INTRODUCTION:

Diabetes mellitus (DM) is the most common endocrine disorder. It affects more than 100million persons worldwide and its incidence is increasing steadily with changes in lifestyles. Complications of diabetes are many and include diabetic nephropathy and retinopathy. Diabetes was discovered as early as 700-200B.C.until the time insulin was invented, this disorder was managed principally by the traditional practices by using medicinal plants^.[3,4] many indigenious Indian medicinal plants have been reported by various authors to have anti diabetic properties.

Ethanobotanical interventions about medicinal plants having beneficial effects on diabetes are reported in approximately 800plants. Discovery of the hypoglycemic drug metformin comes from traditional approaches using Galega ethlinasis^.[1,5]

Reactive oxygen species (ROS) are an important part of the defence mechanisms against infection, but excessive generation of free oxygen radicals may damage tissue. The role of ROS in tissue damage in various human diseases such as cancer, ageing, neurodegenerative disease, diabetes and atherosclerosis has been recognized^.[6,7]

Asystasia gangetica is a straggling herb usually found among short grasses and along pathways. The leaves are green, oval shape with rounded base, very slightly saw edged and smooth (Saunders, 1958). The plant is recognized as a potential food source because the leaves have been shown contain high amounts of proteins, amino acids, minerals and sugars, lipids and fibre (Yeoh and Wong 1993).

The traditional medicine of East Africa (Kenya), Asystasia gangetica is used as anthelmintic. The leaves are crushed boiled in water and decoction drunk as cure for intestinal warms (Kokwro 1976). In nigeria, the leaves of Asystasia gangetica are playing to the highly effect in the local treatment of Asthma (Personal Communication 2000).The fresh leaves are macerated in local gin for 24 hrs or expressed and the extract drunk.^[2]

Over 70 % of Nigeria’s more than 100 million people live in rural areas where traditional medicinal practice is well established and patronized. The success of the practice has continued to reveal the potential of plants therapeutic agents. For further study there are so many activities in this plant which are reported in traditionally used. The present investigation was under taken to study the anti-hyperglycemic and antioxidant effect of Asystasia gangetica T. Adams leafs in Alloxan-induced diabetic rats.

MATERIALS AND METHODS:

Plant material:

The fresh leafs of plant Asystasia gangetica were collected from Tambaram, Tamilnadu, in the month of January. The leaves were authentified by Prof. P. Jayaraman, Director, Plant Anatomy Research Centre (PARC), Medicinal Plant Research Unit, West Tambaram, Chennai.

After authentification the leaves were dried at room temperature until they were free from moisture. The leaves were subjected to size reduction to get coarse powder of desired particle size. The coarse powder was then stored in a clean dry air tight container.

Preparation of extract:

The powdered material was first subjected to de-fatation by Soxhlet apparatus using solvent petroleum ether for fifteen hours. Then it was subjected to extraction by Soxhlet apparatus with methanol and n-hexane for forty eight hours. The obtained extract was finally dried at low temperature under reduced pressure in a rotary evaporator (microwave oven). A crude powder was obtained and used to prepare suspensions 100mg/kg, 200mg/kg (methanolic and n-hexane) concentration using Water: Tween 80 (4:1) for the treatment.

Animals:

Healthy Albino rats (125-250gms) of either sex obtained from central animal house, Madavaram Kanchipuram District were used for the study. Ethical clearance was obtained from Institutional Animal Ethics Committee (No. IAEC.24/2007).Animals were housed individually under standard laboratory conditions and fed with commercial pellet rodent diet and water till the end of the experiment.

Chemicals and Drugs:

1) Alloxan (Sisco Research Lab Pvt. Ltd, Mumbai, India).

2) Tris (Hydroxymethyl) Aminomethane hydrochloride (Sisco Research Lab Pvt. Ltd, Mumbai, India].

3) Pyrogallol (Qualigens Fine Chemicals, Mumbai, India).

4) 1,1,3,3-Tetramethoxy propane (Himedia Ltd., Mumbai, India).

5) Thiobarbituric acid (Loba Chemie Pvt Ltd., Mumbai, India).

6) Trichoro acetic acid (Sisco Research Lab Pvt. Ltd, Mumbai, India)

7) Glycohaemoglobin (A1c) Kit(Biotron Diagnosis Inc, Hernat California, USA).

8) Bun/creatinine/god pod kits (Merck Specialitics Pvt Ltd, Mumbai, India).

9) Erba total protein kit/ albumin kit (Transasia Bio-medical Ltd, Daman, India).

10) Cholesterol kit (Ranbaxy Fine Chemicals Ltd, Diagnostic Division, Baddi, India).

11) Sterile water for injection (Core Health Care Ltd, Mumbai).

12) Strychnos nuxvomica,(Tambaram, Tamil Nadu).

(All the chemicals were of A.R Grade)

Instruments:

1. Autoanalyser (Mayson 500e).

2. Spectrophotometer (Shimadzu).

3. Micro pipettes.

4. Cooling Centrifuge (REMI). Experimental diabetes

Experimental diabetes:

Diabetes was induced by 120 mg/kg of Alloxan administered i.p. in water for two at the interval of twenty 24hrs.twenty four hours after last injection blood glucose levels were measured to confirm the induction of diabetes. Rats with glucose level above 200 mg/dL were selected as diabetic rats and were included in the experiment.^[16]

The methanolic and n-hexane extract was given (100mg/kg, 200mg/kg). This dose was then converted to an equivalent dose in rats using the dose conversion table .

The animals were divided into 7 groups of 6 animals each – normal control, diabetic control, diabetic rats treated with 100 mg/Kg of methanolic-1, 200mg/kg of methonolic-2, 100mg/kg of n-hexane-1, 200mg/kg of n-hexane-2 extracts of Asystasia gangetica and diabetic rats treated with Glipizide (0.5 mg/kg. p.o. daily) for 21 days. Every week (from 0week to 3rdweek) on 1^st_,7^th,14^th,21^st day blood samples were collected by retro-orbital puncture under light ether anesthesia, then serum was separated by centrifugation at 2000rpm for 15min and blood glucose levels were measured. On 21^th day, glucose level was finally measured and antioxidant enzymes levels were measured in liver homogenate solution collected by sacrificing the animals under ether anesthesia.

Estimation of glucose:

Blood glucose was measured by using commercially available GOD POD Kit using auto analyzer.

Estimation of peroxidation product and antioxidant enzymes:

The level of peroxidation product viz. Malondialdehyde (MDA) was measured in liver homogenate where the reaction depends on the formation of a coloured complex between malondialdehyde (MDA) and thiobarbituric acid (TBA) having an absorption maximum at 532 nm.^[8]

After estimating MDA in liver homogenate the remaining solution was further used to check the activities of antioxidant enzymes.

Superoxide dismutase (SOD) activity was measured in liver homogenate. Epinephrine can be autooxidised to adrenochrome by superoxide radicals. The ability of SOD to inhibit the autooxidation of epinephrine to adrenochrome has been used as the basis for the assay of this enzyme.

Catalase (CAT) was measured in haemolysate here the rate of decomposition of hydrogen peroxide by catalase was measured spectrophotometrically at 230 nm^.[17,18]

Histopathology:

At the end of the study, animals from each group were sacrificed, pancreas excised and sent for histopathological examination. The staining was done using H and E stain.

Statistical analysis:

Results were expressed as mean ± SEM and evaluated for statistical significance by ANOVA followed by Dunnet's ‘t' test. Values of P< 0.05 were considered to be statistically significant.

RESULTS:

Table1.1. Effect of methanolic and n-hexane leaf extract of Asystasia gangetica on body weight (g) in Alloxan induced diabetic rats.

S. No	Groups	Initial Body weight (g)	Final body weight (g)
1	Normal control	140.27±1.92	161±3.59
2	Diabetic control	150.2±2.34	115±1.12
3	Standard (Glipizide)	165±2.76	140±2.30
4	Methanolic 1	145±2.12	125±1.72
5	Methanolic 2	140±2.24	122±1.70
6	n-Hexane 1	139±1.97	120±1.65
7	n-Hexane 2	155±2.50	138±1.30

STD: Glipizide 0.5mg/kg treated

MET-1: Extract 100mg/kg treated

MET-2: Extract 200mg/kg treated

n-HEX1: Extract 100mg/kg treated

N-HEX2: Extract 200mg/kg treated

Each value is represented as mean± SEM, No of animals (n) = 6-8,

Nc vs Dc ** P<0.01

Nc vs STD * P<0.05

Nc vs MET1 ns P>0.05

Nc vs MET2 ** P<0.01

Nc vs n-HEX1 * P<0.05

Nc vs n-HEX2 ns P>0.05

Fig. 1.1 Effect of methanolic bark extract of Asystasia gangetica on body weight (g) in Alloxan induced diabetic rats.

Fig..1.3a . Effect of methanolic bark extract of Asystasia gangetica on Total protein in Alloxan induced diabetic rats.

Fig..1.3b. Effect of methanolic and n-hexane leaf extract of Asystasia gangetica on total cholesterol in Alloxan induced diabetic rats

Fig..1.3c .Effect of methanolic and n-hexane leaf extract of Asystasia gangetica on Serum Creatinine in Alloxan induced diabetic rats.

Fig..1.3d .Effect of methanolic and n-hexane leaf extract of Asystasia gangetica on BUN in Alloxan induced diabetic rats.

Table.1.2. Effect of methanolic and n-Hexane extract of Asystasia gangetica on Blood Glucose Level BGL (mg/dl) in Alloxan induced diabetic rats.

S.No	Groups	0 Week (mg/dl)	1Week (mg/dl)	2Week (mg/dl)	3Week (mg/dl)
1	Normal Control	115±5.77	116.5±4.463	118±3.109	122±3.928
2	Diabetic Control	365±2.060	364±2.75	370±2.725	378±2.023
3	Standard	365±8.002	281±4.944	222±3.81	155±2.38
4	Methanolic 1	375±7.051	291±5.43	250±5.833	201±3.029
5	Methanolic 2	375±6.055	287±6.143	232±4.622	184±1.722
6	n-Hexane 1	370±50323	323±4.410	280±5.32	228±2.08
7	n-Hexane 2	369±5.099	309±2.708	260±7.12	195±4.90

Each value is represented as mean± SEM, No of animals (n) = 6-8,

Nc vs Dc ns P<0.01

Nc vs STD * P<0.05

Nc vs MET1 ** P>0.05

Nc vs MET2 ** P<0.01

Nc vs n-HEX1 * P<0.05

Nc vs n-HEX2 * P>0.05

Fig 1.2. Effect of methanolic and n-hexane leaf extract of Asystasia gangetica on Blood Glucose Level BGL (mg/dl) in alloxan induced diabetic rats.

Table.1.3 Effect of methanolic and n-hexane leaf extract of Asystasia gangetica on total protein, total cholesterol, Serum Creatinine, and BUN in Alloxan induced diabetic rats.

S .No	Groups	Total Protein (g/dl)	Cholesterol (mg/dl)	Serum Creatinine (mg/dl)	BUN (mg/dl)
1	Normal Control	7.75±0.077	63±0.9545	0.608±0.0094	41.5±1.08
2	Diabetic Control	3.583±0.108	85.66±1.022	1.031±0.0215	129.66±1.80
3	Standard	6.08±0.068	68.83±0.601	0.7116±4.944	55.86±1.276^*
4	Methanolic 1	4.23±0.076^**	76.33±1.3	0.931±0.008	76.33±1.542
5	Methanolic 2	5.18±0.101	70.66±0.477	0.766±0.010	68.5±0.763
6	n-Hexane 1	3.98±0.047	77±0.733^£	0.96±0.040	77.1±1.470
7	n-Hexane 2	4.8±0.063	70±0.365	0.83±0.013^££	70.5±0.619

Each value is represented as mean± SEM, No of animals (n) = 6-8,

* p<0.01 Vs Normal Control

** p<0.05 Vs Normal Control

£ p<0.05 Vs Diabetic Control

^££ p<0.01 Vs Diabetic Control

Table.1.4. Effect of methanolic and n-Hexane leaf extract of Asystasia gangetica on Antioxidants level in liver homogenate of Alloxan induced diabetic rats.

S .No	Groups	SOD (U/mg Protein)	CAT (U/mg Protein)	LPO (µM/g Protein)
1	Normal Control	16.4±0.193	6.12±0.107	0.456±0.0154
2	Diabetic Control	10.75±0.382	2.866±0.147	1.945±0.0405
3	Standard	14.91±0.153	5.016±0.087	0.68±0.020
4	Methanolic 1	12.95±0.147	3.47±0.1233^£	0.9566±0.0154
5	Methanolic 2	13.85±0.084^££	4.38±0.15	0.801±0.025
6	n-Hexane 1	12.56±0.164^*	3.15±0.084^*	1.02±0.43
7	n-Hexane 2	13.48±0.1302	3.81±0.117	0.835±0.025

Each value is represented as mean± SEM, No of animals (n) = 6-8,

D.C vs * P<0.01 N.C vs £ P< 0.01

D.C vs ** P<0.05 N.C vs ££ < 0.05

The islet of control rat appeared to be circular or oval in shape. Within the islet there were numerous darkly stained cells. At the center of islet, there were several lightly stained cells. Islet of an Alloxan induced diabetic rat showed a different morphology with respect to the islet from a control rat i.e circular shape of the islet was disrupted, limiting membrane between the islet and the surrounding acinar tissue was dissolved .Cells in the of an Alloxan-induced diabetic rat were destroyed. Islet of an of a diabetic rat treated with Asystasia gangetica leaf extract showed better improvement in comparison to Alloxan-induced diabetic rat. The islet was enlarged with more number of cells than that of Alloxan-induced diabetic rat. Diabetic rats treated with standard drug (Glipizide) also shows good improvement in comparison to Alloxan- induced diabetic rat, it depicts a different picture, the islet recovered to that of the control rat. The limiting membrane between the islet and acinar tissue was prominent and numerous cells were visible in the islet. In the islet some cells appeared lightly stained and others were well stained.

DISCUSSION:

Type 1diabetes is one of the most common chronic childhood illnesses, affecting 18 to 20 per 1, 00,000 children a year in the United Kingdom. The American Diabetes Association committee recommends the term type 1A diabetes for immune mediated diabetes with its destruction of the islet β cells of the pancreas. Non immune mediated diabetes with serve insulin deficiency is termed type 1B. At presents, the development of type 1 diabetes is a life sentence to a difficult therapeutic regimen that is only partially effective in preventing acute and chronic complications

The expression of diabetes relates autoantibodies in young children monitored from birth indicate that these markers are a major risk factor for the future development of type 1 diabetes. No treatment has been shown to safely prevent type 1 diabetes in humans, although islet transplantation and new immunosuppressive regiments can be cured (Devasenan D et al 2008).

In Alloxan-induced diabetic rats, increased food consumption and decreased body weight were observed. This indicates polyphagic condition and loss of weight due to excessive breakdown of tissue proteins (Chatterjee and Shinde 2002). Hakim et al (1997) have stated that decreased body weight in diabetic rats could be due to dehydration and catabolism of fats and proteins. Increased catabolic reactions leading to muscle wasting might also be the cause for the reduced weight gain by diabetic rats (Rajkumar et al 1991)

It has been shown that Asystasia gangetica leaf extract markedly improved the glucose tolerance in Alloxan induced diabetes in rats as compared to control (p<0.01).

More over Asystasia gangetica leaf extract show significant reduction in blood urea and creatinine in treated rats as compared to diabetic rats (p<.01) but significantly increased total protein level.

Possible sources of oxidative stress and damage to proteins in diabetes induced free radicals generated by autoxidation reactions of sugars and sugar adducts to protein and by autoxidation of unsaturated lipids in plasma and membrane proteins. The oxidative stress may be amplified by a continuing cycle of metabolic stress, tissue damage, and cell death, leading to increased free radical production and compromised free radical inhibitory and scavenger systems (JW Baynes1991). Under conditions of severe oxidative stress, free radical generation leads to protein modification. Proteins may be damaged directly by specific interaction of oxidants or free radicals with particularly susceptible amino acids. They are also modified indirectly, with reactive carbonyl compounds formed by the auto-oxidation of carbohydrates and lipids, with evenyual formation of advanced glycation/ lipoxidation end products (Gumieniczek 2005).

Lipid peroxidation is a free-radical mediated propagation of oxidative insult to polyunsaturatd fatty acids involving several types of free radicals and termination occurs through enzymatic means or by free radical scavenging by antioxidants (Korkina 1997). Lipidperoxidation end products measured as thiobarbituric acid reactive substances and hydroperoxides were seen increased in plasma of Alloxan-diabetic rats in this study. Drug with antioxidant properties may supply endogenous defense systems and reduce both initiation and propagation of reactive oxygen species.

Under condition of severe oxidative stress, free radical generation leads to protein modification. Proteins may be damaged directly by specific interactions of oxidants free radicals with particular susceptible amino acids. They are also modified indirectly with reactive carbonyl compounds formed by auto oxidation of carbohydrates and lipids, with eventual formation of advanced glycation/ lipoxidation end products (Gumieniczek, 2005). In diabetic mellitus a variety of proteins are subjected to non-enzymatic glycation and is thought to contribute to the long term complications of the disease (Vlassara et al, 1981). The level of total plasma protein was found to be decreased in this study. This could be due to increase lipid peroxidation in the diabetic rats. The decreased in plasma protein may also be ascribed to (i) decreased amino acid uptake. (ii) greatly decreased concentration of variety of essential amino acids, (iii) increased conversion of glycogenic amino acid to co₂and h₂o and (iv) reduction in protein synthesis secondary to a decreased amount and availability of mrna (Ahmed, 2005). Decreased protein content of blood serum in diabetic patients were reported (Mehboob et al, 2005) indicating elevated lipid peroxidation and reduce sod and cat activity and decreased antioxidant defensive system. Asystasia gangetica extract significantly reduced the elevated lpo and significantly improved sod and cat activity.

In histophathological study the light microscopic photograph islet from control rat appeared circular with the granulated beta cells appearing darker. Small and shriken islets and destruction of beta cells were observed in the diabetic condition. Well –formed islets and increased cell number were observed in diabetic rats, after Asystasia gangetica therapy. The data presented in electron micrograph of the beta cell of normal and treated rats showed evidence for increased secretary granule synthesis and there by increased insulin secretion after the administration of seed extract of Asystasia gangetica suggesting possible regeneration /repair of the islets of langerhans in alloxan treated rats.

In diabetes there is a relationship between glucose homeostasis, lipid metabolism later renal function and enzyme activities. We found that a 21 day administration of Asystasia gangetica seeds shows equal effectiveness in controlling diabetics when compared with diabetic rats treated with standard drug (glipizide) methanolic and n-hexane extract of Asystasia gangetica leaf proved to have a hypoglycemic effect on alloxan –induced diabetic rats, a fact that indicated that –there was a repair/regeneration of the beta cells of the islets of langerhans. As a result there was a increase in insulin level, which brought a homeostasis in the above mentioned biochemical parameters such as cholesterol, urea, creatinine, total protein and in the enzyme activities.

CONCLUSION:

In conclusion, the present study indicated a significant anti-diabetic effect of the methanolic and n-hexane leaf extract of Asystasia gangetica and supports its traditional usage in the control of diabetes.

It is also concluded that the leaf extract have strong antioxidant potential activity in vivo studies.

Further, investigation is required for the detailed study in isolation of the compounds and pharmacological actions of the leaf constituents, which have many activities reported in traditionally and its exact mechanism of action.

REFERENCES:

1) Ashok K. Tiwari, and J. Madhusudana Rao. Diabetes mellitus and multiple therapeutic approaches of phytochemicals: Present status and future prospects. Current Science 2002; 83(1): 30-3

2) J.V. Kavitha, Joseph F. Rosaria, Chandran J, Anbu P and Bakkiyanathan. Hypoglycemic and other related effects of Boswellia glabra in Alloxan-Induced Diabetic Rats. Indian J. Physiol Pharmacol. 2007; 51(1); 29-39.

3) Charles R. Craig, Robert E Stitzel. Modern Pharmacology with Clinical Application. 6th Edition, 2003,763-764.

4) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, Tenth edition, 2010, pp. 1679-1715.

5) Goshi S.G, Medicinal Plant 1^st Edition, 2000, pp. 361-362.

6) P.N. Bennett, M.J. Brown, Clinical Pharmacology 9^thEdition, 2003,pp.679-685.

7) Tripathi, K. D., In; Essentials of Medical Pharmacology, 4^th Edn., Jaypee Brothers, Medical Publishers (P) Ltd, New-Delhi, 2001; pp.264.267-68, 273-74.

Received on 16.01.2014 Modified on 14.02.2014

Res. J. Pharmacology & P’dynamics. 6(2): April- June 2014; Page 86-93

Sl. no	Parameter	Reference Range
1.	Glucose(GOD POD )	Serum/plasma 70-110mg/dl
2.	Cholesterol	<5.2 mmol/l
3.	Creatinine	Serum/plasma 62-97 µmol/l
4.	Urea	Serum/plasma 19-44 mg/dl
5.	Total Protein	6.0-803 g/dl

Ethanobotanical interventions about medicinal plants having beneficial effects on diabetes are reported in approximately 800plants. Discovery of the hypoglycemic drug metformin comes from traditional approaches using Galega ethlinasis.[1,5]

MATERIALS AND METHODS:

Preparation of extract:

Animals:

Estimation of glucose:

Estimation of peroxidation product and antioxidant enzymes:

Statistical analysis:

RESULTS:

Histopathological studies:

DISCUSSION:

CONCLUSION:

REFERENCES:

Ethanobotanical interventions about medicinal plants having beneficial effects on diabetes are reported in approximately 800plants. Discovery of the hypoglycemic drug metformin comes from traditional approaches using Galega ethlinasis^.[1,5]