Abstract: Diarun plus a polyherbal formulation containing herbal ingredients of folkloric Antidiabetic effect, was investigated for its effect on glycemic status in rats and mice. In contrast to conventional chemical induced diabetic animal models, changes in glycemic states were induced by physiological maneuvers. Results revealed that in euglycemic animals Diarun plus elicited little change (-10 to +10%) insignificantly. In food deprivation/swim exercise induced hypoglycemia, Diarun plus reduced the degree of hypoglycemia in both rats and mice (From 38 to 27% in rats and 45 to 32% in mice). Similarly, the marked hyperglycemia induced by dextrose (70% in rats and 95% in mice) was reduced markedly to 8 and 25% , respectively. The findings of the present study suggests that the ingredients of Diarun plus have the unique property of maintaining near euglycemic state irrespective of the altered glycemic state and that have no significant effect in euglycemic condition.
INTRODUCTION
Pre-clinical investigation on newer substances for their efficacy against diabetes mellitus is normally carried out in Streptozotocin (STZ) or alloxan induced diabetic animal models, which destroys the cells of β islets of pancreas and induce a chronic hyperglycemic status akin to Diabetes Mellitus (DM). However, it has been documented that these chemicals produce structural and functional abnormalities in the nervous system of these animals (Mohan et al., 2001). Therefore measurement of any parameter involving nerve conduction is likely to generate inconsistent results. Taking this into consideration, in the present study an attempt has been made to identify the effect of a polyherbal formulation, Diarun plus, on the glycemic status in physiologically altered glycemic status where the nervous system is intact.
Diarun plus is a polyherbal formulation containing seeds of Eugenia jambolana, Trigonella foenum-graecum, Momordica charantia leaves of Gymnema sylvestre, fruits of Emblica officinalis, rhizomes of Curcuma longa and roots of Salacia reticulatta. All these ingredients have a traditional claim for their beneficial effects in the management of DM (Anonymous, 1976; Chopra et al., 1956; Nadkarni, 1954). Our earlier preliminary experiments (Anonymous, 2002) with Diarun plus in STZ induced diabetic animal models indicated the inclusion of Diarun plus in the diabetic diet as an adjuvant therapeutic measure in the management of DM. In the present study, attempts were made to attain hypoglycemic status using either food deprivation or swim exercise, which is likely to utilize glucose for energy expenditure while exogenous glucose administration was used to achieve hyperglycemic state.
MATERIALS AND METHODS
Swiss male albino mice (25-30 g) and wistar male albino rats (180-220 g) were housed under normal laboratory conditions with free access to food and water except for one group in which food deprivation was included as a method to elicit hypoglycemia. The study was conducted at the Department Animal House of Tamil University. The study was approved and was conducted in accordance with the Institutional Ethical Committee.
Induction of hypo-and hyperglycemia: Twenty four hour food deprivation prior to experimentation or allowing the animals to swim in water at room temperature (29-30°C) for 3 min in a polypropylene container (40x35x25 cm) filled with water upto 15 cm height was employed to induce hypoglycemia.
Exogenous oral administration of dextrose 2 g kg-1, 15 min prior to experimentation was used to produce hyperglycemia.
Measurement of blood glucose: The blood glucose was measured using Ames glucometer (Bayers Diagnostics with appropriate glucostix) from a drop of blood collected by venipuncture of the tail, which has been validated in earlier study (Rajendran et al., 2001) by comparing with the blood glucose level estimated using auto analyser.
Drug treatment
Diarun plus was administered 60 min prior to glucose estimation as a
1% suspension in carboxyl-methyl cellulose and at the end of 60 min the animals
were allowed either to swim or received dextrose. A separate group of animals
were deprived of food for 24 h. The blood glucose was measured prior to exposing
the animals to food deprivation, swim exercise or dextrose administration and
at 15 min after these maneuvers. The doses of Diarun plus were 100 or
500 mg kg-1; i.p., selected based on pilot studies. Appropriate vehicle
treated animals were served as control. The results were expressed as the actual
blood glucose value and also as the percentage change considering the initial
value as 100%. The data were subjected to statistical analysis by employing
ANOVA followed by Dunnett’s ‘t’ test. A level of p≤0.05 was
considered statistically significant.
Diarun plus was supplied by Rumi Herbals, Mugappair, Chennai, while Dextrose Anhydrous IP, was purchased from Glaxo, Mumbai.
RESULTS
Vehicle treatments in free fed animals, both rats and mice produced an insignificant elevation of only +2% change in blood glucose level. Diarun plus per se in both the doses produced inconsistent and insignificant changes in the glycemic state of both the animals (Table 1). Food deprivation for 24 h produced a significant hypoglycemia to the extent of 31.5% in rats and 33.9% in mice. In Diarun plus supplemented, food deprived animals this fall was attenuated both in rats and mice (Table 2). However, food deprivation induced hypoglycemia was still significant in mice and rats which received Diarun plus (Table 2).
Animals, which swam for 3 min, exhibited hypoglycemia to the extent of 39% in rats and 45% in mice, which was comparable to those observed in food, deprived animals. However, Diarun plus supplementation decreased significantly the hypoglycemia in mice and to a milder extent in case of rats when compared to that of the results observed in food deprived groups (Table 3).
Dextrose in the dose administered elevated significantly the blood glucose level to the extent of 70% in rats and 95% in mice. In Diarun plus pretreated animals dextrose was unable to produce hyperglycemia to the same extent in both rats and mice. A marked and significant decrease was recorded in both the animals. This effect was found to be dose-related (Table 4).
| Table 1: | Influence of Diarun plus on the blood glucose levels in healthy, hypoglycemic and hyperglycemic physiological models in free fed rats and mice |
| Each value represents the Mean±SEM of six observations, Drug was administered 60 min prior to final glucose measurement | |
| Table 2: | Influence of Diarun plus on the blood glucose levels in healthy, hypoglycemic and hyperglycemic physiological models in 24 h food deprivated rats and mice |
| Each value represents the Mean±SEM of six observations, Drug was administered 60 min prior to final glucose measurement, *p<0.05 compared with respective initial value | |
| Table 3: | Influence of Diarun plus on the blood glucose levels in healthy, hypoglycemic and hyperglycemic physiological models by swimming in rats and mice |
| Each value represents the Mean±SEM of six observations, Animals were allowed to swim for 3 min, Drug was administered 60 min prior to final glucose measurement, ** p<0.01 compared with respective initial value † p<0.05 compared with respective vehicle treatment (final value) | |
| Table 4: | Influence of Diarun plus on the blood glucose levels in healthy, hypoglycemic and hyperglycemic physiological models in dextrose fed rats and mice |
| Each value represents the Mean±SEM of six observations, Drug was administered 60 min prior to final glucose measurement, *p<0.05 and ** p<0.01 compared with respective initial value, † p<0.05 compared with respective vehicle treatment (final value) | |
DISCUSSION
The results of the present study wherein the dose related attenuation of the hypoglycemia and hyperglycemia elicited under various physiological circumstances indicate that Diarun plus shows a tendency to maintain the euglycemic status irrespective of any alteration in the glycemic state. This restoration by Diarun plus to the euglycemic status suggest that Diarun plus supplementation will be beneficial in the situations like DM where glycemic status are altered. This effect is considered beneficial since the anti-diabetic agents like insulin or oral hypoglycemic drugs either alone or in combination have a tendency to induce hypoglycemic status. This may be attributed to the difficulty encountered in the assessment of the dose or noncompliance of the food habit advised to the DM patients. This hypoglycemic situation is considered more harmful than the disorder itself. Besides, in the present study, the polyherbal combination investigated failed to modify the glycemic status when administered alone. This is advantageous since the harmful hypoglycemic situation is not seen which is otherwise encountered with conventional hypoglycemic agents.
The herbal ingredients present in the formulation had been proved to exhibit hypoglycemic effect in many experimental diabetic models (Senthilvel et al., 2006a; b) and beneficial effect in diabetic patients (Senthilvel et. al., 2004). Ethanolic extract of C. longa rhizome lowered blood sugar in alloxan-induced diabetic rats by vitalization of pancreatic cells and stimulation of insulin production (Anonymous, 2001). Ethanolic extracts of M. charantia seeds lowered fasting blood glucose and improved glucose tolerance (Karunanayake et al., 1984). Fruits of E. officinalis are potent antioxidant, which are free radicals scavengers and lipid peroxide inhibitors (Jose and Kuttan, 1995). Powdered seeds and aqueous extract of E. jambolana lowered blood glucose in diabetic rabbits (Anonymous, 1976) and produced a marked symptomatic relief in diabetic patients (Kohli and Singh, 1993). Aqueous extract of T. foenum-graceum exhibited potent hypoglycemic effect in alloxan induced diabetic rats (Abdel Barry et al., 1997). The seed extract also induced hyperinsulinemia and hypercholesterolemia (Sauvaire et al., 1996/1997). The alcoholic extract of G. sylvestre lowered blood glucose in diabetic animals and stimulates insulin secretion in rabbits (Chopra et al., 1956). Salacino isolated from S. reticulatta is also been reported to be a potent inhibitory activity against several alpha-glucosidases, such as maltase, sucrase and isomaltase and the inhibitory effects on serum glucose levels in maltose-and sucrose-loaded rats (in vivo) were found to be more potent than that of acarbose, a commercial alpha-glucosidase inhibitor (Yoshikawa et al., 2002). A combination of these herbs can be naturally expected to lower the blood glucose level and exert beneficial effects in DM patients.
The methods employed to achieve alterations in the glycemic status are physiological and transient thereby not causing any structural and functional damage in contrast to chemical models. However a limitation in the present study is that the scope envisages only a transient stage and extrapolation to chronic situation to the disease diabetic state is debatable. Despite this, the present data provide the basic information regarding Diarun plus and its possible clinical utility based on the beneficial observation recorded.
Further detailed studies on its safety and the exact mode of action are warranted.
ACKNOWLEDGMENTS
The authors are thankful to M/s. Rumi Herbals, Mugappair, Chennai for supplying of samples and financial assistance to carry out this study.