Trans Isomerization of

Analytical Letters

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Characterization of the Cis/Trans Isomerization of Resveratrol by High-Performance Liquid Chromatography J. Flieger, M. Tatarczak-Michalewska & E. Blicharska To cite this article: J. Flieger, M. Tatarczak-Michalewska & E. Blicharska (2016): Characterization of the Cis/Trans Isomerization of Resveratrol by High-Performance Liquid Chromatography, Analytical Letters, DOI: 10.1080/00032719.2016.1178756 To link to this article: http://dx.doi.org/10.1080/00032719.2016.1178756

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Date: 31 May 2016, At: 21:30

Supertitle: Liquid Chromatography

Characterization of the Cis/Trans Isomerization of Resveratrol by High-performance Liquid Chromatography

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J. Flieger* M. Tatarczak-Michalewska E. Blicharska1 Department of Analytical Chemistry, Medical University of Lublin, Lublin, Poland *Address correspondence to J. Flieger. E-mail: [email protected] Received 04 February 2016; accepted 11 February 2016. Abstract trans-Resveratrol was evaluated in a stability study. Reverse-phase high-performance liquid chromatography with a diode array detector was employed for comparison of stressed and reference samples. Ethanolic-aqueous solutions were examined under variable conditions. The following parameters were investigated: the time of storage, exposure to sunlight for up to 30 days, temperature from 5 to 80°C, pH from 2.9 to 10.2, trans-resveratrol concentrations from 0.5 to 100 mg L1, and 3, 10, 20, and 50% ethanol. The cis/trans equilibrium position was significantly influenced by the resveratrol concentration. The trans-resveratrol isomer was stable only at solutions more concentrated than 25 mg L1 that were stable for 30 days in a refrigerator

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or at room temperature protected from light. Degradation of no more than 10% was observed at temperatures lower than 50°C and pH values lower 7.43. Keywords: cis/trans isomerization, high-performance liquid chromatography (HPLC), resveratrol

INTRODUCTION


Resveratrol (3,4,5'-trihydroxystilbene) is a polyphenol, a member of the stilben family, widely occurring in higher plants: grains, tea, fruit, berries, and plums. In 1976, it was discovered that its presence in plants is a response to environmental stress (e.g., fungal infection, injury, heavy metal ions, exposure to ultraviolet light) (Langcake and Pryce 1976). Resveratrol possesses cis and trans isomeric forms. Both isomers have different biological properties but trans-resveratrol exhibits more antioxidant and antimicrobial activity (Orallo 2006; Yáñez et al. 2006; Campos-Toimil et al. 2007; Sun et al. 2008; Almeida et al. 2009; Li et al. 2012). Besides large number of phenolic compounds, resveratrol was discovered in grapes Vitis vinifera and wine (Lamuela and Waterhouse 1993; de Quirós et al. 2007; Galeano-Díaz, DuránMerás, and Airado-Rodríguez 2007; Lopez, Dugo, and Mondello 2007; Mercolini et al. 2008; Gonçalves and Câmara 2011). It was confirmed that this key component of wines has the unique ability of preventing, among others, coronary heart disease which has been known as the "French paradox" (Renaud and de Lorgeril 1992). Epidemiological studies have firmly shown an inverse correlation between moderate red wine consumption (20 to 30 g per day) and the incidence of different disorders and overall human health (German and Walzem 2000; Pauwels and Kostkiewicz 2009). Recently it has been noted that, due to diverse physiological and biochemical properties, resveratrol may play a crucial protective role in much wider range of disorders 2

including cancer, diabetes, and even neurodegeneration in aging-related dysfunctions like Alzheimer's or Parkinson's diseases (Kopp 1998; Selkoe 2001; Tanzi and Bertram 2005; Baur and Sinclair 2006; Das and Maulik 2006; Harikumar and Aggarwal 2008; Saiko et al. 2008). A number of papers have described the evaluation of cis- and trans-resveratrol in wine produced in different countries (Perestrelo, Silva, and Câmara 2014). Quantification of resveratrol was carried out using gas chromatography coupled with mass spectrometry (Kanner


et al. 1994; Goldberg et al. 1995; Perestrelo, Silva, and Câmara 2014), capillary electrophoresis (Arce et al. 1998), and high-performance liquid chromatography with photodiode array (Lopez et al. 2001), electrochemical (Zhu et al. 2000) or fluorimetric detection (Vinas et al. 2000; Kolouchova-Hanzlikova et al. 2004). To achieve an appropriate peak shape and resolution of isomers, gradient elution in acids or acidic buffer has been commonly employed. The ionic strength was also enhanced by enrichment of the eluent with neutral ionic compounds. These additives were particularly necessary for methods utilizing electrochemical detection. Besides qualitative and quantitative assays, these methods ensure protection of resveratrol from ultraviolet light transforming the trans-isomer to the cis form (Galeano-Díaz, Durán-Merás, and Airado-Rodríguez 2007). Since ultraviolet light stimulates production of trans-resveratrol as a screen preventing destruction of genetic materials of plant cells mainly through stilbene synthase activation, the cis isomer is not produced in this process, although it has been detected in wine (Mercolini et al. 2008). The light sensitivity of cis-resveratrol has made it difficult to isolate and quantify. In most studies, cis-resveratrol is produced by the irradiation of a trans-resveratrol standard to induce partial isomeric conversion (Ali, Maltese, and Choi 2010). This study focuses on stability of trans-resveratrol under a variety of conditions that have not been previously characterized. Their influence on the isomerization of resveratrol was 3

characterized. The effect of the concentration of trans-reseveratrol, the solvent employed and its concentration, storage temperature, and time of irradiation were carefully investigated for the first time.

EXPERIMENTAL Reagents


trans-Resveratrol was purchased from Sigma (St Louis. MO, USA). cis-Resveratrol was obtained after exposure of a trans-resveratrol ethanolic solution diffused daylight with a radiation intensity of 500 lx, 12 h day length, temperatures of 22/20°C (day/night), and a relative humidity of 50%. HPLC grade methanol, ethanol, and acetonitrile were purchased from Merck (Darmstadt, Germany).

HPLC Apparatus Measurements were performed using a LaChrom HPLC Merck Hitachi (E.Merck, Darmstadt, Germany) equipped with diode array detector, column oven L-7350, and solvent degasser L-7612. The column (150 mm × 4.6 mm I.D.) was packed with 5-μm Zorbax EclipseC18 (pore size: 80 Å, surface area: 180 m2 g1) from Agilent Technologies (Santa Clara, CA, USA). Retention data were recorded at a flow-rate of 1 mL min1. The column was maintained at 20C ± 0.1. The mobile phase consisted of 25% acetonitrile/water and was passed through through a Nylon 66 membrane filter (0.45 μm) Whatman (Maidstone, England) with an appropriate apparatus.

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The detection of the compounds was established at the most sensitive wavelength based on spectra from 220 nm to 400 nm. The optimum wavelengths were 220 nm for cis-resveratrol and 310 nm for trans-resveratrol. Typical injection volumes were 20 μL corresponding to the volume of the Rheodyne injector loop. Duplicate injections were performed.

Determination of trans-Resveratrol For quantification, an external standard calibration curve was obtained from 0.5 to


100 mg L1. trans-Resveratrol was dissolved in 20% ethanol aqueous solution. The square regression coefficient of analytical curve was approached unity (R2 = 0.9998).

RESULTS AND DISCUSSION Optimization of the HPLC Conditions The HPLC analysis was performed by the use of an Agilent Zorbax Eclipse XDB C18 column with 25% acetonitrile/water as the mobile phase for efficient and selective separation of trans- and cis-resveratrol. This eluent composition was advantageous in comparison to a solvent containing methanol, providing superior detection limits and peak shapes. Appropriate chromatograms and spectra of both isomers recorded from 220 to 400 nm are shown in Figures 1 and 2, respectively. The detection limits were evaluated based on a signal-to-noise ratio of three. The limits of detection were 3.73 μg L1 for trans-resveratrol determined in 25% acetonitrile/water and 4.86 μg L1 for trans-resveratrol determined in 35% methanol/water. Acetonitrile-aqueous eluent was selected for subsequent experiments as optimum. This simple and rapid chromatography system provided efficient separation of the isomers with a separation

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factor (α) of 1.75 and a detection limit comparable with electrochemical detection, considered to be a selective and sensitive method.

HPLC Calibration To perform the quantitative determination of resveratrol, the linearity of response was examined for concentrations between 0.5 and 100 mg L1. The quantification of trans-resveratrol isomer was carried out by external standardization using the linear relationship between Downloaded by [La Trobe University] at 21:30 31 May 2016

concentration and peak area. A trans-resveratrol standard was dissolved in 20% (v/v) aqueous ethanolic solution to create conditions similar to wine. The calibration curve was given by y = 75245559(±663475)x + 13993.08(±3415) with n = 5, R2 = 0.9998, se = 51520, and F = 12862 where x is the concentration of trans-resveratrol in mg L1, y is the peak area, the numbers in parentheses denote the standard deviations of the individual regression coefficients, n is the number of measurements employed in deriving the regression equation, R is the correlation coefficient, se is the standard error of estimate of the equation, and F is the value of the Fisher test of significance.

Influence of the Concentration upon the Stability of trans-Resveratrol The trans-resveratrol isomer was transformed to the cis-isomer using ultraviolet light or following a few hours exposure to sunlight (Galeano-Díaz, Durán-Merás, and Airado-Rodríguez 2007). Under these conditions, approximately 80% of the trans-isomer was converted into the cis-isomer (Kolouchova-Hanzlikova et al. 2004). Some papers characterized the effectiveness of the light-induced isomerization with exposition time (Kolouchova-Hanzlikova et al. 2004). Unfortunately, inconsistent data are reported in the literature. In some cases, ten hours exposure of a trans-isomer standard to daylight provides this isomerization yield (Kolouchova-Hanzlikova 6

et al. 2004), but other papers reported that this processes requires up to 30 days (Trela and Waterhouse 1996). Figure 3 shows that concentration appears to be a key factor determining the effectiveness of the light-induced isomerization. trans-Resveratrol at concentrations from 5 to 100 mg L1 were prepared in 20% (v/v) aqueous ethanol and stored at ambient temperature, unprotected from sunlight. Figure 3 shows that the efficiency of the cis/trans isomerization process varied with concentration. While lower concentrations of trans-isomer, up to 5 mg L1,


required only 2 days exposure to the sunlight to be transformed into cis-isomer at 80% efficiency, higher concentrations of 25 and 50 mg L1 only achieved this isomerization efficiency following 10 days of exposition. trans-Resveratrol standards at 100 mg L1 unprotected from light isomerizes by approximately 80% cis-isomer over 30 days. To summarize, lower concentrations of standard trans-resveratrol may isomerize within a few hours while concentrated solutions of trans-resveratrol may require days for complete conversion to the cisisomer. Figure 4 confirms that the cis/trans equilibrium was reached after 3 h exposition to the light for dilute solutions (2.5 mg L1 in 20% ethanol). This concentration was selected to match the conditions in wine. Figure 5 shows that for higher concentrations of trans-resveratrol at 100 mg L1 in 20% ethanol, the equilibrium may require almost 10 days. The stability of standard trans-resveratrol solutions at concentrations from 0.5 to 100 mg L1 following storage for up to 1 month with protection from light at 4C and ambient temperature were evaluated. Figures 6 and 7 show that using ambient temperature conditions at concentrations 25 to 100 mg L1, the solutions were stable for up to 30 days, while more dilute trans-resveratrol solutions were converted into cis-isomer within hours. This process occurred with isomerization efficiencies from 10 to 70% within 15 days depending on the initial concentration. 7

At lower temperatures (Figure 7), higher concentrations of trans-resveratrol (25 to 100 mg L1) were stable for up to 30 days, but half of the trans-resveratrol in more dilute solution was converted into the cis-isomer following 15 days of storage time. These results show that trans-resveratrol is converted into the cis form, even with protection from the sunlight.

Influence of Temperature upon the Stability of trans-Resveratrol To date, the dependence of the trans-resveratrol isomerization has been characterized for Downloaded by [La Trobe University] at 21:30 31 May 2016

temperatures up to 50C (Kolouchova-Hanzlikova et al. 2004). 2 mg L1 trans-resveratrol was prepared in 20% (v/v) ethanol/water solutions and stored away from light. This concentration was selected because it represents the average value in wine. The trans-resveratrol isomerization was independent with temperature up to 50°C. However, at 75–80°C, the trans-resveratrol peak area was reduced to almost 30% after a few hours with an increase in the concentration of the cis-isomer. This observation may be utilized to produce more cis-resveratrol which is of interest because the biological effects of the cis-isomer has not been studied as extensively as the trans form. The repeated confirmed protective and anticancer activity of cis-isomer is strong justification for this study. (Harikumar and Aggarwal 2008; Li et al. 2012).

Influence of Ethanol Concentration The dependence of the trans-resveratrol isomerization on ethanol concentration was measured for 2 mg L1 solutions containing 3, 10, 20, or 50% of ethanol. The samples were analyzed after 24 h. The results in Figure 8 show that the trans-resveratrol isomerization was independent of the ethanol concentration across the examined range of concentrations.

Influence of pH

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trans-Resveratrol at a concentration of 2 mg L1 was stable in solution at pH values less than or equal to 6.12 when protected from light. The half-life for trans-resveratrol at pH 7.43 was 24 h (Figure 9). The trans-resveratrol peak completely disappeared following 1 day of storage with protection from sunlight at pH values from 7.43 to 10.2. These results show that resveratrol was completely decomposed at pH values above 7.43, not at pH10, as previously reported in the literature (Trela and Waterhouse 1996). For pH values from 2.99 to 6.12 with exposure to light,


50% of the trans-resveratrol was converted into cis-isomer after three hours and almost 90% after 30 days.

CONCLUSIONS In the present study, trans-resveratrol was determined using a C18 column and a methanol-aqueous mobile phase. Satisfactory retention, peak efficiency, symmetry, and optimum selectivity from the cis-isomer were achieved. HPLC was employed to characterize the cis/trans isomerization. The storage conditions were examined in detail for the first time. The results showed that the ethanol concentration did not affect the stability of the trans-resveratrol stability. The concentration of trans-resveratrol was a key factor in the light-induced isomerization. Hence, lower concentration of standard trans-resveratrol achieved disomerization equilibrium within a few hours whereas at concentrations exceeding 25 mg L1, trans-resveratrol may require up to a month to be converted into the cis-isomer. When solutions are protected from sunlight, concentrated solutions with pH values less than 7 were stable for up to 1 month at temperatures less than 50C. The decomposition of the trans form was less than 10% in each case.

References

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Figure 1. Typical chromatograms of resveratrol prepared at 2 mg L1 in 20% ethanol/water (a) directly after dissolution and (b) after 1 day storage at room temperature with exposure to light. The peak parameters for trans-resveratrol were a capacity factor of 3.55; an asymmetry factor of 1.52; and 7832 theoretical plates. The peak parameters for cis-resveratrol were a capacity factor of 6.2; an asymmetry factor of 1.59; and 6783 theoretical plates.

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Figure 2. Absorption spectra for trans-resveratrol and cis-resveratrol.

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Figure 3. Stability of trans-resveratrol standard solutions at various concentrations under ambient temperature, unprotected from room light. The samples containing standard transresveratrol were prepared in 20% (v/v) aqueous ethanol.

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Figure 4. Cis/trans isomerization equilibrium as a function of time with exposure to light at ambient temperature. The initial concentration of trans-resveratrol was 2.5 mg L1.

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Figure 5. Cis/trans isomerization equilibrium as a function of time with exposure to light at ambient temperature. The initial concentration of trans-resveratrol was 100 mg L1.

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Figure 6. Stability of trans-resveratrol standard solutions at various concentrations that were protected from sunlight and stored at ambient temperature.

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Figure 7. Stability of trans-resveratrol standard solutions at various concentrations that were protected from the sunlight and stored at 4°C.

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Figure 8. Stability of trans-resveratrol standard solutions at of 2 mg L1 containing variable ethanol concentrations. The samples were protected from sunlight.

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Figure 9. Stability of trans-resveratrol solutions at various pH values. The concentration of trans-resveratrol was 2 mg L1 in 20% ethanol/water. The samples were protected from sunlight (line graph) and unprotected from sunlight (bar graph).

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