Rajiv gandhi university of health sciences, bangalore, karnataka

DEPARTMENT OF PHARMACOLOGY,

K.L.E. SOCIETY’S COLLEGE OF

PHARMACY, J.N.M.C.CAMPUS, NEHRU

NAGAR, BELGAUM.

K.L.E. SOCIETY’S COLLEGE OF

PHARMACY, BELGAUM.

MASTER OF PHARMACY IN

PHARMACOLOGY

JUNE-2008

“SCREENING OF BIOFLAVONOID MORIN FOR BURN WOUND HEALING, HEPATOPROTECTIVE AND ANTIDIABETIC ACTIVITIES IN RATS”

Hepatocytes deaminate amino acids so that the amino acids can be used for ATP production or converted carbohydrate or fats. The liver can detoxify substance such as alcohol or excrete drugs such as penicillin, erythromycin and sulfonamides into bile. It can also chemically alter or excrete thyroid hormones and steroid hormones such as estrogen and aldosterone. liver is also involved in the excretion of bilirubin,synthsis of bile salts, storage of vitamins, phagocytosis.²

Antioxidative agents reduce the effect of dangerous oxidants by binding together with these harmful molecules or scavenging them. The antioxidants by scavenging the oxidants or free radicals it reduces the lipid peroxidation and also prevents the cell damage which also lead to reduce the lysosomal level, which protects liver damage.

Morin is flavonoids with anti oxidant properties and has damage. Morin was compared with two other flavonoids with regard to their cytoprotective effects against oxyradical damage to porcine aortic endothelial cells in vitro.³

Another study showed that when Morin was added to cultured rat glomerular mesangial cells which were attacked by oxyradical, the survival time of the cells were doubled^.

Paracetamol toxicity occurs at over dose. When the liver enzymes catalyzing the normal conjugation reactions are saturated, causing the drug to be metabolized by the mixed function oxidase. The resulting toxic metabolite N-acetyle-p-benzoquinone imine is inactivated by conjugation with glutathione but when glutathione is depleted the toxic intermediate accumulates and reacts with nucleophilic constituents in the cell. this causes necrosis in the liver and also in the kidney tubules.⁴

The bioflavonoid Morin is reported for its hepatoprotective activity against carbon tetra chloride induced liver damage. No reports are available on the activity of Morin on other hepatoprotective models.

Wound may be defined as disruption of anatomic or functional continuity of living tissue. it is cellular injury and death anywhere within the body or on its surface it can be either microscopic or macroscopic.⁵

Diabetes mellitus is chronic form of disease characterized by high blood glucose level due to absolute or relative deficiency of circulating insulin level. It is characterized by hyperglycemia in postprandial and /or fasting state and in its severe form is accompanied by ketosis and protein wasting. Diabetes mellitus is one of the most common metabolic disorders with world wide prevalence. Estimated to be 2.8% the increase in prevalence of diabetes in the world is cause for concern. Diabetes mellitus leads to the abnormalities in carbohydrate, protein, and lipid metabolism and increases the risk of developing atherosclerotic arterial disease. According to WHO projections the prevalence of diabetes is likely to increase by 35% currently there are over 150 million diabetes worldwide and this is likely to increase to 300 millions or more by the year 2025.⁶

Citrus flavonoids have been reported for their Antidiabetic activity. As Morin is a bioflavonoid and antioxidant activity. There are chances of modulation of glucose level. There fore the present study will be designed to evaluate the bioflavonoid Morin for its hepatoprotective, burn wound healing and Antidiabetic activities.

Flavonoids are most abundant polyphenolic compounds present in fruit juices, tea, and red wine.

Among flavonoids morin (3,5,7,2’,4’-pentahydroxy flavone) is found in fig and other moraceae that are used as herbal medicine and exhibits various biological activities including antioxidation, (Hanasaki et al 1994, Kok et al, 2000 and ramanathan et al,1994) anti mutagenesis (bhattacharya and firozi et al,1988, francis et al , 1989) and anti inflammation (Kimet al,1999, raso et al, 2001;fang etal;2003)

Further more the morin inhibits cellular efflux of variousagents (zang and morris 2003) previous studies have reported that morin could modulate the activitiee the activities of metabolic enzymes including cyp.p450.⁷ Flavonoids are also having important property of preventing a wide variety of disease including allergies, hepatic diseases and inflammation. They exhibit various biological activities including, cytoprotection. More over morin acts as chemoprotective agent against oral carcinogenesis in vitro and in vivo.

Oxidative modification of low density lipoprotein (LDL) has been suggested to be a risk factor for the development of atherosclerosis. Agents which can protect LDL from oxidation may be useful in preventing atherogenesis Morin (100 micro mole) effectively inhibits Cu2+ induced oxidation of LDL3 and at 75-100 micromole protects against the oxidation of LDL3 by free radicals produced by 2’,2’- azo-bis (2-aminopropane) dihydrochloride.

Morin exhibits in vitro inhibitory actions on phosphatidylinositol phosphate kinase extracted from rat brain. Morin hydrate or simply Morin is shown here to be an effective hepatoprotector in vitro and in vivo. Between 0.25 micro mole, Morin prolongs survival of rat hepatocytes against free radical damage triggered by xanthine oxidase – hypoxanthine, and substantially better than equivimolar concentrations of Trolox (a vitamin E analogue), Mannitol, and ascorbate.

Mechanistically Morin acts in a two pronged manner as a preventive antioxidant by partially inhibiting xanthine oxidase and partly as a curative antioxidant by scavenging oxyradicals. The role of Morin as an effective free radical scavenger is further evidenced by its ability to protect human red cell membrane from per oxidative attack better than ascorbate, trolox, and Mannitol.⁸

Morin prevents acute liver damage by inhibiting the production of TNF-alpha, IL-1beta, IL-6 and iNOS. Morin is also can be used as antihyperammoninaemic, hepatoprotective and anti oxidant effects of Morin against oxidative stress induced by ammonium chloride.⁹

The administration of Morin facilitated tissue recovery. It exerts beneficial anti-inflammatory effect in the chronic phase of trinitrobenzenesulphonic acid induced rat colitis through the down regulations of some of the mediators involved in the intestinal inflammatory response, including free radicals, cytokines, leukotrienes B4 and nitric oxide.¹⁰

Morin is found in much other plant as Morus alba.

Maclura aurantiaca mundulca sericea,M.serrata, Indigofera hebepetala benth , Machilus bombycina .

Ripe fruits and cut roots of Morus alba are used. A tea made from root is used to treat diarrhea.

Fruit preparation is given for high fever.

Fruit is also a mild laxative.¹¹

Herbs which helps to protect body cells from the damaging effects of oxidation. Factors like stress, aging and pollution high level of free radicals in body which damages DNA & cause heart diseases or cancer or stroke. Substances like vitamin-E or beta carotene protect cell membranes and vitamin-C removes free radicals from inside the cell.

Mechanism: they reduce the free radical energy stop free radical from forming in the first place or interruption an oxidizing chain reaction to minimize the damage caused by free radicals ¹²

Burn wound is caused by transfer of energy into tissues with a resulting disruption in its functional integrity^.­­

Skin is heterogonous in nature and it is composed of collagen, ellastin amorphous ground substances like proteins, polysaccharides, glucoproteins, globular proteins, salts and water. ¹³

Cellular mechanism of wound healing involves different process that has bearing on the understanding of injury, inflammation, regeneration, repair and healing.

Wound healing consists of essentially two discrete phenomenon viz. inflammation and repair.

Inflammation is the first cellular response to tissue injury, further rapid accumulation of polymorph nuclear leucocytes takes place.

The first step in wound healing is proliferation of cells that helps in wound repair. It includes capillary contraction shortly followed by capillary dilation and increased permeability resulting in fluid exudation into wound area from blood and lymph. Constituents of these exudates play role in chemo taxis to attract the cellular elements at the site of wound.

Wound repair

Voluminous knowledge about wound healing process has been divided into distinct interrelated and often concurrently processing event viz

Cellular phase

Immediately after the cellular phase starts. It activates a cascade of chemo attractants and mitogens that recruit cells in neutrophils, macrophages, fibroblasts and endothelial cells.

E.g.: PDGF(Platelet derived growth factor )¹⁴

Wound contraction

The term contraction means diminution of size of an wound which is result of the centripetal movement of the whole thickness of the surrounding skin or deformity resulting from contraction in area where the skin overlies and is attached to the fascia if muscle or to tendon and the end result is resorption and remodeling of scar. Further the wound healing is followed by collagen phase that is collagen synthesis, epitheliazation and scar remodeling (maturation). ¹⁵

Diabetes mellitus is most common endocrine disorders. Caused due to insulin deficiency.

Insulin is the main hormone controlling intermediately metabolism. It’s most obvious acute effect is to lower blood glucose. Reduced secretion of insulin often couple with reduced sensitivity to its action causes diabetes mellitus. The consequences of diabetes are dire such as myocardial infarctions. Hyper glycerin occurs due to uncontrolled hepatic glucose output and reduced uptake of insulin through skeletal muscles with reduced glycogenesis.

When the renal threshold for glucose reabsorption is exceeded then excess of glucose will spill out into the urine (glycosuria) and causes an osmotic diuresis (polyuria) which results in dehydration thirst and increased drinking.

Diabetes are of two types

Type 1 diabetes (insulin dependent diabetes mellitus (IDDM) or juvenile onset of diabetes.

Type 2 diabetes (Non insulin dependent diabetes mellitus or maturity onset diabetes.)

Type 1 diabetes there is an absolute deficiency of insulin resulting from autoimmune destruction of beta cells. Without insulin treatment such patients will ultimately die with diabetic ketoacidosis.⁴

Type 2 diabetes represents 90% of all cases of diabetes, affecting approximately3% of the population worldwide and its incidence is increasing every year.

Flavonoids which are widely distributed in the plant kingdom and present in considerable quantities in common food product, spices and beverages have been used since ancient times by physician and laymen to treat a great variety of human diseases such as diabetes, coronary heart disease and cancers.

Polyphenols from berries have an obvious effect on digestive enzymes catechin is a potent AGI and quercetin is reported ass to alleviate the activity of intestinal and renal disaccharides

Achillea millefolium, adianum capillus azadirachta indica, cordyceps sinensis. Are some other plants used for diabetes.¹⁶
6.3 OBJECTIVE OF STUDY:

The objectives of the present study are :-

• 
  To evaluate the antidiabetic activity.
  
• 
  To investigate the burn wound healing activity of morin.
  
• 
  To investigate the hepatoprotectivity.
  

MATERIALS AND METHODS:

The source of data for this study is based on laboratory experiments on animals. And the data obtained from the literature will be the source of data.

(Including sampling procedure if any)

1. 
   Morin dose: Morin will be administered orally at a dose of 30 mg/kg. this dose is reported by earlier workers on anti oxidant activity.¹⁷
   
2. 
   Drugs used are bioflavonoid Morin, alloxan, sylimarin, paracetamol, Rifampicin, thioacetamide.
   
3. 
   Chemicals required are chloroform anesthetic ether, normal saline, glacial acetic acid, haematoxylene, sodium hydroxide glucose,DNDH colour reagent, ethanol formalin,buffered alanine,ringer lactate.
   

The burn wound healing property of test drug is investigated by burn wound model and dead space wound using rats.

All the starved animals will be employed for assessing wound healing activity by various models. Burn wound will be created by pouring hot molten wax at 800 c into a metal cylinder placed on tha back of the rat.

Actual amount of heat delivered by molten wax to creat burn wound was calculated by the following formula:

∆H/∆A = MS(T1-T2)

area of skin exposed to molten wax(300mm2)

∆H/∆A = amount of heat delivered by molten wax to sq.mm.of exposed skin.

M= mass of molten wax.T1 initial temperature

T2= room temperature

S= specific heat

The degree of wound healing will be calculated as percentage closure in wound area from original wound area using the formula-

Percentage closure = 1- AD/AO X 100

Where AO = wound area on day 0

AD = wound area on corresponding day

The mean and S.E. values of raw wound areas will be calculated. The number of days for complete epithelization was noted.

1)Inflicting burn wound

Healthy Wistar albino rats weighing between 150 and 180g will be used. The dorsum of each rat will be shaved. Burn wounds will be inflicted on overnight-starved animals under anesthesia. A 2 × 2-cm metal cylinder will be placed on the shaven back of the animals. Melted wax at 80°C is poured into the metal cylinder and the wax was allowed to solidify. Eight minutes after this, until the wax will be completely solidified, the metal cylinder containing wax adhering to the skin will be gently removed to inflict a distinctly demarked burn wound.

Assessment of wound healing

After the drug administration, the wound inflicted areas of animals every day from day 1. Animals to be observed for wound healing by measuring the wound contraction (tracing the raw wound area first on a transparent polythene paper and then retraced on graph paper) up to the 12th day post wounding. The wound contraction to be calculated as percentage of original wound size for each animal of a group.

Diabetic rats are prepared by administration of alloxan (150mg/kg) animals are treated with the test drug, after a week of treatment the blood samples are taken from retro orbital plexus puncture method the standardized glucometer is used to determine the concentration of blood glucose level.

Oral glucose tolerance test (Bonner-weir 1988) will be performed in overnight fasted (18 h) normal rats. Blood glucose level can be calculated by the glucometer. The plasma obtained by centrifugation at 3000 rpm. Fasting plasma glucose level will be measured using a glucose estimation kit (ye et al 2002).

Hepatotoxicity studies will be conducted in rats by chronic model. Drug used individually and in combination, administered for one week and rats will be sacrificed and estimated the levels of enzymes (AST, ALT) at regular time interval and histopathology study of liver for Morin will be carried out.

Suspension of test drug and sylimarin administered by gavage at 100 mg/kg once daily for three days. On third day of treatment, paracetamol (3g/kg p.o.) was administered 30 minutes after the administration of test suspension. Control animals received 1ml/kg p.o. of vehicle. After 48 hrs of paracetamol administration, blood was collected from all the animals by puncturing ritro orbital plexus. The blood samples will be allowed to clot for 45 minutes at room temperature. Serum was separated by centrifugation at 2500 rpm at 37⁰ c for 10 minutes and analyzed for various biochemical parameters.

Suspension of drug and sylimarin were administered by gavage at 100 mg/kg p.o. four times at 12 hrs interval for a period of 36 hrs. Rifampicin (1g/kg p.o.) was administered 30 minutes after first dose of test suspension control animals will receive 1ml/kgp.o. Of vehicle. After 48 hrs of Rifampicin administration, blood was collected from all groups of animals and serum was analyzed for various biochemical parameters.

Rats will be divided into groups. One group will serve as control group treated with normal saline. Thioacetamide will be injected intraperitoneally (50 mg/kg) in three consecutive days. For another group (intoxicated control). In other two groups 25 mg/kg of sylimarin and Morin will be administered respectively with Thioacetamide dose of 50 mg/kg intraperitonially.

Diagnostic kit will be used for the determination of levels of aspatate transaminase (AST) and alanine transaminases (ALT) are determined by standard methods.

The animals will be sacrificed and the abdomen will be cut open to remove the liver. Then, 5 mm thick pieces of the liver will be fixed in bovine’s solution (mixture of 75 ml of saturated picric acid 25 ml of 40% formaldehyde and 5 ml of glacial acetic acid) for 12 hrs and then embedded in paraffin, using conventional methods, and cut into 5micrometer thick sections and stained, using hematoxylene. Then the sections will be observed under microscope for Histopathological changes in liver architecture.

• 
  Yes, the above study requires investigations on wistar rats for Antidiabetic, hepatoprotective and burn wound healing activity.
  

• 
  The application has been forwarded to the institutional animal ethical committee for approval.
  

9.

The above information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance.

(in block letters)

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KLE. Society’s College of Pharmacy,