Dose-Dependent Interaction of trans-Resveratrol with Biomembranes

Subscriber access provided by UNIV OF ARIZONA

Article

Dose Dependent Interaction of trans-Resveratrol with Biomembranes: Effects on Anti-Oxidant Property Stalin Selvaraj, Aarti Mohan, Shridhar Narayanan, Swaminathan Sethuraman, and Uma Maheswari Krishnan J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jan 2013 Downloaded from http://pubs.acs.org on January 8, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Dose Dependent Interaction of trans-Resveratrol with Biomembranes: Effects on Anti-Oxidant Property

Stalin Selvaraj1, Aarti Mohan1, Shridhar Narayanan2, Swaminathan Sethuraman1, Uma Maheswari Krishnan1*

1

Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology, SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.

2

Orchid Chemicals & Pharmaceuticals Ltd, Sozhiganallur, Chennai-600119, Tamil Nadu. India

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The present study investigates dose-dependent effects of trans-resveratrol on the membrane fluidity using planar lipid bilayer and liposome models. The complex admittance plots obtained for the lipid bilayer show that resveratrol, below 60 µM preferentially interacts with the polar head groups at the membrane-electrolyte interface leading to enhanced membrane admittance and vice versa at higher concentrations (> 60 µM). This was confirmed using solid-state 13C and 31

P NMR studies and membrane fluidization studies. The localization of resveratrol in the

membrane bilayer was found to alter the membrane rigidity, which resulted in a dose-dependent blebbing and lysis of erythrocytes. The protective effect of trans-resveratrol against DPPH also confirms that its localization in the hydrophobic region prevents lipid peroxidation. The cytotoxic effect of resveratrol on a breast cancer cell line also displays a progressive pattern, indicating possible correlation with its membrane rigidifying properties and localization in the lipid bilayer. Keywords: Resveratrol, planar lipid bilayer, anti-oxidant, membrane interaction

2


Page 2 of 49

Page 3 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.

INTRODUCTION

Trans-resveratrol is one of the most widely studied phytoalexins, naturally present in grapes, berries, peanuts, jackfruit, spruce, legumes, etc 1. Resveratrol has been proven to be a potent antioxidant anti-platelet

2, 3

, anti-cancer agent activity,

4, 5

and anti-inflammatory agent 6. It is also known to exhibit

estrogen-like

growth

promoting

effect,

anti-ageing

property,

immunomodulation, chemosensitization, and chemoprevention effects 1, 7. Moreover, the cardioprotective properties of resveratrol, well documented by several authors

8, 9, 10

are partly

responsible for the phenomenon known as “French Paradox” according to which consumption of wine is said to be beneficial to the heart owing to its rich polyphenol content though the alcohol in wine can produce adverse effects in other tissues 11.

Several diseases like cancer arthritis

14

12, 13

and neuro-degeneration

, inflammation

16

14

, cardiovascular disorders

15

, rheumatoid

have been shown to be related to excessive generation of

reactive oxygen species (ROS) in the physiological system. Excessive ROS can cause damage to DNA, proteins, cell membrane lipids and hence affect the basic functioning of the cell. Although there are inherent radical scavenging mechanisms comprising of the glutathione system, uric acid, vitamins C and E, antioxidant enzymes like superoxide dismutase, catalase, DNA repair enzymes etc., an antioxidant supplement can enhance the scavenging process 16, 17.

Resveratrol is a free radical scavenger and an antioxidant. It possesses three phenolic groups and acts as a free radical scavenger by transferring the proton from its phenolic group to the free radicals 18, 19, 20 (Figure 1). Resveratrol is known to scavenge OH• radicals produced as a result of Fenton’s reaction hence preventing DNA damage. It has also been shown to scavenge O2•-

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 49

(superoxide) radicals, other free radicals produced after exposure to Cr(VI) and also inhibit lipid peroxidation 2. In addition, it also maintains the levels of antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase 21.

The molecular mechanism of action of natural polyphenol antioxidants depends on their structure as well as their interaction with the lipid membranes

22-30

. The structure of the polyphenol

influences its intercalation into the inter-membrane space where it acts as a radical scavenger and protects the membrane from oxidative stress 31-32. Stilbenes preferentially interact with the polar head groups of the lipids while some of its derivatives are reported to penetrate into the inner membrane regions

33-34

. While one study showed that resveratrol interacts with the lipid head

group region of the bilayer 25, another reported that more than 90% of resveratrol permeated into the inter-membrane region of the bilayer 33. A concentration-dependent change was observed on the elasticity of the membrane due to the penetration of polyphenols into the lipid bilayer 35. It has been hypothesized that the polar and non-polar fragments of these compounds interact with the lipid head group and the non-polar acyl chains respectively

36

.

The lipid acyl chains

cooperatively bend to fill the free volumes created by the low concentrations of the intercalated molecules.

On the other hand, higher concentrations of these molecules cause them to

interdigitate resulting in the rigidity of the membrane 27, 36.

While some groups have reported the interaction of low concentrations of resveratrol, others have studied the effects of high concentrations of resveratrol on the lipid bilayers.

25, 27, 33, 34

.

However, the dose-dependent effects of resveratrol on the cell membrane fluidity, structure, and integrity have not been thoroughly investigated. The aim of the current work is to investigate the

4


Page 5 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dose-dependent interactions of resveratrol on the electrochemical parameters of lipid bilayers and its localization within the bilayer membrane in an effort to understand its implications on its anti-oxidant properties. The “painted” lipid bilayer has been used as a model for the cell membrane. The conductance and capacitance was measured across the bilayer in presence and absence of the stilbenoid, resulting in a clear description of the interaction of the polyphenol with the lipid membrane.

2.

RESULTS

2.1

Membrane interaction studies using planar lipid bilayers

The cyclic voltammograms of the lipid bilayers were recorded in the absence and presence of resveratrol between -70 to +70 mV at a scan rate of 0.01V/s. Figure 2 shows the cyclic voltammograms and admittance spectra for the lipid bilayer in the absence and presence of different concentrations of resveratrol.

The characteristic hysteresis pattern in the cyclic

voltammogram with low current flow and no redox peak indicates the capacitive nature for unmodified lipid bilayer (Figure 2A). The addition of resveratrol to the lipid bilayer increases both anodic and cathodic current in a dose-dependent manner without altering the capacitive nature of the lipid bilayer. This indicates that resveratrol may induce conducting pathways in the bilayer architecture with increasing concentrations.

The observed simple admittance plot for unmodified bilayers exhibits a semicircle pattern throughout the frequency ranges studied (10 – 105 Hz) that is indicative of the capacitive nature of lipid bilayers (Figure 2B). It is also observed that the center of the semicircle admittance plot lies on the real axis, which indicates that the planar lipid bilayer possesses a dielectric component

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with slight leakage

37

. The admittance values obtained at high frequencies can be attributed to

charge transfer dynamics while the admittance data obtained at the lower frequencies represent kinetics-controlled current 38. The diameter of the semicircle obtained for unmodified bilayers is high indicating the restricted charge transfer from one compartment to another through the bilayer. Any alteration in the semicircle plot on addition of resveratrol can be attributed to its interaction with the lipid bilayer components either in the surface or interior. In such a case, an increase in the diameter of admittance plot on addition of resveratrol may be mainly attributed to an increase in membrane resistance 37. Contribution to the admittance in planar lipid bilayers is due to four different domains in the lipid bilayer namely acyl chains, glycerol backbone, carbonyl and phosphocholine regions in the phosphatidyl choline molecules forming the lipid bilayer. The total impedance for planar lipid bilayers with respect to an equivalent RC circuit is given by the equation (1). Z( jω ) = Rs +

1 1

RCT

+ jωC dl

------------------------ (1).

where, Z= impedance, Rs = solution resistance, RCT = charge transfer resistance, ω = angular velocity, Cdl = double layer capacitance, j = ion flux. From the admittance plot, the charge transfer resistance (RCT) can be determined from the diameter of the semi-circle. The RCT values for the unmodified bilayer were found to be in the range of 1-2 MΩ. The observed variation in the bilayer RCT for different individual experiments possibly arises because of the heterogeneity in the lipid composition, dynamic nature of the Plateau-Gibbs layer and border leakage in the Perspex membrane support. Because of these technical difficulties, the data is presented in terms of relative alteration with respect to unmodified bilayers to attain uniformity in the results from the representative graph of four independent experiments.

6


Page 6 of 49

Page 7 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The slight distortion in the semi-circle pattern observed in all cases indicates a strong surface interaction between the constituents in the bath medium with the polar head groups of the lipid bilayer at the membrane-electrolyte interface.

The addition of different concentrations of

resveratrol alters the diameter and height of the semi-circle pattern in a dose-dependent manner (Figure 2B). A reduction in the diameter of the semi-circle up to the concentration of 60 µM of resveratrol is observed, following which an increase in the diameter of semi-circle was noted for further additions of resveratrol. Figure 3 shows the relative change in the charge transfer resistance of the bilayer on addition of different concentrations of resveratrol. It is evident that the RCT decrease until the addition of 60 µM of resveratrol implies an increase in the bilayer permeability and the values start to increase beyond resveratrol concentrations of 60 µM.

2.2

Membrane fluidization studies

To understand the effect of resveratrol on the fluidity of the lipid bilayer, membrane fluidization studies were carried out using curcumin-encapsulated liposomes. Figure 4 shows the effect of different concentrations of resveratrol on bilayer fluidity using curcumin as a fluorescent probe. Curcumin is a well-known fluorophore that predominantly localizes in the hydrophobic region of the bilayer.

It can be released from the hydrophobic region either due to increase in the

membrane fluidity or by displacement by a molecule competing for the same location inside the bilayer. It was observed that the addition of different concentrations (20-100 µM) of resveratrol gradually releases the curcumin from unilamellar vesicles in a dose-dependent manner. It was observed that about 17% of curcumin is released from unilamellar vesicles with respect to 100 µM of resveratrol compared to other concentrations (p

Dose-Dependent Interaction of trans-Resveratrol with Biomembranes

Recommend Documents