A Mechanistic Approach to Determination of Anti-diabetic activity of Calystegia sepium R.Br. Flowering plants in normal and Streptozotocin induced rats

 

Kumari Anjali Jain*

Institute of Pharmaceutical Science and Research centre, Bhagwant University, AJMER

*Corresponding Author E-mail:

 

ABSTRACT:

The Hypoglycemic effect of water and ethanol extracts of Calystegia sepium flowering plants  has been evaluated and compared its Hypoglycemic effect of flowering plants with Glibenclamide in Euglycemic and Hyperglycemic Wistar rats. Male Wistar rats were orally administered Alcoholic and Water extracts of Calystegia sepium flowering plant and investigated for Hypoglycemic activity in Normal and Streptozotocin (STZ)-induced diabetic rats  and compared their effect with Glibenclamide  [Single dose one day study and multiple dose15 day study] Blood glucose levels were estimated at 0th, 5th, 10th and 15th day by Kit method and on 15th day lipid profiles were also estimated and other parameters like insulin estimation, insulin tolerance test, and glucose uptake by isolated hemi-diaphragm was estimated. The alcoholic and water extract of Calystegia sepium flower in plant exhibited significant Hypoglycemic and Hypolipidemic effect in Euglycemic and Hyperglycemic rats.  The study reveals the hypoglycemic and hypolipidemic activity of alcoholic and water extract of Calystegia sepium flowering plants in euglycemic and Streptozotocin induced diabetic rats. The extract seems promising for the development of a phyto-medicine for a Diabetes mellitus.

 

KEYWORDS: Diabetes mellitus; Antihyperglycemic; Hypolipidemic; Euglycemic Calystegia sepium.

 


1. INTRODUCTION:

Definition:

Diabetes, often referred to by doctors as diabetes mellitus, describes a group of metabolic diseases in which the person has high blood glucose (blood sugar), either because insulin production is inadequate, or because the body's cells do not respond properly to insulin, or both.

 

Diabetes is defined as a state in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by insulin. This results primarily in elevated fasting and postprandial blood glucose levels. If this imbalanced homeostasis does not return to normalcy and continues for a protracted period of time, it leads to hyperglycemia that in due course turns into a syndrome called Diabetes.Patients with high blood sugar will typically experience polyuria (frequent urination), they will become increasingly thirsty (polydipsia) and hungry (polyphagia).

 

Mellitus.1

The three main types of diabetes are:

·         Type 1 diabetes

·         Type 2 diabetes

·         Gestational diabetes

 

Type 1 diabetes:

Type 1 diabetes commonly called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes. Type 1 diabetes is an autoimmune disease. An autoimmune disease results when the body’s system for fighting infection (the immune system) turns against a part of the body. In diabetes, the immune system attacks and destroys the insulin-producing beta cells in the pancreas. The pancreas then produces little or no insulin. A person who has type 1 diabetes must take insulin daily to live. At present, it is not know exactly what causes the body’s immune system to attack the beta cells, but it is believed that autoimmune, genetic, and environmental factors, possibly viruses, are involved. It develops most often in children and young adults but can appear at any age. Symptoms of type 1 diabetes usually develop over a short period, although beta cell destruction can begin years earlier. Symptoms may include increased thirst and urination, constant hunger, weight loss, blurred vision, and extreme fatigue. If not diagnosed and treated with insulin, a person with type 1 diabetes can lapse into a life-threatening diabetic coma, also known as diabetic ketoacidosis.

Type 2 Diabetes:

Type 2 diabetes is commonly called non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes. The most common form of diabetes is type 2 diabetes. About 90 to 95 percent of people with diabetes have type 2. This form of diabetes is most often associated with older age, obesity, family history of diabetes, previous history of gestational diabetes, physical inactivity, and certain ethnicities. About 80 percent of people with type 2 diabetes are overweight. Type 2 diabetes is increasingly being diagnosed in children and adolescents. When type 2 diabetes is diagnosed, the pancreas is usually producing enough insulin, but for unknown reasons the body cannot use the insulin effectively, a condition called insulin resistance. After several years, insulin production decreases. The result is the same as for type 1 diabetes—glucose builds up in the blood and the body cannot make efficient use of its main source of fuel. The symptoms of type 2 diabetes develop gradually. Their onset is not as sudden as in type 1 diabetes. Symptoms may include fatigue, frequent urination, increased thirst and hunger, weight loss, blurred vision, and slow healing of wounds or sores. Some people have no symptoms.

 

Gestational Diabetes:

Some women develop gestational diabetes late in pregnancy. Although this form of diabetes usually disappears after the birth of the baby, women who have had gestational diabetes have a 20 to 50 percent chance of developing type 2 diabetes within 5 to 10 years. Maintaining a reasonable body weight and being physically active may help prevent development of type 2 diabetes. About 3 to 8 percent of pregnant women develop gestational diabetes. As with type 2 diabetes, gestational diabetes occurs more often in some ethnic groups and among women with a family history of diabetes. Gestational diabetes is caused by the hormones of pregnancy or a shortage of insulin. Women with gestational diabetes may not experience any symptoms.2

 

In contrast to substitution therapy with insulin in IDDM, therapy for type-2 Diabetes relies mainly on several approaches intended to reduce the hyperglycemia itself. Although diet and exercise are the first steps toward achieving treatment goals of diabetics, 90% of patients with Diabetes cannot maintain long term glycemic control with diet and exercise alone. Thus, antihyperglycemic drugs are necessary for the treatment of diabetes.3

 

It is well known that treatment with insulin preparation has many disadvantages viz., needed constant refrigeration, insulin shock, revert hypoglycemia, allergic reactions, lipodystrophy, and gain in weight. Moreover, in the treatment of type 2 diabetes, drugs such as sulfonylureas (insulin secretagogues), metformin (acts to reduce hepatic glucose production) and acarbose (interferes with glucose absorption and metabolism) TZD (improve sensitivity to insulin) were used. These oral hypoglycemics are also not free from adverse effects such as gastrointestinal disturbances, blood dyscrasia, hepatic dysfunction, cardiotoxicity, hypoglycemia, etc.

 

In view of one or the other adverse effects of insulin and oral hypoglycemics, search continues to develop newer agents for the treatment of Diabetes mellitus. Plant derivatives with hypoglycemic properties have been used in folk medicine and traditional healing systems around the world from very ancient time .4

 

The impact of Diabetes:

The global prevalence of diabetes is estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of Diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. Diabetes is a major threat to global public health that is rapidly getting worse and also it is a life threatening condition, at least one in 20 deaths is attributed to diabetes and 8,700 deaths every day, six deaths every minute. Moreover, it is a common condition and its frequency is dramatically rising burden all over the world. Medication often has an important role to play particularly for the control of blood glucose and lipids profiles.5 According to recent estimates, the human population worldwide appears to be in the midst of an epidemic of diabetes. Despite the great strides that have been made in the understanding and management of diabetes, the disease and disease-related complications are increasing. Parallel to this, recent developments in understanding the pathophysiology of the disease process have opened up several new avenues to identify and develop novel therapies to combat the diabetic plague. Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in combating diabetes and diabetes-related complications. Therefore, as the disease is progressing unabated, there is an urgent need of identifying indigenous natural resources in order to procure them, and study in detail, their potential on different newly identified targets in order to develop them as new therapeutics.6

 

In the Indigenous system of medicine like Ayurveda, many herbal medicines have been recommended for the treatment of diabetes or madhumeha and some of them experimentally evaluated.7 Medicinal plants used to treat diabetic conditions are of considerable interest and a number of plants have shown varying degrees of hypoglycemic and antihyperglycemic activity. In recent years, there has been renewed interest in the treatment against different diseases using herbal drugs as they are generally non-toxic and World Health Organization has also recommended the evaluation of the effectiveness of plants in condition where we lack safe modern drugs.

 

Since ancient times, plants have been an exemplary source of medicine. Ayurveda and other Indian literature mention the use of plants in treatment of various human ailments. India has about 45000 plant species and among them, several thousands have been claimed to possess medicinal properties. Research conducted in last few decades on plants mentioned in ancient literature or used traditionally for diabetes have shown anti-diabetic property. That has been mentioned and used in the Indian traditional system of medicine and have shown experimental or clinical anti-diabetic activity. Indian plants which are most effective and the most commonly studied in relation to diabetes and their complications are:

 

Allium cepa, Allium sativum, Aloe vera, Cajanus cajan, Coccinia indica, Caesalpinia bonducella, Ficus bengalenesis, Gymnema sylvestre, Momordica charantia, Ocimum sanctum, Pterocarpus marsupium, Swertia chirayita, Syzigium cumini, Tinospora cordifolia,Trigonella foenum graecum, M. charantia, Eugenia jambolana, Mucuna pruriens, T. cordifolia, T. foenum graecum,  Murraya koeingii, Tapinanthus butungii and Brassica juncea. All these above plants have shown varying degree of hypoglycemic and anti-hyperglycemic activity.

 

Calystegia sepium flowering  are reported to contain Aryl esters8, courmarin glycosides9, p-hydroxycinmamate and scopoletin10, as important phytoconstituents. The flowering  of plant have been reported to posses anti-inflammatory11, wound healing12, antimicrobial13, immunomodulatory14, nootropic15, and hepatoprotective16 activity.

 

Paucity of scientific information regarding the effect of Calystegia sepium on blood glucose and lipid levels, therefore the present study is planned to investigate their hypoglycemic and hypolipidemic activity.

 

2. OBJECTIVES:

The present study has been undertaken to investigate:

*        Hypoglycemic activity of ethanol (CSE) and water (CSW) extracts of Calystegia sepium Sweet. flowerin plant in Normoglycemic rats

*        Effect of CSE and CSW on Oral Glucose Tolerance Test (OGTT) and Oral Sucrose Tolerance Test (OSTT) in Normoglycemic rats  

*        Anti-diabetic activity of CSE and CSW in STZ-induced diabetic rats [single-dose one day study and multiple-dose fifteen-days study] and carry out the following parameters

Ø  Serum glucose, Serum insulin and HOMA

Ø  Lipid Parameters

Ø  Oral Glucose Tolerance Test (OGTT)

ü  AUC glucose

ü  AUC insulin

Ø  Insulin Tolerance Test (ITT) 

*        Mechanistic studies

Glucose uptake by isolated hemi-diaphragm.

 

3. METHODOLOGY:

MATERIAL

Chemicals:

Streptozotocin (STZ) chemical name 2 deosyl-2-(3-methyl-3-nitroso urea) -1-d-glucopyranose (commonly known (N-Methylnitroso carboxyl)-D glucose C8 H15N3O7 Mol.wt   265.2 CAS No- 18883-66-4 Min Assay 98% Purchased from HI-Media Mumbai. Insulin injection biphasic isophane, Ph. Eur. Human mixtard manufactured by Torrent Pharmaceuticals, Ltd Indrad. Standard drug Glidenclamide 5mg (Daonil) obtained from Avents Pharma, Ltd Goa. Acarbose (glucobay) Bayer Pharma, Pvt Ltd. Tragacanth,  Glucose, Sucrose, Sodium Chloride, Potassium Chloride, Calcium Chloride, Magnesium Chloride, Sodium Bicarbonate, Sodium Hypo Phosphate, Citric acid, Trisodium Citrate,  Hi-Media Laboratories, Ltd Mumbai India. Ethanol and other chemicals used were analytical grade obtained from Hi-Media Bangalore, Standard kits for Glucose, Triglycerides, Cholesterol, and HDL-Cholesterol were obtained from Erba Mannheim, Manufactured by Transasia Biomedical Ltd Baddi, India. The instrument used were Soxhlet extracter, centrifuge, pH meter and auto analyzer MS-500E Maysun techcom Ltd.   

Plant and Extraction:

The flowering  of Calystegia sepiumwere collected in the month of May from hilly areas surrounding Dharwad, Karnataka, and authenticated by qualified taxonomist, Dr. G.R Hegde, Department of Botany, Karnataka University Dharwad. The herbarium specimen is present in Department of Pharmacognosy (SETCPD/Ph.cog/Herb/33/1006).

 

The flowering parts were dried under shade and powdered. Dried powder was exhaustively extracted successively using Ethanol (90%) and the marc was macerated with chloroform water respectively. Extracts were concentrated under vaccum at 400c to yield a semisolid mass and stored in desiccators. The percentage yield of ethanol extract (CSE) and water extract (CSW) was found to be 1.93% and 3.69% respectively. Suspension of extracts were prepared using 0.5% tragacanth and used to assess antidiabetic activity.

 

METHODOLOGY

Preliminary Phytochemical Analysis

Preliminary phytochemical analysis was carried out for ethanol (CSE) and water (CSW) extract of Calystegia sepium for detection of phytoconstituents using following standard methods.

 

(I) Test for carbohydrates

Barfoed’s test: Test solution + Barfoed’s reagent, boiled on water bath. Brick red precipitate indicates the presence of carbohydrates.

 

Molisch’s test: Test solution + Few drops of Molisch’s reagent + 2ml of concentrated sulphuric acid along the sides of the test tube. A purple ring formed at the junction of two liquids indicates the presence of carbohydrates.

 

Benedict’s test: Test solution + Benedict’s reagent, boiled on water bath. Reddish brown precipitate indicates the presence of carbohydrates.

 

(II) Test for sterols

Liebermann-Burchard test: Test solution + few drops of acetic anhydride. + concentrated sulphuric acid along the sides of the test tube. Brown ring forms at the junction of the two liquids and the upper layer turns green.

 

Salkowaski test: Test solution + concentrated sulphuric acid, shaken and allowed to stand. The lower layer turns red indicating the presence of sterols.

 

Sulphur test: Sulphur when added to the test solution, it sinks to the bottom indicating the presence of sterols.

 

(III) Test for alkaloids

Wagner’s test: The acidic test solution with Wagner’s reagent (Iodine in potassium iodide) gives brown precipitate.

 

Hager’s test: The acidic test solution with Hager’s reagent (Saturated picric acid solution) gives yellow precipitate.

Mayer’s test: Test solution + Mayer’s reagent (Potassium mercuric iodide) gives cream coloured precipitate.

 

Dragendorff’s test: The acidic test solution with Dragendorff’s reagent (Potassium bismuth iodide) shows reddish brown precipitate.

 

(IV) Test for Proteins/Amino acids

Biuret test: Test solution + 40% sodium hydroxide + dilute copper sulphate solution. Blue colour indicates the presence of protein.

 

Ninhydrin test: Test solution + Ninhydrin reagent gives blue colour.

 

Millon’s test: Test solution + Millon’s reagent, heated on a water bath. Yellow colouration indicates the presence of protein.

 

Xanthoproteic test: Test solution + concentrated nitric acid, on boiling gives yellow precipitate.

 

(V) Test for tannins

Ferric chloride test: Test solution + few drops of ferric chloride solution gives dark red colour.

 

Gelatin test: Test solution + gelatin solution gives white precipitate.

 

(VI) Test for Saponin Glycosides

Foam test: Saponins when mixed with water and shaken shows the formation of froth, which is stable at least for 15 min.

 

(VII) Test for triterpenoids

Haemolysis test: 2 ml each of 18% sodium chloride solution was taken in two test tubes. To one test tube 2 ml of distilled water and to another test tube 2 ml of test sample was added. A few drops of blood was added to both the test tubes, mixed and observed for haemolysis under microscope. Haemolysis of blood cells indicates the presence of saponin glycosides.

 

Salkowaski test: Test solution + few drops of concentrated sulphuric acid, shaken and allowed to stand, lower layer turns yellow indicating the presence of triterpenoids.

 

Liebermann-Burchard test: Test solution + few drops of acetic anhydride. + Concentrated sulphuric acid along the sides of the test tube. Development of deep red colour indicates the presence of triterpenoids.

 

(VIII) Test for flavanoids

Zinc-Hydrochloric acid reduction test: Test solution + zinc dust + few drops of hydrochloric acid shows magenta red colour.

 

Ferric chloride test: Test solution + few drops of ferric chloride solution give intense green colour.

 

Shinoda test: Test solution + few fragments of magnesium ribbon + concentrated hydrochloric acid, shows pink to magenta red colour.

Lead acetate solution test: Test solution + few drops of lead acetate (10%) solution gives yellow precipitate.

 

Alkaline reagent test: Test solution + sodium hydroxide solution shows increase in the intensity of yellow colour which becomes colourless on addition of few drops of dilute acid.

 

Animals:

Young adult male Wistar rats 7-8 weeks old, weighing 150-200g were obtained from inbred animal house Venkateshwara enterprises, Bangalore, Karnataka. The animals were housed in polypropylene cages in standard environmental conditions, 12 h light and 12 h dark cycle at 25 ± 2 oC. Before and during the experiments, the rats were fed with standard laboratory pellet diet and water ad libitum obtained from Venkateshwara enterprises, Bangalore. Animals were acclimatized to the laboratory condition for at least 15 days prior to the experiment and were maintained in a well ventilated animal house. The experimental protocol was approved by the Institutional animal Ethical Committee (IAEC) animals and the care of the laboratory was taken as per the CPCSEA regulation.

 

Pharmacological Evaluation

Preparation of extract dose 

Weighed quantity of ethanol (CSE) and water (CSW) extracts of Calystegia sepiumwere suspended in water using 1.0 % tragacanth and administered orally to experimental animals. Suspension of extract was prepared freshly. The extracts were administered at a constant volume of ~5ml/kg for each animal.

 

Determination of acute Toxicity

Acute toxicity study was carried out using Swiss albino mice (50-60 g) by up and down staircase method as per CPSCEA guidelines. Both the extracts were orally administered to different groups of mice at the doses of 50 mg, 300 mg, 500 mg and 2500 mg/kg body weight respectively. Animals were observed for 48 hrs to study the general behavior of animals, sign of discomfort, nervous manifestation and mortality.

 

Effect of extracts in normoglycemic rats

Oral Glucose Tolerance Test (OGTT) in normal rats

Experimental protocol

The experimental rats were divided into six groups of five rats each and treated as follows.  

Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o.)

Group 2: NC rats treated with CSE (50 mg/kg, p.o.)

Group 3: NC rats treated with CSE (100 mg/kg, p.o.)

Group 4: NC rats treated with CSW (50 mg/kg, p.o.)

Group 5: NC rats treated with CSW (100 mg/kg, p.o.)

Group 6: NC rats treated with glibenclamide (10 mg/kg, p.o.)

 

Different doses of CSE/CSW/glibenclamide (GLB) and vehicle were given orally to normoglycemic rats fasted for 18 h. Thirty minutes later, glucose (2 g/kg in distilled water) was administered orally. Blood samples were collected from the retro-orbital plexus at 0 min (i.e. immediately after glucose load), 30, 60 and 120 mins after glucose administration. Serum glucose (SG) was estimated by the enzymatic glucose oxidase method using diagnostic reagent kit. The results are expressed as integrated area under curve for glucose (AUCglucose) calculated by trapezoid rule.

Experimental design for Multiple-dose fifteen-day study in Normoglycemic rats

The animals treated with respective doses of CSE, CSW and GLB were further treated for fifteen consecutive days [Multiple-dose fifteen-day study]. The following groups of animals were further treated with single daily doses for another 15 days in order to evaluate the chronic effect of extracts/GLB treatment

 

Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o./day)

Group 2: NC rats treated with CSE (50 mg/kg, p.o./day)

Group 3: NC rats treated with CSE (100 mg/kg, p.o./day)

Group 4: NC rats treated with CSW (50 mg/kg, p.o./day)

Group 5: NC rats treated with CSW (100 mg/kg, p.o./day)

Group 6: NC rats treated with glibenclamide (0.5 mg/kg, p.o./day)

 

Oral Sucrose Tolerance Test (OSTT) in normal rats

Experimental protocol

The experimental rats were divided into six groups of five rats each and treated as follows.  

Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o.)

Group 2: NC rats treated with CSE (50 mg/kg, p.o.)

Group 3: NC rats treated with CSE (100 mg/kg, p.o.)

Group 4: NC rats treated with CSW (50 mg/kg, p.o.)

Group 5: NC rats treated with CSW (100 mg/kg, p.o.)

Group 6: NC rats treated with Acarbose (7 mg/kg, p.o.)

 

Different doses of CSE/CSW/Acarbose and vehicle were given orally to normal rats fasted for 18 h. Thirty minutes later, sucrose (2 g/kg in distilled water) was administered orally. Blood samples were collected from the retro-orbital plexus at 0 min (i.e. immediately after sucrose load), 30, 60 and 120 mins post sucrose administration. SG was estimated by the enzymatic glucose oxidase method using diagnostic reagent kit. The results are expressed as integrated area under curve for glucose (AUCglucose) calculated by trapezoid rule using formula as mentioned above.85

 

Evaluation of anti-diabetic effect of CSE and CSW in standardized STZ-induced diabetic rats

Induction of Diabetes mellitus

Diabetic condition was induced in male Wistar rats by single intravenous injection of STZ (50 mg/kg) [Chosen optimum dose: In house data] after overnight fasting for 12 h.86 Rats showing SG level > 100 mg/dl seven days after STZ administration were considered diabetic and included in the study. Diabetic rats were randomized into different groups based on their SG levels.

 

Experimental design for Single-dose one-day study

The experimental rats were divided into seven groups of five rats each and treated as follows.  

Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o.)

Group 2: Diabetic control (DC) received 0.5% tragacanth (10 ml/kg, p.o.)

Group 3: DC rats treated with CSE (50 mg/kg, p.o.)

Group 4: DC rats treated with CSE (100 mg/kg, p.o.)

Group 5: DC rats treated with CSW (50 mg/kg, p.o.)

Group 6: DC rats treated with CSW (100 mg/kg, p.o.)

Group 7: DC rats treated with Glibenclamide (10 mg/kg, p.o.)

 

Blood samples were collected at 0, 2 and 4 h after extracts/GLB administration [single-dose one-day study]. SG was estimated by the enzymatic glucose oxidase method. Percent reduction in glycemia was calculated with respect to the initial (0 h) level by above mentioned formula.

 

Experimental design for Multiple-dose fifteen-day study

The animals treated with respective doses of CSE, CSW and GLB were further treated for fifteen consecutive days [Multiple-dose fifteen-day study]. The following groups of animals were further treated with single daily doses for another 15 days in order to evaluate the chronic effect of extracts/GLB treatment on hyperglycemia.

 

Group 1: Normal control (NC) received 0.5% tragacanth (10 ml/kg, p.o./day)

Group 2: Diabetic control (DC) received 0.5% tragacanth (10 ml/kg, p.o./day)

Group 3: DC rats treated with CSE (50 mg/kg, p.o./day)

Group 4: DC rats treated with CSE (100 mg/kg, p.o./day)

Group 5: DC rats treated with CSW (50 mg/kg, p.o./day)

Group 6: DC rats treated with CSW (100 mg/kg, p.o./day)

Group 7: DC rats treated with glibenclamide (0.5 mg/kg, p.o./day)

 

Oral glucose tolerance test (OGTT)

On 10th day, glucose tolerance of various groups was estimated by a simple OGTT. Glucose (2 g/kg) was administered to 12 h-fasted rats and blood samples were collected from the retro-orbital plexus at 0 (before glucose load), 30, 60 and 120 mins after glucose administration. SG was estimated by the enzymatic glucose oxidase method. The results were expressed as integrated area under curve for glucose (AUCglucose), which was calculated by trapezoid rule,

 

Also serum insulin was estimated 0 (before glucose load), 30 and 60 mins after glucose administration. Serum insulin (SI) was estimated by radioimmunoassay method using the kit from Bhabha Atomic Research Centre, Mumbai, India. The results were expressed as integrated area under curve for insulin (AUCinsulin), which was calculated by trapezoid rule.

 

Insulin tolerance test (ITT)

Insulin tolerance test is a measure of the extent of peripheral utilization of glucose. On 13th day, insulin (2 U/kg, i.v) was administered to six h-fasted rats. Blood samples were collected from the retro-orbital plexus at 0 (just before insulin load), 10, 20 and 30 mins after insulin injection. SG was estimated by the enzymatic glucose oxidase method. The results were expressed as integrated area under curve for glucose (AUCglucose), which was calculated by trapezoid rule using formula as give above.

 

Estimation of biochemical parameters

At the end of the treatment schedule, blood samples were collected from retro-orbital plexus. Serum was separated and analysed spectrophotometrically for triglyceride (STG), total cholesterol (STC), HDL-cholesterol (HDL-c), using diagnostic reagent kit ERBA diagnostics Mannheim GMBH, Germany. Serum insulin (SI) was estimated by radioimmunoassay method using the kit from Bhabha Atomic Research Centre, Mumbai, India. Homeostatic Model Assessment (HOMA) as a measure of insulin resistance was calculated by the following formula.

 

The markers of dyslipidemia such as TC/HDL-c and LDL-c/HDL-c ratios were also calculated.

Glucose uptake by isolated hemi-diaphragm of diabetic rats

Isolation of diaphragm

At the end of the study, the overnight fasted experimental rats were killed by cervical dislocation. The diaphragms were dissected out quickly with minimal trauma and divided into two equal halves. The hemi-diaphragms were then rinsed in cold Tyrode solution (without glucose) to remove any blood clots.88 Two diaphragms of five animals (10 diaphragms) in each group were used for the study.

 

Experimental design

Seven sets of ten graduated test tubes each, were grouped as follows,

Group 1: 2 ml of Tyrode solution with 2g% glucose [Normal control]

Group 2: 2 ml of Tyrode solution with 2g% glucose [Diabetic control]

Group 3: 2 ml of Tyrode solution with 2g% glucose + insulin (Nova Nordisk) 0.62ml of 0.4 U/ml solution [Insulin treated group]

Group 4: 2 ml of Tyrode solution with 2g% glucose + CSE (50mg/ml)

Group 5: 2 ml of Tyrode solution with 2g% glucose + CSE (50 mg/ml)

Group 6: 2 ml of Tyrode solution with 2g% glucose + CSW (50mg/ml)

Group 7: 2 ml of Tyrode solution with 2g% glucose + CSW (50 mg/ml)

 

The volumes of all graduated test tubes were made upto 4 ml with distilled water and estimated the initial glucose concentration by commercially available glucose kit based on glucose-oxidase method (ERBA diagnostics Mannheim GMBH, Germany). The hemi-diaphragms were placed in test tubes and incubated for 30 min at 37° C in bubbled with oxygen with continuous shaking. Glucose uptake per gram of tissue was calculated as the difference between the initial and final glucose content in the incubated medium.88, 89

 

Statistical analysis

The data were expressed as Mean ± S.E.M for six rats in each group. Statistical comparisons were performed by one-way ANOVA followed by Tukey’s post-test using GraphPad Prism version 4.0, USA.

 

4. RESULTS:

Preliminary Phytochemical Investigation

Preliminary phytochemical investigation on ethanol (CSE) and water (CSW) extracts of Calystegia sepium flower in plant are shown in Table .1

 

Table 1. Preliminary phytochemical investigation of Calystegia sepium flowerin plant extracts.

S r. No.

Name of the Phytoconstituent

Ethanol (95%)     extract

Aqueous

extract

01

Carbohydrates

+

+

02

Steroids

_

_

03

Alkaloids

-

_

04

Amino acids

+

+

05

Proteins

+

+

06

Tannins

+

+

07

Saponins

+

+

08

Triterpenoids

_

_

09

Flavanoids

+

+

 

Pharmacological Evaluation

Acute oral toxicity studies

The CSE and CSW were found devoid of mortality of animals at the dose of 2500 mg/kg body weight. Hence the 1/10th of the dose selected for the screening of adaptogenic activity as follows,

1.        CSE 50 mg/kg 

2.        CSE 50 mg/kg

3.        CSW 50 mg/kg

4.        CSW 100 mg/kg

 

Effect of extracts in normoglycemic rats

Oral Glucose Tolerance Test (OGTT) in normal rats

Administration of glucose (2g/kg) produces significant change in SG level of normal rats. Treatment with lower dose of CSE (50mg/kg) and CSW (50 mg/kg) and GLB (10mg/kg) significantly (P<0.01; P<0.001) improve the glucose tolerance Whereas, treatment with higher dose of CSE and CSW (100 mg/kg) did not significantly reduced the AUC glucose compared to normal control group.

 

SG levels were measured prior to, and after p.o. administration of glucose alone (2g/kg body weight), or in combination with CGE, CGW or Glibenclamide [GLB].

Treatment with CSE (50 and 100 mg/kg) and CSW (50 and 100 mg/kg) once daily for fifteen days showed hypoglycemic activity (P<0.05; P<0.01; P<0.001) (Table 5.2). CSE (50 mg/kg) showed significant (P<0.05; P<0.01) percentage reduction (15.14% and 30.39%) in SG levels at 5th and 15th days, compared to basal values (0 day) and activity is better than CSE (100 mg/kg). Whereas, higher dose of CSW (100 mg/kg) exhibited higher activity than CSW (50 mg/kg) at all tested days (Fig 1) (Table 2).

 

Table 2. Effect of CSE, CSW on Serum glucose levels in normoglycemic  rats [Multiple-dose fifteen-day study]

Treatment [dose/kg b.w]

SGL levels [mg/dl]

0 day

5th day

15th day

Normal

Control

93.64

91.47 (2.17)

87.77 (5.87)

CSE

[50 mg]

90.02

74.25 (15.77)

62.04 (27.98)

CSE

[100 mg]

71.22

61.52 (9.7)

55.57 (15.65)

CSW

[50mg]

85

77.25 (7.75)

63.31 (21.69)

CSW

[100 mg]

100.98

74.25 (26.73)

66.02 (34.96)

GLB

[0.5 mg]

91.25

68.2 (23.05)

56.56 (34.69)

Each value represents Mean ± S.E.M., n=5. Values in parentheses indicate Percent reduction in glycaemia and*P<0.05;**P<0.01; ***P<0.001 compared to basal values [0 day] of the same group. One-way ANOVA followed by tukey post-test.

 

Fig.1. Effect of ethanol (CSE) and water extract (CSW) of Calystegia sepium in Normoglycemic rats.

 

Bar graph represents the percentage reduction in glycemia with respect to the initial (0 day) level. Each value represents Mean ± S.E.M., n=5. b P<0.01,compared to normal control of the same time interval. One-way ANOVA followed by Tukey’s post-test.

 

Estimation of Lipid parameter

Treatment with CSE (50 and 100 mg/kg) and CSW (50 and 100 mg/kg) once daily for fifteen days showed reduction in tested lipid parameters but activity was not significant (Table 3).

 

Oral Sucrose Tolerance Test (OSTT) in normal rats

Animals subjected to Sucrose (2g/kg) load produces significant change in SG level of normal rats. Treatment with different dose of CSE (50 and 100 mg/kg), CSW (50 and 100 mg/kg) and Acarbose (7mg/kg) exhibited significant (P<0.05; P<0.01) reduction in SG level over the period of 120 min compared to normal control group (Fig 2 A). Further, treatment of CSE/CSW/Acarbose significantly (P<0.05; P<0.001) improve the Sucrose tolerance (Fig 2 B) suggested that the components of extracts may inhibit a-glucosidase. Moreover, treatment of lower dose of CSE and CSW were better than higher dose to improve sucrose tolerance in normal rats.

 

Fig.2 Effect of ethanol (CSE) and water extract (CSW) of Calystegia sepium on sucrose tolerance in fasted normal rats.

[A] SG levels were measured prior to, and after p.o. administration of sucrose alone (2g/kg body weight), or in combination with CGE, CGW or Acarbose. [B] Area under curve for glucose (AUCglucose) values for 0-120 min post sucrose load. Data represent the mean ± S.E.M., for 5 rats. a P < 0.05; b P < 0.01; c P < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).

 

 


Table.3. Effect of ethanol (CSE) and water extract (CSW) of  Calystegia sepi um on lipid profile in Normoglycemic rats model.  (multiple dose-fifteen-day study)

Serum parameter

Normal control

CSE50

mg/kg

CSE 100

mg/kg

CSW 100

mg/kg

CSW 50

mg/kg

GLB 10

mg/kg

STG (mg/dl)

75.98±14.22

52.54±2.95

78.23±8.77

80.63±10.02

52.42±8.41

60.20±15.90

STC (mg/dl)

56.14±6.16

70.28±12.46

59.50±15.13

71.42±5.10

73.69±9.84

52.07±4.70

HDL-c (mg/dl)

17.68±3.67

12.07±1.50

16.80±3.19

15.55±2.06

16.96±1.28

16.15±2.88

VLDL-c (mg/dl)

14.58±2.84

11.41±0.59

14.77±1.75

15.53±2.00

11.78±1.68

11.54±3.18

LDL-c (mg/dl)

25.88±8.18

48.60±11.67

27.83±14.31

38.14±7.01

44.84±9.02

22.18±5.66

TC/HDL -c ratio

2.91±0.92

5.30±0.46

3.76±1.31

1.04±0.57

4.15±0.50

3.37±0.61

LDL-c/HDL- c ratio

1.70±0.75

3.40±0.56

1.80±1.15

2.39±0.65

2.57±0.52

1.50±0.45

Each value represent Mean S.E.M n=5 aP<0.05, bP<0.01,cP<0.001 compared to normal control. One way ANOVA followed by Tukey’s post test.


 

Evaluation of anti-diabetic effect of CSE and CSW in standardized STZ-induced diabetic rats

Single-dose one-day study

A single dose of CSE (50 and 100 mg/kg) and CSW (50 and 100 mg/kg) treatment exhibited reduction in SG levels at different time intervals compared to basal levels (0 h). However, administration of  GLB showed significant (P<0.05; P<0.001) reduction is SG levels with maximum reduction (50.94%) at 4 h post GLB treatment compared to their basal levels (Table 5.4). Whereas, CSE treated animals showed dose dependent percentage reduction in SG levels compared to their basal levels (Table 4) (Figure 3).

 

Table.4. Effect of CSE, CSW on SGL levels in STZ-induced MD rats [Single-dose one-day study]

Treatment

[dose/kg b.w]

SGL levels

[mg/dl]

0 h

2h

4h

Normal

Control

86.38±3.15

87.38±3.31

[-1.0]

87.4±2.8

[-0.1]

Diabetic control

(DC)

340.01±29.26

342.5±29.3

[-1.37]

342.65±29.2

[-1.98]

DC+CSE

[50 mg]

337.5±27.7

329.2±24.2

[7.6]

325.2±20.9

[11.3]

DC+CSE

[100 mg]

329.7±24.3

311.9±22.8

[17.8]

307.6±26.8

[22.1]

DC+CSW

[50 mg]

284.3±24.0

275.4±14.3

[8.9]

267.8±23.6

[16.1]

DC+CSW

[100 mg]

281.31±19.4

271.3±18.1

[10.8]

268.3±24.2

[13.0]

DC+GLB

[10 mg]

342.3±28.3

313.8±18.25*

[29.5]

292.04±8.29***

[50.94]

Each value represents Mean ± S.E.M., n=5. Values in parentheses indicate Percent reduction in glycaemia and ***P<0.001, **P<0.01 AND *P<0.05 compared to basal values [0 hr] of the same group. One-way ANOVA followed by Tukey’s post test.

 

Fig. 3. Effect of ethanol (CSE) and water extract (CSW) of Argeria speciisa on SG levels in STZ-induced rats [Single-dose one-day study]. Bar graph represents the percentage reduction in glycemia with respect to the initial (0 h) level. Each value represents Mean ± S.E.M., n=5. aP<0.05; bP<0.01;cP<0.001 compared to diabetic control of the same time interval. One-way ANOVA followed by Tukey’s post-test.

 

Multiple-dose fifteen-day study

Repeated administration of CSE (50 and 100mg/kg) and CSW (50 and 100mg/kg) for 15 days, showed significantly (P<0.05; P<0.01) reduced levels of SG compared to respective basal values (0 day) (Table 5). On 15th day, tested doses of CSE and CSW showed significantly (P<0.001) greater percentage reduction in glycemia (24.6%: 24.7% and 23.9%:21.9% respectively) compared to diabetic control (Fig 4).


 

Table 5. Effect of CSE, CSW on SG levels in STZ-induced MD rats [Multiple-dose fifteen-day study]

Treatment [dose/kg b.w]

SGL levels [mg/dl]

0 day

5th day

10th day

15th day

Normal Control

87.38±3.15

88.95±4.2 [-1.77]

92.45±3.7 [-6.75]

99.98±4.9 [-15.64]

Diabetic control (DC)

343.01±29.6

323.7±18.64 [5.89]

345s.42±15.95 [-0.78]

388.97±12.98 [-14.76]

DC+CSE [50 mg]

330.5±20.4

259.0±23.6 [20.8]

250.7±19.4 [23.4]

246±14.9* [24.6]

DC+CSE [100 mg]

329.7±18.5

265.9±18.8 [20.2]

232.4±11.5** [28.5]

236.3±10.5** [24.7]

DC+CSW [50mg]

280.3±21.5

236.2±9.6 [15.5]

210.1±14.0* [24.8]

210.3±9.5* [23.9]

DC+CSW [100 mg]

285.31±11.3

239.73±12.1 [17.4]

234.62±10.2* [18.6]

225.6±8.4* [21.9]

DC+GLB [0.5 mg]

335.3±28.3

270.5±8.22 [19.2]

240.9±8.9* [27.57]

182.0±10.4 *** [43.0]

Each value represents Mean ± S.E.M., n=5. Values in parentheses indicate Percent reduction in glycaemia and *P<0.05;**P<0.01; ***P<0.001 compared to basal values [0 day] of the same group. One-way ANOVA followed by Tukey’s post test.


 

Fig.4  Effect of ethanol (CSE) and water extract (CSW) of Argeria speciisa on SG levels in STZ-induced diabetic rats [Multiple-dose fifteen-day study]. Bar graph represents the percentage reduction in glycaemia with respect to the initial (0 day) level. Each value represents Mean ± S.E.M., n=5. a P<0.05; b P<0.01, c P<0.001 compared to diabetic control of the same time interval. One-way ANOVA followed by Tukey’s post-test.

 

Oral glucose tolerance test (OGTT)

On 10th day, oral administration of glucose (2g/kg) did not produced significant change in SG level of normal control rats and AUC for the 120 min interval was not altered. The diabetic rats exhibited significant elevation in fasting SG (at time zero) and showed significant impairment in glucose tolerance to exogenously administered glucose compared to Normal rats (Figure 5 A). Treatment with different dose of CSE, CSW (50 and 100 mg/kg), and GLB (10mg/kg) significantly (P<0.05; P<0.01) improve the glucose tolerance (Fig 5 B).  Further, treatment of CSE and CSW exhibited significant (P<0.05; P<0.01) reduction in SG level over the period of 120 min compared to diabetic control group (Fig 5 A).

 

Administration of glucose (2g/kg) stimulated the release of higher levels of insulin in normal control rats, whereas glucose load was ineffective in stimulating the release of insulin in diabetic rats, suggesting that these diabetic rats resembled severe diabetic (type I) condition in which a maximum pancreatic damage occurred. Whereas, treatment of CSE and CSW to diabetic rats enhanced the glucose stimulated insulin release from pancreatic β-cells but response was not significant (Fig 6 A). Integrated areas under the insulin curve over 60 min (AUCinsulin) of diabetic group was significantly lower (P<0.001) compared to normal control. Treatment with GLB produced a significantly (P<0.05) increased AUCinsulin compared to diabetic control, whereas, administration of different doses of CSE and CSW fail to increCSe significantly (Figure 7 B).

 

 

Fig.5. Effect of ethanol (CSE) and water extract (CSW) of Argeria speciisa on glucose tolerance in fasted diabetic rats.

[A] SG levels were measured prior to, and after p.o. administration of glucose alone (2g/kg body weight), or in combination with CGE, CGW or Glibenclamide [GLB]. [B] Area under curve for glucose (AUCglucose) values for 0-120 min post glucose load. Data represent the mean ± S.E.M., for 5 rats. a P < 0.05; bP < 0.01; c P < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).

]

 

Fig.6. Serum insulin (SI) levels [A] Incremental AUC insulin values for 0-60 min[B] post glucose (2g/kg body weight) challenge performed on tenth day of treatment with ethanol (CSE) and water extract (CSW) of Argeria speciisa

Data represent the mean ± S.E.M., for 5 rats. a P < 0.05; bP < 0.01; c P < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).

 

Blood glucose, serum insulin and HOMA

On 10th day, diabetic rats exhibited significant (P< 0.001) hyperglycemia (354.42±15.95; SG levels rose to between 296 to 406 mg/dl) and hypoinsulinemia (8.25±1.1) as compared to normal control rats. The degree of insulin resistance as calculated by HOMA values were similar in both diabetic and normal control rats suggested that, the diabetic rats were not under insulin resistance condition (i.e. peripheral utilization of glucose was not compromised) (Figure 3).

Oral administration of CSW (50 mg/kg) to diabetic rats, significantly (P< 0.05) decreased SG levels, whereas all other tested doses of CSE and CSW fail to produce significant effect. Moreover, tested doses of CSE and CSW did not significantly increase SI levels. However, HOMA values were near to normal levels (Figure 7).

 

Fig.7. Effect of ethanol (CSE) and water extract (CSW) of Argeria speciisa on [A] Serum glucose (SGL) [B] Serum insulin (SI) [C] Homeostatic model assessment (HOMA) levels in diabetic rats.

 

Insulin tolerance test (ITT)

On 13th day, SG levels were measured following insulin challenge (2 U/kg, i.v). Surprisingly, in contrast to established reports, severe (type I) diabetic rats subjected to insulin challenge did not exhibit a marked fall in SG levels suggested that, these diabetic rats were not able utilize the exogenously administered insulin to reduce the SG levels. This contrary observation may be due to the marginal loss of insulin sensitivity in diabetic rats, even though these diabetic rats were in type I diabetic condition (as evident by lower insulin levels after glucose challenge and HOMA values).      

 

Moreover, the blood glucose levels  and AUC glucose in diabetic rats treated with CSE and CSW were significantly (P<0.01; P<0.001) lower at 10, 20 min compared to the glucose levels at the corresponding time points in the diabetic rats receiving the vehicle (Fig 8A and B).

 

Fig.8. Effect of ethanol (CSE) and water extract (CSW) of Calystegia sepium on insulin tolerance 6hr in fasted diabetic rats. [A] SG levels were measured prior to, and after p.o. administration of insulin alone (2U/kg body weight), or in combination with CGE, CGW or Glibenclamide [GLB]. [B] Area under curve for glucose (AUCglucose) values for 0-30 min post insulin injection. Data represent the mean ± S.E.M., for 5 rats. a P < 0.05; b P < 0.001; c P < 0.001 as compared with normal rats (one way ANOVA followed by Tukey’s post-test).


Table.6. Effect of ethanol (CSE) and water extract (CSW) of Cassia glauca on Lipid Profile in STZ-induced model  (Multiple Dose fifteen-days Study

Serum parameter

Normal control

Diabetic control

CSE 50mg/kg

CSE100mg/kg

CSW50mg/kg

CSW100mg/kg

GLB 10mg/kg

STG (mg/dl)

82.9+3.13

125.77±7.5

81.9+3.13***

79.04+5.5***

60.2+13.0***

82.9+4.3***

86.9±6.7***

STC (mg/dl)

67.41+3.9

125.62±4.7

68.31+3.13***

68.68+3.8***

59.9+8.9***

72.5+3.2***

81.9±6.9***

HDL-c (mg/dl)

32.1+2.0

12.52±1.8

28.13+3.24

25.75+3.8

16.3+1.6

22.2+3.2

23.6±1.9

VLDL-c (mg/dl)

10.4+0.7

23.34±1.5

13.97+0.63***

14.71+1.1***

10.9+2.6***

15.2+0.9***

16.4±1.3***

LDL-c (mg/dl)

15.2+3.3

83.70±3.4

19.30+5.46***

22.15+6.5***

25.5+5.8***

26.8+5.6***

34.9±8.5***

TC/HDL-cratio

1.2+0.1

8.17±1.2

1.85+0.24***

1.97+0.5***

2.6+0.4***

2.2+0.5***

2.7±0.4***

LDL-c/HDL-c ratio

0.5+0.1

5.76±0.9

0.67+0.21***

0.76+0.4***

1.2+0.3***

0.9+0.4***

1.2±0.4***

Each value represent Mean± S.E.M n=5 aP<0.05,bP<0.01,cP<0.001 compared to Diabetic control. One way ANOVA followed by Tukey’s post test.


 

Estimation of Lipid parameter

Diabetic rats showed significantly (P<0.001) increased levels of STG, STC, VLDL-c and LDL-c levels, whereas HDL-c was decreased in diabetic rats compared to normal rats (Table 8 and Figure 9). The markers of dyslipidemia such as TC/HDL-c and LDL-c/HDL-c ratios were significantly elevated in the diabetic group. Oral administration of different doses of CSE and CSW for fifteen-days exhibited significant reduction (P<0.001) in all tested lipid parameters and restoring them to near-normal values (Table 6, Figure 9).

 

Fig.9. Effect of Fifteen-day treatment with ethanol (CSE) and water (CSW)  extract of Calystegia sepium on [A] Serum TG [B] Serum TC [C] Serum HDL-c  [D] VLDL-c [E] LDL-c [F] TC/HDL-c [G] LDL-c/HDL-c levels in STZ-induced diabetic rats. Each bar represent the Mean ± S.E.M. (n = 5). a P < 0.05; c P < 0.001 compared with diabetic control.

Glucose uptake by isolated hemi-diaphragm of diabetic rats

Normal rats showed 2.154 mg glucose uptake per gram of diaphragm tissue, whereas fifteen-day post STZ (50 mg/kg, i.v) treated rats showed significant (P<0.01) reduction in glucose uptake (0.328 mg/g of tissue) (Figure 10). This observation suggested that these STZ administered rats were under insulin resistance condition (even though these STZ rats exhibited type In diabetic condition as evident by lower insulin levels post glucose challenge and HOMA values). Furthermore, this observation (reduction in glucose uptake by diaphragm of diabetic rats) was in line with data of insulin tolerance test.

 

Treatment of insulin (62 mU) caused significant (P<0.001) stimulation of glucose uptake leading to ~5 fold increase compared to diabetic control values (Figure 10). Whereas, treatment of different doses of CSE (50 and 50 mg/ml) and CSW (50 and 50 mg/ml) failed to stimulate glucose uptake.

 

 

Fig.10. Glucose up take by isolated hemi- diaphragm of Normal and STZ-induced diabetic rats. Each bar represent the Mean ± S.E.M. (n = 10). b P < 0.01; c P < 0.001 compared with diabetic control.

 

4. DISCUSSION:

Epidemological evidence suggests that a diet high in fruits and vegetables is associated with a decrease in the incidence of cancer and cardiovascular disease and possibly of other degenerative or age related disease.

 

Much progress in the understanding of functional relationship between nutritional factors has been provided by epidemiological and diet intervention studies and relevant in-vivo studies of progression and inhibition. This concept is based on the mechanism of action of the chemical compound and/or micronutrients in foods and their toxicity and antidiabetic effect.

 

The effect of extract of ethanol (CSE) and water (CSW) extract of Calystegia sepium flowerin plant has endowed with anti-diabetic (single-dose one-day study and multi-dose fifteen-day study), anti-hyperlipidaemic activity in standardized STZ-induced diabetic rats, justifying its use in the traditional system of medicine.

 

In this study, It is observed that different concentration of CSW and CSE having consistent effect on STZ induced diabetic rats. The % reduction in sugar level is increased as concentration increased and it ih higher in CSW than CSW.

Effect of CSE and CSW on Oral Glucose Tolerance Test (OGTT) and Oral Sucrose Tolerance Test (OSTT) in Normoglycemic rats   also detrmined and it shows that CSW controls the Glucose level at below the normal level.

 

Lipid parameters and anti lipidemic activity was also measured and also shows that both have CSE and CSW have antihyperlipidemic activity.

 

Hence, The study reveals the hypoglycemic and hypolipidemic activity of alcoholic and water extract of Calystegia sepium flowering plants in euglycemic and Streptozotocin induced diabetic rats. The extract seems promising for the development of a phyto-medicine for a Diabetes mellitus.

 

5. CONCLUSION:

We conclude that the ethanol (CSE) and water (CSW) extract of Calystegia sepium flower in plant has endowed with anti-diabetic (single-dose one-day study and multi-dose fifteen-day study), anti-hyperlipidaemic activity in standardized STZ-induced diabetic rats, justifying its use in the traditional system of medicine.

 

6. SUMMARY:

*        The flowering  of Calystegia sepiumwere collected in the month of May from hilly areas surrounding Dharwad, Karnataka, and authenticated by qualified taxonomist, Dr. G.R Hegde, Department of Botany, Karnataka University Dharwad. The herbarium specimen is present in Department of Pharmacognosy (SETCPD/Ph.cog/Herb/33/1006).

*        Shade dired, powdered flowering of Calystegia sepium were successively macerated with 95% ethanol (CSE) and chloroform water (CSW)

*        Phytochemical studies of the extracts were carried out and extracts showed the presence of carbohydrates, alkaloids, tannins, saponins, flavonoids.

*        Effective dose of the extracts were determined after carrying out acute oral toxicity studies in mice.

*        Lower dose of CSE and CSW (50 mg/kg) improve the oral glucose tolerance in normal rats.

*        Both the tested doses (50 and 100 mg/kg) of CSE and CSW exhibited significant anti-hyperglycemia on 15th day in Normoglycemic rats [Multiple-day fifteen-day study]. However, administration of CSE and CSW to normal rats did not alter lipid profiles.

*        Administration of CSE and CSW to post sucrose challenged rats improve the sucrose tolerance, suggested that the components of extracts may inhibit a-glucosidase enzyme.

*        Both the extracts were subjected to comparative evaluation of anti-diabetic and anti-hyperlipidaemic activity in in-house standardized STZ (50mg/kg, i.v) induced diabetics rats. The following biochemical parameters were analyzed

*    Fasting serum glucose levels

*    Serum Insulin

*    HOMA [Homeostatic Model Assessment: a measure of insulin resistance]

*    Oral Glucose Tolerance Test [OGTT]

*        Integrated Area Under Curve for glucose [AUC glucose]

*        Integrated Area Under Curve for glucose  [AUC insulin]

*    Lipid Parameters

*        Serum triglyceride (TG)

*        Serum total cholesterol (TC)

*        Serum HDL-c

*        VLDL-c, LDL-c

*        TC/HDL-c and LDL-c/HDL-c.

*    Glucose uptake by isolated hemi-diaphragm of Normal and STZ-induced diabetic rats.

 

*        The ethanol and water extract showed anti-diabetic in both single-day one-day study and multiple-dose fifteen-day study (in STZ-induced diabetic model).

*        Treatment of CSE and CSW for fifteen day exhibited reduced levels of lipid profiles in STZ-induced diabetic animals, indicated its hypo-lipidaemic property.

*        Treatment of insulin (62mU/ml) showed greater stimulation of glucose uptake by diaphragm of diabetic rats, whereas, CSE and CSW treatment failed to stimulate. This indicated that pre-treatment of CSE and CSW and for 15 days not able to combat the insulin resistance (reduced peripheral utilization of glucose) induced by STZ (50 mg/kg, i.v). 

*        Major upshot of the present study is that ethanol (CSE) and water (CSW) extract of Calystegia sepiumexhibited anti-hyperglycemic in normoglycemic rats, anti-diabetic and anti-hyperlipidaemic in standardized severe (Type 1) diabetic rats.

 

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Received on 15.04.2014          Accepted on 20.06.2014        

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Asian J. Res. Pharm. Sci. 4(2): April-June 2014; Page 55-70