Annexure II

Sri Balaji Nilaya,

Behind Balaji Paints and Hardware,

N.H. - 206,

Nittur - 572223

Tumkur (Dist.)

Al-Ameen College of Pharmacy,

Bengaluru-560027

Karnataka.

Course of Study and Subject

Master of Pharmacy (M. Pharm) in

Pharmacology

Date of Admission to Course

Diabetes Mellitus is a complex metabolic disorder primarily characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Changes in human behavior and lifestyle over the last century have resulted in a dramatic increase in the incidence of Diabetes Mellitus worldwide. Approximately 200 million people around the world have been diagnosed with diabetes thus making diabetes the sixth leading cause of disease-related death. This creates a huge economic burden related to the management of its complications, which are micro- and macro-angiopathic. Oxidative stress is believed to play a central role in the development of diabetic complications in many tissues.¹

It has been clearly demonstrated that Diabetes Mellitus is associated with an increased production of free radicals and lipid peroxidation² produced by glucose oxidation.³ Insulin and synthetic drugs used for diabetes treatment, are helpful in controlling hyperglycemia, but these drugs also have prominent side effects, such as hypoglycemia, dropsy, drug resistance, weight gain and so on. On the other hand , plant products are generally considered to be less toxic with fewer side effects than synthetic products.⁴

Diabetes of type I and II induces serious complications related to functional and structural changes in the central nervous system.⁶ The cerebral complications of both type I and type II Diabetes may be referred as ‘diabetic encephalopathy’, a concept introduced several years ago (Reske-Nielsen et al., 1965). Oxidative stress and inflammation play a very important role in diabetic tissue damage (Mastrocola et al., 2005). Alongside enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels were found to be increased in brain mitochondria. Oxidative damage to various regions of brain results in long term complications, morphological abnormalities and memory impairment.

Fruits from lycium barbarum L, belonging to family Solanaceae commonly called as Wolf Berry (China), Goji Berry (India) is a well-known in traditional Chinese herbal medicine. Nowadays, it is widely used as a popular functional food, with a large variety of beneficial effects, such as reducing blood glucose and serum lipids, anti aging, immune-modulating, anti-cancer, anti-fatigue and male fertility facilitating.⁵ The earliest Chinese medicinal monograph documented the medicinal use of Lycium barbarum around 2300 years ago. The berries are eaten raw, drunk as juice, wine or tea. They can be processed as tinctures, powders and tablets. The recommended dosage of dried berries varies between 5 and 12 g ^18. The polysaccharide fraction referred to as Lycium barbarum polysaccharides or LBP consists of a complex mixture of highly branched and only partly characterized polysaccharides and proteoglycans. The glycosidic part accounts in most cases for about 90-95 % of the mass and consists of arabinose, glucose, galactose, mannose, rhamnose, xylose and or galacturonic acid.¹⁹ It has been proven that the polysaccharides from Lycium barbarum possess antioxidant property. The antioxidant capacity was assessed using trolox equivalent antioxidant capacity and oxygen radical absorbance capacity.²⁰

Nutritional factors including antioxidants have great influence in the management of Diabetes Mellitus and its complications.⁶ An imbalance between oxidative stress and anti-oxidative defense mechanisms in diabetes can result in cell and tissue damage and accelerate diabetic complications. Diabetes could conceivably lead to cognitive impairment through chronic hyperglycemia,⁷ which is strongly related to several complications, including brain and vascular disorders.⁸ These impairments have been characterized mainly by moderate deficits in learning and memory, psychomotor slowing and reduced mental flexibility⁹. Diabetic patients also seem to double the probability of developing Alzheimer’s disease, effective disorders and other dementias.^{10, 11} Diabetes is characterized by hyperglycemia due to insufficient availability of or insensitivity to insulin and is associated with slowly progressive damage in the eyes, kidneys, blood vessels, heart and peripheral nerves, as well as in the brain.^12,13 The mechanism causing brain damage in diabetes mellitus has not been fully elucidated, but appears to be multifactorial process which involves fluctuation in the blood glucose level, as well as acute and chronic metabolic and vascular disturbances, such as decrease in cerebral blood flow¹⁴ and alterations in cellular calcium homeostasis¹⁴ moreover, it has been demonstrated that hyperglycemia induces oxidative stress in various brain regions¹⁵ and also alters activities of enzymes that are considered critical for normal CNS functioning, such as Na⁺-K⁺-ATPase,¹⁶ catalase, NTPDase and 5’-nucleotidase. ¹⁷ In addition to some studies using experimental diabetes have found an increase in acetyl cholinesterase (AChE) activity which may indicate alterations in cholinergic neurotransmission and consequently be associated to cognitive impairments observed in Diabetes Mellitus.

Studies on hypoglycemic action of various plant products been carried out by investigators from long time.

1. 
   Hypoglycemic effect of whortle berry, straw berry, walnut and burdock roots were studied by Kit et al., (1972).
   
2. 
   Hypoglycemic action of Eugenia jambolana (rotra) has been intensively studied.
   
3. 
   Juice expressed residue of Onion (Allium cepa) was found to control hyperglycemia effectively.
   

4. 
   The influence of Chinese fruit on blood sugar levels of alloxan diabetic rabbits was studied by Lee et al., (1975).
   
5. 
   The hypoglycemic, hypolipidemic and antioxidant property of Lycium barbarum was studied by Qiong Luoa, Yizhong Cai.
   

1. 
   This study is planned to investigate the effect of Lycium barbarum in diabetes induced cognitive disorder in streptozotocin mediated diabetic rat models.
   
2. 
   Extraction and characterization of Lycium barbarum, for the application in the treatment of cognitive disorder caused in diabetic rats.
   
3. 
   Comparison of cognitive enhancing efficacy of Lycium barbarum with Piracetam.
   

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

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

1. 
   The dried L. barbarum fruit were ground to a fine powder, placed in boiling water and decocted for 2 h. The juice was then centrifuged at 9000 r/min for 20 minute to remove the precipitate. The decoction was left to cool at room temperature, filtered and then freeze-dried to obtain crude polysaccharides. The dried crude polysaccharides were refluxed three times to remove lipids with chloroform/methanol (2:1, v/v). After filtering the residues were air-dried and then refluxed again with 80% ethanol. The resultant product was extracted three times in hot water (90oC) and then filtered. The combined filtrate was precipitated using 95% ethanol, 100% ethanol and acetone, respectively. After filtering and centrifuging, the precipitate was collected and vacuum-dried, affording the desired polysaccharides. The content of the purified polysaccharides was measured by phenol-sulfuric method (Robyt and Bemis, 1967). Absorbance was measured at 490 nm with standard glucose solutions from 0 to 100 mg/L spectrophotometrically. Result showed that the content of the polysaccharides in the extract may reach 96.27 %
   
2. 
   Wolfberry fruits (20 g dry solids equivalent) were soaked in 100 mL distilled water overnight and then blended for 3 min. This was followed by the addition of an additional 100 mL of distilled water to the blended mixture. The mixture was then extracted using continuous stirring on a stir plate for 2 h at 100 ºC (Li et al., 2007). After centrifugation at × 8,627 g for 15 minute, the residues were then extracted two more times using the same process. The supernatants were combined and concentrated to about 1/4^th volume of the original using a rotary evaporator at 40 ºC. Four volumes of anhydrous ethanol were added to the solution to give an 80.00 % final concentration of ethanol, and the mixture was kept overnight at 4 ºC to precipitate the polysaccharides. After centrifugation at × 8,627 g for 15 minute, the precipitate was washed with 95 % ethanol, 100 % ethanol and acetone three times, respectively (Li et al., 2007). The residues were dried in a vacuum oven at 40 ºC.
   

Male Albino rats of Wister strain in the weight range of 150 – 200 g will be included for the study.

Animals will be housed in polypropylene cages on clean paddy husk bedding. Animals will be maintained under controlled temperature at 25 ± 2 ºC with 12 h light/dark cycle, provided with standard pellet diet and water ad libitum.

A single dose of Streptozotocin (65mg/kg) was administered intraperitonially. The animals’ blood glucose level was measured after 2 days of administration of STZ and animals with blood glucose level more than 200 mg/dL are selected for study.

1. 
   Open field test for Behavioral Analysis.
   
2. 
   Elevated plus maze method for Memory and Learning.
   
3. 
   Step down passive avoidance can be used as a model for evaluating the diabetes
   

The rats were randomised into seven groups as below. Lycium barbarum polysaccharides (LBP) was administered orally in normal saline once per day.

ml Saline (8 rats)

(8 rats)

Blood samples of about 0.2 ml will be collected from Retro-Orbital sinus and the serum will be separated from blood by centrifuging at 6000 rpm for 15 minute.

1. 
   Blood glucose
   
2. 
   Estimation of total protein content.
   
3. 
   Estimation of cholinergic neurotransmitter (ACh) and enzyme (AChE) by Hestrin methods.
   
4. 
   Measurement of Glutathione (GSH)
   

The effect of polysaccharides derived from Lycium barbarum will be evaluated using an animal model of cognitive disorders due to diabetes in rodents.

YES in animals

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

Has ethical clearance been obtained from your institute?

YES

1. 
   Celik S, Erdogan S.Caffeic acid phenethyl ester (CAPE) protects against oxidative stress and inflammation induced in diabetic rats.Ml Cell Biochem 2008; 39-46.
   
2. 
   Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidents in normal physiological functions and human disease. The Int J Biochem Cell Bio 2007; 44-84.
   
3. 
   Goodarzi MT, Navidi AA, Rezaei M, Babahmadi-Rezaei H. Oxidative damage to DNA and lipids correlation with protein glycation in patients with type I diabetes. J Clin Lab Anal 2010; 72-76.
   
4. 
   Gao D, Li Q, li Y, Liu Z, Fan , et al. Antidiabetic effect and antioxidant effects of oleanolic acid from ligustrum licidum Ait in alloxane induced diabetic rats. Phytother Res 2009;23: 1257-1262.
   
5. 
   Gao et al., 2000; Peng et al., 2001 Wang et al.,2002a,b; an and Zhang, (2003). Alberti et al., 1997; Packer et al., 2000. Stewart R, Liolitsa D. type 2 diabetes mellitus, cognitive impairment and dementia. Diabetic Medicine 1999; 16 (2): 93-112.
   
6. 
   Rosenbloom AL. Hyperglycemic crisis and their complications in children. J Pediatr Endocrinal Metab 2007;20:5-18.
   
7. 
   Branda AM, Biessels GJ, Kappelle LJ, Haan de EH., Valk de HW, Algra A, Kessels RPC. Cognitive functioning and brain MRI in patients with type I and II diabetes mellitus: a comparative study. Dement Geriatr Cogn Disord 2007;23:343-350.
   
8. 
   Arvanitakis Z Wilson RS, Bienias JL, Evans DA, Bennett DA. Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function. Arch Neurol 2004;61:661-666.
   
9. 
   Biessels GJ Staekenborg S, Brunner E Brayne C, Scheltens P. Risk of dementia in diabetes mellitus: a systemic review. Lancet Neurol 2006;5:64-74.
   
10. 
    Northam E Rankins D, Cameron FJ. Therapy insight: the impact of type I diabetes on brain development and function. Nat Clin Pract Neurol 2006;2: 78-86.
    
11. 
    Gispen WH, Biessels GJ, Cognition and synaptic plasticity in diabetes mellitus. Trends Neurosci 2000;23:542-49.
    
12. 
    Manschot SM, Biessels JG, Cameron NE, Cottor MA, KamalA, Kapelle LJ. Et al. Angiotensin converting enzyme inhibition partially prevents deficits in water maze performance, hippocampal synaptic plasticity and cerebral blood flow in streptozotocin diabetic rats. Brain Res 2003; 966:274-82.
    
13. 
    Cameron NE, Eaton SEM, Cotter MA, Tesfaye S. vascular factors an metabolic interactions in the pathogenesis of diabetic neuropathy, Diabetologia 2001;44:1450-1458.
    
14. 
    Yuzcu M, Baydas G. Effect of melatonin and vitamin E on diabetes induced learning and memory impairment in rats. Eur J Pharmacol 2006;537:106-110.
    
15. 
    Frazon R, Chiarani F, Mendes R, Bello-Klein A, Wyse A. Dietary soy prevents brain Na, K-ATPase reduction on streptizotocinn induced diabetic rats, Diabetes Res Clin Pract 2004;69:107-112.
    
16. 
    Zhufan X. Practical traditional Chinese medicine. Beijing: Foreign language Press; 2000.
    
17. 
    Olivier Potterat., Goji (Lycium barbarum and L. chinense):Phytochemistry, Pharmacology and Safety in the Perspective of Traditional Uses and Recent Popularity: Division of Pharmaceutical Biology, University of Basel, Basel, Switzerland Planta Med 2010; 76: 7–19.
    
18. 
    Qiong Luoa, Yizhong Caib, Jun Yana, Mei Sunc, Harold Corkeb., Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium barbarum ., Life Sciences ;76 (2004): 137–149.
    
19. 
    Jing et al., Evaluation of hypoglycemic activity of the polysaccharides extracted fromlycium barbarum .Afr. J. Trad. CAM (2009); 6 (4): 579 – 584.
    
20. 
    Huiping Fan1, 2, 3, G. Mazza2*, X. Liao. Purification, composition and antioxidant activity of polysaccharides from wolfberry, cherry, kiwi and cranberry fruits. Croat. J. Food Sci. Technol. (2010); 2 (1): 9-17.
    

“Pharmacological evaluation of cognitive enhancement effect of polysaccharides extracted from Lycium barbarum in streptozotocin induced diabetes in rat”

Professor and Vice-Principal,

Al-Ameen College of Pharmacy,

Bangalore-560027

Head of the Department,

Department of Pharmacology,

Al-Ameen College of Pharmacy,

Bangalore-560027

Recommended for approval.

Principal,

Al-Ameen College of Pharmacy,

Bangalore-560027