EVALUATION OF THE EFFECT OF FLAVONOID EXTRACT OF Ginkgo biloba LEAVES AND GLIMEPHAN ON OXIDATIVE STRESS AND RETINA DEGENERATION IN DIABETIC MALE RABBITS INDUCED BY STREPTOZOTOCIN

This study was carried out in animal house of Collage of Veterinary Medicine/ University of Basrah. An attempt has been done to induce diabetic by streptozotocin in male rabbits and investigation of the diabetes was induced oxidative stress and retina degeneration. Moreover, the purpose of this study was to isolate and evaluate the ameliorating effect of flavonoid extract of Ginkgo biloba leaves and glimephan in the prophylaxes or delay the development of diabetic retina degeneration and scavenging free radical induced oxidative stress and diabetic retina degeneration in male rabbits.The study was done on (32) adult male rabbits, their weight ranged between (2000-2500g) and aged between 7-8 months. The male rabbits were divided randomly into four groups, each group consists of eight rabbits as the following: also in addition, this extract improved the retina degeneration.Histological examination observed many pathological changes in pancreas and retina in diabetic group but in treated with flavonoid extract of Ginkgo biloba , the histological changes were near to the normal status. It is concluded that good anti-diabetic activity, hypoglycemia effect and regeneration of retina. Based on these results, we suggested the possible utilization of Ginkgo biloba as a therapy to prevent diabetic complication and improved the retina degeneration compared with another treated such as glimephan drugs. study aimed to determine the effects of the of biloba on oxidative and retinal degeneration in male GPx serum of diabetic rabbits treated with glimephan with (+ve)


INTRODUCTION
Diabetes mellitus is a metabolic disorders that is characterized by hyperglycemia and insufficiency in production or action of insulin produced by the pancreas within the body (1) . Insulin is a hormone synthesized in β-cells of pancreas in response to various stimuli such as glucose, sulphonylureas and arginine however glucose is the major determinant (2) . The complications consist of both microvascular (neuropathy, nephropathy, and retinopathy) and macrovascular (atherosclerotic) disease (3) . There are several factors that play great role in pathogenesis of diabetes such as hyperlipidemia and oxidative stress leading to high risk of complications (4) .
Diabetes in animals can be induced by partial pancreatectomy or by the administration of diabetogenic drugs such as Streptozotocin causes destruction of pancreatic β-cells which results in reduced insulin production. As a result, fatty acid is released from adipose tissues accompanied by increase ketone synthesis and progression to ketosis (5) .
Ophthalmic complications of hyperglycemia are most effective in retina, represented by Diabetic retinopathy (DR) that is a vascular disease of the retina (6) . It is the most common cause of blindness in people over the age of 50 years (7) .
The synthetic drugs for diabetes treatment are valuable in the treatment of diabetes mellitus, limited pharmacokinetic characteristic, secondary failure rate and accompanying adverse effects such as cardiovascular disease, hypoglycemia, coma and damage of the kidney and liver that can endanger the life of diabetic patient (8) .
There are many anti-diabetic plants, which might provide useful source for the development of drugs in the treatment of diabetes mellitus, such as Ginkgo biloba (9) . G. biloba extract is used for treatment of diseases such as retinopathy, Alzheimer's disease, cardiovascular disease, dementia, memory loss, and cerebral ischemia (10) .
The pharmacological modes of action include antioxidant effects, radical scavenging, inhibition of platelet activating factor, alterations in membrane fluidity (signal transduction), and inhibition of glucocorticoid synthesis (11) . also protect retinal tissue from oxidative stress. These might prevent progression of tissue degeneration in patients with D.M (12) . The study aimed to determine the effects of the flavonoid extract of Ginkgo biloba on oxidative stress and retinal degeneration in diabetic male rabbits.

MATERIAL AND METHODS
Experimental Animal Thirty two healthy adult male domestic rabbits with body weight ranged (2000g-2500g) about (7-8 months) age old were brought from local markets.). The animals were allowed to acclimatize in animal house in Veterinary Medicine Collage / Basrah University for two weeks before experimentation. The experimental animals were kept in well ventilated cages with (23±2 ºC) under controlled conditions about 12 hour light and 12 hour dark [13]. Provided with ration composed fodder which consists of dry bread and lettuce and drinking water.

Preparation of Flavonoid Extract of Ginkgo biloba
Leaves.This method according (14) Experimental Desig:Thirty two rabbits were divided randomly into four equal groups each group consistof eight animals as following: The first group: served as (negative control)(-ve), administration of normal saline1ml daily for 30 days.
The second group: served as(positive control) (+ve)injected of single dose of STZ (65mg/kg B.W) [15] into marginal ear vein after over fasted night. STZ dissolve in citrate buffer pH4.5 and administration D.W 1ml for 30 days. Then give after STZ injected water contain 5% glucose instead of drinking water to overcome the high insulin released to all rabbits injected with STZ and let it without treated for 30 days. Histological Techniques.:The organ samples were removed from animals after the end of experimental as pancreas and eyeball and this method according to (15) .

Statistical Analysis :
The mean values ± SD. Statistical analysis by using multivalent analysis of variance. All statistical analyzes were performed using SPSS statistical version 23 software package. Least significant different test (LSD) was used to test the difference between means (groups) p≤ 0.05 was considered significant (16) .

Streptozotocin treated with Flavonoid Extract of Ginkgo biloba Leaves and
Glimephan.
The results in Table (

Glucose and Insulin Concentrations in serum of Diabetic Male Rabbits Induced by Streptozotocin Treated with Flavonoid Extract of Ginkgo biloba Leaves and
Glimephan.
The obtained results in Table (

Treated with Flavonoid Extract of Ginkgo biloba Leaves and Glimephan.
The results in Table (  n=number of animals., Capital letters denote differences between groups,P≤0.05 vs. control.

Rabbits Induced by Streptozotocin Treated with Flavonoid Extract of Ginkgo biloba Leaves and Glimephan.
The obtained results in Table (  biloba leaves while the results showed a non-significant change (P˃0.05) SOD in serum of diabetic male rabbits treated with glimephan compared with (+ve) control.
The results of Gpx revealed a significant height (P≤0.05) in diabetic male rabbits (+ve) compared with (-ve)control and treated flavonoid extract of Ginkgo biloba leaves while the results showed a non-significant change (P˃0.05) GPx in serum of diabetic male rabbits treated with glimephan compared with (+ve) control.

Grossly Examination of lenses Opacification:
The results of grossly examinations in Table (5   The section of pancrease of (-ve) control male rabbits is composed of two major types of tissues, the acini and the islets of Langerhans. Fig.(2) and (6) Fig. (3) and (7).
But the pancrease of male rabbits treated with flavonoid extract of Gingo biloba leave showed amelioration of architecture of islets langerhan's compared to pancreas treated with streptozotocin alone as shown in Fig. (4) and (8). while the pancrease of male rabbits treated with glimephan showed clear revealed histopathological changes. The changes included vacuolation of langerhan's islets as shown in Fig. (5) and (9).

Retina :
The section of retina of (-ve) control male rabbits is composed of the

DISCUSSION
Diabetes mellitus was caused by dysfunction in pancreas in male rabbits (17) . This leads to partial or complete insulin deficiency in diabetic rabbits that appears to have side effects on all systems, including eyes lead to retina degeneration (18) . The present study concludes that sterptozotocine at 65mg/kg B.W weight induces the diabetes and the rabbits are shown higher glucose levels and reduced body weight and organs weight. This is associated with catabolism of fats and proteins due to proteolysis on muscular tissues occurred in insulin deficiency states (19) .
The animals treated with STZ appeared very weak with loss of their body weights because of injurious effects of STZ which caused alkylation of DNA and produced hyperglycaemia and necrotic lesions. Present observations are in agreement with the findings of (20,21,22,23) . Our results also showed that treated with flavonoid extract of Ginkgo biloba lead to improve the body weight and body weight gain in male rabbits.
Through its contents which were flavonoids several flavonoid subclasses have been shown to decrease energy intake, increase glucose uptake in muscle in vivo, and decrease glucose uptake in adipose tissue in vitro.
In this study, glucose concentration of serum diabetic male rabbits elevate concentration above the normal concentration, and this elevation in glucose concentration was approximately constant through the course of diabetes. Reduction in pancreatic β-cell mass is associated with development of diabetes (24). Treatment with the flavonoid extract of Ginkgo biloba revealed reduction in blood glucose concentration diabetic male rabbits, due to the presence of flavonoid in Ginkgo biloba leaves. These compounds can decline glucose level by stimulating pancreatic β-cells to secret more insulin in blood circulation (25,26) . It is possible that the flavonoid extract of Ginkgo biloba may increase glucose removal from blood, through reducing the release of glucagon or elevate that of insulin. Flavoinod extract Ginkgo biloba is one of the most widely used herbs in the world, and is known for its numerous health benefits (28) . Previous studies showed that Ginkgo biloba leaves contains high amounts of narengin (193.15 ppm), rutin (108.49 ppm), hisperdin (107.21ppm) and quercitrin (94.86 ppm).
Ophthalmic examinations revealed diabetic rabbits only developed retina degeneration caused by oxidative stress (18) . The flavonoid content of Ginkgo enables the compound to scavenge the free radicals more effectively. The flavonoids in Ginkgo help protect cells against free radical contact. In addition, these flavonoids provide protection to blood vessels against the damaging effect of plaque build-up.
The Ginkgo leaf extract is also known to improve coronary blood flow through antiplatelet activity and by improving contractile functions which are due to elevate release of catecholamines from endogenous liver tissue reserves by flavonoids (29) . In this study found significantly elevated in the level of MDA and GPx but significantly The pharmacological activity of flavonoid extract Ginkgo biloba was attributed to synergistic action of flavonoids such as terpene and trilactones (30). Previous studies indicated the use of the Ginkgo extract in the treatment of heart, short term memory loss, diabetes, depression and dementia (31)(32)(33). Also, acting as a powerful antioxidant (34,35). In addition, Ginkgo biloba extracts were found to protect rats against different eye diseases such as age related macular degeneration (36) . diabetic retinopathy (37) .
Gingko biloba leaves contain are full of flavonoids, which act as antioxidants and these flavonoids are known to help with retinal problems (38) .
The major causes of blindness are diabetic retinopathy and trauma. Among these alternations, cataract is the foremost cause of blindness globally and is responsible for 50, 51% of total blindness (39) . Therefore an alternative medicine treatment for the control/delay of diabetic retina degeneration could make a great impact in these parts of the world. Recognition of the role played by natural antioxidant in delaying the onset of diabetic retina degeneration has opened new avenues for treatment retinpathy (40) Recent investigations have shown that phytochemicals antioxidants can scavenge free radicals and prevent various diseases like retina degeneration. Both of synthetic and plant origin that could be effective in a delaying or preventing the formation of retina degeneration (41) .
The present study was designed to determine the possible protective effects of Ginkgo biloba against oxidative damage induced by streptozotocin. The excess oxidative stress was previously reported to induce extensive oxidative modifications on lens proteins especially α-crystalline protein, a major protein component of the lens resulting in the enhanced lens opacity. Ginkgo biloba extract is considered an alternative medicine for the treatment and / or the prevention of different eye diseases.
Flavonoid extract Ginkgo biloba can enhance the antioxidant ability of retina and partially inhibit the apoptosis of photoreceptors (42) . Ginkgo biloba has also been shown to prevent diabetic retinopathy in diabetic rats. Ginkgo biloba may act as a neuroprotective and prevent damage to retinal ganglion cells, this plant extract would be an interesting component for prevention and treatment of ocular diseases and other major neurodegenerative retinal pathologies (43) .
The results revealed an increase in the serum of total cholesterol and LDL and VLDL in male rabbits treated with streptozotocin. In streptozotocin-induced diabetes, the rising in serum cholesterol and reducing in HDL concentrations is usually associated with an increase in glucose concentration (44)(45)(46) .
These results indicate the relationship between hypercholestermia and retina degeneration. The most significant ocular findings, such as the accumulation of lipids in the choroid, retinal disorganization, and lipid keratopathy. With respect to the retinal macroglia, the synthesis of the apoE by the Müller cells, its subsequent secretion in vitro, and its being taken up by the axons and transported by the optic nerve enabled the detection of apoE in the latter geniculate body and in the superior colliculus (47) . These optically empty spaces, with an elongated or needle shape, were previously occupied by crystals of cholesterol monohydrate or crystals of cholesterol esters (50) In other studies, the analysis in the form adopted for the crystallizations of the different types of lipids revealed that the needles corresponded to esterified cholesterol, and the short, thin ones to triglycerides (49) . It had been recently reported that hypercholesterolaemic rabbis had a build-up of lipids (foam cells and cholesterol clefts) mainly at the suprachoroidea and to a lesser extent at the choroidal vascular layers. This lipid compressed the choroidal vessels and causes hypertrophy of the vascular endothelial-and vascular smooth-muscle cells. The ultra-structural analysis of these vascular structures demonstrated numerous sings of necrosis and a severe damage of the cytoplasmic organelles and caveolar system (48,51) Recently, it has been reported that in comparison with normal control animals, hypercholesterolaemic rabbits had a reduction of the amplitudes of the first negative peak of the visually evoked potentials, the density of the RGCs, and the thickness of the INL and photoreceptor-cell layer. Enhanced activity of iNOS in hypercholesterolaemic rabbits might be involved in impaired visual function and retinal histology. Down regulation of eNOS activity might be one of the causes for impairment of the autoregulation (52).
The formation of foam cells is a consequence of phagocytes from the macrophageoxidized LDL (48) , with the retention of cholesterol in the vascular wall and the activation of ACAT (acetyl-cholesterol-acyl-transferase) (53) .