Improved Stability of trans-Resveratrol in Aqueous Solutions by

Jan 27, 2014 - NOOS s.r.l., v. Campello sul Clitunno 34, 00181 Roma, Italy. J. Agric. Food Chem. , 2014, 62 (7), pp 1520–1525. DOI: 10.1021/jf404155...
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Improved Stability of trans-Resveratrol in Aqueous Solutions by Carboxymethylated (1,3/1,6)-β‑D‑Glucan Antonio Francioso,† Paola Mastromarino,‡ Rossella Restignoli,§ Alberto Boffi,† Maria d’Erme,† and Luciana Mosca*,† †

Department of Biochemical Sciences and ‡Department of Public Health and Infectious Diseases, Section of Microbiology, Sapienza University, p.le Aldo Moro 5, 00185 Roma, Italy § NOOS s.r.l., v. Campello sul Clitunno 34, 00181 Roma, Italy ABSTRACT: Resveratrol is a polyphenolic compound endowed with multiple health benefits. However, its limited bioavailability and poor stability in solution hamper its use in pharmaceutical applications. Due to its low solubility in water, solvents such as ethanol and dimethyl sulfoxide are often used to dissolve resveratrol. However, these solvents have adverse effects on cultured cells or in vivo. The purpose of this study was to develop an aqueous liquid formulation of resveratrol in combination with a modified glucan, the carboxymethylated (1,3/1,6)-β-D-glucan (CM-glucan). The proposed liquid formulation conferred stability to resveratrol without affecting its antioxidant capability. Shelf-life measurements revealed that resveratrol in aqueous solution is degraded within a few weeks, due to spontaneous oxidation. In contrast, the combination with CM-glucan matrix exerted a strong stabilizing effect in aqueous medium and increased resveratrol stability up to 12 months at 25 °C. These data provide evidence of a stable resveratrol formulation in liquid suspensions and support the possible development of pharmaceutical applications of this association in biopharmaceutics and drug delivery. KEYWORDS: resveratrol, carboxymethylated glucan, stability, shelf life, antioxidant activity



INTRODUCTION Natural products, useful in treating and/or preventing various diseases, have been sought throughout the history of man. In recent years, due to the exponential increase in the economic burden for the development of new drugs, natural compounds are actively sought as potential therapeutic agents. Bioactive molecules occur in plants as secondary metabolites and as defense mechanisms against predation, herbivores, fungal attack, microbial invasion, UV irradiation, and viral infection. Polyphenols represent one of the most attractive families of bioactive molecules endowed with well-known multiple health beneficial effects. Resveratrol (trans-3,5,4′-trihydroxystilbene) is a nonflavonoid polyphenolic compound abundant in grapes, peanuts, and other foods that are commonly consumed as part of the human diet (Figure 1). It displays wide pharmacological

autoxidation and photosensitivity, features that limit its use in pharmaceutical preparations.3,4 Resveratrol has been utilized in the solid form, as capsules or dermatological preparations, in most clinical trials and experimental settings. However, the oral bioavailability of small molecule drugs is widely thought to be determined by the aqueous solubility, membrane permeability, and metabolic stability of the given compound.3 Some trials have used liquid formulations of resveratrol in ethanol or polyethylene glycol (PEG), which seriously limited clinical use due to the short shelf life. In recent years, many attempts have been made to stabilize and protect resveratrol from degradation, to increase its solubility in water in order to improve its bioavailability, to achieve a sustained release, and ultimately to target resveratrol to specific locations via multiparticulate forms and colloidal carriers, as excellently reviewed by Amri and co-workers.3 Various types of excipients, stabilizers, or carriers have been used, including cyclodextrins, pectinate beads, nanoparticles, or liposomes.5−11 However, in these studies the long-term shelf life or accelerated stability of liquid formulations of resveratrol complexed with high molecular weight polysaccharides have not been examined. There is only one paper that describes the encapsulation of resveratrol in yeast cells, with the aim of protecting resveratrol from photodegradation induced by UV light and conferring stability in the short term.12 The authors demonstrated that the encapsulation of resveratrol confers a higher solubility in water, slower photodegradation, and better

Figure 1. trans-Resveratrol chemical structure.

activities and is well-known for its antioxidant, anti-inflammatory, cardioprotective, neuroprotective, chemopreventive, and antiaging properties.1 Starting from the 1990s the number of scientific papers investigating resveratrol’s biological activity has increased sharply, denoting a strong interest of the scientific community in this molecule.2 However, despite its multiple well-documented beneficial effects on human health, resveratrol use as a drug is strongly limited by its poor solubility and low bioavailability and for its tendency to be unstable due to © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1520

September 19, 2013 January 23, 2014 January 27, 2014 January 27, 2014 dx.doi.org/10.1021/jf404155e | J. Agric. Food Chem. 2014, 62, 1520−1525

Journal of Agricultural and Food Chemistry

Article

Figure 2. Carboxymethylated-(1,3/1,6)-β-D-Glucan chemical structure.

storage stability under wet and light stresses within 10 days. Furthermore, the authors demonstrated that no chemical modification occurs on resveratrol, which retains unaltered antioxidant and physicochemical properties following encapsulation in yeasts. In this paper we aimed at studying a new association of resveratrol with a high molecular weight polysaccharide, the carboxymethylated (1,3/1,6)-β-D-glucan (CM-glucan), to be used in liquid formulations (Figure 2). β-Glucans are a class of natural polysaccharides that are endowed with well-known pharmacological activities and are increasingly utilized in pharmaceutical applications and in the food industry.13 The term β-glucans indicates noncellulosic polymers of β-glucose, with glycosidic bonds in position β(1→3) and with a significant portion of β(1→6)-bound glucose molecules. They are isolated from fungi, cereals, bacteria, or seaweeds.14−16 The most important quality of β-glucans, and the reason so much attention has been devoted to these compounds, is that they are typical biological response modifiers with pronounced immunomodulating activity, which confer anticancer, antioxidant, antiviral, and wound-healing capacities to these molecules.13,17 These biopolymers are also widely utilized in view of their structural and drug delivery properties for the nanoencapsulation of lipophilic pharmaceutical compounds and for poorly bioavailable drugs.18,19 However, despite their very interesting pharmaceutical and pharmacological profile, β-glucans have limited applications in liquid formulations due to their poor water solubility. Hence, for our aims we opted for the use of the carboxymethylated derivative of β-glucan, CM-glucan, showing a higher degree of solubility in water due to the presence of the easily ionizable carboxyl residues (Figure 2).20 This chemical modification does not affect the pharmaceutical and pharmacological properties of the product, which still exerts outstanding anticancer and antioxidant properties.21,22 The purpose of the present investigation was to evaluate the effect of CM-glucan on the long-term stability and solubility of resveratrol in aqueous solutions. To this purpose, we have developed a liquid formulation in which resveratrol is complexed with CM-glucan and resuspended in a buffered saline solution. The stability and antioxidant activity of resveratrol were further confirmed with spectroscopic methods.



MATERIALS AND METHODS

Reagents. trans-Resveratrol was purchased from Shanghai Novanat Co., Ltd. (Shanghai, China) and CM-glucan from Nutraceutica s.r.l. (Bologna, Italy). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and benzalkonium chloride were purchased from Sigma-Aldrich (Milan, Italy). Gradient grade solvents used for chromatographic analyses were purchased from Carlo Erba Reagents (Milan, Italy). All other reagents were analytical grade products from Sigma-Aldrich. Sample Preparation. Samples for the stability study were prepared by dissolving resveratrol at a final concentration of 0.05% in different aqueous solvents (0.9% NaCl or phosphate-buffered saline (PBS)) containing 0.1% CM-glucan. Sonication or extensive magnetic stirring was employed for evenly dispersing the preparations. The samples were prepared in triplicate and then split in two aliquots, one of which was maintained at 25 °C and the other at 40 °C for accelerated stability determinations. Samples were stored in the dark at over a relative humidity (RH) range of 40−45%. At different times, an aliquot of each sample was taken and analyzed for resveratrol content. In a similar manner, control solutions were prepared without CMglucan to evaluate the stability of 0.05% resveratrol, which was brought in solution by using surfactant agents, such as 0.5% PEG and 0.1% Tween-20. Benzalkonium chloride (0.04%) was employed as a preserving agent to inhibit microbial growth during long-term storage in stability analyses both in samples and in controls. For specific purposes, such as for spectrofluorometric measurements, samples were centrifuged at 12000g for 10 min before analysis. HPLC Determination and Quantification of Resveratrol. The HPLC consisted of a Waters apparatus equipped with a 600 pump and pump controller, a Waters autosampler model 717, a Symmetry C18 column (reversed phase, 3.9 × 150 mm, 5 μm particle size, with a 10 mm guard column of the same material matrix), and a UV−visible photodiode array detector model 2996. Solvent A was 10% acetic acid in water, and solvent B was acetonitrile. The elution was performed in isocratic conditions at a flow rate of 1 mL/min at 25 °C with 80% A and 20% B. Resveratrol had a retention time of ≃7.0 min, and its quantitation was performed by automatic peak area integration using dedicated software (Millennium,32 Waters). Before the analysis, each sample was resuspended, diluted in mobile phase 1:10, and filtered onto 0.2 μm filters, and then 50 μL was injected onto the column. Stock solutions of resveratrol were freshly prepared immediately before use in DMSO at a concentration of 20 mM. Working solutions were prepared by diluting stock resveratrol solutions to the desired concentration in appropriate solvents. Calibration curves in the range of 1−12 nmol were prepared by replicate autosampler injections of different volumes of a standard solution of 0.219 mM resveratrol. The curves (six data points, in duplicate) were linear with R2 values of ≃1.00. Peak detection was carried out at 306 nm, and the purity of the peak was checked with the diode array detector to verify the characteristic absorption spectrum of the polyphenol. The limit of 1521

dx.doi.org/10.1021/jf404155e | J. Agric. Food Chem. 2014, 62, 1520−1525

Journal of Agricultural and Food Chemistry

Article

Figure 3. Stability analyses of the resveratrol/CM-glucan suspension. (A) Long-term stability at 25 °C: 0.05% resveratrol was resuspended in aqueous solutions in the presence (white squares) or in the absence (black squares) of 0.1% CM-glucan. Data points at 9 and 12 months are significantly different between resveratrol and resveratrol with CM-glucan (∗∗ = p < 0.01). (B) Accelerated stability at 40 °C: 0.05% resveratrol was resuspended in aqueous solutions in the presence (white squares) or in the absence (black squares) of 0.1% CM-glucan. Data points at 2 and 3 months are significantly different between resveratrol and resveratrol with CM-glucan (∗∗ = p < 0.01). detection (4 pmol) was calculated as the analyte concentration giving a signal 3 times higher than the baseline value. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectra were measured with a Nicolet Magna 760 (Thermo Scientific, Waltham, MA, USA) infrared spectrometer equipped with an ARK attenuated total reflectance device. The internal reflection element was ZnSe. Spectra were recorded at 4 cm−1 resolution with a DTGS detector. Samples were evaporated onto the ATR crystal by N2 stream. The IR spectrum of the preserving agent utilized in the long-term measurements (i.e., benzalkonium chloride) was subtracted from spectrum of each sample analyzed. Spectrofluorometric Analyses. Fluorescence emission spectra were recorded on a SPEX-Fluoromax spectrofluorometer (Horiba Scientific, Horiba Ltd., Kyoto, Japan) from 360 to 500 nm with excitation at 350 nm using a 0.1 cm path length quartz cuvette, under continuous stirring. The excitation and emission slits were both set to 5 nm, and scan speed was 120 nm/min. Free Radical Scavenging Assay. Samples were tested for their free radical scavenging capacity by using the DPPH• test. DPPH•, a stable nitrogen-centered free radical, shows an absorption maximum at 517 nm, which decreases in the presence of H-donor molecules. Ten microliters of each sample was added to 1 mL of 30 μM DPPH• in ethanol, vortexed, and left for 15 min in the dark. Absorbance was measured at 517 nm on a Hitachi U-2000 spectrophotometer (Hitachi Ltd., Tokyo, Japan). The DPPH radical scavenging activity was estimated as described by Shi et al.12 Statistical Analyses. Experiments were repeated at least in triplicate, and all of the results are expressed as the mean value ± standard deviation (SD). Statistical comparison between groups was made using Student’s t test. p values