|Year : 2015 | Volume
| Issue : 2 | Page : 63-68
Pharmacological evaluation of antidiabetic activity of Urginea indica in laboratory animals
Ankit Gupta, Sandeep Kumar Singh, Ashutosh Kumar Yadav
Department of Pharmacology, School of Pharmacy, Babu Banarasi Das (BBD) University, Lucknow, Uttar Pradesh, India
|Date of Submission||16-Dec-2014|
|Date of Acceptance||31-Jan-2015|
|Date of Web Publication||23-Mar-2015|
Ashutosh Kumar Yadav
Department of Pharmacology, School of Pharmacy, Babu Banarasi Das (BBD) University, BBD City, Faizabad Road, Chinhat, Lucknow - 226 028, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The present study was designed to investigate the antidiabetic potential of the bulbs of Urginea indica (Roxb.) Kunth. (U. indica), which has been utilized traditionally to cure diabetes mellitus. Materials and Methods: An acute toxicity study was done to check the toxicity of U. indica ethanolic extract (UIEE) and an oral glucose tolerance test (OGTT) was carried out in a study population of normoglycemic rats. Diabetes was induced by single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg body weight). Animals were orally treated with the extracts and standard drug (glibenclamide 10 mg/kg), and vehicle daily for 14 days. Hypoglycemic effects, change in body weight, lipid profile, and feed and water intake of the diabetic rats were assessed for normal, diabetic control, standard, and extract-treated groups. Histopathology was also carried out for the pancreatic tissue. Results: The acute toxicity study revealed the nontoxic nature of U. indica ethanolic extract. A considerable decrease in blood glucose was observed within 120 min on glucose tolerance testing among normoglycemic rats treated with a high dose of extract UIEE (1.5 g/kg). Daily oral treatment with the extract and the standard drug for 14 days significantly reduced blood glucose, total cholesterol (TC), and triglyceride (TG) levels. High-density lipoprotein (HDL) levels were found to be improved compared to the diabetic control group. The feed and water intake in diabetic rats was markedly reduced and weight loss was minimized. Histopathological analysis confirmed the biochemical findings. Conclusion: The results of the experiments suggest that UIEE has significant antidiabetic effects on STZ-induced diabetic rats.
Keywords: Diabetes, insulin, streptozotocin, Urginea indica
|How to cite this article:|
Gupta A, Singh SK, Yadav AK. Pharmacological evaluation of antidiabetic activity of Urginea indica in laboratory animals. Int J Nutr Pharmacol Neurol Dis 2015;5:63-8
|How to cite this URL:|
Gupta A, Singh SK, Yadav AK. Pharmacological evaluation of antidiabetic activity of Urginea indica in laboratory animals. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2020 Nov 25];5:63-8. Available from: https://www.ijnpnd.com/text.asp?2015/5/2/63/153795
| Introduction|| |
Diabetes is a complex disorder involving abnormalities in carbohydrate, fat, and protein metabolism caused by an absolute or relative lack of insulin, reduced insulin activity, or both, which in turn lead to hyperglycemia.  It is recognized as a major global chronic health problem, accounting for significant morbidity and mortality; it is associated with microvascular (retinopathy, neuropathy, and nephropathy) and macrovascular (heart attack, stroke, and peripheral vascular disease) complications.  The International Diabetes Federation (IDF) estimates that in 2013, the number of people suffering from diabetes was 382 million, while 5.1 million died, and that this prevalence will rise to 592 million by 2035.  Based on etiology, there are two main basic types of diabetes: Type 1 or insulin-dependent diabetes mellitus (IDDM), which is autoimmune or hereditary and characterized by a deficiency of insulin-producing beta cells in the pancreas, and type 2 diabetes or non-insulin-dependent diabetes mellitus (NIDDM), which is due to insulin resistance or reduced insulin sensitivity.  Though there are various modern drugs available for improving the hyperglycemic state, such as sulfonylureas, biguanide, thiazolidinedione, α-glucosidase inhibitors, and glinides, patients continue to suffer from the many side effects of these modern drugs due to which the search for new pharmacological approaches is ongoing and concerted efforts are being made to develop suitable alternative effective remedies for diabetes. , Presently, there is a growing interest in herbal remedies that are apparently efficient, produce minimal side effects, and are rich sources of antidiabetic, antihyperlipidemic, and antioxidant agents such as flavonoids, gallotannins, amino acids, and other related polyphenols. 
Urginea indica (Roxb.) Kunth. (U. indica), belonging to the family Liliaceae, is a perennial, glabrous herb commonly known as the "Indian squill" and locally as "Junglipiyaz," found in the drier sub-Himalayan tracts of the western Himalayas, in Bihar, on the Konkan Coast, and on the Coromandel Coast.  In the Indian indigenous traditional system of medicine, U. indica is reputed for a number of therapeutic benefits, for which bulbs are the most commonly employed plant parts reportedly used as cardiotonics and, in small doses, as an expectorant and digestives as well as in the treatment of many diseases, especially asthma, dropsy, rheumatism, leprosy, and skin ailments.  The extract of the bulbs has also been traditionally reported to possess hypoglycemic properties. , Fresh squill yields two glycosides, scillarin A and scillarin B. It also contains flavonoids, carbohydrates, antifungal glycoproteins, steroids, alkaloids, tannins, coumarins, and saponins.  To our knowledge, there is no scientific evidence in the support of antidiabetic activity of U. indica. Hence, the present study was aimed to ascertain the scientific basis for the use of U. indica in the management of diabetes using streptozotocin (STZ)-induced diabetic rats.
| Materials and methods|| |
Bulbs of U. indica were purchased from N.N. Aushadhalaya, Lucknow, Uttar Pradesh, India. The plant was identified and authenticated by the Department of Botany, Lucknow University. A voucher specimen was deposited at the herbarium of the School of Pharmacy, BBD University, Lucknow for future reference.
STZ was purchased from Sigma-Aldrich Corporation (St. Louis, IL, USA). Glibenclamide was obtained as a gift sample from Sun Pharmaceutical Industries Limited (Ankaleshwar, Gujarat, India). All other commercial reagents used during the experiments were of analytical grade.
The collected plant bulbs were sliced transversely and subjected to air drying under shade for 4 days. The air-dried, sliced pieces were then crushed to form a powder. Extraction of dried and powdered bulbs of U. indica was done by cold extraction process. The resulting plant material (350 g) was soaked at room temperature in 70% aqueous ethanol (v/v) for 3 days with occasional shaking. The soaked material was passed through double-layered muslin cloth to remove vegetative debris and the fluid obtained was subsequently filtered through filter paper. The residue was resoaked for the next 3 days and the procedure was repeated twice. The filtrates obtained were then evaporated to a thick, semisolid paste of dark brown color, the crude Urginea indica ethanolic extract (UIEE) (yielding 8%). 
Healthy albino Wistar rats of both sexes weighing about 150-250 g were acquired from the animal house of BBD Northern India Institute of Technology. Before the initiation of the pharmacological study, the rats were kept for 7 days to acclimatize them to the laboratory environment. They were housed in clean, ventilated polypropylene cages at an ambient temperature of 22 ± 2°C and 45-55% relative humidity, with a dark and light cycle (12 h each). The rats were fed with a standard pellet diet and water ad libitum. The experimental protocol was approved by the animal house Institutional Animal Ethics Committee (BBDNIIT/IAEC/023/2014).
Assessment of acute toxicity study
Healthy adult Wistar rats, starved overnight, were divided into two groups, each consisting of six rats, and were orally fed with the extracts at the doses of 750 mg/kg and 1.5 g/kg body weight, respectively.  The animals were then observed continuously for 2 h under the following profiles:
- Behavioral profile: Alertness, restlessness, irritability, and fearfulness.
- Neurological profile: Spontaneous activities, reactivity, touch response, pain response, and gait.
- Autonomic profile: Defecation and urination. 
After every 24 h, up to 7 days, they were observed for any signs of toxicity or lethality.
Oral glucose tolerance test (OGTT)
The rats were divided into four groups of six animals (n = 6) each. Group I served as control and received distilled water; Group II received glibenclamide (10 mg/kg body weight); Group III was treated with UIEE (750 mg/kg body weight); and Group IV was treated with UIEE (1.5 g/kg body weight) orally. The rats were on overnight fast and glucose tolerance was tested by oral administration of glucose (2 g/kg body weight) to normoglycemic rats 30 min after treatment. Blood glucose levels were estimated at 0 min, 30 min, 60 min, and 120 min after glucose administration. 
Induction of diabetes
Diabetes was induced using STZ at a dose of 40 mg/kg body weight. The drug was freshly prepared by dissolving in 0.1 M citrate buffer (pH 4.5). The solution was then administered intraperitoneally to rats that had been on overnight fast prior to administration.  After 72 h,, the animals were screened for diabetes, and the rats that showed fasting blood glucose (FBG) ≥200 mg/dL were included in the study. 
All the diabetic animals were further, randomly divided into five groups of six animals each and treated orally once a day for 14 days, as follows:
Group 1: Served as normal control and received distilled water.
Group 2: Served as diabetic control and received distilled water.
Group 3: Diabetic rats treated with standard glibenclamide (10 mg/kg body weight).
Group 4: Diabetic rats treated with UIEE (750 mg/kg body weight).
Group 5: Diabetic rats administered UIEE (1.5 g/kg body weight).
Changes in body weight, feed and water intake were monitored during the experimental periods. After 14 days of treatment, blood samples were collected by retro-orbital sinus puncture using capillary tubes under light diethyl-ether anesthesia. The serum was separated by centrifuging the blood samples at 3000 rpm for 10-15 min for biochemical analysis.
Blood glucose was measured using one touch glucometer (Accuchek, Roche Diagnostics) at weekly intervals, that is, 0 days, 7 days, and 14 days after the daily administration of extract orally to ascertain the diabetic status of each group. Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein HDL levels were also evaluated in normal and STZ-induced diabetic rats.
After termination of the experiment, pancreatic tissues from all groups were subjected to histopathological studies. The whole pancreas from each animal was removed after sacrificing under anesthesia. A portion of the pancreas was fixed in 10% neutral formalin fixative solution. After fixation, tissues were embedded in paraffin; thick sections were cut at 4-5 μm and stained with hematoxylin and eosin for histological examinations. The sections were examined under light microscope. 
All values of results are presented as mean ± standard error of mean (SEM). The statistical significance was evaluated by one-way analysis of variance (ANOVA), followed by Tukey's post test using GraphPad Prism software (GraphPad Software, Inc., USA). A value of P < 0.05 was considered to be significant.
| Results|| |
0Acute toxicity study
An acute toxicity study of UIEE did not reveal any behavioral, neurological, or autonomic changes, and all the rats survived during the whole experimental period. There was no lethality or any toxic reaction found at any of the doses selected until the end of the study period.
Effect on oral glucose tolerance of normoglycemic rats
On OGTT, at 60 min, a rise in blood glucose levels was observed in all the groups (due to glucose load), with maximum increase in the control group and the least increase in the standard group. Due to the administration of UIEE and standard drug, the decrease in blood glucose levels was observed within 120 min. The results are depicted in [Figure 1].
|Figure 1: : Effect of ethanolic extract of Urginea indica on oral glucose tolerance of STZ induced diabetic rats|
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Effect on blood glucose level
Single-dose STZ (40 mg/kg body weight) significantly (P < 0.0001) increases the blood glucose, as shown in [Table 1]. After the daily oral administration with UIEE, for 14 days, a significant reduction (P < 0.0001) in the blood glucose levels was observed in the diabetic rats compared to the diabetic control rats. At the end of the experiment, on the 14th day, the blood glucose level was (156.00 ± 4.35) mg/dL and (153.67 ± 6.07) mg/dL of the groups treated with doses of UIEE of 750 mg/kg and 1.5 g/kg respectively.
|Table 1: Effect of UIEE on blood glucose level in STZ induced diabetic rats|
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Effect on body weight
[Table 2] shows the changes in body weight of the experimental rats at zero and final day of treatment. The body weights of the diabetic control rats decreased when compared with the normal control rats. However, an improvement in body weight was observed in the UIEE-treated groups when compared with diabetic control rats.
Effect on feed and water intake
Significant increases in the feed intake (P < 0.001) and water consumption (P < 0.0001) were observed throughout the study period, compared to the normal control rats. However, after administration of UIEE, the feed and water intakes were markedly reduced compared to the diabetic control rats as shown in [Figure 2] and [Figure 3].
|Figure 2: Effect of ethanolic extract of Urginea indica on the feed intake of STZ induced diabetic rats|
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|Figure 3: Effect of ethanolic extract of Urginea indica on the water intake of STZ induced diabetic rats|
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Effect on lipid profile
The diabetic control group showed significant elevation in serum TG (P < 0.0001) and TC (P < 0.001) levels, compared to normal control [Table 3]. UIEE supplementation at 750 mg/kg and 1.5 g/kg for 14 days administered to the STZ-induced diabetic rats resulted in significant diminution in TG (P < 0.05) and TC (P < 0.05) compared to the diabetic control group. A marked improvement was found in the HDL-cholesterol level when compared with diabetic control.
|Table 3: Effects of UIEE on triglycerides (TG), total cholesterol (TC), and high density lipoproteins (HDL) level of STZ induced diabetic rats|
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Histopathological study of rat pancreas
The microscopic structures of the pancreas of rats in different groups are shown in [Figure 4]. The pancreas of normal control rats showed normal islets and abundant pancreatic β-cells. However, in diabetic control rats, there was extensive destruction of the islet cells. The pancreas of diabetic rats treated with glibenclamide showed minimal damage of the cellular population in the islets. However, UIEE 2 (1.5 g/kg body weight)-treated rats showed partial restoration of normal cellular population as compared to diabetic control rats.
|Figure 4: Histological examinations of rat pancreas after 14 days treatment (a) Normal rat pancreas showing abundant pancreatic ƒÀ cells (b) Pancreas of diabetic control rat with destruction in the ƒÀ cells (c) Pancreas of diabetic rat after treatment with glibenclamide (d) Pancreas of diabetic rat after treatment with UIEE 2 (1.5 g/kg) (All fi gures are in 10x magnifi cation)|
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| Discussion|| |
The incidence of diabetes mellitus is increasing at alarming rates worldwide, and it can be mainly attributed to sedentary lifestyles and calorie-rich diets. Hyperglycemia, the most important feature of diabetes, is a well-known cause of elevated free radical levels, followed by the production of reactive oxygen species (ROS), which can lead to increased lipid peroxidation, alter antioxidant defense, and further impair glucose metabolism in biological systems. Diabetes is often linked to abnormal lipid metabolism and is considered a major risk factor for the premature development of atherosclerosis and cardiovascular complications.  Effective blood glucose control is the key to preventing or reversing diabetic complications and improving the quality of life in patients with diabetes. 
Acute toxicity studies revealed the nontoxic nature of UIEE. No lethality or toxic reaction was found at any of the doses selected for the toxicity study.
UIEE contains various phytochemicals, including saponins, steroids, flavonoids (quercetin), dietary fiber, and vitamins C and K, which are likely to exhibit hypoglycemic activity. 
STZ, a nitrosurea derivative produced by Streptomyces achromogenes, is one of the most commonly used diabetogenic agents for inducing diabetes in experimental animals, which causes selective β-cell cytotoxicity mediated through the release of nitric oxide (NO). , These toxins cause the death of pancreatic β-cells by alkylation of DNA, resulting in reduced synthesis and release of insulin.  Based on the above perspectives, the present study was undertaken to evaluate the antidiabetic potential of UIEE in diabetes-induced rats. The antidiabetic effects of 750 mg/kg and 1.5 g/kg of UIEE were comparable to 10 mg/kg of glibenclamide, a standard antidiabetic drug. A significant decrease in the blood glucose level was observed after treatment with U. indica. This might be due to the presence of the flavonoid quercetin,  which positively affects pancreatic tissues subjected to STZ-induced oxidative stress by directly quenching lipid peroxides and indirectly enhancing the production of endogenous antioxidants.  Furthermore, the possible mechanism for the antioxidant activity could be through the chelation of transition metals in the body and by breaking the chain reaction of lipid peroxidation by removing the hydroxyl radicals. 
STZ-induced diabetes is characterized by a severe loss in body weight,  which could be due to poor glycemic control or the excessive catabolism of proteins to provide amino acids for gluconeogenesis during insulin deficiency, resulting in muscle wasting and weight loss in diabetic, untreated rats.  Improvement in body weight was observed after the treatment with UIEE.
STZ caused a significant increase in the feed and water intake in the rats. After treatment with UIEE for 14 days, a marked decrease in the food and water intake was observed in the diabetic rats. This decrease may have occurred due to the decreased concentration of glucose in circulating blood, causing a reduction of osmotic effects in urine. Insulin deficiency resulting from STZ causes unrestrained lipolysis and proteolysis, leading to extreme hunger, which was allayed by U. indica.
Abnormalities in lipid profile are one of the most common complications in diabetes mellitus. At normal state, insulin activates lipolytic hormone action on the peripheral fat depots, which hydrolyzes triglycerides and prevents the mobilization of free fatty acids. However, insulin deficiency inactivates the lipoprotein lipase, which promotes liver conversion of free fatty acids into phospholipids and cholesterol and their final discharge into blood, resulting in elevated serum phospholipid levels.  Urginea indica supplementation led to a decrease in the TG and TC levels and an increase in the HDL levels.
Histopathological studies of pancreatic tissues of the rats were made, and STZ-induced rats showed damage in the pancreatic β-cells. UIEE 2 (1.5 g/kg body weight) showed comparable restoration of these cells, which might be due to its antioxidant properties.
Based on this study it can be postulated that the UIEE shows potential antidiabetic activity in STZ-induced diabetic rats, providing evidence to support the traditional claim. However, more efforts are needed for the isolation, characterization, and biological evaluation of the active principle(s) of UIEE to elicit aforementioned antidiabetic activity.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]
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