Mechanism of Action of Ahypoglycemic Principle Isolated from Fenugreek Seeds
D. PURI*, K .M. PRABHU AND P S MURTHY**
Department of Biochemistry,
University College of Medical Sciences,
Dilshad Gardan, Delhi – 110 095
(Received on January 16, 2002)

 

Abstract: Mechanism of action of an orally active hypoglycemic principle isolated from water extract of seeds of Trigonella foenum graecum (fenugreek) was investigated in alloxan induced subdiabetic and overtly diabetic rabbits of different severities. The active principle was orally administered to the subdiabetic and mild diabetic rabbits (five in each group) at a dose of 50 mg/kg body weight for 15 days. The treatment produced significant attenuation of the glucose tolerance curve and improvement in the glucose induced insulin response, suggesting that the hypoglycemic effect may be mediated through stimulating insulin synthesis and/or secretion from the beta pancreatic cells of Langerhans. Prolonged administration of the same dose of the active principle for 30 days to the severely diabetic rabbits (n = 5) lowered fasting blood glucose significantly, but could elevate the fasting serum insulin level to a much lower extent, which suggests an extra-pancreatic mode of action for the active principle. The effect may also be by increasing the sensitivity of tissues to available insulin. The hypoglycemic effect was observed to be slow but sustained, without any risk of developing severe hypoglycemia.

 

Key words:     trigonella foenum graecum                     fenugreek

alloxan – diabetic                                  glucose tolerance test                insulinotropic

 

 

Introduction
Methods
Results
Discussion
References

 

INTRODUCTION

Type II diabetes (non-insulin diabetes mellitus or NIDDM) is a major health problem because of its high frequency, long duration and high risk of chronic complications. At present, second and third generation sulfonylureas are the oral pharmacological agents used to counteract insulin secretion deficiency in this disease (1). But their long-term use produces undesirable side effects, such as skin rashes, dilutional hyponatremia, transient leukopenia, thrombocytopenia, skin rashes, myocarditis, severe hypoglycemia and increased chances of cardiovascular death of unknown mechanism. This highlights the importance of searching for an alternative disease therapy strategy with drugs which not only have insulinotropic effect but increase insulin sensitivity also. Plants have long been a source of traditional antidiabetic medicines (2, 3). Evaluation of these plants and especially their natural active substances is a logical way of developing new drugs to treat NIDDM. We have isolated an orally active principle called G_II from fenugreek (Trigonella foenum graecum) seeds, which elicits significant hypoglycemic effects in alloxan-diabetic animal models of varying severities: alloxan recovered, mild diabetic and sever diabetic (Radha Moorti et al; Puri et al, unpublished data). Mode of action of the active principle is reported in the present study.

Most hypoglycemic agents act in more than one way. Stimulation of synthesis/release of insulin from pancreatic beta cells (i.e., insulinotropic effect) is an important mechanism of action in a number of plant products (4). Possible insulinotropic effect of the G_II fraction was investigated in the present study by orally feeding the G_II fraction to the alloxan diabetic rabbits for fifteen days and comparing glycemic control and serum insulin levels before and after the treatment. Serum insulin and blood glucose assays were made in the fasting state and during a glucose tolerance test, and the pretreatment and the post-treatment values were compared to observe how the treatment influences serum insulin and blood glucose levels.
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METHODS

Plant material: A potent hypoglycemic principle isolated from water extract of fenugreek seeds through a series of chromatographic techniques was used in the present study. The active principle termed G_II was found to be different from trigonelline. The details of the method of isolation of G_II is not given here since patent is applied for the isolation.

Animals: Inbred male albino rabbits weighing between 700 g and 1000 g and age 2 - 2.5 months, used in the present study, were screened for any abnormality of glucose homeostasis by an oral glucose tolerance test before induction of diabetes by alloxan. Aqueous solution of alloxan monohydrate was injected intravenously through the marginal ear vein of the animals, fasted overnight, at a dose of 80 mg/kg body weight. Fasting blood glucose (FBG) was determined and glucose tolerance test (GTT) was performed once every week for a period of 4 weeks to monitor the hyperglycemic state. The rabbits that developed hyperglycemia were arbitrarily divided in three groups of five each; the subdiabetic, the mild diabetic and the severely diabetic.

• The subdiabetic or the alloxan-recovered rabbits (AR) have near normal or slightly elevated FBG (below 120 mg/dl) but have elevated glucose tolerance curve (5).
• The mild diabetic rabbits have elevated FBG (120-250 mg/dl) and abnormal glucose tolerance as well.
• The severely diabetic rabbits (SD) have severe hyperglycemia (> 250 mg/dl). GTT was avoided in these rabbits because they showed high mortality with this test.

Biochemical estimations: Blood glucose was estimated by glucose oxidase-peroxidase method (6). Serum was obtained and stored at –20°C prior to determination of insulin level. Serum insulin was determined by ELISA (7) using a commercially available kit (Boehringer Mannheim Immunodiagnostic, Bombay).

Drug trial: Since aim of the present study is to observe how G_II fraction would influence serum insulin level, insulin assays were made before and after treatment with G_II fraction and the corresponding values were compared. A daily oral dose of 50 mg/kg body weight of the G_II fraction, which was found to be maximally active in our preliminary studies in lowering blood glucose, was administered in the present study. Initially, the pre-treatment basal values of blood glucose and serum insulin were estimated in the fasting state and during a glucose tolerance test in the subdiabetic and the MD rabbits. A 10% solution of D-glucose at a dose of 3 g/kg body weight was orally administered after the withdrawal of blood sample (for glucose and insulin assay) at the end of 16 hours of fasting. More samples were collected for these estimations after 1 h and 2.5 h of the glucose load. This was followed by 15 days of G_II therapy where daily oral administration of G_II fraction (50 mg/kg body weight) was carried out for the next 15 days. The post-treatment values of blood glucose and serum insulin were then determined in the fasting state and during OGTT. The corresponding pre-treatment and post-treatment values were compared: fall in blood glucose level if any would indicate hypoglycemic effect, and elevation of serum insulin response would indicate beta-cryotropic (or insulinotropic) effect of the G_II fraction.

As stated above, OGTT was not performed in SD rabbits. Therefore blood glucose and serum insulin levels in these animals were determined in the fasting state only, before and after the G_II therapy.
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RESULTS

Table I shows the levels of blood glucose and serum insulin in normal rabbits during glucose tolerance test, i.e., after oral glucose load; and Table II shows these parameters in the subdiabetic and mild diabetic rabbits. Serum insulin levels, estimated in each of the fasting and post-oral glucose load  samples on the first day of the study( day 1,) showed that the fasting insulin levels were nearly the same in the alloxan recovered (AR or subdiabetic) rabbits as those of the normal rabbits. The same is true of fasting blood glucose values. Since abnormalities in the AR rabbits is only in the glucose tolerance, the 1 h and the 2.5 h blood glucose values (on day 1) were significantly higher than the corresponding values of the normal rabbits, while the serum insulin levels were decreased compared to normal rabbits. But treatment with the G_II fraction for fifteen days brought about significant fall in blood glucose in the 1 h sample (220  ± 19 mg/dl to 140 ± 16.7 mg/dl) and the 2.5 h sample (188 ± 12.3 mg/dl to 108 ± 14.2 mg/dl), which were significant (P < 0.01). Serum insulin levels improved from 19.8 ± 2 µU/ml to 29.8 ± 2.1 µU/ml in the 1 h sample and from 10.9 ± 2.1 µU/ml to 28.4 ± 5.5 µU/ml in the 2.5 h sample: these elevations were also significant (P<0.01). Thus, administration of G_II fraction for fifteen days brought down the blood glucose levels considerably, and this was associated with elevation of the serum insulin levels both in the fasting and the post-prandial conditions. Similar results were observed in the mild diabetic rabbits also where the blood glucose levels in the fasting as well as the post prandial condition were significantly brought down (40%) to normal values by 15 days of treatment with G_II (Table II). Moreover, the post-treatment levels of serum insulin in the fasting, 1h and 2.5h samples were significantly higher than the corresponding pre-treatment levels. Thus, administration of the G_II fraction does elicit a significant blood glucose lowering effect. An important observation is that it does not bring about any dangerous hypoglycemic reaction, which is an additional advantage.

TABLE I: Glucose induced insulin response in the normal rabbits.

 

TABLE II:  Effect of 15 days administration of GII on glucose induced insulin secretion in the subdiabetic (AR) and mild diabetic (MD) rabbits.

Group and Parameters		(Mean !SD of five animals in each group)
		Fasting	1-hour	2.5-hour
Subdiabetic
Serum Insulin	(mU/ml)
	Day 1	11.6±1.4	19.8±2	10.9±2.21
	Day 15	11.6±21	29.8±1.1	28.4±5.6
Plasma Glucose	(mg%)
	Day 1	90±5.6	220±19	188±12.3
	Day 15	86±8.2	140±16.7	108±14.2
Mild diabetic
Serum Insulin	(mU/ml)
	Day 1	3.4±0.5	4.02±0.5	3.8±0.4
	Day 15	8.9±1.6	19.3±2	16.3±2
Plasma Glucose	(mg%)
	Day 1	150±11.7	246±19.4	198±14.3
	Day 15	89±9.8	140±11.6	110±10.2

In the severely diabetic rabbits, the G_II treatment for 30 days elicited significant hypoglycemic effect – the FBG decreased from 42.8 ± 46 mg/dl to 110.1 ± 11 mg/dl (74% fall), but without a corresponding increase (15%) in the serum insulin level (Fig. 1). Although the FBG level after treatment reached normal level, the serum insulin level was still much lower than in normal animals.

Fig.1 click for full view

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DISCUSSION

A short period of drug therapy improved glucose tolerance in subdiabetic and MD rabbits in 15 days, and brought down FBG to normal levels in 15 and 30 days respectively in MD and SD rabbits. The improvements in glucose tolerance in subdiabetic and MD rabbits after the treatment occurred simultaneously with an enhancement of serum insulin levels. The serum insulin increased to normal levels in subdiabetic rabbits. But in MD rabbits the increased insulin level was still below that in normal. This suggests that the improvement in subdiabetic rabbits is mediated via increased insulin secretion. Among the factors capable of stimulation of insulin secretion, glucose enjoys a prominent position and GTT is still considered to be the best test for endocrine pancreatic function.

A direct relationship between levels of serum insulin and glucose is well known. The administration of the G_II fraction to diabetic animals has a significant influence on the serum insulin response to an oral glucose load: the response is greater in the treated than the untreated diabetic rabbits. The improved response shows that the active principle possibly mimics the effects of sulphonylureas in stimulating serum insulin levels in diabetes with residual pancreatic beta cell integrity.

In the MD rabbits, the favourable response of bringing down the FBG to normal levels, even through increase in serum insulin was not upto the normal level, suggests two mechanisms of actions occur side by side. One is increase in insulin levels and the other is probably increase in sensitivity of the tissues to the available insulin. The latter is a great advantage in case of type II diabetes mellitus. The combined response of increased secretion after oral glucose and probably increase in insulin sensitivity lead to better glucose utilization in G_II treated rabbits. It can, however, not be stated whether increased synthesis or increased release of insulin from beta cells accounts for increased serum insulin level. An added advantage with the G_II therapy is that, unlike sulfonylureas which may lead to severe hypoglycemic episodes, us of G_II is not associated with any such complication.

In severely diabetic rabbits, after daily administration of G_II for one month, even though there was 74% fall in the fasting blood glucose levels from 427.8 ± 46 mg/dl to 110.1 ± 11 mg/dl, the increase in the insulin levels were marginal only (Fig. 1). Such small increase in serum insulin levels can cause only a mild reduction in blood glucose level but cannot account for the observed hypoglemic effect of the drug, which is quite pronounced.

This suggests that the hypoglycemic effect of G_II is mainly due to extra-pancreatic factors in the severely diabetic animals and to a slight extent to increased sensitivity of tissues to available insulin (as see in MD rabbits). Slight increase in the serum insulin levels might be due to stimulation of few surviving beta cells. The extrapancreatic effects may be due to enhanced insulin-receptor binding, or induction/stimulation of activities of enzymes of glucose utilization. Further biochemical and histopathological studies are necessary to clarify the point whether G_II treatment reverses the necrotic action of alloxan, as shown in case of catechin (8), a plant flavonoid.

In conclusion, our results show that the purified compound G_II brings about its hypoglycemic effect by a combination of three mechanisms: 1) increasing the levels of serum insulin, 2) increasing the sensitivity of tissues to insulin action, and 3) stimulating the activity of enzymes of glucose utilization depending on the needs in subdiabetic, mild diabetic and severe diabetic situation. Thus it seems to cover a broad range of situations in diabetes mellitus.
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REFERENCES

1. Melander A. Non insulin dependent diabetes mellitus treatment with sulfonylureas. In: Clinical endocrinology and metabolism. Eds Natras M and Hale P, Ballere Tindal, London 1988; p. 443-453.

2.      Mark Bluementhal, Goldberg A, Brinckmann J. Herbal Medicine. Ist Edn. Integrated Medicine Communication, Newton 2000 MA 02464.

3.      Kirtikar KR, Basu BD. In : Indian Medicinal plants, volume II. Eds. E Balthr, Caius JF, Basu LM, Allahabad 1688; 3^rd edn.

4.      Shukla R, Sharma SB, Puri D, Prabhu KM, Murthy PS. Medicinal plants for the treatment of diabeties mellitus. Indian Journal of Clinical Biochemistry 2000; 15(supp): 169-177.

5.      Babu BV, Radha Moorty, Pugazhenthi S, Prabhu KM, Murthy PS, Alloxan recovered rabbits as animal models for screening the hypoglycemic activity of compounds. Indian Journal of Biocemistry and Biophysics 1998; 25: 714-718.

6.      Burtis CA, Ashwood ER. Eds. Tietz Text book of clinical Chemistry 3^rd Edn. Philadelphia: W.B. Saunders and Co: 1976.

7.      Anderson L, Dinesen B, Jagerson PN. Enzyme immunoassay for insulin in serum or plasma. Clinical Chemistry 1993; 38:578-582.

8.      Chakravarthy BK, Gupta S, Gode KD. Functional beta cell regeneration in the islets of pancreas in alloxan induced diabetic rats by (-)-epicatechin. Life Sciences 1983; 31(24):2693-2697.
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