Evaluation of Antidibetic Potential of Hydromethanolic Extract of Tridax procubens (Linn) in Mice

 

Raman Chandak¹, Harshada Tayade²

¹Department of Pharmacognosy, School of Pharmacy, G. H. Raisoni University. Nagpur. M.S.

²Department of Pharmacology, Dr. VPPC of Pharmacy, Aurangabad. M.S.

 

ABSTRACT:

The aqueous leaves extract of Tridax procumbent (Linn) was evaluated for its antidiabetic potential on normal and streptozotocin (STZ)-induced diabetic rats. In the chronic model, the hydromethanolic leaf extract was administered to normal and STZ- induced diabetic rats at the doses of 200 and 400mg/kg body weight (b.w.) p.o. per day for 14 days. The fasting Blood Glucose Levels (BGL), serum insulin level and biochemical data such as glycosylated hemoglobin, Total Cholesterol (TC), Triglycerides (TG), High Density Lipoproteins (HDL) and Low Density Lipoproteins (LDL) were evaluated and all were compared to that of the known anti-diabetic drug glibenclamide. The statistical data indicated significant increase in the body weight, liver glycogen, serum insulin and HDL levels and decrease in blood glucose, glycosylated hemoglobin, total cholesterol and serum triglycerides when compared with glibenclamide. Thus the hydromethanolic leaves extract of Tridax procumben had beneficial effects in reducing the elevated blood glucose level and lipid profile of STZ-induced diabetic rats.

 

 

 

INTRODUCTION:

Diabetes mellitus is one of the largest global health problems demanding preventive and new therapeutic interventions. Currently, there is a need for safe, effective, and less costly antidiabetic medications, and investigating medicinal plants for new antidiabetic medication is an interesting research area. Thus, the present study was done to evaluate the antidiabetic activities of 80% methanolic leaf extract of Tridax procumbens (Linn) in mice.

 

Hypoglycemic and antihyperglycemic activity of the three doses (200 mg/kg, and 400mg/kg) of crude hydromethanolic leaf extract was studied on normoglycemic, oral glucose loaded, and streptozotocin-induced diabetic mice models.

 

The effect of the extract on body weight and diabetic dyslipidemia was also studied on streptozotocin-induced diabetic mice. Glibenclamide (5mg/kg) was used as a standard drug in all cases. A glucose meter and an automated chemistry analyzer were used to measure blood glucose and serum lipid level respectively. Data were analyzed using one-way analysis of variance followed by Tukey’s post hoc multiple comparison test. All the three doses of the plant extract (100mg/kg, 200 mg/kg, and 400mg/kg) showed a significant (p<0.05) antihyperglycemic activity in the diabetic mice at the 7th and 14th day of repeated daily dose administration as compared the negative diabetic control. But, the extract did not show significant blood glucose lowering activity in normoglycemic, oral glucose loaded, and diabetic mice after single dose administration, and it did not significantly improve the body weight loss and diabetic dyslipidemia of diabetic mice after repeated daily dose administration for 14 days. This study revealed that the hydromethanolic extract of Tridax procumbens leaves possesses significant antihyperglycemic activity justifying the traditional use of the plant for diabetes. Present study was done to evaluate the antidiabetic activity of 80% methanolic leaf extract of Tridax procumbent (Linn) in mice.

 

MATERIALS AND METHODS:

Drugs, Chemicals, and Instruments. Methanol absolute (Nice Chemicals, India), streptozotocin (Sigma Aldrich, Germany), glibenclamide (Julphar Pharmaceuticals, Ethiopia), citric acid monohydrate (Lab Tech Chemicals, India), trisodium citrate dihydrate (Blulux Labratories, India), sterilized water for injections (Nirma Ltd., India), 40% glucose solution (Reyoung Pharmaceuticals, China), analytical balance, pH meter,  distilled water, mindray BS-240 clinical chemistry analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd,, China) were all of analytical grade.

 

Plant Material Collection and Preparation:

Fresh leaves of Tridax procumben were collected. After collection, taxonomic identification and authentication were done and the specimen of the plant was kept at the Herbarium of Biology Department, Dr. BAMU, and Aurangabad.

 

Preparation of Plant Material Extract:

The leaves of the plant were first thoroughly washed with distilled water and allowed to dry under shade with optimal ventilation. The dried leaves were then chopped to coarse powder. Nine hundred gram of the coarse powdered plant material was macerated in 80% methanol for 72 hours and then the extract was filtered using Whatman filter paper No. 1. Then, the residue was remunerated two times with fresh solvent, each for 72 hours, and the filtrates obtained from the successive maceration were dried in a hot air oven at 40 degree centigrade. The dried extract was then kept in a desiccator to maintain dryness till use.

 

 

Experimental Animals:

Healthy Swiss albino mice (weighing 25-30g and age of 8-12 weeks) were purchased. The animals were then kept in the animal house, using polypropylene cages. The animals were maintained under standard conditions (12 h light and 12 h dark cycle) and allowed free access to standard pellet laboratory diet and water ad libitum. Animals were acclimatized to the laboratory conditions for 1 week before the initiation of the experiment.

 

 

 

 

Induction of Experimental Diabetes:

Streptozotocin (STZ) was used to induce experimental diabetes. STZ was first dissolved in 0.1M cold citrate buffer (pH=4.5). Then, the freshly prepared solution was given intraperitoneally to the mice at a dose of 150 mg/kg [34]. Mice were fasted overnight for 16 hours prior to STZ administration. Food and water were allowed to the animals thirty minutes after the administration of STZ. Six hours after STZ administration, animals were allowed to drink 5% glucose solution for the next 24 hours to prevent hypoglycemic shock and death. Four days after STZ injection, animals were screened for diabetes. Mice with fasting blood glucose level > 200mg/dl were included in the study as diabetic. Immediately after screening, STZ-induced diabetic animals were assigned randomly into different groups to perform the experiment.

 

Assessing Antihyperglycemic Activity, Effect on Body Weight, and Antidyslipidemic Activity of Repeated Daily Doses of the Extract in Streptozotocin-Induced Diabetic Mice. After overnight fasting for 16 hours, STZ-induced diabetic mice and normal mice were randomly assigned into 6 groups (5 groups of diabetic mice and 1 group of normal mice, 6 animals per group). Then, mice were treated with distilled water, plant extract, and glibenclamide once daily for 14 days according to their respective grouping as explained above. Blood glucose level and body weight of mice were measured just before starting treatment on the 1st day of treatment (four days after STZ injection) as baseline and then on the 7th and 14th day of treatment following overnight fasting for 16 hours. On day 15, overnight fasted mice were first sacrificed using overdose of an anesthetic, sodium pentobarbitone at a dose of 150 mg/kg IP, and then blood samples were collected from each animal in a sterile gel tube via cardiac puncture. The blood samples were left at room temperature for 2 hours and then centrifuged. The supernatant was immediately separated from the pellet to prepare serum samples in order to determine the level of triglyceride (TG), total cholesterol (TC), and high density lipoprotein cholesterol (HDL-C) using automated chemistry analyzer.

 

RESULT:

Percentage Yield of Plant Material Extraction:

A total of 154 grams of dried dark-brown gummy extract was harvested at the end of the extraction process. The extract was found to be better soluble in water than organic solvent. The percentage yield of the extract was found to be 17.11% (w/w).

 

 

Table 1: Hypoglycemic activity of hydromethanolic leaf extract of Tridax procumben in normoglycemic mice

Each value represents mean ± SEM; n=6 for each treatment. a Compared to the negative control, compared TPLE 200mg/kg, compared to TPLE 400mg/kg, and compared to baseline blood glucose level. 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001. TPLE = Tridax Procumben leaf extract, DW = distilled water, and GLC = glibenclamide

 

Table 2: Effect of Tridax procumben leaf extract on oral glucose tolerance in normal mice.

Each value represents mean ± SEM; n=6 for each treatment. a Compared to the negative control, c compared to TPLE 200mg/kg, compared to baseline blood glucose level, and compared to the blood glucose level at 30 minute. 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001. TPLE = Tridax procumben leaf extract, DW = distilled water, and GLC = glibenclamide. Time refers to time after oral glucose loading.

 

Table 3: Antihyperglycemic activity of single dose of Tridax procumben leaf extract in STZ-induced diabetic mice.

Each value represents mean ± SEM; n=6 for each treatment. a Compared to the negative control and compared to baseline blood glucose level. 1.p < 0.01, and 2 p < 0.001. TPLE = Tridax procumben leaf extract, DW = distilled water, and GLC = glibenclamide.

 

Table 4: Antihyperglycemic activity of repeated daily doses of Tridax procumben leaf extract in STZ-induced diabetic mice.

Each value represents mean ± SEM; n=6 for each group. a Compared to the diabetic control, compared to the normal control, and compared to baseline blood glucose level. 1 p < 0.05, 2 p < 0.01,. TPLE = Tridax procumben leaf extract and GLC = glibenclamide.

 

Antihyperglycemic Activity of Single Dose of the Hydromethanolic Leaf Extract of Tridax procumben in Streptozotocin-Induced Diabetic Mice:

Between and within group analysis were performed to see BGL differences across the various groups and time points, respectively (Table 3). The between group analysis indicated no significant difference in baseline fasting BGL across all groups. Similarly, there was no significant difference in BGL across all groups at the 2^nd hour after treatment. Compared to the negative control, plant extract treated groups did not show a statistically significant reduction in BGL at all time points. Similarly, there was no significant difference in BGL at all time points when groups treated with plant extract were compared to each other and compared to the positive control.

 

Within a group comparison showed that there was no significant BGL reduction observed in TPLE 200mg/kg, and TPLE 400mg/kg treated groups at all time points compared to the baseline fasting BGL. However, percent reduction in BGL was recorded as 21.59% in TPLE 200 mg/kg treated group, and 22.75% in TPLE 400mg/kg treated group at the 8th hour compared to the respective baseline fasting level. The standard drug (glibenclamide, 5mg/kg) produced a significant BGL reduction at the 4th, 6th, and 8th (p<0.001) hours compared to the initial level.

 

Antihyperglycemic Activity of the Repeated Daily Doses of the Hydromethanolic Tridax procumben Leaf Extract in Streptozotocin-Induced Diabetic Mice:

The effect of repeated daily doses of the plant extract on blood glucose level of diabetic mice is shown in Figure 1. Between group analysis indicated no significant difference in baseline fasting BGL across all groups of diabetic mice, but the baseline BGL of the diabetic groups was significantly (p<0.001) higher than the baseline BGL of the normal control (Table 4). All the three doses of TPLE significantly (P<0.05) reduced the BGL on the 14th day of treatment compared to the diabetic control.

Table 5: Effect of repeated daily doses of the hydromethanolic leaf extract of Tridax procumben on body weight of STZ-induced diabetic mice.

Each value represents mean±SEM; n=6 for each group. a Compared to the diabetic control, ncompared to the normal control, and compared to baseline body weight. 1 p < 0.01, and 2 p < 0.001. TPLE = Tridax procumben leaf extract and GLC = glibenclamide.

 

Table 6: Effect of repeated daily doses of hydromethanolic Tridax procumben leaf extract on serum lipid level of diabetic mice.

Each value represents mean ± SEM; n=6 for each group. a Compared to the diabetic control, compared to TPLE 200 mg/kg, compared to CALE 400mg/kg, and compared to the normal control. 1 p < 0.05,, and 2 p < 0.001. TPLE = Tridax procumben leaf extract, GLC = glibenclamide, TC = total cholesterol, TG = triglyceride, and HDL-C = high density lipoprotein cholesterol.

 

The GLC treated group also showed significant reduction in blood glucose level on the 7th and 14th day of treatment compared to the diabetic control. But, GLC treated group showed no significant difference in BGL at all time points when compared to plant extract treated groups. There was no statistically significant difference in BGL at all time points when groups treated with plant extract were compared with each other.

 

Within a group analysis revealed that 200 mg/kg TPLE significantly (P<0.05) reduced the BGL on the 14th day of treatment compared to the baseline level, but the diabetic control and the normal control did not show a significant change in BGL on 7th and 14th days compared to the respective baseline level. The standard drug reduced the BGL significantly (P<0.001) on the 7th and 14th days compared to the baseline level.

 

DISCUSSION:

Diabetes is one of the largest global health emergencies of the 21st century. There is a need for safer, more effective, and less costly treatment as currently available drug regimens of DM have limitations. Novel compounds with pan-target antidiabetic activity and long-term safety should be targeted for patients with coexisting diabetes and dyslipidemia. Thus, investigating plant derived compounds for DM is an attractive research area as they are believed to be safe and easily accessible and do not require laborious pharmaceutical synthesis.

 

Previous acute oral toxicity tests were done on the hydromethanolic extract of Tridax procumben leaves, although there is discrepancy among the results of the studies. The present study revealed that the median lethal dose (LD50) of the plant extract is greater than 2000 mg/kg showing a wide margin of safety. In the present study, there were no significant differences in baseline BGL across groups as observed in all animal models. Similarly, the vehicle treated groups did not show detectable reduction in BGL compared to the baseline level in all animal models. But, significant BGL reduction was observed in the diabetic mice after repeated daily dose administration of the hydroalcoholic leaf extract, indicating the change in BGL was attributed to the treatment received.

 

The antidiabetic activity of medicinal plants is due to the presence of T alkaloids, tannins, flavonoids, and carotenoids thus, the antihyperglycemic activity of TPLE may be due to the presence of these different secondary metabolites known to have blood glucose lowering activity with possible synergistic effects.

 

The antihyperglycemic activity of CALE may be due to potentiation of insulin effect either by increasing the secretion of insulin from beta cells of pancreas or by increasing the peripheral glucose uptake. However, detailed molecular studies are required to identify the exact mechanism for the antihyperglycemic activity of CALE observed in the study.

 

Induction of experimental diabetes using STZ causes severe body weight loss in mice. Studies have shown that severe hyperglycemia in mice is associated with an increased body weight loss after STZ treatment. Similarly, our study revealed that STZ-induced diabetes caused significant body weight loss in the diabetic control. The induction of diabetes with STZ leads to loss of body weight due to increased wasting of fat stores, muscle, and tissue proteins. Lipid abnormality is also one of the complications of diabetes mellitus, manifested mainly by high serum TG, TC, and low HDL-C. Insulin deficiency causes activation of hormone sensitive lipase that can lead to increased lipolysis and increased secretion of VLDL from the liver. Decreased activity of lipoprotein lipase, secondary to insulin deficiency, also leads to decreased clearance of chylomicrons and VLDL. In addition, hypertriglyceridemia stimulates the enzymatic action of cholesteryl ester transfer protein which leads to an increase in triglyceride content of LDL and HDL. Triglyceride-enriched HDL particles easily undergo catabolism, and Triglyceride-enriched LDL particles undergo subsequent hydrolysis via hepatic lipase or lipoprotein lipase resulting in reduced LDL particle size.

 

CONCLUSION:

The Present study revealed that the hydromethanolic extract of Tridax procumben leaves has significant antihyperglycemic activity, but it did not significantly improve body weight loss and diabetic dyslipidemia in diabetic animals.

 

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Received on 09.12.2022          Modified on 30.12.2022

Accepted on 13.01.2023     ©AandV Publications All right reserved