Title of the Topic:
“COMBINED EFFECT OF QUERCETINWITH ORAL HYPOGLYCEMICS IN DIABETES INDUCED INCREASE IN SUSCEPTIBILITY TO ULCEROGENIC STIMULI”
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Ulceration is due to imbalance between offensive acid-pepsin secretions versus impaired mucosal resistance. Diabetic state has deleterious effect on various functions of gastrointestinal tract1. Starvation or stress in insulin dependent diabetic rats leads to gastric mucosal ulcerogenic responses2-3. Diabetes increases the mucosal susceptibility to ulcerogenic stimuli and predisposes gastric ulceration4. Recent studies indicate that peptic ulcers related to diabetic state are more severe and often associated with complications such as gastrointestinal bleeding5. Role of free radicals and antioxidant enzymes in gastric ulcerations is well known6. Lipid peroxidation has been postulated to be one of the important factors in ulcerogenesis7. Increased back diffusion of acid plays an important role in the formation of acute hemorrhagic ulceration. Mucous-bicarbonate barrier plays an important role in preventing back diffusion of H+ ion by neutralizing H+ ion from the luminal side of stomach. Marked increase in acid back diffusion and mucosal H+ permeability and impairment of HCO3—secretion have been reported in diabetic rats8-9 .Neuronal dysfunction has been reported to alter under diabetic conditions and that can lead to aggravated vulnerability of the gastric mucosa10. Functional ablation of these neurons has been reported to worsen the mucosal ulcerogenic response to a variety of noxious agents and impaired the healing of gastric lesions11.
NIDDM rats showed enhanced propensity of ulceration in all the gastric ulcer models studied. Even though the causative factors for ulcerogenesis may be different, the net imbalance in offensive and defensive factors brought about by them is thought to be the cause for ulcerogenesis12.Proper glycemic control and attainment of other nonglycemic management targets (e.g., lipids, antioxidant status) are essential for the prevention of long-term complications of diabetes. Exogenous antioxidants can compensate for the lower plasma antioxidant levels often observed in NIDDM and in pre-diabetic individuals. Consumption of fruits and vegetables rich in vitamins and other antioxidants can increase overall antioxidant status13-14.
Diabetes may lead to decrease in the mucosal defensive factors with concomitant increase in propensity of ulceration in response to various physical and chemical agents. The changes induced by diabetes mellitus could be reversed by using drugs which can correct the blood glucose levels15.
Flavonoids like quercetin causes regeneration of the pancreatic islets and probably increases insulin release in streptozotocin-induced diabetic rats16. Hence the objective of the present study is required to study the effect of quercetin alone and in combination with oral hypoglycemic on the susceptibility of gastric mucosa of NIDDM rats subjected to various ulcerogenic stimuli.
6.2 Review of Literature:
Increased lipid peroxidation is thought to be aconsequence of oxidative stress, which occurs when the dynamic balance between prooxidant andantioxidant mechanism is impaired. Glipizide can simultaneously decreasehyperglycemia, restore insulin secretion, and inhibit oxidative stress produced by high glucose seems to be an interesting therapeuticprospect for the prevention of vascular complications of diabetes.Glipizidehas also been investigated for antioxidantproperties. It seems to play a prominent role in scavengingfree radicals and restoring antioxidant activities in thetissues of diabetic animals17. Quercetin, improves the neuronal blood flow through reactive oxygen species scavenging or by its direct vasorelaxant properties18. Quercetin helps in insulin release and thus suggesting its role in type 2 diabetes19. Pterocarpus marsupium did not prevent gastric ulcers in normal rats but protected the gastric mucosa of NIDDM rats which may be due to its antidiabetic activity20. nnnnn
SPECIFIC OBJECTIVES:
6.3 Objective of study:
The objectiveof the present study is to investigate effect of quercetinalone and in combination with oral hypoglycemic agents in rats with type 2 diabetes induced increase in susceptibility to ulcerogenic stimuli.
  1. Primary outcome measures:
1.To study the effect of quercetin at different doses in oral glucose tolerance test in rat.
2.To study the effect quercetin (single & multiple dose) in normal and diabetic rats.
3.To study the role of oral hypoglycemics in diabetes induced increase in susceptibility to ulcerogenic stimuli.
4.To study the combined effect of quercetin with oral hypoglycemics in diabetes induced increase in susceptibility to ulcerogenic stimuli.
  1. Secondary outcome measures:
To study the effect of quercetin on antioxidant defense status in stomach homogenate.
To study the combined effect of quercetin with oral hypoglycemic agents on antioxidant defense status in stomach homogenate.
MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of Krupanidhi College of Pharmacy, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research Publications and Journals.
7.2 Method of Collection of Data: 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.
7.3 EXPERIMENTAL MODELS:
A. Oral Glucose tolerance test21.
The oral glucose tolerance test will be performed on overnight fasted Sprague dawley rats. Rats will be divided into different groups as follows:
Group 1: Normal control, rats receive saline/vehicle.
Group 2: Diabetic control.
Group 3: Quercetin (50 & 100 mg/kg)22 .
Group 4: Pioglitazone (10 mg/kg)23 .
Group 5: Glipizide (5mg/kg)24 .
Group 6: Acarbose(5mg/kg)25 .
Glucose will be fed 30 min and 1hr after the administration of extracts. Blood will be withdrawn from the retro orbital sinus under ether inhalation at 30, 60, 90 and 120 min of glucose administration. Glucose levelswill be estimated.
B. Single and multiple dose study in normal and diabetic rats26.
C. Development of high fat diet-fed/low dose streptozotocin-treated type 2 diabetic
rats27.
The animals were fed high fat diet (HFD), once a day for 2 weeks followed by IP injection of streptozotosin (35mg/kg) dissolved in 1M/L citrate buffer (ph 4.4) after overnight fasting. Blood sample was collected from tail vein and glucose was measured using glucose diagnostic kit (Accucheck, India). The rats with non fasting plasma glucose level of >300 mg/dl will be considered diabetic.
7.4 Treatment protocol:
Group 1: Normal control, rats receive saline/vehicle.
Group 2: Diabetic control.
Group 3: Diabetic rats treated with quercitin.
Group 4:Diabetic rats treated with Pioglitazone.
Group 5: Diabetic rats treated with glipizide.
Group 6: Diabetic rats treated with acarbose.
Group 7: Diabetic rats treated with glipizide + Pioglitozone.
Group 8: Diabetic rats treated with glipizide + acarbose.
Group 9: Diabetic ratstreated with quercitin+ pioglitazone(low dose).
Group 10: Diabetic ratstreated with quercitin+ glipizide(low dose).
Group 11: Diabetic ratstreated with quercitin + acarbose (low dose).
Antiulcer evaluation will be done by following models in experimental animals
1.Acetic acid induced chronic gastric ulcers.
The method described by Asad et al 28 will be followed. Briefly, the rats will be anaesthetized using ether followed by laporatomy, glacial acetic acid 0.05 ml will be added onto the serosal surface of the stomach through a cylindrical mould of 6.0 mm diameter. The acid solution will be removed after 1 min and the mould will be rinsed with normal saline to avoid damage to surrounding tissues.The abdomen will be closed and the animals will be treated with the drug for 10 days. On the 11th day, the animals will be sacrificed and ulcer index and ulcer score will be determined. The tissues will also be subjected to histopathological studies.
2.Healing of indomethacin induced gastric ulcers, a model to study free radical scavenging activity29.
The gastric ulcers will be induced by administering indomethacin (5mg/kg .p.o) for 5 days to the normally fed rats. The animals were then treated with the drug for 5 days after induction of ulcer while the control group will receive only vehicle. The last dose of indomethacin will be considered as 0th day. Rats will be sacrificed on the 0th day and 5th day. The stomach will be removed; ulcer score and ulcer index will be determined. The glandular portion of the stomach will be used for the estimation of mucin content30, total proteins31, antioxidant factors like superoxide dismutase activity32, total tissue sulphydryl groups 33 and catalase activity34.
3.Ethanol induced gastric ulcers35.
Albino rats will be fasted for 36hr before administration of 90% ethanol (1ml/200gm).The drug will be administered 1 hr before ethanol administration. One hour after ethanol administration, the animals will be sacrificed, stomach will be isolated and ulcer index will be determined.
4. Stress induced gastric ulcers36.
The rats will be placed in a restraint cage and the cage will be placed at a temperature of 20 c for 3hr, drug will be administered prior to subjection of stress. The animals will be sacrificed, stomach will isolated and ulcer index was determined.
7.5 Does the study require any investigation or interventions to be conducted on
patients or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug / extract will be studied on various parameters using rats as experimental animal model.
7.6 Has ethical clearance been obtained from your institute:
Ethical Committee approval letter is enclosed.
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