ANTIDIABETIC AND HYPOGLYCEMIC EFFECT OF ISOLATED FLAVONOIDS FROM FICUS RACEMOSA STEM BARK IN STREPTOZOTOCIN DIABETIC RATS

DEPARTMENT OF PHARMACEUTICAL SCIENCESBABA SAHEB BHIMRAO AMBEDKAR

UNIVERSITY LUCKNOW, U.P.

SESSION 2014-2015

A thesis presentation submitted to

Babasaheb Bhimrao Ambedkar University in partial fulfillment of the requirements for the

degree of

MASTER OF PHARMACY In

pharmacologySUPERVISOR

Dr. Sudipta SahaAssistant professor

PRESENTED BY

GhanendraRoll No- 32006

INTRODUCTION Diabetes is a global epidemic with an estimated worldwide prevalence of 246

million people in 2007 and forecasts to rise to 300 million by 20251.

Diabetes is a metabolic disorder.

It characterized by chronic hyperglycaemia with disturbance of carbohydrate,

fat, and protein metabolism resulting from defects in insulin secretion, insulin

action or both.

There are two main types of diabetes -

I. IDDM (insulin-dependent diabetes mellitus) - It results from the

body’s failure to produce insulin

II. NIDDM (non-insulin-dependent diabetes mellitus)- It occurs due to

insulin resistance, a condition in which cells fail to use insulin properly

CONT... There are symptom of diabetes –

Blurred vision, unusual thirst, Polydepsia, Polyurea Rapid weight loss, Erectile dysfunction

It is diagnosis by the several parameters –

Fasting plasma glucose level ≥ 7.0 mmol/l (126 mg/dl).

Plasma glucose ≥ 11.1 mmol/l (200 mg/dl) two hours after a

75 g oral glucose load as in a glucose tolerance test.

Glycosylated hemoglobin (Hb A1C) ≥ 6.5%.

CONT... Treatment -

In type 1 diabetes require insulin.

In type 2 diabetes treatment will vary depend on blood sugar level.

Prevention –

Good nutrients for normal body weight

sensible exercise

Test sugar level

LITERATURE REVIEW Eleazu et al (2013) reported that streptozotocin (STZ) produced

cytotoxicity through DNA methylation, nitric oxide production, free radical

generation and alteration NF- κB (nuclear factor kappa-light-chain-

enhancer of activated B cells) cell signal transduction pathway. Arunachalam K et al (2013) stated that ficus amplissima smith: bark

extract improved the diabetic condition in STZ induced diabetic rats and

significantly enhanced the level of antioxidant enzyme.

Peibo Li et al (2013) reported that the 13-week subchronic oral toxicity

study, daily doses of 50, 250, and 1250 mg/kg of naringin were well

tolerated and did not cause either lethality or toxic clinical symptoms and

changes in both sexes of rats except for slight body weight decrease.

CONT... Cheng D et al (2012) repoted that Ginkgo biloba extract (GBE) possesses

antihyperglycemic, antioxidant, and antihyperlipidemic activities in STZ-induced

chronic diabetic rats, which promisingly support the use of GBE as a food

supplement or an adjunct treatment for diabetics.

Mahmoud M A et al (2012) stated that antihyperglycemic efficacy of the citrus

flavonoids, hesperidin and naringin, may be mediated via up-regulating adipose

tissue PPARγ and adiponectin in conjunction with down-regulating adipose

tissue resistin.

Rahigude A et al (2012) stated that both narigenin and pioglitazone showed

improvement in cognitive behavior against diabetes induce memory dysfunction

via depleting elevated lipid peroxide and nitric oxide and inhibition of elevated

ChE activity.

RESEARCH ENVISAGEDprevious report suggested that

flavonoids produced antidiabetic action in streptozotocin induced

diabetic rats.

No detail study had been performed regarding their hypolipidemic and

toxicological action.

The overall objectives of this project –Toxicological, hypoglycemic and molecular docking study via PPARγ and GLUT1 receptor

PLAN OF WORKDrugs: FR7, FR8 and glibenclamide

Toxicant: streptozotocin (STZ)

Animal study (n=5)

Biochemical estimation

CONT...S.NO GROUP DOSE ROUTE OF

ADMINISTRATION1 Normal

control0.25mg/kg, CMC Oral

2 Diabetic control

50mg/kg, STZ I.P

3 D + N 10mg/kg, Glibenclamide

Oral

4 D + FR7 50mg/kg, FR7 Oral5 D + FR8 50mg/kg, FR8 Oral

CONT...

Biochemical estimation

Glucose level and weight

variation on 1st 3rd 5th &7th day

1. Oxidative parameter: SOD, CAT,

GSH, TBARS, PC

Lipid profile:

HDL LDL, TC, TG &

VLDL SGOT & SGPT

level in plasma

Histopathology and docking

study

Statistical analysis: ANOVA

RESULTS AND DISCUSSION Antidiabetic effect of isolated

flavonoids: Diabetic rats treated with FR7 (Quercetin), FR8

(Naringenin) & G (glibenclamide) showed reduction of glucose level in comparison to D (diabetic control).

N Contro

l

D Contro

lD + G FR

7FR

80

200400

1 day3 day5 day7 day

7 day

mg/

dL

Absorpti-on of

glucose from

intestine

Neogluc-

ogenesis

Increase glucose level in diabetic

ratsFR7

RESULTS AND DISCUSSION Effect of isolated flavonoids on body weight:

D group showed the reduction in body weight in STZ(streptozotocin) diabetic rats. Orally administration of FR7 and FR8 displayed improvement in body weight and treated with G showed increased in body weight.

N Contro

l

D contr

olD + G FR

7FR

80

50100150200

1 day3 day5 day7 day

7 day

Chan

ge in

bod

y w

t.

RESULTS AND DISCUSSION Effect of flavonoids on glycogen content in liver:

Glycogen content was massively decreased in D control than N control.

Again, we found that glycogen content was improved by FR7 and FR8 treatment.

N Contro

l

D Contro

lD + G FR

7FR

80

204060

glycogen

7 day

mg/

g

RESULTS AND DISCUSSION Effect flavonoids lipid profile in plasma:

We observed that TC, TG, LDL, VLDL levels were increased and HDL level was deceased in toxic control rats (D control).

lipid profile improved via FR7, FR8 and G treatment.

N Contol

D Contro

lD + G FR

7FR

80

50100150200

TCTGHDLLDLVLDL

7 day

mg/

dL

RESULT AND DISCUSSION Oxidative parameter in pancreas:

We observed that GSH, SOD & CAT level were decrease in D control than N control. The level of GSH, SOD & CAT were restored via 7 day treatment therapy of FR7 and FR8.

N Control D Control D + G FR7 FR80

20406080

100120

SODCATGSH

RESULTS AND DISCUSSION Oxidative parameter in liver

According to observation, the formation of TBARS and PC were higher in D control as compared to N control. Both formations were decreased when flavonoids treated to STZ rats.

N ControlD Control D + G FR7 FR80

20

40

60

80

100

TBARSPC

RESULTS AND DISCUSSION CAT is mainly responsible for the catalytic

decomposition of H2O2 to oxygen and water.

SOD is responsible for catalytic dismutation of free radicals and decrease superoxide level.

Reduced GSH converted to disulfide glutathione during oxidative damage.

TBARS and PC level reduced by inhibition of lipid peroxidation protein carbonylation.

RESULTS AND DISCUSSION Effect of FR-7 & FR-8 on AST and ALT level:

Isolated flavonoids also showed protective action on AST, ALT levels in plasma. Serum enzyme levels were increased during STZ treatment which was normalized during FR7 and FR8 administration .

N Control D Control D + G FR7 FR80

50

100

150

200

ASTALT

7day

u/dL

RESULTS AND DISCUSSION Histopathology of pancreatic tissue

N Control

D Control

D + N FR7 FR8

RESULTS AND DISCUSSION

GLUT 1

1SUK: TYR28,GLY31,

ARG126, TRP412FR7

PDB

Target

Exothermic reaction

Hydrogen bond formationAnd interaction energy (-6.2)

Docking study of FR7, FR8 on GLUT1 & PPARγ

RESULTS AND DISCUSSION

2PRG:LEU353,

MET348, ILE341, CYS285, ARG288

PPARγFR7

Target

PDB

Exothermic reaction

Hydrogen bond formationAnd interaction energy(-9.6)

Docking study ...

RESULTS AND DISCUSSION

FR8

GLU

T 1

GLU261, ALA405, VAL406, PHE81, SER80, MET77,

Exothermic reaction

Hydrogen bond formationAnd interaction energy(-5.9)

Docking study ...

RESULTS AND DISCUSSION

VAL248, PHE347, LEU237

PPARγ

Exothermic reaction

Hydrogen bond formation And interaction energy(-6.2)

Docking study ... FR8

SUMMARY AND CONCLUSION

Whole plant have antidiabetic property

Previous literature suggested that flavonoid has antidibetic activity

We isolated four flavonoids from whole plant and performed antidiabetic activity of two compound – FR7 and FR8

REFERENCES A report of WHO named diabetes.

Sapiro M, Welss JP. Diabetes insipidus: A review. Diabetes and metab.2012; S8:001.

Cheng D, Liang B, Li Y. Antihyperglycemic effect of Ginkgo biloba extract in

Streptozotocin - induced diabetes in rats. BioMed research international.2012; 2013.

Chukwuma S, Essien NU. Review of the mechanism of cell death resulting from

streptozotocin challenge in experimental animals, its practical use and potential risk

to humans. Journal of diabetes & metabolic disorders. 2013; 12:60.

Mahmoud AM, Ahmed OM, Monein AA, Ashour MB. Upregulation of PPARγ

mediates the antidiabetic effects of flavonoids in type 2 diabetic rats. International

journal of bioassays. 2012; 02(05): 756- 761.

CONT... Kushwaha PS, Raj V, Singh AK, Keshari AK, Saraf SA, Mandal SC, et al.

Antidiabetic effects of isolated sterols from Ficus racemosa leaves. RSC

Advances. 2015;5(44):35230-7. Yadav RK, Keshari AK, Kaithwas G, Maity S, Saha S. Antidiabetic and

hypolipidemic effect of Ficus racemosa petroleum ether extract in streptozotocin induced diabetic Albino Rats. BioMed Research International. 2014

Saha S, Chan DSZ, Lee CY, Wong W, New LS, Chui WK, et al. Pyrrolidinediones reduce the toxicity of thiazolidinediones and modify their anti-diabetic and anti-cancer properties. European journal of pharmacology. 2012;697(1):13-23.

Sophia D, Manoharan S. Hypolipidemic activities of Ficus racemosa Linn. bark in alloxan induced diabetic rats. African Journal of Traditional, Complementary and Alternative Medicines. 2008;4(3):279-88.

Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiological research. 2001;50(6):537-46.