K.s.d green tech conference

ANTIDIABETIC ACTIVITY FROM DIFFERENT EXTRACTSOF SGWO ON STZ INDUCED RATS

K.S.DAYANANDA ACHARYA INSTITUTE OF TECHNOLOGY BANGALORE 560090

KSD/Green Technologies April 5th 2012

Diabetes• Diabetes:

– Derived from the Greek a word that literally means "passing through," or "siphon“.

• Diabetes Mellitus:– Diabetes mellitus is a group of metabolic diseases characterized by

high blood sugar levels, which result from defects in insulin secretion, action, or both

• Gestational Diabetes:– Increased Blood Sugar during Pregnancy.

• Diabetes Insipidus:– Diabetes insipidus is caused by the inability of the kidneys to conserve

water, which leads to frequent urination and pronounced thirst.


History

• Diabetes is one of the first diseases describedwith an Egyptian manuscript from c. 1500 BCE mentioning “too great emptying of the urine.” The first described cases are believed to be of type 1 diabetes.Indian physicians around the same time identified the disease and classified it as madhumeha or honey urine noting that the urine would attract ants. The term "diabetes" or "to pass through" was first used in 230 BCE by the Greek Appollonius Of Memphis. The disease was rare during the time of the Roman empire with Galen commenting that he had only seen two cases during his career. Type 1 and type 2 diabetes where identified as separate conditions for the first time by the Indian physicians Sushruta and Charaka in 400-500 AD with type 1 associated with youth and type 2 with being overweight.The term "mellitus" or "from honey" was added by the Britain John Rolle in the late 1700s to separate the condition from diabetes insipidus which is also associated with frequent urination.


http://en.wikipedia.org/wiki/Circa

http://en.wikipedia.org/wiki/Common_Era

http://en.wikipedia.org/wiki/Apollonius_(physician)

http://en.wikipedia.org/wiki/Apollonius_(physician)

http://en.wikipedia.org/wiki/Roman_empire

http://en.wikipedia.org/wiki/Galen

http://en.wikipedia.org/wiki/Diabetes_insipidus

http://en.wikipedia.org/wiki/Diabetes_insipidus

Insulin• Produced in Pancreas by B-Cells of islets of

langerhans• Activates the Glucose transport proteins located in

2/3 of the body’s cells.– Skeletal Muscle and Adipose tissue


Diabetes Mellitus

• Type 1 Diabetes– The body stops producing insulin or produces too

little insulin to regulate blood glucose level• Type 2 Diabetes

– The pancreas secretes insulin, but the body is partially or completely unable to use the insulin (Insulin Resistance)

Type 1 Diabetes• Decreased Insulin Production• Comprises 10% of all Diabetic Patients • 15/100,000 population• Early onset

– Childhood/ Adolecence• 1.5 times more likely to develop in American whites than

in American blacks or Hispanics • All patients are Insulin Dependant• Increased risk of Infections, Kidney Disease, Ocular

Disease, Nerve injury, HTN, CAD, CVA

Type 2 Diabetes• Insulin resistance• Comprises 90% of all Diabetic Patient• 6.2% population in 2002• Related to Obesisty• Affects All Ages

– Becoming more common among adolescents• More prevalent among Hispanics, Native Americans, African

Americans, and Asians• Increased risk of infections, Kidney Disease, Ocular Disease,

Nerve injury, HTN, CAD, CVA• Can Be Controlled with Diet, Exercise, Weight Lose• Patients frequently take Oral Medications and/or Insulin.


Serum Glucose Levels

– Normal: • 100 mg/dL • This fluctuates from 70-150 mg/dL

– Pre-Diabetic • 100-125mg/dL Fasting Serum Glucose test

– Fasting indicates no oral intake for 6 hours prior to test

– Diabetic • >125mg/dL for Fasting Serum Glucose Test

– Fasting indicates no oral intake for 6 hours prior to test


Diabetic Emergencies

• Hyperglycemic– HHNC: Hyperosmolar Hyperglycemic Nonketotic Coma – DKA: Diabetic Ketoacidosis

• Hypoglycemic– Diabetic Coma or Insulin Reaction


HHNC: Hyperosmolar Hyperglycemic Nonketotic Coma

• Effects Type 2 Diabetics• Prominent later in life• Elevated Blood Glucose lead to increases

serum osmolarity• This results in Diuresis and Fluid Shift.• Increased Urination causes body wide

depletion of Water and Electrolytes.– Extreme Dehydration


DKA: Diabetic Ketoacidosis• Dereased Insulin or Insulin resistance leads to Elevated Blood

Glucose levels• However, Cellular Glucose is Low without insulin

– Equivalent to Starvation• As a result the body attempts to Compensate

– Uses Glucose stores– Breaks Down Fat and Protein

• In an attempt to save the Heart and Brain, the body produces Ketone Bodies from fatty acids– Acetoacetate, Beta-hydroxybutyrate, And Acetone

• Excessive Ketones lead to Acidosis– Beta-hydroxybutyrate is a carboxylic Acid


DKA: Diabetic Ketoacidosis• Physical Signs

– Altered mental status without evidence of head trauma– Tachycardia– Tachypnea or hyperventilation (Kussmaul respirations)– Normal or low blood pressure– Increased capillary refill time– Poor perfusion– Lethargy and weakness– Fever– Acetone odor of the breath reflecting metabolic acidosis

• Symptoms– Often insidious– Fatigue and malaise– Nausea/vomiting– Abdominal pain– Polydipsia– Polyuria– Polyphagia– Weight loss– Fever


Hypoglycemia• Effects Type 1 & 2 Diabetic• Secondary to Insulin or Oral Hypoglycemic Medication

– More Common with Insulin Use• Serum Glucose Levels Fall Below Normal Levels• Serum Glucose Levels

– Normal: • 100 mg/dL

– Hypoglycemia:• <50gmg/dL in men• <45 mg/dL in women• <40 mg/dL in infants and children

– Protocol: <80 mg/dl


Hypoglycemia• Physical Signs

– Sweating– Tremulousness– Tachycardia– Respitory Distress – Abdominal Pain– Vomiting– Combative or agitated

• ComaSymptoms– Anxiety – Nervousness– Confusion– Personality changes– Nausea


Blood Glucometry

• Measurement of Blood Glucose levels– Hospital labs evaluate Serum Glucose (10-15% higher)

• Requires a small sample of blood– No IV’s or Phlebotomy

• Only seconds to obtain results


GLOBAL STATISTICS OF DIABETESDescription English: Prevalence of diabetes worldwide in 2025 (per 1000 inhabitants). World average will be 29.23‰. no data less than 7.5 7.5-15 15-22.5 22.5-30 30-37.5 37.5-45 45-52.5 52.5-60 60-67.5 67.5-75 75-82.5 more than 82.5


VARIATION OF BG-LEVEL


CAUSES

• Hereditary or Inherited Traits : It is strongly believed that due to some genes which passes from one generation to another, a person can inherit diabetes. It depends upon closeness of blood relationship as mother is diabetic, the risk is 2 to 3%, father is diabetic, the risk is more than the previous case and if both the parents are diabetic, the child has much greater risk for diabetes.

• Age : Increased age is a factor which gives more possibility than in younger age. This disease may occur at any age, but 80% of cases occur after 50 year, incidences increase with the age factor.

• Poor Diet (Malnutrition Related Diabetes) : Improper nutrition, low protein and fiber intake, high intake of refined products are the expected reasons for developing diabetes.

• Obesity and Fat Distribution : Being overweight means increased insulin resistance, that is if body fat is more than 30%, BMI 25+, waist grith 35 inches in women or 40 inches in males.

• Sedentary Lifestyle : People with sedentary lifestyle are more prone to diabetes, when

compared to those who exercise thrice a week, are at low risk of falling prey to diabetes.

http://diabetesinformationhub.com/


• Stress : Either physical injury or emotional disturbance is frequently blamed as the initial cause of the disease. Any disturbance in Cortiosteroid or ACTH therapy may lead to clinical signs of the disease.

• Drug Induced: Clozapine (Clozaril), olanzapine (Zyprexa), risperidone

(Risperdal), quetiapine (Seroquel) and ziprasidone (Geodon) are known to induce this lethal disease.

• Infection : Some of the strephylococci is suppose to be responsible factor for infection in pancreas.

• Hypertension : It had been reported in many studies that there is direct relation between high systolic pressure and diabetes.

• Serum lipids and lipoproteins : High triglyceride and cholesterol level in the blood is related to high blood sugars, in some cases it has been studied that risk is involved even with low HDL levels in circulating blood.


COMMON CONSEQUENCES OF DIABETES• According to the WHO, Over time, diabetes can damage the heart, blood vessels, eyes,

kidneys, and nerves.• Diabetes increases the risk of heart disease and stroke. 50% of people with diabetes die

of cardiovascular disease (primarily heart disease and stroke). • Combined with reduced blood flow, neuropathy in the feet increases the chance of foot

ulcers and eventual limb amputation. • Diabetic retinopathy is an important cause of blindness, and occurs as a result of long-

term accumulated damage to the small blood vessels in the retina. After 15 years of diabetes, approximately 2% of people become blind, and about 10% develop severe visual impairment.

• Diabetes is among the leading causes of kidney failure. 10-20% of people with diabetes die of kidney failure.

• Diabetic neuropathy is damage to the nerves as a result of diabetes, and affects up to 50% of people with diabetes. Although many different problems can occur as a result of diabetic neuropathy, common symptoms are tingling, pain, numbness, or weakness in the feet and hands.

• The overall risk of dying among people with diabetes is at least double the risk of their peers without diabetes


Preparation of material and experimental animals: stevia raubdeana,gymnema sylvestre,withania somnifera and occimum sanctumauthenticated by Taxonomist, was freeze dried at – 40°C to get a powder. About hundred and twenty five male albino Wistar rats of body weight of 150-200 g, were housed under standard environmental conditions (25 ± 2°C temperature, 50 ± 5 % humidity with a 12 h each of dark and light cycle) and maintained with free access to water and a standard laboratory diet ad libitum. The Institutional Ethical Committee approved the study.


Induction of diabetes in rats: A single intraperitonial injectionof two different doses of 45 and 65 mgkg-1 of Streptozotocin(STZ) (Sigma Aldrich Chem. Co. USA.) were used for induction

of diabetes of type II and type I respectively in over night fasted animals and were dived into sub, mild and

severely diabetic models depending upon their FBG levels after 3 days of administration. Type II model: Sub diabetic:

FBG 80-100 mgdl-1; PPG 210-310 mgdl-1; Mild diabetic: FBG120-250 mgdl-1; PPG 210-310 mgdl-1. Type I models: Severelydiabetic: FBG > 350 mgdl-1; PPG >550 mgdl-1.


Estimation: Blood glucose level (BGL) (12), total cholesterol(TC), high density lipoprotein (HDL) and triglyceride (TG)(13,14) asparate transferase (AST) and alanine transferase(ALT) (15), alkaline phosphatase (ALP) (16), total protein (TP)(17), creatinine (CRE) (18) and total haemoglobin (19) weremeasured using standard kits (Bayer Diagnostics India, Ltd.)by following known procedures. However, very low densitylipoprotein (VLDL) and low density lipoprotein (LDL) wascalculated by the formula. (20). Urine sugar (US) and urineprotein (UP) were detected by reagent based Uristix of BayerDiagnostics. All the parameters were measured initially beforethe treatment and then monitored regularly every week uptotwo weeks in case of severely diabetic animals.


Experimental Design: Hypoglycemic effect was studied innormal healthy rats by conducting fasting blood glucose (FBG)and glucose tolerance test (GTT) studies. Antidiabetic effectwas assessed in sub diabetic as well as mild diabetic modelsof Type II by conducting similar set of GTT studies. The mosteffective dose identified in sub and mild diabetic cases wasused for evaluating the antidiabetic, hypolipidemic andhepatoprotective potential in severely diabetic animalsconsidered as type I models.


Assessment of hypoglycemic activity in normal healthyrats – FBG & GTT studies: Four groups of five rats each,fasted over night, were used in each of the experiments ofFBG and GTT studies. Group I served as untreated controlGroups II, III and IV received orallythe doses of 150, 200 and

250 mg kg-1 respectively of theefreeze driedSWGO.Fasting blood samples were collected from tail vein initiallybefore the treatment and then at 1.5, 3, 4.5 and 6 h after thetreatment for FBG study. Whereas, for GTT study the effectof the above mentioned doses on FBG was studied initially at2 h considered as ‘0’ h value. The animals were then orallyadministrated with 2 g kg-1 of glucose and their glucosetolerance was studied at 1 h interval for the next 3 h.


Evaluation of antidiabetic activity in sub diabetic and milddiabetic rats – GTT studies: The antidiabetic effect of SGWOwas

alsoassessed byimprovement of glucose tolerance in sub and mild diabeticrats. The overnight fasted rats were divided in to five groupsof five rats each. Group I was control, whereas variable doses of 150, 200 and 250 mg kg-1 offreeze dried SGWOwere given orally to group II, III and IV respectively afterchecking their FBG. Blood glucose levels were further checkedfirstly after 2 h of treatment considered as ‘0’ h value and then2 g/kg glucose was given orally to all the groups and theirglucose tolerance was studied three hours at regular intervalsof 1 h each, considered as 1 h, 2 h and 3 h values. The resultswere compared with group V treated with 0.5 mg kg-1 ofGlipizide (hypoglycemic agent).


STATISTICS

• Statistical analysis: Data were statistically evaluated using

• one-way ANOVA, followed by a post hoc Newman-Keuls

• Multiple Comparison Test. The values were expressed as

• mean ± SD and considered significant at (P<0.05).


: Hypoglycemic effects of freeze driedSWGO on FBG of normoglycemic rats

• Blood glucose levels (mg/dl)• Pretreatment Post treatment (hours)• Groups Treatment Doses (mg kg-1) FBG 1.5 3.0 4.5 6.0• I 70.2 ± 3.9 70.5 ± 3.2 70.8 ± 4.6 69.4 ± 3.8 69.1 ± 4.2• II 150 71.5 ± 3.2 68.2 ± 4.4 65.6 ± 4.6** 62.1 ± 5.1 62.3 ±

3.8*• 200 70.3 ± 3.2 66.9 ± 4.4 63.4 ± 4.6 57.8 ± 5.1** 58.1 ±

3.8*• IV 250 72.1 ± 3.2 67.4 ± 4.4 65.1 ± 4.6 59.3 ± 5.1 60.1 ±

3.8*• Values are in mean±SD; **P<0.01, *P<0.05 as compared

with initial


• Hypoglycemic effects of freeze dried sgwo on BGL during GTT of normoglycemic rats

• Blood glucose levels (mg/dl)• Pretreatment Post treatment (hours)• Groups Treatment Doses (mg kg-1) FBG 0 1 2 3• I 72.3 ± 4.7 72.5 ± 4.1 106.9 ± 4.6 102.5 ± 3.2 94.8 ± 4.3• II 150 71.8 ± 42 69.6 ± 4.6 88.9 ± 4.9 80.6 ± 4.4** 73.8 ± 3.8• III 200 71.4 ± 3.5 69.1 ± 3.6 83.2 ± 4.9 74.3 ± 4.6** 68.3 ± 3.7• IV 250 72.8 ± 4.2 70.5 ± 5.6 85.4 ± 4.9 76.1 ± 4.8* 70.0 ± 4.4*• Values are in mean±SD; **P<0.01, *P< 0.05 as compared with

control


• Table 3: Effect of most effective dose of SWGO on BGL and lipid profile of severely diabetic rats• Groups Treatment Pre treatment level Post-treatment levels• 7 days 14 days• FBG (mg/dl)• 342 ± 4.5 354.1 ± 5.8 361.8 ± 6.1• Insulin 372.6 ± 5.7 290.7 ± 5.4*** 234.8 ± 5.1***• SWGO377.8 ± 7.5 316.2 ± 8.6** 233.4 ± 6.8**• PPG (mg/dl)• 549.9 ± 10.2 550.1 ± 8.7 554.4 ± 7.8• Insulin 563.4 ± 9.8 419 ± 8.1* 302.5 ± 7.8• SWGO 553.7 ± 9.5 414.9 ± 9.0** 307.2 ± 8.7**• Triglycerides (mg/dl)• 178.7 ± 6.4 179.2 ± 6.9 176.9 ± 7.4• Insulin 172.6 ± 8.4 132.6 ± 5.1* 94.5 ± 3.5*• SWGO 180.2 ± 5.2 142.8 ± 4.6*** 89.5 ± 4.8**• Total cholesterol (mg/dl)• 119.9 ± 4.2 118.5 ± 6.5 117.4 ± 5.8• Insulin 123.7 ± 7.8 109.2 ± 5.2* 91.6 ± 6.8• SWGO 122.5 ± 6.3 111.2 ± 5.2*** 93.8 ± 5.9***• HDL cholesterol (mg/dl)• 19.2 ± 4.8 19.0 ± 5.2 17.6 ± 2.8• Insulin 20.9 ± 5.2 23.6 ± 4.8* 26.8 ± 4.8• SWGO 20.7 ± 3.2 24.3 ± 4.4** 26.5 ± 3.8**• Values are in mean±SD; ***P<.001 as compared to pretreatment levels; **P<0.01 as compared to pretreatment levels;

*P<0.05 as compared to• pretreatment levels.


: Impact of the most effective dose of freeze dried SWGOon serum enzymes• of severely diabetic rats on two weeks treatment (mean ± SD)• Groups Treatment Pre treatment level Post-treatment levels• 7 days 14 days• SGOT (U/L)• 28.7 ± 4.5 29.4 ± 3.8 30.2 ± 5.5• Insulin 29.9 ± 4.1 24.1 ± 3.9** 21.5 ± 3.2**• SWGO30.5 ± 4.5 27.9 ± 3.8* 23.6 ± 4.6**• SGPT (U/L)• 31.6 ± 3.5 32.1 ± 2.8 32.9 ± 2.1• Insulin 30.8 ± 4.5 24.5 ± 5.1* 19.6 ± 3.4*• 30.1 ± 4.7 26.8 ± 3.9** 20.7 ± 4.1**• ALP (U/L)• 144.5 ± 6.4 144.8 ± 6.8 145.3 ± 2.8• Insulin 158.5 ± 4.4 131.2 ± 5.6** 103.6 ± 5.1**• SWGO155.8 ± 5.8 135.2 ± 4.6** 105.6 ± 4.8***• CRE (U/L)• ± 0.2 2.2 ± 0.1 2.3 ± 0.2• Insulin 1.2 ± 0.6 1.1 ± 0.3# 1.1 ± 0.8#• SWGO2.1 ± 0.5 1.9 ± 0.3* 1.5 ± 0.4**• Values are in mean±SD; **P<0.01 as compared to pretreatment levels. ***P<0.001 as compared

to pretreatment levels. *P<0.05 as compared to• pretreatment levels. # Not Significant as compared to pretreatment levels.


• Impact of the most effective dose of SWGOon Hb, TP, UP, US and bw of severely diabetic rats on two weeks treatment

• Groups Treatment Pre treatment level Post-treatment levels• 7 days 14 days• Haemoglobin (mg/dl)• 9.6 ± .7 8.7 ± .5 8.0 ± .8• Insulin 7.8 ± .5 8.0 ± .6** 8.2 ± .7**• SWGO 9.7 ± .5 9.9 ± .5*** 10.4 ± .4***• Total protein (mg/dl)• 6.1 ± .5 5.9 ± .9 5.4 ± .8• Insulin 6.4 ± .6 6.9 ±.3** 7.3 ± .5**• SWGO6.5 ± .6 6.7 ± .8* 6.9 ± .4*• Urine protein• +++ +++ +++• Insulin +++ ++ +**• SGWO+++ ++** ++**• Urine Sugar• ++++ ++++ ++++• Insulin ++++ +++ ++**• SWGO++++ ++** ++**• Body weight (g)• 150 ± 8.0 150 ± 7.5 145± 5.5• Insulin 150 ± 5.0 150 ± 6.5* 150± 4.5*• SWGO145 ± 2.5 150 ± 3.0** 155± 4.0**• Values are in mean±SD; *** P<0.001 as compared to pretreatment levels; **P< 0.01 as compared to

pretreatment levels; *P< 0.05 as compared to pretreatment levels; # Not Significant as compared to pretreatment levels.


DISCUSSION

The present study was under taken to explore the vision of creativity scientifically by assessing the antidiabetic, hypolipidemic and hepatoprotective potential, of freeze dried SWGOpowderonboth type II as well as type I diabetic models. Glipizide a potent inhibitor of tyrosine phasphatase and insulin were used as reference drugs for type II and type I models respectively. It is known that Glipizide mimics several insulin actions in vivo such as


the stimulation of hexose cellular uptake and lipogenesis and the inhibition of lipolysis . The maximum fall observed during FBG and GTT studies was 17.7 and 27.9 % respectively with the dose of 200 mgkg-1 of SWGOin normal rats. The hypoglycemic activity proposed is due to the inhibition of key enzymes involved in the gluconeogenesis and glucogenolysis pathway. The same dose showed a marked improvement in GTT of sub and mild diabetic animals by 34.9 and 35.8 % respectively. These falls are comparable with the fall of the synthetic drug Glipizide hence, the dose of 200 mgkg-1 was identified as the most effective dose for the long term treatment of two week of severely diabetic animals and it reduced the level of FBG by 38.2 % and PPG by 44.5 % in case of severely diabetic rats. On the other hand blood glucose levels of its control group were increased significantly. This increment could be due to reduced glucose clearance apparently arising from a defect in glucose transport .


Diabetes mellitus is usually associated with elevated serum lipid levels considered as risk factors for coronary heart diseases Lowering of these elevated levels either though drug or dietary therapy seems to be associated with a decrease in the risk of vascular disease The present study of severely diabetic animals showed significantly declined levels of TC, LDL, and VLDL by 23.4, 24.9 and 50.2 % respectively and enhanced HDL cholesterol level by 28.4 % after two weeks treatment with SWGO powder.

Many antihypercholesterolemic drugs do not decrease TG levels though TG plays an independent role in increasingly the risk of coronary heart disease However, this long-term treatment of SWGOfor two weeks lowered the TG levels too by 50.3 %.


Moreover, the enhanced levels of hepatoprotective enzymes due to severe diabetes such as SGOT, SGPT, ALP and CRE were also reduced by 28.0, 34.6, 34.6, and 8.3% respectively as an additional advantage of this study. The SWGOpowder decreased the glucose concentration in urine by 50 %, which is generally high in case of chronic diabetic cases Other important parameters such as Hb, TP and bw, which are generally lower than their normal values in chronic diabetic cases, were also controlled by the treatment of SWGOand managed to raise by 6.7, 8.6 and 3.4 % respectively. A scientific and systemic exploration reveals the antidiabetic, hypolipidemic and hepatoprotective effects of SWGOfreeze dried powder which could be used as an effective and safe antidiabetic dietary supplement of high potential. The study concludes that this first reporting of the scientific exploration of triple action potential of SWGOfreeze dried powder with special reference in type I diabetes models is not only interesting but very encouraging too for medicinal chemists as well as diabetic patients globally


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