diabetes

Evaluation of hypoglycemic affect of plant extract

What is Diabetes?

• The term diabetes mellitus describes a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long-term damage, dysfunction and failure of various organs.

(K.G.M.M. Alberti et al, 1998)

• DM consists of a group of disorders characterized by hyperglycemia; altered metabolism of Lipids, carbohydrates, and proteins and an increased risk of complications from vascular disease.

Prevalence of diabetes in Pakistan

• Cross-sectional survey conducted earlier in the rural and urban areas of all the four provinces of Pakistan. Newly diagnosed diabetes was 5.1% in men and 6.8% in women in urban areas and 5.0% in men and 4.8% in women in rural areas

( F. Jawad et al,2006)

• Prevalence of diabetes is high ranging from 7.6 to 11% in Pakistan

(R. Hakeem et al ,2010)

Global Prevalence

• The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030.

(SARAH WILD et,al 2004)

Cause: • Genetic Susceptibility

• Autoimmune Destruction of Beta Cells

(Alexandra E. Butler et al, 2004)

• Environmental Factors

• Viruses and infections

• Obesity and Physical Inactivity

• Insulin Resistance(NIH Publication No. 09–3873, November 2008)

• Abnormal Glucose Production by the Liver

• Metabolic Syndrome Cell Signaling and Regulation Beta Cell Dysfunction deficit in -cell mass, increased -cell apoptosis, and impaired insulin secretion.

• Endocrinopathies.

(AMERICAN DIABETES ASSOCIATION,2006)

• Drug- or chemical-induced diabetes.

• Diseases of the exocrine pancreas.

• Point mutations in mitochondrial DNA have been found to be associated with diabetes mellitus and deafness

• Uncommon forms of immune-mediated diabetes.

(Expert Committee on the Diagnosis and Classification of Diabetes,2006)

Types of diabetes

The three main types of diabetes are

• Type 1 diabetes

• Type 2 diabetes

Type 1 Diabetes • Type 1 indicates the processes of beta-

cell destruction that may ultimately lead to diabetes mellitus in which ‘insulin is required for survival’ to prevent the development of ketoacidosis, coma and death.

• Type 1 is usually characterized by the presence of anti-GAD, islet cell or insulin antibodies which identify the autoimmune processes that lead to beta-cell destruction.

(K.G.M.M. Alberti et al, 1998)

Type 1

Type 2 Diabetes

• Type 2 is the most common form of diabetes and is characterized by disorders of insulin action and insulin secretion, either of which may be the predominant feature. Both are usually present at the time that this form of diabetes is clinically manifest.

(K.G.M.M. Alberti et al, 1998)

Pathophysiology

• Several pathogenetic processes are involved in the development of diabetes. These include processes which destroy the beta cells of the pancreas with consequent insulin deficiency, and others that result in resistance to insulin action. The abnormalities of carbohydrate, fat and protein metabolism are due to deficient action of insulin on target tissues resulting from insensitivity or lack of insulin.

(Kahn SE et al, 2000)

• Diabetes mellitus type 2 (formerly noninsulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes) is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency Virtually all forms of DM result from a decrease in the circulating concentration of insulin (insulin deficiency) and a decrease in the response of peripheral tissues to insulin (insulin resistance).

• These abnormalities lead to alterations in the metabolis of carbohydrates, lipids, ketones, and amino acids , the central feature of the syndrome is hyperglycemia.

• Insulin lowers the concentration of glucose in blood by inhibiting hepatic glucose production and by stimulating the uptake and metabolism of glucose by muscle and adipose tissue.

• These two important effects occur at different concentrations of insulin. Glucose production is inhibited half maximally by an insulin concentration of about 20 μunits/mL, whereas glucose utilization is stimulated half maximally at about 50 μunits/mL.

• Lack of GIP amplification of the latephase plasma insulin response to glucose seems to be a consequence of diabetes mellitus, characterizing most, if not all, forms of diabetes.1 beta-cell dysfunction and insulin resistance are two central, interrelated defects in the pathophysiology of type 2 diabetes

( T. VILSBØLL et al, 2003)

• Type 2 diabetes is a progressive disease and that this progression is due to declining beta-cell function.

(The American Journal of Medicine ,2000)

• Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired b-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology.

(Rona J. Strawbridge et al ,2003)

Diabetes Research & Wellness Foundation

Role of insulin in glucose regulation

• Insulin lowers the concentration of glucose in blood by inhibiting hepatic glucose production and by stimulating the uptake and metabolism of glucose by muscle and adipose tissue

( Goodman and Gillman’s Pharmacology)

REGULATION OF GLUCOSE TRANSPORT

• Stimulation of glucose transport into muscle and adipose tissue is a key response to insulin. Glucose enters cells by facilitated diffusion through one of a family of five glucose transporters, GLUTs 1–5, that mediate Na+-independent facilitated diffusion of glucose into cells

• Insulin stimulates glucose transport by promoting translocation of intracellular vesicles that contain the GLUT4 and GLUT1 glucose transporters to the plasma membrane .The transporters return to the intracellular pool on removal of insulin

Symptoms

• Polyuria , polydipsia, and unexplained weight loss) and a random plasma glucose concentration of greater than 200 mg/dL , a fasting plasma glucose concentration of greater than 126 mL/dL or a plasma glucose concentration of greater than 200 mg/dL 2 hours after the ingestion of an oral glucose load.

(K.G.M.M. Alberti et al, 1998)

• In its most severe forms, ketoacidosis or a non-ketotic hyperosmolar state may develop and lead to stupor, coma and, in absence of effective treatment, death occur.

(K.G.M.M. Alberti et al, 1998)

Complications 5• Diabetic cardiovascular disease

(Dongjuan wang et al, 2013)• Insulin resistance • Beta-cell defect• Metabolic syndrome

These are the pathologic problems leading to hyperglycemia

• Heart attack• Stroke• Cardiovascular death

( Collins FM. Et al, 2013 )

• Oxidative stress causes diabetic complications that are undefined.

• Elevated FFA result in the generation of ROS and RNS, leading to increased oxidative stress. In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes overwhelmed (redox imbalance), leading to the activation of stress-sensitive signaling pathways, such as NF-B, p38 MAPK, JNK/SAPK, PKC, AGE/RAGE

(JOSEPH L.et al, 2002)

Current treatment

• Sulfonylureas

• Insulin secretagogues

• Biguanides

• Alpha-glucosidase inhibitors

• Thiazolidinediones

• Insulin

( Collins FM. Et al, 2013 )

Possible targets for diabetes type 2

• Adenosine 58- monophosphate-activated protein kinase (AMPK) now appears to be a metabolic master switch, phosphorylating key target proteins

(W. W. WINDER et al.2013)

• Several G protein-coupled receptors (GPCRs) expressed in islet β-cells are known to be involved in the regulation of islet function

(Bo Ahrén et al 2009)

• Several mechanisms have been proposed, including increased non-esterified fatty acids, inflammatory cytokines, adipokines, and mitochondrial dysfunction for insulin resistance, and glucotoxicity, lipotoxicity, and amyloid formation for β-cell dysfunction

( Michael Stumvoll et al, 2013)

• Genes have been identified so far: genes for calpain 10, potassium inward-rectifier 6·2, peroxisome proliferator-activated receptor γ, insulin receptor substrate-1

( Michael Stumvoll et al, 2013)

Current targets

(David E. Moller et al, 2001)

(David E. Moller et al, 2001)

Enhancing glucose-stimulated insulin secretion

(David E. Moller et al, 2001)

Targeting the insulin signalling pathway

(David E. Moller et al, 2001)

AMP-activated kinase and acetyl-CoA carboxylase

(David E. Moller et al, 2001)

Need

• Patients with diabetes have CVD-related mortality that is 2 to 4 times higher than in non-diabetics.

• Despite all the new treatments that have emerged in the last 20 years, diabetes sufferers remain challenged in effectively managing their disease over the long term and some drugs lose efficacy as the disease progresses. Oral drugs eventually fail to control blood glucose, and insulin injections become unavoidable.

• The challenge will be to advance these agents while meeting the demands of patients for better-tolerated drugs, optimal efficacy, and safety in the face of heightened testing requirements to assess CV risk.

• Metformin was approved by the US food and drug administration (FDA) in 1994. Preferred first-line drug for the treatment of type 2 diabetes.

• Additional agents are often prescribed in combination with metformin.

• Alpha-glucosidase inhibitors; various novel insulins with rapid onset of action or long duration of effect; thiazolidinediones (tzds); meglitinides; glucagon-like peptide-1 receptor agonists (GLP-1 RA); and dipeptidyl peptidase-4 (DPP-4) inhibitors.

• Rosiglitazone/avandia published in 2007 raised concerns about cv safety

• Rosiglitazone had an increased risk of myocardial infarction and a borderline increased risk of death from cv events.

• Taking insulin glargine had no effect (negative or positive) on the risk of a heart attack or stroke.

• Treatment of diabetes requires a multi-faceted approach and there are no “one-size-fits- all” solutions.

• Some of the newer drugs such as glp-1 ra, dpp-4 inhibitors, and sodium-glucose cotransporter 2 (sglt2) inhibitors may have favourable CV effects.

• Novel drug class is oral inhibitors of sglt2, the protein responsible for at least 90 per cent of the glucose reabsorption in the kidney.

• Sglt2 compounds have also shown significant blood pressure-lowering effects, but it remains uncertain whether these benefits will reduce CV events.

• Dapagliflozin was approved for use in europe, becoming the first sglt2 entrant to gain regulatory approval for type 2 diabetes.

• Reductions in weight and blood pressure.

• Dapagliflozin was denied approval by the fda in early 2012 due to concerns over apparent bladder and breast cancer risks.

• Other novel drug classes in development for type 2 diabetes are G-protein-coupled receptor (GPCR) agonists and interleukin-1-receptor (IL-1) antagonists. GPCRs are cell surface receptors that mediate a variety of important cellular signals related to control of blood pressure and blood glucose.

New targets

• Aldosterone and MR signaling represent an ideal candidate pathway linking early promoters of diabetes, especially overnutrition and obesity, to vascular insulin resistance, dysfunction, and disease

(Shawn B. Bender et, al 2013 )

• Preserve b-cell function and the impact on clinical care and outcomes

(Judith E et, al 2013)

• An increased b-cell workload (insulin resistance) is a risk factor for T2DM, most individuals adaptively increase insulin and IAPP expression and secretion without b-cell failure.

(Safia Costes et, al 2013)

• Several nonpeptide, except DPP-4 inhibitors, binding G protein-coupled receptors (GPCRs) have been deorphanized recently and are currently being evaluated as candidate GLP-1 secretagogues for T2DM

(Xiaoyun Zhu et al , 2013)

(Xiaoyun Zhu et al , 2013)

• A characteristic feature of type 2 diabetes is delayed wound healing, which increases the risk of recurrent infections, tissue necrosis, and limb amputation.

• Diabetes impairs resolution of wound healingstimulating resolution with proresolving lipid mediators could be a novel approach to treating chronic, nonhealing wounds in patients with diabetes

(Yunan Tang et, al 2013 )

• Damage to mitochondrial DNA (mtDNA), may result in Glucotoxicity due to metabolic oversupply

(Martin Picard et al, 2013)

(Martin Picard et al, 2013)

• Brown adipose tissue (BAT) as therapeutic strategies for preventing and treating obesity and type 2 diabetes 20

(Harold Sacks et, al 2013)

• Islet transplantation as a treatment

(R. Paul Robertson et, al 2004)

(R. Paul Robertson et, al 2004)

• Renoprotective Agents

(Macaulay Onuigbo et al ,2013)

• Blood pressure as targets for treatment, the efficacy of blocking the renin-angiotensin system (RAS) pathway,

(EBERHARD RITZ et, al 2011)

• Postprandial hyperglycemia is a direct and independent risk factor for cardiovascular disease (CVD) Correcting the postprandial hyperglycemia may form part of the strategy for the prevention and management of CVDs in diabetes

(Antonio Cerielloet et,al 2005)

• The acute phase of insulin release is a major determinant of the efficiency of glucose clearance,

• cAMP potentiates both acute-phase and sustained-phase insulin secretion and provides a therapeutic target to restore glucose control.

(Kelly A. Kaihara et,al 2013)

• Oleanolic acid (OA), improves insulin response, preserves functionality and survival of b-cells, and protects against diabetes complications.

(Jose M. Castellano et,al 2013)

• Vascular endothelial growth factor (VEGF) are revolutionizing the treatment of diabetic retinopathy (DR) and diabetic macular edema (DME).

(Paul M. Titchenell et, al 2013)

• Inhibition of hepatic glycogen phosphorylase is a promising treatment strategy for attenuating hyperglycemia , glycogen phosphorylase inhibition aimed at attenuating hyperglycaemia is unlikely to negatively impact muscle metabolic and functional capacity.

(David J. Baker et, al 2005)

• Pharmacological inhibition of hepatic glycogen phosphorylase has the potential to be an effective therapeutic strategy for the treatment of type 2 diabetes

• A novel peptide has been named ‘irisin’ acts on the cells of white adipose tissue. Irisin increases total energy expenditure and, in certain animal models, prolongs life expectancy, reduces body weight, and mitigates diet-induced insulin resistance, thus reducing obesity and insulin resistance

(Fabian Sanchis et, al 2012)

• Use of antioxidants may be very important in preventing activation of oxidative stress

(JOSEPH L et,al 2012)

Molecular targets

• Reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.

(Silvia Del Guerra et,al 2005)

• Nitrotyrosine and 8-hydroxy-2-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment

Plants having hypoglycemic activity (Nidhi Aggarwal et al , 2011)

• Eugenia jambolana (EJ) commonly known as Jaman

• Lawsonia alba Lam commonly known as henna is a perennial plant of the family Lythraceae, henna leaves (mehendi)

• Momordica charantia

• Morus alba (synonyms Morus alba Linn) (MA) known aswhite mulberry

• Nigella sativa Linn. (NS), known as black cumin Kalonji

• Trigonella foenum graecum Linn. (TF) commonly known as methi (M. SAEED ARAYNEetal, 2007)

Plant having hypoglycemic activity

Botanical name : Allium cepa

Family : (Liliaceae)

Common name :Onion

• Allium cepa has some anti-diabetic that b may be due to the antioxidant properties of its essential oil components, which can signify its anti-diabetic and antihyperlipidaemic activity

(DK Patel et,al 2012)

Why this plant…!!!

• Hypoglycaemic and hypolipidaemic activity

• Antioxidative activity

(Ozougwu et al , 2011)

Methods

1. Streptozotocin-induceddiabetes

2. Alloxan-induced diabetes

3. Virus-induced diabetes

4. Pancreatectomy in dogs

5. Growth hormone-induced diabetes

6. Corticosteroid-induced diabetes

7. Insulin deficiency due to insulin antibodies

Hans Gerhard Vogel

3rd edition

Streptozotocin Induced Diabetic

Aim :

This is apropos to investigate the antioxidative effect of allium cepa essential oil in streptozotocin induced diabetic albino rats

(Neveen Abou El-Soud et,al 2010)

Materials

• Essential oil of red onion (0.05%) will be prepared and isolate it according to Harborne

(Harborne JB et, al 1984)

• Streptozotocin (STZ) will be purchased from ABC company Pakistan

• Chloroform, Methyl alcohol, ether will be purchased from GHK chemicals

Animals:

• Thirty male albino rats weighing 150-200g will be obtained from UOS animal house

• Rats will be caged under controlled temperature 20-24°C and 12 h light/dark cycle. They will be fed with standard laboratory chow and water ad libitum.

Induction of Diabetes

• Rats will be kept on fasting prior to streptozotocin injection.

• On the day of administration, STZ will be freshly dissolved in 50 Mm sodium citrate (pH 4.5) solution containing 150 mM NaCl and subcutaneous injection will be given at the dosage of 60 mg/kg b.w.

• Blood glucose concentration will be checked by the glucose oxidase method (Trinder P. Et, al 1969) after 3 days of STZ injection. The animals with glucose concentration exceeding 200 mg /dl will be considered diabetic.

Rats will be divided into 3 groups 10 rats in each

• Group: group I: normal control rats

• Group II: diabetic control rats

• Group III: diabetic rats received onion essential oil (100 mg/kg b.W. Orally)

• The dose will be chosen according to its LD50 (the medium 50 lethal doses after acute toxicity).

Groups FBG (mg/dl) Serum insulin (µu/ml)

I Normal control

II Diabetic control

III Onion essential oil control (100 mg/kg b.W. Orally)

Samples Collection• After 21 days from the beginning of the

experiment

• Rats will be fasted for 12 hours

• Then blood samples will collected

• Blood will be collected retro-orbitally from the inner canthus of the eye under ether anaesthesia using capillary tubes containing sodium fluoride

(Madway W et, al 1969)

• Serum and plasma will be separated and centrifuged at 3000 rpm for 5 minutes.

• The serum and plasma will be separated for measurement of glucose, insulin, cholesterol, triglycerides, HDL, LDL, nitric oxide and tbars in different study groups.

ALLOXAN-INDUCED DIABETES

(Ozougwu et al ,2011)

Animal Model• Sixty three (63) adult white wistar strain albino

rats (R. norvegicus) weighing 200 to 250g, bred in the animal house of the Faculty of Pharmacology UOS will be used for the study.

• They will be fed ad labium with 30% crude protein (Guinea feed) commercial feed.

• They will be allowed to acclimatize under standard photoperiodic condition in a clean rat cage Research Laboratory.

• All animals will be maintained under the standard laboratory condition for temperature (26 ± 20C) and light (12 hours day length) and allowed free access to food and water.

Preparation of Plant Extracts

• The methods of Habib MY et al 2005 and Battu GR et al, 2005 will be used.

• Fresh health plant of A. cepa (2000 g) will be washed, cut into small pieces and homogenized in a warring blender.

• The resulting mixture will be soaked in 2L of distilled water.

• The mixture will be allowed to stand for twenty four hours with intermittent shaking.

• Following filtration, the filtrates will be heated to dryness in a water bath and the weight of the crude extract will be determined.

• The extract will be kept in refrigerator (40 C) thereafter.

• The extract will be reconstituted in normal saline (0.85% NaCl) at a concentration of 1g/ml before administration.

Induction of Diabetes Mellitus

• The methods of Osinubi AA et al , 2006 and Battu GR et al , 2007 will be used to induce diabetes in the rats.

• 150 mg of alloxan per kg body weight of rat will be administered intraperitoneally after overnight fast (access to only water) of twelve hours to make them more susceptible to developing diabetes.

• Rats with serum glucose levels between (250 – 400 mg/dl) after two weeks will be considered diabetic and used for the experiment.

Experimental Design• The study will be carried out on alloxan-

induced diabetic rats for six weeks.

• The animals will be fasted for sixteen hours before each experiment and blood sample collected from the eye of the rats.

• All parameters assessed will be determined before the extract treatments of the animals (initials) and subsequently will be evaluated weekly for six weeks.

• The experimental design will be three by three Latin square design using 63 rats divided into two major groups

• Group I: nine non diabetic rats (non diabetic control)

• Group II: fifty four alloxan induced diabetic rats.

• Group I rats were divided into 3 subgroups (Ia, Ib, Ic) of 3 rats each in different cages and receives 1.0ml of normal saline intraperitoneally daily.

• Group II (fifty - four alloxan induced diabetic rats) will be divided into 2 subgroups (IIa , IIb). Subgroups IIa, (twenty seven rats) will be divided into 3 replicates (IIa1, IIa2, IIa3).

• Each replicate will have three rats and received 200 mg/kg, 250 mg/kg or 300 mg/kg of A. cepa aqueous extracts intraperitoneally daily respectively.

• The subgroups IIb will be diabetic control (twenty- seven rats) and will be divided into 3 replicates (IId1, IId2 and IId3) each replicate will have three rats and will be administered 2.5mg/kg/, 3.8mg/kg and 5.0mg/kg of standard antidiabetic drug (glibenclamide) daily for six weeks

Parameters

• Determination of Blood Glucose

• Determinations of Total Serum

• Cholesterolose Level Determination

• Determination of Total Lipids in Serum

INSULIN DEFICIENCY DUE TO INSULIN ANTIBODIES:

• Bovine insulin, will be dissolved in acidified water (pH 3.0) and incorporated in a water-oil emulsion based on complete Freund’s adjuvant or a mixture of paraffin oil and lanolin.

• A dose of 1 mg insulin will be injected in divided doses subcutaneously to male guinea pigs weighing 300–400 gm.

• Injections will be given at monthly intervals and the guinea pigs will be bled by cardiac puncture two Weeks after the second and subsequent doses of antigen.

• It is possible to get 10 ml blood from every animal once a month. Intravenous injection of 0.25–1.0 ml guinea pig antiinsulin serum to rats will induce a dose-dependent increase of blood glucose reaching values up to 300 mg%.

• This effect is unique to guinea pig anti-insulin serum and is due to neutralization by insulin antibodies of endogenous insulin secreted by the injected animal. In this way a state of insulin deficiency will be induced.

• It persists as long as antibodies capable of reacting with insulin remain in the circulation. Slow rate intravenous infusion or intraperitoneal injection prolongs the effect for more than a few hours.

• However, large doses and prolonged administration accompanied by ketonemia, ketonuria, glucosuria, and acidosis will proove to be fatal to the animals.

• After lower doses, the diabetic syndrome will be reversible after a few hours (Moloney and Coval 1955; Wright 1968).

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