gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Analysis of resveratrol incorporated in liposomes containing vitamin C and E

Meeting Contribution

  • M. Atanackovic - Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Serbia
  • J. Cvejic - Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Serbia
  • D. Güzel - Institute of Physiology, University of Tubingen, Germany
  • V. Kenchappa - Institute of Physiology, University of Tubingen, Germany
  • H. Heinle - Institute of Physiology, University of Tubingen, Germany
  • corresponding author L. Gojkovic-Bukarica - Department of Pharmacology and Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Belgrade, Serbia

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf11

DOI: 10.3205/10dgaf11, URN: urn:nbn:de:0183-10dgaf110

Published: March 23, 2011

© 2011 Atanackovic et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

Resveratrol, a polyphenol mainly present in grapes and red wine, demonstrated interesting biomedical properties for its cardio protective action due to inhibition of the oxidation of low-density lipoprotein (LDL) and of platelet aggregation, inhibitory effects on cancer promotion and propagation and anti-inflammatory activities. These potential therapeutic and prophylactic applications are limited by the low bioavailability caused by its physical chemical properties. Additionally, resveratrol has low water solubility and stability making its clinical success a formidable technological and medical challenge. Liposomal formulations have been proposed as a means of improving the therapeutic efficacy of poorly bioavailable drugs. The aim of the study was to determine amount of resveratrol loaded into liposomes in presence of vitamin C and vitamin E. Liposome preparations containing pure resveratrol, resveratrol with vitamin C and resveratrol with vitamin E were prepared using thin film hydration method. Resveratrol content was analyzed using HPLC with UV/DAD detection. Resveratrol was detected in all analyzed samples. Results showed that increase in amount of resveratrol used for preparation of liposomes, lead to increase of amount of resveratrol loaded into liposomes. Also, addition of natural antioxidants such as vitamin C and vitamin E increased incorporated amount of resveratrol.


Introduction

Trans-resveratrol (trans-3,5,4’-trihydroxystilbene) is a naturally occurring phytoalexin isolated for the first time from the root of Veratrum grandiflorum in 1940. Since then, it has been isolated and indentified in over 70 different plant species [1]. It is used in traditional medicine for treatment of different bacterial and fungal infections, inflammations, dermatitis, hearth, liver and blood vessels diseases. Grapes and related products are considered one of the most important dietary sources of resveratrol. It is present in leaves, grape skins and wine [2], [3]. In recent years, resveratrol has become interesting because of its biological activity. Results of many different publications showed that resveratrol possesses numerous biological properties which include inhibition of lipid peroxidation, prostaglandine synthesis and thrombocyte aggregation, free radical quenching, vasodilatation, antineoplastic and estrogenic activity [2], [4], [5], [6], [7]. Still, resveratrols’ physical properties are limiting factor for its application. First of all, it has low water solubility (<0.001 mol/l) and converts to less active cis-form when irradiated with UV light [8]. In buffer solutions, its solubility is still low, but it increases with increase of pH [9]. Most of the experiments use resveratrol in its free form, dissolved in organic solvent which are inappropriate for humans. Some suggested ways for improvement of resveratrol low solubility include micellar and liposomal solubilization [8], [10], [11]. Liposomes are optimal carriers for the entrapment and cellular delivery of drugs because they can incorporate a lipophilic drug within the membrane bilayers and protect it from light and other degradative processes. The aim of the study was to determine amount of resveratrol loaded into liposomes in presence of vitamin C and vitamin E. Since vitamins C and E are natural antioxidants, they could improve quality of obtained preparations, and potentially increase resveratrol content in liposomes.


Methods

Preparation of liposomes: Liposomes containing resveratrol (pure, and in combination with vitamin C and E) were prepared by using thin film hydration method. Lipid phase consisting of appropriate weight of Phospholipon 90H, cholesterol, di-cetyl phosphate dissolved in Chloroform:Methanol (2:1) was transfered into dry round bottom flask. Organic solvents were evaporated using vacuum evaporator and thin lipid film was obtained. Lipid film was hydrated with 100 ml of water (double distilled) and appropriate quantity of resveratrol and vitamin C or E was added. Preparation was rotated without vacuum for one hour.

Quantification of resveratrol: Liposomal samples were centrifugated (40000 rpm, 50 minutes, +6°C). After centrifugation, water supernatants were decanted. The obtained pellets were consecutively extracted with methanol. More specifically, 5 ml of methanol was added on pellet, vortexed, centrifugated (35 min, 3500 rpm). Methanol supernatant was decanted, filtered through 0,45 µm filter and analyzed using High Performance Liquid Chromatography (HPLC). This procedure was repeated until the extraction was completed and the pellet was dissolved. The quantities of resveratrol in pellets (liposomal fractions) are presented Table 1 [Tab. 1].

Resveratrol was determined using HPLC Agilent 1100 series with binary pump, autosampler and a diode array UV/Vis detection using modified method proposed by Trela and Waterhouse [12]. Quantification was accomplished by comparison with a standard response curve prepared from solutions of resveratrol in methanol. Correlation coefficient for resveratrol calibration curve was 0.99983.


Discussion

Resveratrol was detected in all analyzed samples. Results showed that increase in amount of resveratrol used for preparation of liposomes, lead to increase of amount of resveratrol loaded into liposomes (Table 1 [Tab. 1]). More specifically, increase of added resveratrol from 100 µM to 150 µM increases incorporation efficiency for 29.21% in preparations containing pure resveratrol. For preparations with resveratrol and vitamin C or E, this enlargement of incorporation is 29.65% and 21.23% respectively. Also, addition of natural antioxidants such as vitamin C and vitamin E increased incorporated amount of resveratrol. Preparation with pure resveratrol (100 µM) had incorporation efficiency 13.15%, while these values for those with vitamin C and E were 35.5% and 53.47%. Presence of vitamin E in liposomal preparation showed the biggest effect on resveratrols’ incorporation, and preparation made with addition of 200 µM of resveratrol and vitamin E had the highest amount of resveratrol (Figure 1 [Fig. 1]).

Obtained results suggest that resveratrol incorporation in liposomes might be dose dependent and that presence of natural antioxidants improves incorporation probably due to enhancement of systems’ stability. These conclusions could help development of safe and effective resveratrol preparation for topical delivery.


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