Effect of Naringin on myocardial potency of Resveratrol against ischemia reperfusion induced myocardial toxicity in rat

Journal Pre-proof Effect of Naringin on myocardial potency of Resveratrol against ischemia reperfusion induced myocardial toxicity in rat Manodeep Chakraborty, Ananya Bhattacharjee, Mohammed Gulzar Ahmed, Sindhu Priya, Haleema Shahin, Tahreen Taj

PII:

S2213-7130(20)30001-8

DOI:

https://doi.org/10.1016/j.synres.2020.100062

Reference:

SYNRES 100062

To appear in:

Synergy

Received Date:

10 October 2019

Revised Date:

17 December 2019

Accepted Date:

6 January 2020

Please cite this article as: Chakraborty M, Bhattacharjee A, Ahmed MG, Priya S, Shahin H, Taj T, Effect of Naringin on myocardial potency of Resveratrol against ischemia reperfusion induced myocardial toxicity in rat, Synergy (2020), doi: https://doi.org/10.1016/j.synres.2020.100062

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Original Research Article Effect of Naringin on myocardial potency of Resveratrol against ischemia reperfusion induced myocardial toxicity in rat

of

Dr. Manodeep Chakraborty M.Pharm, Ph.D Associate Professor, Pharmacology Department, Yenepoya Pharmacy College and Research Centre, Mangalore, India-575018

ro

Mrs. Ananya Bhattacharjee M.Pharm Assistant Professor, Pharmacology Department, Srinivas College of Pharmacy, Mangalore, India- 574 143

re

-p

Dr. Mohammed Gulzar Ahmed M.Pharm, Ph.D Principal, Pharmaceutics Department, Yenepoya Pharmacy College and Research Centre, Mangalore, India-575018

lP

Mrs. Sindhu Priya E.S M.Pharm Assistant Professor, Pharmacology Department, Yenepoya Pharmacy College and Research Centre, Mangalore, India-575018

ur na

Ms. Haleema Shahin M.Pharm Assistant Professor, Pharmacology Department, Yenepoya Pharmacy College and Research Centre, Mangalore, India-575018

Jo

Mrs. Tahreen Taj Assistant Professor, Pharmacology Department, Yenepoya Pharmacy College and Research Centre, Mangalore, India-575018 Running Title: Effect of Naringin on myocardial potency of Resveratrol. Corresponding Author Dr. Manodeep Chakraborty M.Pharm, Ph.D Associate Professor, Pharmacology Department, Yenepoya Pharmacy College and Research Centre,

Mangalore, India-575018 E.mail: [email protected] Mobile: +91-9663417306 Phone: +91-824 - 2206000 Fax: +91-824 - 2204667 Graphical abstract

ro

of

Lagendorff’s Technique

Combination group showed better recovery of Heart rate & developed tension

re

-p

Combination group reported improved restoration of biomarker & Antioxidant level

Jo

ur na

lP

Ischemic Heart

Resveratrol

Naringin

Combination improved kinetic parameters of Resveratrol Inhibition of P-glycoprotein mediated efflux pump and microsomal enzyme CYP3A

Abstract: Objective Resveratrol (RES) is a well known cardioprotective phytoconstituent, but the poor bioavailability provides further scope for research to improve its therapeutic efficacy. The present study was designed to address this challenge by combining with bio-enhancer like Naringin (NAR) in the

of

prevention of ischemia reperfusion injury (IRI) induced myocardial toxicity in rats.

ro

Methods

Rats (n = 8) were treated with RES (20 mg/kg, p.o.) alone and combination of NAR (15 mg/kg,

-p

p.o.) and RES (20 mg/kg, p.o.) for 30 days. Twenty four hour after last treatment Ischemia reperfusion injury was induced by modified Lagendorff apparatus, and the effect of different treatments was evaluated by percentage recovery in terms of heart rate and developed tension,

re

biomarkers, heart tissue antioxidant levels and a histopathological examination. The Influence of NAR on the pharmacokinetics of RES was studied by HPLC. Results were assessed by one‑way

lP

analysis of variance followed by Tukey–Karmer multiple comparison test.

ur na

Results

Combination of RES and NAR demonstrated significant (P<0.01) restoration of biomarker, antioxidant, tension and heart rate compared to RES alone treated group. Significant (P<0.01) increase in bioavailability and half life, along with significant (P<0.001) decrease in clearance

Jo

was observed for RES in combination group compared to RES alone treated group.

Conclusion

The combination of RES and NAR exhibited profound protection compared to RES alone treated group against IRI induced myocardial toxicity. Findings of pharmacokinetic interaction support the results of a pharmacodynamic interaction.

Key words: Cardioprotective, Ischemia reperfusion induced injury, naringin, resveratrol, Interaction

Introduction Ischemic conditions in myocardial cells are responsible for the development of acute or chronic

of

forms of cardiac disability such as Myocardial infarction (MI). MI causes irreversible necrosis of heart tissue. It is one of the prime causes of mortality throughout the globe including both

ro

developed and developing countries.1 From the beginning of the civilization, in many societies, herbs and herb-based therapy played an important role to treat different diseased conditions and

-p

to improve the quality-of-life. In recent years, drug -drug, herb -drug or herb -herb interactive studies are gaining popularity as such combinations may influence the pharmacokinetic and

re

pharmacodynamic profile of each other, and can mimic, magnify, or oppose the action of each other.2

lP

Resveratrol belongs to the stilbene family of compounds having two aromatic C rings joined together by an alkene bridge. It is considered a phytoalexin and, environmental stresses or microbial attacks are responsible for synthesis of this metabolite. Resveratrol possesses multiple

ur na

biological and pharmacological activities. Resveratrol has been reported to possess cardioprotective,

protective

in

ischemic

injuries,

anti-diabetic,

neuroprotective,

anti-

inflammatory, immunomodulatory, chemopreventive, anti-ageing, antioxidant properties.3,4 The poor bioavailability associated with resveratrol is a major problem in translating its effects in humans. The 6-8 P-glycoprotein mediated efflux pump and CYP 3A4 mediated metabolism are

Jo

the prime reasons for the poor bioavailability associated with resveratrol.5 Naringin is a flavanone glycoside, widely available in citrus fruits and grapefruits. Naringin has the ability to inhibit the P-gp efflux pump and also to inhibit metabolic enzymes belonging to the cytochrome P450 family particularly CYP3A4.6 It was found that naringin has antiviral, anticancer, hepatoprotective, antiinflammatory, anti-ulcer and antioxidant activities.7 Naringin is responsiblefor ascorbic acid induced lipid peroxidation and a potent inhibitor of the acyl CoAcholesterol-Oacyltransferase (ACAT). ACAT-promotes the esterification of cholesterol in blood,

macrophage-lipid complex accumulation activity.8 Naringin is able to alter the pharmacokinetics of co-administered drugs such as verapamil, diltiazem, paclitaxel, tamoxifen those are substrates of P-gp and/orCYP3A.6,8Therefore the present study was designed to evaluate the influence of Naringin on the effect of Resveratrol against ischemia reperfusion induced myocardial toxicity. Moreover the effect of Naringin on pharmacokinetic parameters of Resveratrol were also determined.

of

Methods

ro

Chemicals

All chemicals used were of analytical grade and purchased from standard companies such as R L

-p

Fine Chem, Bengaluru and Rankem, Mumbai. Biochemical kits were procured from Crest Biosystems (Goa, India). Pure samples of Resveratrol and Naringin were procured from Sigma-

re

Aldrich, India.

lP

Experimental Animals

Healthy adult Wistar albino rats of either sex weighing 175–250 g were housed in polypropylene

ur na

cages, maintained under standardized conditions (12h light: Dark cycles, 25° ±5°C) with paddy husk bedding at the Central Animal House, of the institute and were provided with standard pellet food and had free access to purified drinking water. The guidelines of the Committee for

Jo

the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India were followed and prior permission was sought from the Institutional Animal Ethics Committee for conducting the study (SDCP/IAEC 02/2013-14). Dose selection

From earlier literature review, the dose of Resveratrol (RES) was found to be 20 mg/kg.9 The same dose was selected for the present study. It has been found that Naringin (NAR) with the dose of 5-15mg/kg through oral route can significantly alter the pharmacokinetic of co administered drug. In this study 15 mg/kg dose was selected for the present study. Experimental protocol

were termed as Normal control and Toxic control

ro

II received vehicle (1% Tween -80)

of

The animals were divided into four different treatment groups of eight animals each. Group I &

respectively Group III received Resveratrol (RES) (20 mg/kg) Group IV received Naringin Group V received combination of RES (20 mg/kg) and NAR (15 mg/kg).

-p

(NAR) (15 mg/kg)

re

All the treatments were given for thirty days through oral route. Experimental procedure

lP

Two hours after the last treatment, heart was isolated from the animals of different groups under ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia. Isolated heart was subjected

ur na

to modified Lagendroff apparatus as mentioned in earlier reported article. The isolated heart was perfused with Kreb-Henseleit (K‑H) solution, gassed with carbogen (95% O2 and 5% CO2) at 37°C, at a constant flow rate of 5 mL/min. The composition of K‑H solution was (mM) NaCl

Jo

118, KCl 4.7, NaHCO3 25, NaHPO4 1.0, MgSO4 7H2O 0.57, CaCl2 2.5, and glucose.11 The pH of K‑H solution was adjusted to 7.4 to avoid K‑H buffer acidosis that may occur after prolonged gassing with carbogen. The heart was allowed to equilibrate for 10 min and then regular recordings were taken for a perfusion period of 15 min. The heart was kept for equilibrium for a period of 10 min and then regular reading was taken for 15 min. The measurement of contractile force was determined with the help of force displacement transducer and recording was taken in

digital physiograph (model no-DI-2, INCO, Ambala city, India). After the initial preischemic period, the isolated heart was subjected to no flow global ischemia by stopping the flow of carbogenated K‑H solution for 15 min. Then, the heart was reperfused for a period of 15 min. The heart rate and force of contraction was determined during the pre‑ and post‑ischemic period, and the percentage recovery of heart rate and force of contraction was calculated. The

of

levels of creatine kinase‑MB (CK‑MB), creatine kinase‑N‑acetylcysteine (CK‑NAC), lactate dehydrogenase (LDH) activity were determined in perfusate collected during postischemic

ro

condition. Then half of the isolated hearts from all the groups were subjected for the preparation

-p

of heart tissue homogenate (HTH) using sucrose. Superoxide dismutase (SOD), catalase and thiobarbituric acid reactive substances (TBARS) were measured in HTH. For remaining heart

re

samples, histological study was carried out.11,1

lP

Histological analysis

Heart sections were prepared from the heart samples in each group, stained with hematoxylin

ur na

and eosin and change in histology were observed. The myocardial damage was determined by scoring method depending on the severity as follows, no change 0 score, mild 1 score (focal myocytes damage or small multifocal degeneration with a slight degree of inflammation), moderate 2 score (extensive myofibrillar degeneration) and marked 3 score (necrosis with diffuse

Jo

inflammation).2

Statistical Analysis Results are expressed as mean ± standard error. Statistical significance was assessed using oneway analysis of variance followed by Tukey–Karmer multiple comparison tests. P < 0.05 was considered statistically significant.

Effect of naringin on pharmacokinetics of resveratrol The rats were divided into two groups of six animals each (n = 6, each) Group I was treated with Resveratrol (RES) (20 mg/kg) and Group II was treated with RES along with Naringin (NAR) (15 mg/kg). Both the treatments were administered through oral route. NAR was administered 30 min prior to oral administration of RES. A blood sample (0.45-mL aliquot) was collected into

of

heparinized tubes via the femoral artery at 0, 0.1, 0.25, 0.5, 1, 2, 3, 4, 8, 12, and 24 h intervals. To prevent hypovolemia whole blood (approximately 1 mL) collected from untreated rats was

ro

infused via the femoral artery at 0.25, 1, 3, and 8 h, respectively, to replace blood loss due to

-p

blood sampling. The blood samples were centrifuged at 13,000 rpm for 5 minutes to separated plasma. 200-μL aliquot of separated plasma samples was stored at −40◦C for HPLC analysis.12,13

re

Results

lP

Effect on enzyme and biomarkers concentrations

All treatment groups Resveratrol (RES), Naringenin (NAR) and the combination group of RES

ur na

& NAR showed a significant restoration of enzyme and biomarker concentrations in the perfusate compared to the Ischemia reperfusion group (IRI). Among all the treatment groups the strongest effect was found for the combination group of RES and NAR. Effect on SOD, catalase and TBARS activities

Jo

All treatment groups (RES, NAR and RES + NAR) showed a reported significant increase in both SOD and catalase activities compared to the IRI positive control group. The group treated with the combination of RES + NAR showed a significant increase in both SOD and catalase compared to RES alone treated group (Table 2). Tension and heart rate

All treatment groups showed significant improvements in the percentage of recovery in terms of developed tension and heart rate compared to the IRI positive control group. Among all groups the combination group of RES and NAR was found to be the most effective group. Effect on the histopathology Under ischemia, the isolated hearts showed different pathological changes such as severe myocardial oedema, separation of fibers, and a loss of striation. The treated tissues ( RES, NAR,

of

RES + NAR) showed significant decreases in the histopathological scores compared to the ISO

ro

positive control. The tissue of the group treated with RES + NAR showed a significant reductions in the histopathological scores compared to the RES alone treated group (Table 2)

-p

(Figure 1).

re

Effect on pharmacokinetic parameters

The RES and NAR combination group showed significant increases in Cmax and AUCtotal.

lP

Cmax was remarkably high for RES in the presence of NAR indicating an enhanced extent of absorption. Vd was found to be the almost the same for both the groups. The combination group

ur na

showed a significant decrease in clearance and Tmax indicating a significant increase in the rate of absorption (Ka) compared to the RES alone treated group. (Table 3)

Jo

Discussion

The present study was designed to investigate the pharmacodynamic interaction of Resveratrol (RES) with Naringenin (NAR) against ischemia reperfusion injury (IRI) induced myocardial toxicity. More over the effect of a NAR treatment on the pharmacokinetic profile of RES was also investigated. Observed results suggest that RES and NAR alone and the combination group

of RES and NAR showed significant beneficial results. Apart from that, RES when combined with NAR indicated improved results compared to the RES alone treated group against IRI induced myocardial toxicity. From the observed results of the pharmacokinetic study it is also suggested that the co-administration of RES and NAR can improve the bioavailability of RES. Resveratrol exists in two isomers, the cis and trans diastereoisomers. The trans isoform is responsible for the biological activity.14 Different approaches already have been adopted to

of

tackle the problem of poor bioavailability associated with RES. Combining RES with

ro

bioenhancers has been found to be beneficial to improve the bioavailability. It has been found earlier that the combination of Resveratrol and piperine showed synergistic anti-depressant,

-p

cognitive activities. 15, 16Liposome encapsulation of the combination of curcumin and resveratrol

re

was reported to reduce prostate cancer occurrence.17 The combination of Resveratrol and quercetin showed synergistic anti-diabetic, cardioprotective, anti-inflammatory, hepatoprotective

lP

and antioxidant activities.18

For the induction of myocardial toxicity the model of ischemia reperfusion injury (IRI) by the

ur na

Langendorff setup was adopted. Ischemia, which is an acute or chronic form of cardiac disability, occurres due to the imbalance between the supply and demand of oxygen in blood. In the IRI model, global ischemia is associated with immediate biological alterations, such as an increase in intracellular Na+, which results in an increase in intracellular Ca2+ via Na+/Ca2+

Jo

exchange. Increased intracellular Ca2+ is made responsible for the formation of irreversible damage in the cardiac cell at the end of 15 min of global ischemia.11 Similar types of results were evident in this study. In the IRI control group significant decreases in the percentage of recovery in terms of developed tension and heart rate were observed. Data for the RES-treated group alone and for the combination group of RES and NAR revealed significant protection, however the

strongest effect was evident for the combination group of RES and NAR. Myocardial cells subjected to IRI are responsible for generation of tremendous amounts of oxygenfree radicals. 10 These released oxygen free radicals are extensively associated with the loss of membrane integrity, which causes the leaking of different biomarker enzymes from the cell. Free radical induced tissue damage is responsible for release of tissue antioxidants such as SOD and catalase resulted in unavailability of these tissue antioxidants for neutralization free radicals. As a result,

of

further accumulation of free radicals takes place, leading to exaggerated tissue damage.11,17

ro

In this study the IRI control group showed significant increases in cardiac biomarkers in the perfusate such as lactate dehydrgenase (LDH), creatinine kinase-MB (CKMB) and creatinine

-p

kinase-NAC (CKNAC). Simultaneously a decrease in superoxide dismutase (SOD), catalase and

re

an increase in thiobarbituric acid reactive substances (TBARS) in the heart tissue homogenates (HTH) were observed.

lP

Groups treated with RES and the combination of RES and NAR showed restored antioxidant and biomarker levels but the strongest effect was found to be for combination group of RES and

ur na

NAR. Cardio-protection associated with RES may be due to improved post ischemic ventricular function and reduced infarct size on isolated rat heart. The effect may be attributed to the peroxyl radical scavenging activity of the treatment. Moreover coronary vasodilatation and the reduction of myocardial oxygen consumption via up regulation of cGMP may be the contributory factors

Jo

for cardioprotective activity associated with RES.18 Poor bioavailability associated with RES is a major therapeutic challenge. This is due to poor absorption, rapid metabolism, and rapid systemic elimination. That NAR is able to increase the efficacy of RES may be due to the inhibition of the P-glycoprotein mediated efflux pump and microsomal enzyme CYP3A as shown earlier.8

The findings of the pharmacokinetic study support the potency of NAR to increase the bioavailability of RES. The study shows that the co administration of NAR leads to significant increases in Cmax, AUC total, an increase in the rate of absorption and the prolongation of elimination half lifeT1/2(h). Conclusion It can be concluded that Resveratrol and Naringin in combination have a cardio-protective

of

potential against ischemia reperfusion induced myocardial injury. Apart from that, the Naringin

ro

co-treatment leads to a significant improvement in bioavailability of Resveratrol. Future studies with combinations of different concentrations and ratios can be carried out to identify the most

Jo

ur na

lP

re

-p

suitable and synergistically acting formulation of the combination of Resveratrol and Naringin.

References 1. Chakraborty M, Bhattacharjee A, Kamath JV. Cardioprotective effect of curcumin and piperine combination against cyclophosphamide-induced cardiotoxicity. Indian J Pharmacol 2017 JanFeb;49(1):65-70. 2. Chakraborty M, Kamath JV, Bhattacharjee A. Pharmacodynamic Interaction of Green Tea

of

Extract with Hydrochlorothiazide against Cyclophosphamide-Induced Myocardial Damage. Toxicol Int 2014 May;21(2):196-202.

ro

3. Chih-Yang Huang, Wei-Jen Ting, Chih-Yang Huang, Jing-Yi Yang, Wan-Teng Lin. Resveratrol attenuated hydrogen peroxide-induced myocardial apoptosis by autophagic flux. Food Nutr Res

4.

-p

2016 May; 60: 10.

Jin-Fang Ge, Ya-Yun Xu, Gan Qin, Jiang-Qun Cheng, Fei- Hu Chen1. Resveratrol Ameliorates

re

the Anxiety- and Depression-Like Behavior of Subclinical Hypothyroidism Rat: Possible

May; 7: 44.

lP

Involvement of the HPT Axis, HPA Axis, and Wnt/β-Catenin Pathway. Front Endocrinol 2016

5. Chow SHH, Garland L, Hsu C, Donna R, Wade MV, Jessica CA. Miller et al. Resveratrol

ur na

Modulates Drug and Carcinogen Metabolizing Enzymes in a Healthy Volunteer Study. Cancer Prev Res 2010 Sep;3(9):1168-75.

6. Kesarwani K, Guptan R. Bioavailability enhancers of herbal origin: An overview. Asian Pac J Trop Biomed 2013 Apr; 3(4): 253–266.

Jo

7. Alam MA, Subhan N, Rahman MM, Uddin SJ, Reza HM, Sarker SD. Effect of Citrus Flavonoids, Naringin and Naringenin, on Metabolic Syndrome and Their Mechanisms of Action. Adv Nutr. 2014 Jul; 5(4): 404–417.

8. Choi JS, Han HK. Enhanced oral exposure of diltiazem by the concomitant use of naringin in rats. Int J Pharm. 2005 Nov 23;305(1-2):122-8.

9. Juan ME, Vinardell MP, Planas JM. The daily oral administration of high doses of transresveratrol to rats for 28 days is not harmful. J Nutr 2002 Feb;132(2):257-60.

10. Yeum CH, Choi JS. Effect of naringin pretreatment on bioavailability of verapamil in rabbits. Arch Pharm Res. 2006 Jan;29(1):102-7. 11. Chakraborty M, Kamath JV. Pharmacodynamic interaction of green tea extract with

of

hydrochlorothiazide against ischemia-reperfusion injury-induced myocardial infarction. J Adv Pharm Technol Res 2014 Jul;5(3):134-9.

ro

12. Huang H, Zhang J, Chen G, Lu Z, Wang X, Sha N et al. High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and

-p

resveratrol in rat plasma after oral administration of Smilax china extract. Biomed Chromatogr.

re

2008 Apr;22(4):421-7.

13. Chakraborty M, Ahmed MG, Bhattacharjee A. The potential for interaction of tolbutamide with

Dec;6(4):354-360.

lP

pomegranate juice against diabetic induced complications in rats. Integr Med Res. 2017

ur na

14. Shindikar A, Singh A, Nobre M, Kirolikar S. Curcumin and Resveratrol as Promising Natural Remedies with Nanomedicine Approach for the Effective Treatment of Triple Negative Breast Cancer. J Oncol 2016 May; 2016: 1-13. 15. Johnson JJ, Nihal M, Siddiqui IA, Scarlett CO, Bailey HH, Mukhtar H. Enhancing the

Jo

bioavailability of resveratrol by combining it with piperine. Mol Nutr Food Res. 2011 Aug;55(8):1169-76.

16. Huang W, Chen Z, Wang Q, Lin M, Wu S, Yan Q. Piperine potentiates the antidepressant-like effect of transresveratrol: involvement of monoaminergic system. Metab Brain Dis. 2013 Dec;28(4):585-95.

17. Narayanan NK, Nargi D, Randolph C, Narayanan BA. Liposome encapsulation of curcumin and resveratrol in combination reduces prostate cancer incidence in PTEN knockout mice. Int J Cancer. 2009 Jul 1;125(1):1-8. 18. Hung LM, Chen JK, Huang SS, Lee RS, Su MJ. Cardioprotective effect of resveratrol, a natural

Jo

ur na

lP

re

-p

ro

of

antioxidant derived from grapes. Cardiovasc Res. 2000 Aug 18;47(3):549-55.

Fig 1: Hematoxylin and eosin (H&E) stained sectioned of heart against Ischemia-reperfusion injury (IRI) in isolated rat heart.

1. b. (H&E) (x400) stained microscopic section of (RES) treated group (myofibrillar degeneration, inflammation); RES (Resveratrol)

Jo

ur na

1. a. (H&E) (x400) stained microscopic section of IRI control group (Necrotic cells with degeneration of myofibril, increased interstitial space, diffuse inflammation);

lP

re

-p

ro

of

Photographed at magnification 400X

1. c. (H&E) (x400) stained microscopic section of (RES + NAR) treated group (Small multifocal degeneration, slight inflammation, fall in interstitial space); RES (Resveratrol), NAR (Naringin)

Enzyme level in Perfusate (U/L) CKMB

LDH

-p

CKNAC 571.14 ± 12.86

487.10 ± 11.26

568.03 ± 5.18

RES

273.33 ± 14.53###

309.00 ± 4.26###

NAR

328.90 ±81.14###

358.00 ± 5.86###

RES +

156.23 ± 7.86###••

215.26 ± 8.53###••

ur na

NAR

AST

ALT 364.03 ± 5.18

659.03 ± 5.18

376.10 ± 3.28###

302.20 ± 4.28###

175.10 ± 4.28###

342.10 ± 3.28###

429.63 ± 5.15###

345.63 ± 5.01###

209.63 ± 6.01###

389.43 ± 6.01###

163.20 ± 5.81###••

82.21 ± 4.81###••

205.30 ± 4.81###••

152.30 ± 4.81###••

All values are mean ± SEM, n=8, ###P <0.001, compared to IRI control ••P<0.01, compared to RES. RES (Resveratrol), NAR (Naringin).

Jo

ALP

525.03 ± 4.18

lP

IRI

re

Treatment

ro

of

Table 1: Effect on marker enzymes in perfusate against Ischemia-reperfusion injury (IRI) induced myocardial toxicity

Table 2: Effect on antioxidant and percentage recovery of developed tension and heart rate against Ischemia-reperfusion

CATALSE

TBARS

(unit/mg)

(unit/mg)

(unit/mg)

HR

-p

Treatment

Percentage recovery

ro

SOD

of

injury (IRI) induced myocardial toxicity

DT

Histological Score

25.73 ± 1.13

23.44 ± 1.07

119.46 ± 2.99

44.19 ± 2.95

47.79 ± 2.82

2.85 ± 0.04

RES

45.23 ± 0.54###

49.63 ± 2.52###

57.33 ± 2.64###

69.20 ± 1.82###

79.46 ± 1.57###

0.93 ± 0.01###

NAR

35.32 ± 0.38###

45.33 ± 1.50###

65.42 ± 2.17###

64.13 ± 2.78###

62.27 ± 4.34###

1.15 ± 0.02###

RES +

63.73 ± 0.27###••

69.16 ± 1.29###••

lP

ur na

NAR

re

IRI

45.34 ± 3.72###••

86.50 ± 0.37###•• 89.49 ± 3.46###•• 0.67 ± 0.01###••

All values are mean ± SEM, n=8, ###P <0.001, compared to IRI control ••P<0.01, compared to RES. RES (Resveratrol), NAR (Naringin). HR: Heart Rate; DT:

Jo

Developed Tension

Table 3: Effect of Naringin on Pharmacokinetic parameters of Resveratrol

Parameters

RES

RES + NAR

4.17±0.39

12.86±1.23***

T max (min)

20.22±0.42

11.65±0.58**

AUC total (µg /mL/min)

13.43±0.33

23.49±1.59**

CL (L/kg/min)

1.48±0.04

0.76±0.01***

Vd (L/kg)

32.47±2.34

34.46±1.25

Ka (1/min)

0.22±0.03

0.91±0.05***

ro

of

Cmax (µg/mL)

-p

All values are mean±SEM, n=8, ** P<0.01, and *** P<0.001, when compared to RES. RES. RES (Resveratrol),

Jo

ur na

lP

re

NAR (Naringin).