Plant Material
Tridax procumbens, whole plant was collected from the Udaipur district during the month of September-October, and was identified and authenticated from the department of Botany, University of Rajasthan, Jaipur. A voucher specimen (RUBL No. 20534) was retained in the Herbarium of the department of Botany, University of Rajasthan, Jaipur.
Preparation of Extract
The shed-dried plant material was coarsely powdered (6.9 kg) and extracted with 50% methanol using soxhlet apparatus for 36 hrs. The resulting mixture was filtered and the filtrate was evaporated in an oven at 40°C to get the dry residue (7.86 g). The resultant residue was used as 'drug' in experiments.
Chemicals
Alloxan monohydrate was obtained from Sigma Chemical Co. (St. Louis, MO., USA). Glibenclamide tablets (Daonil; Aventis Pharma. Ltd., India) were procured from the authorized distributor of the company. All other chemicals used were of analytical grade.
Experimental Animals
Adult healthy male albino rats of Wistar strain weighing 140-160 g, obtained from IVARI Izatnagar, Bareli (U.P.) were used after acclimatization for 14 days for this study. The animals were housed in polypropylene cages under standard husbandry conditions (12 hrs light/dark cycle: 25 ± 3°C). Rats were provided water and pellet diet (Hindustan Lever Ltd., Bangalore, India.) ad libitum. The study was conducted after the approval from the institutional ethical committee for animal care.
Induction of Experimental Diabetes in Rats
After fasting, diabetes was induced by a single intraperitoneal injection of 120 mg/kg body weight of 'Alloxan monohydrate' in distilled water. The animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycemia. These animals were tested for diabetes after 15 days and animals with blood glucose (fasting) range 300 - 450 mg/dl were selected for experimentation.
Experimental Protocol
Animals were divided into seven groups of 6 rats each.
Group I: Rats served as normal-control and received the vehicle (0.5 ml distilled water/day/rat)
Group II: Rats (normal) were administered T. procumbens (250 mg/kg b.wt./day) in distilled water as a fine aqueous suspension orally.
Group III: Rats (normal) were administered T. procumbens (500 mg/kg b.wt./day) in distilled water as a fine aqueous suspension orally.
Group IV: Rats served as diabetic-control and received the vehicle (0.5 ml distilled water/day/rat)
Group V: Rats (diabetic) were administered T. procumbens (250 mg/kg b.wt./day) in distilled water as a fine aqueous suspension orally.
Group VI: Rats (diabetic) were administered T. procumbens (500 mg/kg b.wt./day) in distilled water as a fine aqueous suspension orally.
Group VII: Rats (diabetic) were administered Glibenclamide (10 mg/kg b.wt./day) in distilled water as a fine aqueous suspension orally.
All the rats were fasted for 16 hr. before experimentation, but allowed free access to water.
Acute Dose Study
All the rats received single dose treatment in all groups.
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Blood Sugar Estimation: Blood samples were collected by tail vein puncture just prior to drug administration and at 1/2, 1, 2, 4, 6 and 8 hrs. The blood glucose was estimated by 'One touch-ULTRA' glucometer (Johnson & Johnson company, USA).
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Acute Toxicity Evaluation in Rats: The methanolic extract was tested for its acute and short-term toxicity (if any) in normal rats. To determine acute toxicity of a single oral administration of herbal drug, different doses of the drug (0.25--5.0 g/kg) were administrated to different groups of rats (8 rats in each group with 4 male and 4 female). Mortality and general behavior of the animals were observed periodically for next 48 h. The animals were observed continuously for the initial 4 h and intermittently for the next 6 h and then again for 24 h and 48 h after the drug administration. The parameters observed were grooming, mood, hyperactivity, sedation, loss of righting reflex, respiratory rate and convolutions.
Sub-chronic Dose study
All the rats received treatment for 30 days in all groups.
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Body weight: Body weight was measured at the time of alloxan-dosing. After 15 d of alloxan-dosing, the body weight of all the rats was measured once a week with at sacrifice (30 d). Before blood collection and at sacrifice day experimental animals were overnight fasted (water was not restricted) to reduce the erratum of feeding.
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Fasting Blood Glucose (FBG) Estimation: Fasting Blood Glucose was measured at the time of alloxan-dosing. After 15 d of alloxan-dosing, the Fasting Blood Glucose of all the rats was measured once a week with at sacrifice (30 d). Blood samples were collected by tail vein puncture just prior to drug administration and at 7, 15, 21 and 30 days. The blood glucose was estimated by 'One touch-ULTRA' glucometer (Johnson & Johnson company, USA). The results were expressed in terms of mg/dl of blood.
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Oral Glucose Tolerance Test (OGTT): Prior to an OGTT all the rats were fasted for 16 h Distilled water (control), a reference drug glibenclamide (10 mg/kg b.wt.) or each of the two different doses of Tridax procumbens extract (250 and 500 mg/kg b.wt.) were then orally administered to respective groups of 6 rats each. 30 min. later, glucose (3 g/kg) was orally administered to each rat with a feeding syringe. Blood samples were collected from the tail vein by tail milking at -30(just before the extract and glibenclamide administration), 0 (just before the oral administration of glucose), 30, 60, 90 and 120 min. after glucose load.
Statistical Analysis
Results were expressed as mean ± SEM. Data were analyzed with one way ANOVA for the comparison between groups, followed by Tukey as a post hoc test. The significance level was set at p < 0.05.