Glycemic Properties of Trichosanthes dioica Leaves

Abstract

In continuation of our previous work on Trichosanthes dioica Roxb. (Cucurbitaceae), the aim of this study was to screen the glycemic attributes of an aqueous extract of Trichosanthes dioica leaves in normal as well as various diabetic models. The variable doses of 250, 500, and 750 mg kg^{− 1} body weight (bw) of the extract were administered orally to normal and streptozotocin (STZ)-induced sub- and mild-diabetic rats in order to define its glycemic potential. The dose of 500 mg kg^{− 1} bw was identified as the most effective dose which brings down the blood glucose level (BGL) by 32.9% (P < 0.001) at 6 h during fasting blood glucose (FBG) studies in normal rats. However, glucose tolerance test (GTT) showed the maximum reduction of 30.9% (P < 0.001) in BGL at 5 h in normal rats with the same dose, whereas the reduction observed was by 40.3% and 88.6% (P < 0.001) in sub- and mild-diabetic rats, respectively, at 3 h of glucose administration only. This evidence clearly indicates that the aqueous extract of Trichosanthes dioica leaves has good hypoglycemic potential along with a high anti-diabetic profile.

Keywords::

• Anti-diabetic
• glucose tolerance test
• glibenclamide
• Trichosanthes dioica leaves
• streptozotocin

Introduction

India leads the world with the largest number of diabetic subjects (Balasubramanyam & Mohan, 2007) earning the dubious distinction of being termed the “diabetes capital of the world” (Mohan et al., 2007). A number of Indian medicinal plants have been used for thousands of years in the traditional system of medicine for treating various diseases. The medicinal property of plants has now been investigated scientifically throughout the world, due to their potent pharmacological activities, low toxicity, and economic viability (Singh et al., 2007; Gupta et al., 2005). More than 100 medicinal plants are mentioned in the Indian systems of medicine, including folk medicines, for the management of diabetes (Kar et al., 2003). Ayurveda terms its clinical specialty as Rasayana, and it has been reported that the Rasayanas are rejuvenators (Sharma et al., 1992), nutritional supplements and possess strong anti-diabetic activity (Rai et al., 2008). In recent years, a number of plants commonly used to treat diabetes in the traditional system of medicine have been explored scientifically by our research group for the investigation of their chemical constituents, elemental analysis, their role in diabetes management, and pharmacological activities (Kesari et al., 2004; Rai et al., 2007a, 2007b).

Pointed gourd [Trichosanthes dioica Roxb. (Cucurbitaceae)] is a tropical vegetable crop with its origin in the Indian subcontinent. It is one of the most nutritive cucurbit vegetables and holds a coveted position in Indian markets during the summer and rainy season. The fruits are very rich in protein and vitamin A (Kumar et al., 2003). It has a number of medicinal properties and many reports are available regarding its role in the circulatory system, especially in lowering blood sugar and lipid profile (Sharma & Pant, 1988a,1988b; Sharma et al., 1989; Mukharjee, 1996). Direct intake of seeds of the plant was also found to be effective in the serum lipid profile of normal and mild-diabetic human subjects and rabbits (Sharma et al., 1990; Sharma & Pant, 1988c). Seeds of the plant were also found to possess anti-fungal and anti-bacterial activity and are widely used in the treatment of acid dyspeptic disease (Harit & Rathee, 1996). The leaves and tender shoots have been used in the Ayurvedic system of medicine since ancient times (Sharma et al., 1989). Thus, the present investigation was undertaken to evaluate the glycemic profile of the aqueous extract of Trichosanthes dioica leaves on blood glucose level (BGL) of normal and streptozotocin (STZ) induced sub- and mild-diabetic rats during fasting blood glucose (FBG) and glucose tolerance test (GTT) studies, so that a novel oral anti-diabetic agent can be identified with high nutritive value.

Materials and Methods

Preparation of crude drug

Fresh leaves of Trichosanthes dioica (7 kg) were collected in the month of June from the local area of Sherpur Kalan Ghazipur-U.P. (India) and shade-dried. It was authenticated by Prof. Satya Narayan, Taxonomist, Department of Botany, University of Allahabad, India. A voucher specimen has been submitted. The dried leaves (2 kg) were mechanically crushed and extracted with distilled water at 70°C using a Soxhlet, up to 54 h. The extract was filtered and concentrated in a rotary evaporator at 35 ± 5°C under reduced pressure, to obtain semisolid material, which was then lyophilized to yield a powder (about 11.3% w/w).

Experimental animals

More than a hundred male albino Wistar rats of the same age group and body weight, 150–200 g, were selected for all the experiments. Animals obtained from the National Institute of Communicable Disease (NICD), New Delhi, India, were housed in polypropylene cages at an ambient temperature of 25–30°C and 45–55% relative humidity with a 12 h dark and light cycle. Animals were fed pellet diet (Golden Feed, New Delhi) and water ad libitum. The study was approved by the Institutional Ethical Committee.

Induction of diabetes

Diabetes was induced by a single intraperitonial injection of freshly prepared streptozotocin (50 mg kg^{− 1} bw) in 0.1 M citrate buffer (pH = 4.5) to rats fasted overnight. After 3 days of streptozotocin (STZ) administration, rats with marked hyperglycemia (fasting blood glucose > 150 mg dl^{− 1}) were selected for the study. The rats with hyperglycemia were divided into two groups of 30 rats each: sub-diabetic animals with normal FBG and abnormal PPG levels; mild-diabetic animals with FBG 150–200 mg dl^{− 1}.

Estimation

Blood glucose level (BGL) was estimated by the glucose oxidase method (Brahm & Trinder, 1972) using standard kit of Bayer Diagnostics India Ltd.

Experimental design

Initial screening of the aqueous extract for the hypoglycemic activity was done with a range of variable doses in normal healthy rats by conducting fasting blood glucose (FBG) and glucose tolerance test (GTT) studies. The antidiabetic effect was assessed in sub- as well as mild-diabetic models with the same range of doses based on similar studies of FBG and GTT (Rai et al., 2008).

Evaluation of glycemic management in normal healthy rats

Five groups of six rats each were used in the experiment; group I serving as untreated control received vehicle (distilled water) only, and animals of groups II, III, and IV received aqueous leaf extract suspended in distilled water at doses 250, 500, and 750, mg kg^{− 1}, respectively. Blood samples were collected from the tail vein at 2, 4, and 6 h after giving the extract.

Assessment of hypoglycemic activity by GTT in normal healthy rats

The aqueous extract was given orally to different groups of normal healthy animals in the same fashion as above and their effect on FBG was studied hourly up to 2 h. The BGL value at 2 h was treated as “0” h value for GTT. The animals were then orally treated with 4 g kg^{− 1} of glucose and their glucose tolerance was studied at 1 h intervals for another 3 h. Thus, the total period of blood collection was up to 5 h.

Study of anti-diabetic activity by GTT in sub- and mild-diabetic rats

The anti-diabetic effect of aqueous extract of Trichosanthes dioica leaves in sub- and mild-diabetic rats was also assessed by improvement in glucose tolerance. The rats were divided into six groups. Group I control, received vehicle (distilled water) only, whereas variable doses of 250, 500, and 750 mg kg^{− 1} of leaf extract was given orally to group II, III, and IV respectively. Blood glucose levels were first checked after 90 min of treatment, considered as “0” h value, and then 2 g kg^{− 1} glucose was given orally to all the groups. Blood glucose levels were further checked up to 3 h at regular intervals of 1 h each, considered as 1, 2, and 3 h values. The results were compared with group V rats, which were treated with 2.5 mg kg^{− 1} of glibenclamide (synthetic hypoglycemic agent).

LD₅₀ experiment

Toxic effect of the water extract was also studied by a LD₅₀ experiment. Two groups of rats of both sexes (6 animals per group, 3 females and 3 males), weighing about 180–200 g, were orally treated with a single dose of 5 and 7.5 g of the aqueous extract of Trichosanthes dioica leaves. Then, rats were observed for gross behavioral, neurologic, autonomic, and toxic effects continuously. Food consumption, faeces and urine were also examined at 2 h and then at 6 h intervals for 24 h.

Statistical analysis

Data were statistically evaluated using two-way ANOVA, followed by a post hoc Scheffe's test using the statistical package PRISM 3.0 version. The values were considered significant when P < 0.05.

Results

Impact on FBG in normoglycemic rats

Table 1 describes the hypoglycemic effect of a single oral treatment of variable doses of 250, 500, and 750, mg kg^{− 1} of aqueous leaf extract in normal healthy rats. Treated rats showed a regular fall of 27.9 and 32.9% from the doses of 250 and 500 mg kg^{− 1}, respectively, after 6 h. However, a fall of only 31.6% was observed with the dose of 750 mg kg^{− 1} after the same interval of time.

Table 1.  Effect of variable doses of Trichosanthes dioica leaf aqueous extract on BGL during FBG test of normoglycemic rats (mean ± SD).

	Blood glucose levels (Mg dl^−1)
	Pre-treatment		Post-treatment (h)
Experimental groups	Treatment (mg kg^− 1 bw)	FBG	2	4	6
Control	Distilled water	75.9 ± 3.6	75.4 ± 4.2	75.7 ± 3.5	75.1 ± 3.9
Extract	250	74.3 ± 3.4	63.9 ± 4.5	57.1 ± 3.7	54.6 ± 3.5*
Extract	500	76.8 ± 4.1	61.2 ± 3.6	55.4 ± 4.2	51.5 ± 3.7*
Extract	750	72.2 ± 3.2	62.3 ± 3.8	54.7 ± 4.3	51.4 ± 3.9*

* P < 0.05 as compared with control.

Effect on FBG in normal rats during GTT

Table 2 deals with the study of aqueous extract of Trichosanthes dioica leaves on BGL levels and glucose tolerance of normal healthy rats. Different doses of 250, 500, and 750 mg kg^{− 1} of extract were given orally to overnight fasted healthy rats. The fall observed with doses of 250, 500, and 750 mg kg^{− 1} in BGL after 2 h of administration was 14.0, 17.2, and 14.9%, respectively, considered as ‘0’ h value. However, the fall was observed up to 3 h after glucose administration at 1 h intervals and the results reveal that the percentage fall in BGLs was regular up to the dose of 500 mg kg^{− 1} and reaches a maximum of 30.9%. Moreover, the fall of 27.5% was observed with the dose of 750 mg kg^{− 1} at the same time.

Table 2.  Effect of variable doses of Trichosanthes dioica leaf aqueous extract on BGL during GTT of normoglycemic rats (mean ± SD).

Blood glucose levels (Mg dl^−1)
Pre-treatment			Post-treatment (h)
Experimental Groups	Treatment (mg kg^− 1 bw)	FBG	0	1	2	3
Control	Distilled water	76.7 ± 3.7	76.1 ± 4.5	112.4 ± 4.1	105.6 ± 4.3	98.7 ± 3.9
Treated 1	250	73.2 ± 3.8	65.4 ± 3.5	88.8 ± 3.7	80.8 ± 4.5	74.1 ± 3.8*
Treated 2	500	74.7 ± 4.1	63.0 ± 3.9	82.1 ± 4.2	75.7 ± 3.6*	68.2 ± 3.9*
Treated 3	750	75.9 ± 3.6	64.7 ± 4.2	84.8 ± 3.6	78.4 ± 3.5*	71.5 ± 3.7*

* P < 0.05 as compared with control.

Effect on diabetic rats during GTT

Figure 1 and Figure 2 demonstrate the anti-diabetic effect of aqueous extract of Trichosanthes dioica leaves on sub- and mild-diabetic animals, respectively. Different doses of aqueous extract as mentioned above along with the standard drug glibenclamide 2.5 mg kg^{− 1} was given orally to the groups as defined in the experimental design. A fall of 23.5, 40.3, and 36.0% in BGLs of sub-diabetic animals was observed after 3 h of glucose administration with doses of 250, 500, and 750 mg kg^{− 1}, respectively. However, the dose of 2.5 mg kg^{− 1} of glibenclamide reduced BGL by 41.8% at 3 h during GTT in sub-diabetic rats. Thus, the results clearly reveal that the dose of 500 mg kg^{− 1} of aqueous extract is almost the same as the dose of 2.5 mg kg^{− 1} of glibenclamide during GTT in sub-diabetic rats. The fall observed after 3 h of glucose administration was 25.7%, 38.6%, and 36.7% in BGLs of mild-diabetic animals with the doses of 250, 500, and 750 mg kg^{− 1}, respectively. However, the dose of 2.5 mg kg^{− 1} of glibenclamide reduced BGL by 37.5% at 3 h during GTT in mild-diabetic rats, which is less effective than the dose of 500 mg kg^{− 1} leaf aqueous extract.

Figure 1.  Effect of variable doses of Trichosanthes dioica leaf aqueous extract on BGL during GTT in sub-diabetic rats. **P < 0.01 as compared with control. Control: Distilled water, Treated 1: 250 mg kg^{− 1}, Treated 2: 500 mg kg^{− 1}, Treated 3: 750 mg kg^{− 1}, Glibenclamide: 250 mg kg^{− 1}.

Figure 2.  Effect of variable doses of Trichosanthes dioica leaf aqueous extract on BGL during GTT in mild-diabetic rats. **P < 0.01 as compared with control. Control: Distilled water, Treated 1: 250 mg kg^{− 1}, Treated 2: 500 mg kg^{− 1}, Treated 3: 750 mg kg^{− 1}, Glibenclamide: 250 mg kg^{− 1}.

LD₅₀

The experiment was carried out on normal healthy rats. The behavior of the treated rats appeared normal. No toxic effect was reported at doses up to 10 and 15 times the effective dose of the aqueous extract, and there was no death in any of these groups.

Discussion

Anti-hyperglycemic effect of Trichosanthes dioica seeds was reported previously by our research group (Rai et al., 2008). Hence, the present study was carried out on its leaves and the dose of 500 mg kg^{− 1} of leaf extract was found 12.5% more active than the dose of 1,000 mg kg^{− 1} of seed extract. The leaves of Trichosanthes dioica are eaten as a vegetable and have the following composition: moisture, 80.5%; protein, 5.4%; fat, 1.1%; fiber, 4.2%; other carbohydrates, 5.8%; mineral matter, 3.0% and calcium, 5.31 mg 100 g^{− 1} and phosphorus, 73 mg 100 g^{− 1} (Gopalan et al., 1989). β -Sitosterol, its glucosides, and luteolin-7-glucoside have also been isolated from its leaves (Gopal & Ramchandra, 1979). A steroidal saponin, 24-α -ethyl-20-ene-7-hydro-stigmast-8 β: 14 β-di-3-O−β-d-xylofuranoside, has been isolated from MeOH soluble fraction of 95% EtOH extract of the leaves of Trichosanthes dioica (Saxena & Dave 1995).

Since no data have been reported so far for the glycemic profile of the aqueous extract of Trichosanthes dioica leaves in normal as well as STZ-induced diabetic animals models, this study deals with complete screening of its glycemic attributes based on FBG and GTT studies in vivo. The observed difference between initial and final BGLs of different groups of animals during these studies revealed a significant elevation in blood glucose in the control group as compared with treated groups. The maximum hypoglycemic effect (Table 1) was produced within 6 h during FBG studies in normal rats. In glucose-loaded normal rats the maximum hypoglycemia was observed at 5 h on oral administration of the extract of Trichosanthes dioica leaves (Table 2). It is generally accepted that the sulphonyl ureas, including glibenclamide, produce hypoglycemia in normal as well as diabetic animals by stimulating the pancreatic β -cells to release more insulin (Goth, 1985; Larner, 1985). Hence, the significant decrease in BGLs of diabetic rats treated with extract as well as glibenclamide (Figure 1 and Figure 2) may be by stimulation of the residual pancreatic mechanism, probably by increasing peripheral utilization of glucose as postulated by Earth et al. (1996). This vindicates the efficacy of the extract to control elevated blood sugar levels. Maintenance of blood sugar levels in normal and diabetic rats was observed throughout the period of study. The data suggests that the active ingredients of the aqueous extract or their metabolites need about 2 h to exhibit their hypoglycemic and anti-diabetic effect by reaching target tissues through circulation. The data also show that the effect remains significant even after 3 h of glucose administration. The dose of 500 mg kg^{− 1} of aqueous extract was found to be more effective than the dose of 2.5 mg kg^{− 1} glibenclamide in case of mild-diabetic rats. Whereas, in the case of sub-diabetic rats, the effect of the same dose of aqueous extract as well as of glibenclamide is practically the same. Moreover, the effectiveness of the aqueous extract in both the cases of sub- as well as mild-diabetic rats is comparable with the synthetic drug glibenclamide.

In conclusion, the aqueous extract of Trichosanthes dioica leaves was found to exhibit a hypoglycemic effect in normoglycemic rats and an anti-diabetic effect in the STZ-induced diabetic model. The antidiabetic effect of extract was greater than glibenclamide. Enzymatic studies are in progress in order to elucidate the detailed mechanism of action at cellular and molecular levels. Isolation and characterization of compounds of leaves responsible for lowering of BGL is also processing.

Acknowledgements

The authors are grateful to the NMPB (National Medicinal Plants Board) for financial assistance and the first author (PKR) thanks the ICMR (Indian Council of Medical Research) for the award of SRF.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.