and aqueous extract at doses 400 mg/kg significantly (P<0.01) reduced the blood glucose level after 2h while 200 mg/kg doses of extracts showed P<0.01 significant result after 4h. The maximum reduction observed at 10 h. The maximum percentage reduction of standard drug glibenclamide is 57.25%. It is 29.27 and 51.11% for ethanol extract at doses 200 and 400 mg/kg respectively and 21.87 and 46.95% for aqueous extract at doses 200 and 400 mg/kg respectively [Figure-3].
Streptozotocin is cytotoxic nitrosoureido glucopyranoside derivative isolated from fermentation of Streptomyees achromogenes and has been widely used for inducing type-I diabetes in a variety of animals by affecting the degeneration and necrosis of pancreatic β-cells.29 The present data indicated that the ethanolic and aqueous extracts of Verbesina encelioides Benth. roots decreased the blood glucose in streptozotocin- diabetic mice. This suggests the said effect be due to extraintestinal action of the test extracts.30 The extracts decreased blood glucose without stimulating insulin secretion, in addition the extract may also have exerted hypoglycaemic effect by other mechanism such as stimulation of glucose uptake by peripheral tissues, inhibition of endogenous glucose production or activation of gluconeogenesis in liver and muscles, as similar mechanism have been proposed for different plant drugs which were reported for antidiabetic activity in streptozotocin induced diabetic animals.31
The results of the In-vitro study on glucose utilization by isolated mice hemidiaphargm are given in Table-4. The data revealed that glucose uptake by the extracts significantly similar to that of insulin. These findings suggest that the extracts may have direct insulin like activity which enhances the peripheral utilization of glucose and have extra-pancreatic effect.32
The authors are grateful to Dr. Madhu Chitkara, Director, Chitkara
Institute of Engg.
and Technology, Rajpira,
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