Antidiabetic and Antioxidant effect of. Patrick N. Okechukwu Ph.D UCSI University, Malaysia

Transcription

1 Antidiabetic and Antioxidant effect of Palmatine on STZinduced Diabetic Rats. Patrick N. Okechukwu Ph.D UCSI University, Malaysia

2 CONTENT Introduction and literature review Research question/hypothesis/objectives Methodology Results and discussion Conclusion References 2 CONTENT

3 Introduction & Lit. Review

4 Type 1 diabetes T-lymphocytes target and kill off insulin producing cells. Insulin dependence Type 2 diabetes Insulin resistance 4

5 Causes of Diabetes Obesity Diet: High Carbohydrates, Fats & Protein Age Stress Smoking 5

6 Complications Eye (retinopathy) Brain and cerebral circulation Heart & coronary circulation Kidney Peripheral Nervous System Lower Limbs Diabetic Foot Amputation 6

7 Coscinium fenestratum Classification: Kingdom: Plantae Phylum: Tracheophyta Class: Magnoliopsida Order: Menispermales Family: Menispermaceae Genus : Coscinium Species: Fenestratum Found in Western Malaysia, India, Vietnam and Thailand Traditional Use: anti-inflammatory, antiseptic, stomachic, dressing wound, snake bite, intermittent fever Its other names include darvi, daruharidra, peetadaru, peetadru, tree Turmeric, daruhaldi, jharihaldi, maramanjal 7

8 Literature review Coscinium fenestratum Hypotensive Crude Extract Antiinflammatory Antioxidant Anti-diabetic 8

9 Sample Extraction and Partial Purification Fraction A Fraction B Maceration 3 days Occasional stirring 500g LP : 6L DCM 5X filtration Concentration and drying Previous: Column (42 cm x 2.5 cm) preparation Gas pressure 100% Hexane 90:10 Hexane: Ethyl acetate - 100% Ethyl acetate 90:10 Ethyl acetate: Methanol - 100% Methanol Fraction C Fraction D Fraction E 9

10 Analysis of Fraction E Antidiabetic Fraction E Antioxidant 10

11 Characterization of Fraction E and Validated Method TLC HPTLC HPLC HPLC-UV During column chromatography Comparison amongst phenol compounds Validated Method development Characterization of fraction E Methanol: ethyl acetate (60:40) TLC Silica gel 60F₂₅₄ (Aluminium) (Merck) Validated Method Development Linearity Precision Accuracy Quantification of absorbed drug in rat plasma for bioavailability study HPLC-UV shows 2 gram of Palmatine is contained in Fraction E 11

12 Literature review on palmatine Palmatine is a protoberberine alkaloid found in several plants 12

13 Research question Is Palmatine the actual bioactive compound in Fraction E Is the antidiabetic effect of Palmatine due to its antioxidant property? Hypothesis Palmatine may be the main bioactive compound in Fraction E responsible for its antidiabetic activity. Anti-diabetic activity of Palmatine may be due to its antioxidant properties Objectives To determine the antidiabetic and in vivo antioxidant effect of Palmatine 13

14 Methodology

15 Experimental Design Group 1: (Non-diabetic control) 1ml of saline Group 2: (Diabetic negative control) 1ml of saline Group 3: (Positive control) Tolbutamide-treated group (100mg/kg) Group 4: Palmatine-treated group (2mg/kg) Group 5: Purified Fraction E-treated group (100mg/kg) 15 N=6, where n is the number of rats in each group

16 Induction and treatment Baseline measurement (plasma glucose level and Weight) Overnight fasting Diabetes induction (50mg/kg STZ) 1 week Blood glucose level and weight Experimental group (n=6) Higher than 10 mmol/l 16

17 Rats sacrificed Pancreas harvested Liver harvested Blood collected Histology Antioxidant Biochemical analysis 17

18 18 ANTIOXIDANT TEST

19 19 Hematology, biochemical and lipid profile

20 20 Histology

21 Results and discussion

22 AVERAGE BLOOD GLUCOSE LEVEL (mmol/l) AVERAGE WEIGHT OF RATS (g) Diabetic Test * * Normal Negative control Tolbutamide Palmatine Fraction E GROUPS ** * ** Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week ** ** ** ** Normal Negative control Tolbutamide Palmatine Fraction E GROUPS ** Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Mean blood glucose level and weight of rats per group. Rats were treated over a period of 90 days. All data were presented as mean (±) standard error mean (SEM) (n = 6) using SPSS. The data were statistically analyzed by two-way ANOVA followed by Dunnet s test. Values were considered statistically significant when p<

23 Palmatine, Fraction E and Toulbutamide reduced the plasma glucose level. they also increased the body weight of the diabetic animals. the effect of Palmatine, Fraction E and Toulbutamide was similar. Block K+ channel and stimulation of insulin release by activating Ca2+ channels. 23 July 22, 2012 Footer text here

24 In vivo antioxidant test CONCENTRATION 1,6 1,4 ** GSH (nmol/mg) LPO(nmol/mg) CATALASE (μmol/mg) SOD (U/mg) 1,2 ** *** 1 * * 0,8 0,6 * * ** * 0,4 0,2 * * 0 Normal Negative control Tolbutamide Palmatine Fraction E GROUPS In vivo antioxidant activities. Values were considered statistically significant when p< Rats were treated over a period of 90 days. 24

25 LPO was kept low in Palmatine, Fraction E and Toulbutamide treated group. GSH, CAT were increased in Palmatine, Fraction E and Toulbutamide treated group. SOD was low in Fraction E and Toulbutamide treated group but was increased in Palmatine. 25 July 22, 2012 Footer text here

26 CONCENTRATION CONCENTRATION Hematology test Hemoglobin (g/l) 200 * 150 * * * NORMAL NEGATIVE TOLBUTAMIDE PALMATINE FRACTION E GROUPS Red blood cell (RBC) (g/l x 10^12 ) White cell count (10^9)/L 10 8 ** * *** * *** *** ** NORMAL NEGATIVE TOLBUTAMIDE PALMATINE FRACTION E GROUPS Hemoglobin, red blood cell count and white blood cell count. The data were statistically analyzed by two-way ANOVA followed by Dunnet s test. Values were considered statistically significant when p<

27 Hemoglobin was increased in the Negative control group but the level in Palmatine, Fraction E and Toulbutamide group was almost apar with normal group. Acute dehydration can cause high hemoglobin (clinical report) Hb has been reported to be involved in superoxide radical formation and the inactivation of glutathione peroxidase (Falcioni et al 1987; Grelloni et al 1991) 27 July 22, 2012 Footer text here

28 CONCENTRATION CONCENTRATION Biochemical test (renal/liver function test) 20 Urea (mmol/l) ** * * ** 5 0 NORMAL NEGATIVE TOLBUTAMIDE PALMATINE FRACTION E GROUPS 80 Creatinine (umol/l) 60 *** *** *** 40 ** 20 0 NORMAL NEGATIVE TOLBUTAMIDE PALMATINE FRACTION E GROUPS Renal and liver function test. The data were statistically analyzed by two-way ANOVA followed by Dunnet s test. Values were considered statistically significant when p<

29 Urea was observed to be high in negative control group but the level in Palmatine, Fraction E and Toulbutamide group was almost apar with normal group. An increase in the urea level is known as azotemia, and it may be caused by either or combination of the below factors: 29 July 22, 2012 Footer text here

30 Impaired renal function Congestive heart failure as a result of poor renal perfusion Dehydration Shock Hemorrhage into the gastrointestinal tract Acute myocardial infarction Stress Excessive protein intake or protein catabolism 30 July 22, 2012 Footer text here

31 CONCENTRATION Lipid profile test Alkaline Phosphatase (U/L) Aspartate transaminase (AST) (U/L) Alanine aminotransferase (ALT) (U/L) * NORMAL NEGATIVE TOLBUTAMIDE PALMATINE FRACTION E GROUPS Lipid profile. The data were statistically analyzed by two-way ANOVA followed by Dunnet s test. Values were considered statistically significant when p<

32 AP, AST and ALP were in creased in negative control and toulbutamide, but they were maintain in Palmatine and Fraction E. AP reflects liver synthetic function while AST and ALP measures the concentration of intracellular hepatic enzymes that have leaked into the circulation and also serve as a marker of hepatocyte injury (Marsano et al 2003). 32 July 22, 2012 Footer text here

33 33 July 22, 2012 Footer text here Palmatine and Fraction E may have maintained the AP, AST and ALP because of their antioxidant property

34 Lipid profile. The data were statistically analyzed by two-way ANOVA followed by Dunnet s test. Values were considered statistically significant when p< July 22, 2012 Footer text here

35 There was an observed decreased in level of HDL and elevated levels of triglycerides and LDL in negative group. Mooradian et al 2009 reported that the distinguishing features of abnormal lipids levels in diabetes are high plasma triglyceride concentration, low HDL cholesteriol concentration and increased concentration of small dense LDL-cholesterol particles. However the reverse was the case in Palmatine toulbutamide, and Fraction E treated group. 35 July 22, 2012 Footer text here

36 The changes in lipid function associated with diabetes mellitus may be due to increased free fatty acid flux secondary to insulin resistance (Marsano et al 2003). 36 July 22, 2012 Footer text here

37 Histopathology (A.) (B.) Islet of Langerhans (C.) (D.) (E.) H&E stained pancreatic sections. (A.) Non Diabetic - Normal, (B-E.) Diabetic rats; Negative, Tolbutamide, Palmatine and Fraction E, respectively. Rats were treated over a period of 90 days. 32

38 Revealed degenerative and necrotic changes in the negative control. The pancreatic cells were regenerated with the treatment of palmatine, toulbutamide and Fraction E. 38 July 22, 2012 Footer text here

39 Palmatine, Toulbutamide and Fraction E was able to protect the cells because of their antioxidative ability to suppress apoptosis in β-cells without changing the rate of β-cells proliferation, it also preserves the insulin content and insulin mrna. The result is in line with reports by Kaneto et al July 22, 2012 Footer text here

40 Conclusion: 1. Palmatine possess antidiabetic property and it is able to protect the β-cells, kidney and liver against induced oxidative stress by STZ. 2. Palmatine effect may be due its antioxidant property. 3. Palmatine maybe the main bioactive compound present in Fraction E. 4. The exact molecular mechanism of how palmatine protects β- cells, kidney and liver requires further investigation. 40 July 22, 2012 Footer text here

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43 Lipid metabolizing proteins Pancreatic lipase (Pnlip): Lipolytic protein that breaks down triglycerides Bile Salt-activated lipase (cel): aids fats digestion Enoyl CoA hydratase (Echs1): aids in fatty acid metabolism

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46 Antioxidant proteins Peroxidoxin 4 (prdx4): removal of peroxides from metabolism Glutathione-s-transferase (GST): detoxification enzyme which provide protection against products of oxidative stress Serum albumin (Alb): free radical trapping activity

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50 ACKNOWLEDGEMENT This work was supported by funds from Centre of Excellence for Research, Value, Innovation and Entrepreneurship Research Grant Scheme UCSI University (UCSI-CERVIE-RGS Proj-in-FAS 017). 35

51 References 1. Wild S, Roglic G, Green A, Sicree R, King H. Global Prevalence of Diabetes, Estimates for the year 2000 and projections for Diabetes Care 2004;27(5): Paonessa F, Durlach A, Malloy MJ, Kane JP, Heiner SW, Filocamo M. Functional Variants of the HMGA1 Gene and Type 2 Diabetes Mellitus. Am. Med. Assoc. 2011;305(9): Ozougwu JC, Obimba KC, Belonwu CD, Unakalamba CB. The pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. J. Physiol. Pathophysiol. 2013;4(4): doi: /jpap Jakus V. The role of free radicals, oxidative stress and antioxidant systems in diabetic vascular disease. Bratisl lek List. 2000;10(101): Maritim a C, Sanders R a, Watkins JB. Diabetes, oxidative stress, and antioxidants: a review. J. Biochem. Mol. Toxicol. 2003;17(1): doi: /jbt Rahimi R, Nikfar S, Larijani B, Abdollahi M. A review on the role of antioxidants in the management of diabetes and its complications. Biomed. Pharmacother. 2005;59(7): doi: /j.biopha Shalaby EA, Shanab SMM. Antioxidant compounds, assays of determination and mode of action. African J. Pharm. Pharmacol. 2013;7(10): doi: /ajpp Vrba J, Papouskova B, Pyszkova M, et al. Metabolism of palmatine by human hepatocytes and recombinant cytochromes P450. J. Pharm. Biomed. Anal. 2015;102: doi: /j.jpba Wang C, Li J, Lv X, et al. Ameliorative effect of berberine on endothelial dysfunction in diabetic rats induced by high-fat diet and streptozotocin. Eur. J. Pharmacol. 2009;620(1-3): doi: /j.ejphar Yin J, Xing H, Ye J. Efficacy of berberine in patients with type 2 diabetes mellitus. Metab. Exp. 2008;57(5): doi:doi: Bagheri M, Jahromi BM, Mirkhani H, et al. Azelnidipine, a new calcium channel blocker, promotes skin wound healing in diabetic rats. J. Surg. Res. 2011;169(1):e doi: /j.jss

52 52 THANK YOU