a) 6 BRIEF RESUME OF THE INTENDED WORK:

6.1 Need of study:

Type-2 diabetes is a complex and heterogeneous disorder presently affecting more than 100 million people worldwide and causing serious socio-economic problems1.Diabetes mellitus has become a problem of great magnitude and a major public health concern. Studies have demonstrated that, in some countries, diabetes affects up to 10% of the population aged 20 years and older. The disease develops if the pancreas does not make enough insulin or the cells in the muscles, liver and fat do not use insulin properly, or both2.Diabetic Nephropathy is the major cause of end stage renal disease. Approximately 30% of patients with diabetes experience diabetic nephropathy, which gradually develops to final renal failure3. It is considered to be one of the most frequent complications of diabetes mellitus. Renal disease is usually attributed to metabolic consequences of abnormal glucose regulation4. The pathology of the complication is attributed by a progressive accumulation of extracellular matrix components in glomerular mesangium and tubular interstitium, which eventually leads to proteinuria and renal failure5. Elevation of significant markers such as serum urea and creatinine are related to renal dysfunction in diabetic hyperglycemia6. HbA1c has been used as marker 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.Boerhaavia diffusa (B.diffusa) is commonly known as punarnava7.

This plant possesses diuretic, cardiotonic8, immunomodulatory9, antioxidant10, antihemorrhagic 11, antispasmodic12, antimicrobial13, cytotoxic14 and anticancerous activity. It showed that ethanolic root extract of B.diffusa showed the presence of alkaloids, saponins and flavonoids, which were considered to have anti diabetic and antioxidant effect25.Several herbal preparations are often used by the diabetic patients along with the routine antidiabetic agents with an intention to produce better glycemic control, lipid profile and ultimately to reduce diabetic complications. Such herbal preparations often contain bioflavonoids that are antioxidant in nature and thus help to eliminate hyperglycemia-induced oxidative stress15. Boerhaavia diffusa leaf extract has alpha glucosidase inhibitory activity16 and may be responsible in attenuating the hyperglycemia and reversal of diabetic nephropathy. To attain adequate glycemic control combinational therapy is preferred but at the same time the combination therapy should not produce hypoglycemia. Apart from combining two or more oral hypoglycemic agents, physicians also recommend use of anti-diabetic herbal drugs along with oral hypoglycemic agents. Caution should be taken while prescribing drug-herb therapy and their interactions must be carefully considered when herb is added as an adjuvant to Acarbose because the combination may produce hypoglycemia.

The present investigation is being done to determine whether bioflavonoid rich extract of Boerhaavia diffusa leaf by virtue of its hypoglycemic and hypolipidemic activity would modulate the reversal of diabetes induced nephropathy in experimental animals.In the present study we are trying to whether its combination of Boerhaavia diffusa leaf extract with Acarbose in the progression of Diabetes induced nephropathy in Rats.

6.2 Review of Literature:

Diabetic nephropathy is a clinical syndrome characterized by the occurrence of persistent microalbuminuria in concomitance with insulin- or non–insulin dependent diabetes17.Diabetic nephropathy has been classically defined by the presence of proteinuria_0.5 g/24 h. This stage has been referred to as overt nephropathy, clinical nephropathy, proteinuria, or macroalbuminuria18.Type-2 Diabetes mellitus is often associated with hyperlipidemia and tends to influence renal function. Studies in type 2 DM patients suggests that , although poor metabolic control is the most important determinant of the development of nephropathy, hyperlipidemia is also considered to be involved19,20. Treatment of hypercholesterolemia has been shown to reduce diabetic nephropathy. Diabetic patients with nephropathy with a low serum cholesterol concentration are reported to exhibit a lower degree of kidney lesions than those patients with a high serum cholesterol concentration21.

Chloroform extracts of leaves of Boerhaavia diffusa showed anti diabetic activity in Streptozotocin induced diabetic rats which mainly act by reducing blood glucose level and increasing insulin sensitivity22. Hypoglycemic and antihyperglycemic activity of aqueous leaf extract Boerhaavia diffusa 200 mg/kg p.o. for 4 weeks in normal and Alloxan induced diabeticrats showed to increase plasma insulin level23. Glycemic control is the most important approach in the management of diabetic complications24.

Boerhaavia diffusa leaves showed that it inhibits the activity of the alpha amylase16 and possesses an antioxidant activity, which may be attributed to its carbohydrate metabolism inhibitory action and to the enhancing effect on cellular antioxidant defense contributing to the protection against oxidative damage in Streptozotocin induced diabetes.

The combinations of the above two class of drugs such as Acarbose and fenofibrate along with Boerhaavia diffusa, may play a role in decreasing the adverse effects seen in monotherapy of conventional combination of drugs.

In the present study we will evaluate the combined effect of Boerhaavia diffusa with Acarbose alone and in combination with fenofibrate in the progression of diabetes induced nephropathy and may help in reversal of diabetic nephropathy.

6.3 Objective of study:

The objectiveof the present study is to evaluate whether the combination of Boerhaavia diffusa leaf extract with Acarbose could attenuate the progression of Diabetes induced nephropathy in Rats.

Specific Objectives:

Primary Objectives:

  • To study the role of Aqueous Leaf extract of Boerhaavia diffusain diabetic nephropathy.
  • To study whether Acarbose and fenofibrate can play a vital role in preventing the progression of diabetic nephropathy.
  • To study the effect of the Aqueous leaf extract of Boerhaavia diffusa alone and in combination with Acarbose and fenofibrate on antioxidant enzyme levels in kidney.
  • To study the effect of Aqueous Leaf extract of Boerhaavia diffusa in combination with Acarbose and fenofibrate in diabetic nephropathy.

Secondary Objectives:

  • To study the effect of Aqueous Leaf extract of Boerhaavia diffusaalone and in combination with Acarbose and fenofibrate on structural integrity of kidney by microscopic examination.

b) 7MATERIALS AND METHODS:

7.1 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, Government College of Pharmacy etc., and other Research Publications and Journals.

7.2 Method of Collection of Data:

Extractions of Boerhaavia diffusa leaf extract25:

500g of Boerhaavia diffusa leaves were chopped into small pieces extracted with 1500 ml water by the method of continuous hot extraction at 60 ºC for 6 h and evaporated. The residual extract was dissolve in water and used in this study. A dark semi-solid (greenish-black) material was obtained (22.5 g). It was stored at 4 ºC until used. When needed the residual extract was suspended in distilled water and used in the study.

7.3 Experimental Model:

1. Experimental animals:

Male Sprague dawley rats weighing between 200 and 250 gm will be used. The experimental protocol was approved by the Institutional Animal Ethics Committee. The animals will be maintained under standard conditions in an animal house approved by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).

2. Estimation of blood glucose:

Blood samples were collected from the tail vein of the rat and the blood glucose level will be estimated by the GOD-POD method (Accucheck, India).

3.Inclusion and exclusion criteria:

Inclusion criteria

  • Male rats will be selected for the present study.
  • The rats with non fasting plasma glucose level of >250 mg/dl will be considered as diabetic.
  • Water and feed intake by the experimental animals.
  • Weight variation during the study period.

Exclusion criteria

  1. Female rats are excluded from the study because the hormonal imbalance will interfere in the present study.
  2. Rats showing too much of variations in the blood glucose levels will be excluded from the study.
  3. Rats showing abnormal nephropathy will be excluded from the study.
  1. Oral glucose tolerance test26 :

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: Boerhaavia diffusa extract (200 mg/kg)25.

Group 3: Acarbose (5 mg/kg)32.

Glucose will be fed 30 min after the administration of extracts. Blood will be withdrawn from the tail tip at 0, 30, 60, 90 and 120 min of glucose administration. Glucose levelswill be estimated by using (Accucheck, India).

2.Single and repeated dose study in normal and diabetic rats27:

Single dose study

Normal and diabetic rats will be administered with a single dose of plant extract and the selected oral hypoglycemic agents (OHA). The blood glucose level will be estimated just prior to administration of plant extract and OHA and at 1, 2 and 4 h after administration. Glucose levels will be estimated as mentioned above.

Repeated dose study

The same groups (Single dose study) of normal animals will be continued with the same dose levels of plant extract and OHA once daily, for 11 days. The glucose levels of all the animals will be measured on 3, 5, 7, 9 and 11th day of the treatment period.

3.Antioxidant activity of tissue-Invitro:

  1. Preparation of kidney homogenate28 :

About 100mg of kidney tissue will be homogenized in 10 volume of 100mM KH2PO4 buffer containing 1mM EDTA, pH 7.4 and centrifuged at 12000g for 30min at 4ºC. The supernatant will be collected and will be used for the experiments.

  1. Superoxide Dismutase (SOD)33:

Estimation of SOD will be done by detecting O2 by oxidation of hydroxylamine hydrochloride yielding nitrite, which will be measured colorimetrically at 560 nm. O2 will also be detected during auto-oxidation of hydroxylamine at pH 10.2, nitro blue tetrazolium is reduced and nitrite is produced in the presence of EDTA, which can be detected colorimetrically.

  1. Catalase34:

The enzyme is found in the peroxisomes and converts hydrogen peroxide to water and oxygen.Catalase will be estimated by determining the decomposition of H2O2 at 240 nm in an assay mixture containing phosphate buffer.(0.25M, pH 7)

  1. Estimation of lipid peroxidation35,36:

Plasma concentration of thiobarbituric acid reactive substance is the index of oxidative stress and lipid peroxidation. Elevated levels of TBARS are associated with increased risk of cardiovascular diseases. Oxidative stress during reperfusion leads to lipid peroxidation because direct measurement of liberated ROS is difficult due to their instability, malondialdehyde(MDA), a stable lipid peroxidation end–product, is frequently used as amarker of ROS production and measured at 532nm.

4.Development of high fat diet-fed/low dose streptozotocin-treated type 2 diabetic rats29:

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 >250 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 as mentioned above.

5.Effect of Boerhaavia diffusa leaf extract alone and in combination with Acarbose and fenofibrate in streptozotocin induced diabetic nephropathy.

7.4 Treatment Protocol:

Rats will be divided into different groups as follows:

(The study consists of 14 groups and each group is consist of 8-10 rats)

The Drugs and extract will be suspended in 0.5% w/v of carboxy methyl cellulose (CMC).

Group 1: (Normal Control), Rats will be maintained on standard food and water and no treatment will be given.

Group 2: (Diabetic Control), Rats will be administered high fat diet with streptozotocin (35 mg/kg, i.p., once) dissolved in citrate buffer (pH 4.5).

Group 3: (Fenofibrate per se), the normal rats will be administered fenofibrate (30 mg/kg p.o.) for 7 weeks.

Group 4: (Acarbose per se), the normal rats will be administered Acarbose (5 mg/kg p.o.) for 7 weeks.

Group 5: (Quercetin per se) the normal rats will be administered Quercetin (50 mg/kg p.o.)37for 7 weeks.

Group 6: (Leaf extract of Boerhaavia diffusa)normal rats will be administered Leaf extract of Boerhaavia diffusa (200 mg/kg p.o.) for 7 weeks.

Group 7: (Fenofibrate Treated), the diabetic rats, after one week of streptozotocin administration, were treated with low dose of fenofibrate (30 mg/kg p.o.) for 7 weeks.

Group 8: (Acarbose Treated), the diabetic rats, after one week of streptozotocin administration, were treated with low dose of Acarbose (5 mg/kg p.o.) for 7 weeks.

Group 9: (Quercetin Treated) the diabetic rats will be administered Quercetin (50 mg/kg p.o.) for 7 weeks.

Group 10: (Leaf extract of Boerhaavia diffusa) the diabetic rats will be administered Leaf extract of Boerhaavia diffusa (200 mg/kg p.o.) for 7 weeks.

Group 11: (Fenofibrate+Acarbose Treated), the diabetic rats, after one week of streptozotocin administration, will treated with the combination of low dose of fenofibrate (30 mg/kg, p.o.) and low dose of Acarbose (5 mg/kg p.o.) for 7 weeks.

Group 12: (Fenofibrate+ Leaf extract of Boerhaavia diffusa Treated), the diabetic rats, after one week of streptozotocin administration, will treated with the combination of low dose of fenofibrate (30 mg/kg, p.o.) and Leaf extract of Boerhaavia diffusa (200 mg/kg p.o.) for 7 weeks.

Group 13: (Leaf extract of Boerhaavia diffusa +Acarbose Treated), the diabetic rats, after one week of streptozotocin administration, will be treated with the combination of Leaf extract of Boerhaavia diffusa (200 mg/kg, p.o.) and low dose of Acarbose (5 mg/kg p.o.) for 7 weeks.

Group 14: (Lisinopril Treated), the diabetic rats after one week of streptozotocin administration, will be treated with lisinopril (1 mg/kg p.o.) for 7 weeks.

7.5 Estimation of Diabetic Nephropathy:

The diabetes mellitus-induced nephropathy was assessed Biochemically by estimating serum creatinine, blood urea nitrogen and proteinuria30.

The treatment period will be between 8 -10 weeks. Body weight will be measured after every week. The blood and 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) 30 levels will be measured using standard kits. Urine samples will be used to measure creatinine (uCr), protein (uPro), albumin and glucose content. Urinary excretion of sodium and potassium will be estimated31.

Histopathology: At the end of the treatment period the kidneys will be weighed and fixed in10% phosphate-buffered formalin. Paraffin embedded tissues will be sliced into 4 mm sections and stained with periodic acid-Schiff reagent (PAS) for microscopic examination.

Statistical Analysis: All the data are expressed as mean ± SD. Comparisons among the groups were analyzed with ANOVA followed by Dunnett’s test.

7.6 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 animal model.

7.7 Has ethical clearance been obtained from your institute?

Ethical Committee approval letter is enclosed.

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