Interactive Study of Trigonella Foenum Graecum Seed Extract with Oral Hypoglycemic Agents

“INTERACTIVE STUDY OF TRIGONELLA FOENUM GRAECUM SEED EXTRACT WITH ORAL HYPOGLYCEMIC AGENTS IN DIABETES INDUCED NEPHROPATHY”.

a) BRIEF RESUME OF THE INTENDED WORK:

Need of study:

Diabetic nephropathy (DN) is the major cause of end stage renal disease. Approximately30% of patients with diabetes experience diabetic nephropathy,which gradually develops to final renal failure1. Diabetic nephropathy is one of the most frequent complications of diabetes mellitus. Renal disease is usually attributed to metabolic consequences of abnormal glucose regulation2.Diabetic nephropathy is characterized by a progressive accumulation of extracellular matrix components in the glomerular mesangium and tubular interstitium, which eventually leads to proteinuria and renal failure3.Elevation of significant markers such as serum urea and creatinine are related to renal dysfunction in diabetic hyperglycemia4. HbA1c has been used widely to assess glycemic control in patients as well as in diabetic mice. Studies in diabetic mice are consistent with the validity of the use of HbA1c to assess glycemic control. Diabeticrats treated with fenugreek seeds,significantly decreased the serum glucose level, triglycerides,cholesterol, urea, uric acid, LONG FORM (AST) and LONGFORM (ALT) when compared to untreated diabetic rats5. Trigonella foenum graecum seeds possess significant hypoglycemic6and antiatherosclerotic7 activity. Different flavonoids, namely, vitexin, tricin, naringenin, quercetin, and tricin-7-O-β-D-glucopyranoside, are reported to be present in fenugreek seeds8. Anti-oxidant activity of fenugreek is also been reported9. Pravastatin is reported to be effective in controlling the hypercholesterolemia present in diabetic hyperlipidemic animals10.

Combined pharmacological therapy is preferred to attain adequate glycemic control but at the same time the combination therapy should not produce hypoglycemia. Glycemic control of various combinations of oral hypoglycemic agents has been reported11. Apart from combining two or more oral hypoglycemics, physicians also recommend use ofanti-diabetic herbal drugs along with oral hypoglycemics. Drug-herb interactions must be carefully considered when herb is added as an adjuvant to oral hypoglycemic agents because the combination may produce hypoglycemia.

In the present investigation, we are planning to determine whether polyphenolic rich extract of fenugreek seeds by virtue of its hypoglycemic, hypolipidemic and antioxidant activity would modulate the renallesions associated with diabetes in experimental animals. The study also investigates the combined effect of fenugreek seed extract with oral hypoglycemic agents in diabetes induced nephropathy.

Review of literature:

Type 2 Diabetes Mellitus (DM) is often associated with hyperlipidemia and tends to influence renalfunction. Studies in type 2 DM patients suggestthat, although poor metabolic control is themost important determinant of the developmentof nephropathy, hyperlipidemia is also considered to be involved12,13.

Glycemic control is the mostimportant approach in the management of diabetic nephropathy14 and other strategies have been proposed to offer specificadvantages. Treatment of hypercholesterolemia has beenshown to reduce diabetic nephropathy. Diabetic patients withnephropathy with a low serum cholesterol concentrationare reported to exhibit a lower degree of kidneylesions than those patients with a high serum cholesterol concentration15.

Leaching of lactate dehydrogenase (LDH), aspartate aminotransferase, alanine aminotransferase as well as acid and alkaline phosphatases were increased in urine samples of diabetic rats. The beneficial amelioratinginfluence of dietary onion on diabetic nephropathy may be mediated through hypolipidemic and hypoglycemic property of onion16.

In diabetic patients the administration of moderate amounts of redwine and a polyphenol-enriched diet slowed the progression of diabetic nephropathy17.Oxidative stress and endothelial dysfunction, which areinter-related, play a role in many diseases, including atherosclerosisand several nephropathies18.

Endothelial dysfunction is present in subjects with chronic renal failure (CRF) and isinter-related to oxidative stress19.

The glomerular oxidative damage leading to glomerulosclerosisis also mediated by oxidized forms of low-densitylipoprotein (LDL) and LDL frequently undergoes glycation, oxidation and carbamoylation in CRF20.

After long-term exposure to polyphenol-rich red wine, anenhancement of antioxidant defenses was demonstrated inrat plasma and kidney21.

Quercetin administration reduced oxidativestress and improved renal function22Quercetin inhibits apoptosis of mesangial cells by blockingtyrosine phosphorylation23, as was confirmed in vitro and in vivo24, and protects the rat kidney against oxidativestress-related damage caused by cyclosporine25.

The influence of polyphenols on NO-dependent vasodilationmight be particularly beneficial in CRF, which isaccompanied by an impairment of NO-signaling mechanisms in peripheral arterioles26.

Fenugreek seeds(Trigonella foenum-graecum Fam:Fabaceae) also called as birds foot is a condiment widely used in India, china, Ukraine, and other parts of world. It was used for thousands of years in Arabian, Greek, Indian and Chinese medicine. Externally it is useful for boils, hives, ulcers and eczema. Internally fenugreek seeds have been used in folk medicine to reduce blood glucose level, increased lactation, treat pellagra, appetite loss, indigestion, bronchitis, fever, dyspepsia, hernia and stomach ulcer. Medicinal properties andprotective effects of fenugreek seeds have been well established by epidemiological studies and animal experiments. The hypoglycaemic potency of fenugreek seeds has been attributed to the 4- hydroxy isoleucin, lysine and tryptophan. The mechanism ofhypoglycaemic action probably involves direct or indirect stimulation of insulin secretion. It also acts by delayed gastric emptying, slow carbohydrate metabolism, improve peripheral glucose utilization and shows potential pancreatic and extra pancreatic effects. Treatment of diabetic rats with fenugreek seeds extract decreased serum acid and alkaline phosphatase, serum alanine and asparate transferases, as well as serum amylase in diabetic rats. Fenugreek also have secretolytic and mild antiseptic property27.

OBJECTIVE OF STUDY:

The objective of the present study is to evaluate the actions of fenugreek seed extract with oral hypoglycemic agents in type II diabetic nephropathy rat models.

SPECIFIC OBJECTIVES:

Primary outcome:

• To study the role of fenugreek seed extract in diabetic nephropathy.
• To study whether oral hypoglycemics can play a vital role in diabetic nephropathy.
• To study the effect of the fenugreek seed extract in combination with standard oral hypoglycemic agents (OHA) in diabetic nephropathy.
• To study the effect of Trigonella foenum graecum seed extract alone and in combination with oral hypoglycemic agents on antioxidant status.

Secondary outcome:

• To study the effect of fenugreek seed alone and in combination with oral hypoglycemic agents on structural integrity by microscopic examination of kidney.

b) MATERIALS AND METHODS:

Source of Data:

Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of Krupanidhi College of Pharmacy, Indian Institute of Sciences, etc., and other Research Publications and Journals.

Method of Collection of Data:

The data will be from animal experimentation as per the parameters studied under each animal model, which are mentioned under the objectives of the study.

Fenugreek seed extraction28:

Soxhlet apparatus will be used to obtain polyphenolic rich extract of fenugreek seed.

EXPERIMENTAL MODELS :

1. oral glucose tolerance test29 :

The oral glucose tolerance test will be performed on overnight fasted Sprague dawley rats. Rats will be divided into different groups as follows:

Group 1: Normal control, rats receive saline/vehicle.

Group 2: Diabetic control.

Group 3: Trigonella foenum graecum extract(200 mg/kg)

Group 4: Pioglitazone (10 mg/kg)

Group 5: Glipizide (5 mg/kg)

Group 6: Acarbose (5 mg/kg)

Glucose will be fed 90 min after the administration of extracts or drug. Blood will be withdrawn from the retro orbital sinus under ether inhalation at 30, 60 and 120 min of glucose administration and glucose levels will be estimated by using Accuchek glucometer.

2. Single and multiple dose study in normal and diabetic rats30.

3. Antioxidant activity - Invitro and invivo31.

4. Development of high fat diet-fed/low dose streptozotocin-treated type 2

diabetic rats32.

The animals will be fed high fat diet (HFD), once a day for 2 weeks followed by i.p injection of streptozotocin (35 mg/kg) dissolved in 1M/l citrate buffer (pH 4.4) after overnight fasting. The rats with non fasting plasma glucose level of >300 mg/dl will be considered diabetic and will be used for study. Blood sample will be collected from tail vein and glucose will be measured using glucose diagnostic kit (Accucheck, India).

5. Effect of fenugreek seed extract alone and in combination with oral

hypoglycemic agents in streptozotocin induced diabetic nephropathy

Rats will be divided into different groups as follows:

Group 1: Normal control, rats receive saline/vehicle.

Group 2: Diabetic control.

Group 3: Diabetic rats treated with Trigonella foenum graecum seed extract33.

Group 4: Diabetic rats treated with pioglitazone34.

Group 5: Diabetic rats treated with glipizide35

Group 6: Diabetic rats treated with acarbose36.

Group 7: Diabetic rats treated with pioglitazone and glipizide.

Group 8: Diabetic rats treated with glipizide plus acarbose.

Group 9: Diabetic rats treated with Trigonella foenum graecum seed extract

plus pioglitazone (low dose).

Group10: Diabetic rats treated with Trigonella foenum graecum seed extract

plus glipizide (low dose).

Group 11: Diabetic rats treated with Trigonella foenum graecum seed extract

plus acarbose (low dose).

TREATMENT PROTOCOL:

The following are the new rat models of diabetic nephropathy induced by

sreptozotocin:

ANIMAL MODELS:

Streptozotocin induced diabetic rat model (both the kidneys are intact).

Streptozotocin induced diabetic rat model (Heminephrectomy37 rat model where one kidney is removed and the other is intact).

Body weight will be measured every week end. The blood and 24 hrs urine samples will be collected once in 15 days of the treatment period. Hemoglobin A1c(HbA1c) will be measured to assess the glycemic control. Blood glucose (Glu),creatinine (Cr), urea nitrogen (BUN), total cholesterol(TC), HDL-cholesterol (HDL), and triglyceride(TG) levels will be measured using standard kits. Urine samples will be used to measure creatinine(uCr), protein (uPro), albumin and glucose content (US). Urinary excretion of sodium and potassium will be estimated.

At the end of the treatment period the kidneys will be weighed and fixed in 10% phosphate-bufferedformalin. Paraffin embedded tissues will be slicedinto 4 mm sections and stained with periodic acid-Schiff reagent (PAS) for microscopic examination.

Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:

YES

Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animals.

Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed

c)List of References:

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