Article pubs.acs.org/JAFC
β‑Cyclodextrin-Assisted Extraction of Polyphenols from Vine Shoot Cultivars Hiba N. Rajha,*,†,‡ Stephanie Chacar,† Charbel Afif,† Eugene Vorobiev,‡ Nicolas Louka,† and Richard G. Maroun† †
Centre d’Analyses et de Recherche, Unité de Recherche Technologie et Valorisation Alimentaire (UR TVA), Faculté des Sciences, Université Saint-Joseph, B.P. 11-514 Riad El Solh, Beirut 1107 2050, Lebanon ‡ Sorbonne Universités, Université de Technologie de Compiègne, Laboratoire Transformations Intégrées de la Matière Renouvelable (UTC/ESCOM, EA 4297 TIMR), Centre de Recherche Royallieu, CS 60 319, Compiègne 60 203 CEDEX, France ABSTRACT: This work optimized the β-cyclodextrin (β-CD)-assisted extraction process of polyphenols from vine shoots. The efficiency of β-CD was compared to that of ethanol in terms of the quantity and antioxidant capacity (AC) of the extracted polyphenols. Response surface methodology permitted the optimization of the β-CD concentration, time, and temperature. The optimal polyphenol content (PC) [5.8 mg of gallic acid equivalent (GAE)/g of dry matter (DM)] and AC [3146 micromolar trolox equivalent per milliliter (μMTE)] were initially obtained with Syrah cultivar after an extraction of 48 h at 66.6 °C with a 37.7 mg/mL aqueous β-CD solvent. The same PC (5.8 mg of GAE/g of DM) was reached with 50% ethanol/water solvent after 1.65 h. However, a lower AC was found with ethanol (2000 μMTE) compared to β-CD. A comparison of the PC and AC of four different vine shoot cultivars was realized. Our results clearly show the capacity of β-CD to amplify polyphenol extraction from vine shoots. KEYWORDS: polyphenols, β-cyclodextrin, response surface methodology, high-performance liquid chromatography, vine shoots
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conducted on the β-CD-assisted extraction of polyphenols from vine shoots. The current study was carried out with the aim of defining the optimal β-CD concentration, time, and temperature for the recovery of polyphenols from vine shoots, comparing the efficiency of β-CD to organic solvent extraction, studying polyphenol diversity and antioxidant activity of the extracts, and comparing polyphenol contents of four diffirent vine shoot cultivars.
INTRODUCTION Viticulture is an agricultural activity important worldwide because of its valuable economic and health benefits.1 For highquality grape production, cultural practices are annually performed in vineyards, such as pruning. The latter affects vine form, shape, and size. It also influences the production of grapes in terms of quantity and quality. Vine shoots are pruning wastes thrown or burned in the field for their disposal, thus causing environmental problems.2 Vine shoots are value-added industrial products regarding their content in high-value phytochemicals, such as polyphenols.3 The extraction and purification processes are primordial steps for the extraction of biomolecules from natural sources. They permit the utilization of bioactive compounds in pharmaceuticals, nutraceuticals, cosmetics, etc.4 Organic solvents are commonly used in the conventional extraction processes for polyphenol recovery from plants.5 Alternative environmentally friendly extraction processes, such as high-voltage electrical discharges, supercritical fluid extraction, pulsed electric fields, ultrasounds, etc., were conceived to diminish organic solvent use and their toxicological and safety concerns.6−8 However, the availability of these methods is limited by their requirement of advanced and costly equipment. Cyclodextrins (CDs) are cyclic oligosaccharides generally recognized as safe (GRAS), widely used in food industries for their capacity to form inclusion complexes with bioactive compounds, increasing thus their stability, solubility, and bioavailability.9 The use of aqueous βcyclodextrin (β-CD) was studied for the recovery of polyphenols from grapes and their pomace.10 Extraction parameters, such as time, temperature, and solvent mixture, are likely to influence the process, having possible interactions among the variables.3,4 To our knowledge, no research has been © 2015 American Chemical Society
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MATERIALS AND METHODS
Reagents. All reagents were of analytical grade. Sodium carbonate (Fluka, Buchs, Switzerland), Folin’s phenol reagent (Scott Science, U.K.), and gallic acid (GA, Sigma Chemical Co., St. Louis, MO) were used in the Folin−Ciocalteu assay. 2,2-Diphenyl-picrylhydrazyl (DPPH) radical and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (Trolox) were purchased from Sigma Chemical Co. (St. Louis, MO). The high-performance liquid chromatography (HPLC) solvents were acetonitrile (Merck, Darmstadt, Germany), formic acid (Scharlau, Barcelona, Spain), and water (Scharlau, Barcelona, Spain) of HPLC ultra gradient grade. Phenolic compound standards of GA, protocatechuic acid (PA), ferulic acid (FA), transcinnamic acid (TCA), 4-hydroxybenzoic acid (HBA), and resveratrol (R) were from Sigma Chemical Co. (St. Louis, MO). Raw Material. Vitis vinifera var. Syrah vine shoots were used for the optimization process of polyphenol extraction by response surface methodology (RSM). However, other varieties were also studied: Tempranillo, Cabernet Sauvignon, and Cabernet Franc. Vine shoots were pruned in 2013 and stored at room temperature until use. Vine Received: Revised: Accepted: Published: 3387
February 4, 2015 March 18, 2015 March 18, 2015 March 18, 2015 DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
Article
Journal of Agricultural and Food Chemistry
Table 1. RSM Central Composite Design of Three Factors and Five Levels and the Experimental Responses (PC and AC) of βCD-Assisted Extraction from Vine Shoots run factorial design
center points
star points
β-CD concentration, β (mg/mL) 20 40 20 40 20 40 20 40 30 30 30 30 30 30 13.82 46.82 30 30 30 30
(−1) (+1) (−1) (+1) (−1) (+1) (−1) (+1) (0) (0) (0) (0) (0) (0) (−α) (+α) (0) (0) (0) (0)
time, t (h) 1.5 1.5 5 5 1.5 1.5 5 5 3.25 3.25 3.25 3.25 3.25 3.25 3.25 3.25 0.30 6.19 3.25 3.25
temperature, T (°C)
(−1) (−1) (+1) (+1) (−1) (−1) (+1) (+1) (0) (0) (0) (0) (0) (0) (0) (0) (−α) (+α) (0) (0)
40 40 40 40 60 60 60 60 50 50 50 50 50 50 50 50 50 50 33.18 66.82
PC (mg of GAE/g of DM)
AC (μMTE)
0.43 0.69 0.93 0.96 1.43 1.65 1.75 2.48 1.04 1.29 1.14 1.38 1.20 1.15 0.85 1.05 0.36 2.02 0.56 1.76
800.96 1190.57 1250.79 1130.34 1340.51 1710.88 1540.44 1740.37 1220.06 1390.49 1370.00 1270.04 1240.55 1410.99 1120.72 1390.49 1090.60 1460.97 940.66 1740.37
(−1) (−1) (−1) (−1) (+1) (+1) (+1) (+1) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (−α) (+α)
0.1% (v/v) formic acid in water and mobile phase B consisting of 100% acetonitrile were used. The elution profile had the following proportions (v/v) of solvent B: 5 min, 15−20%; 45 min, 20−44.5%; 1 min, 100%; kept at 100% of B for 9 min; and followed by 5 min of stabilization at 15%. Spectrophotometric detection was performed at 280 nm. Experimental Design. The optimization of the β-CD-assisted extraction process of polyphenol from vine shoots was conducted by RSM. The independent variables were β-CD concentration, time, and temperature. They were coded at five levels (−α, −1, 0, 1, and +α), in a central composite design (23 + star) of 20 experiments, including six center points. Table 1 (first four columns) shows the central composite design with the range of variables and their coded values. Two responses were studied: PC and AC. Data analyses were carried out using STATGRAPHICS Plus 4.0 for Windows and were fitted to a second-degree regression equation of the form
shoots were cut into cylinders of 1 cm height and a mean diameter of 5 mm. Dry Matter (DM) Content. Vine shoots were dried in a convictive oven at 105 °C for 48 h for the measurement of the DM content. The DM of Syrah, Cabernet Sauvignon, Cabernet Franc, and Tempranillo vine shoots was found to be 60, 65, 57, and 69%, respectively. Solid−Liquid Extraction. The solid−liquid extraction process of polyphenols from cylindrical vine shoots was performed with a solid/ liquid ratio of 1:20 (w/v).8 After β-CD aqueous extraction at different concentration, time, and temperature combinations, extracts were centrifuged at 5000 rpm for 15 min. β-CD aqueous concentrations were prepared by dissolving the required weight of β-CD in the specific water volume in a 50 °C water bath under stirring. Total Polyphenol Content (PC). The total PC was determined by the Folin−Ciocalteu colorimetric assay.11 A total of 1 mL of Folin− Ciocalteu reagent (10-fold diluted) was mixed to 200 μL of extract. Then, 800 μL of sodium carbonate (75 g/L) was added. The mixture was incubated for 10 min at 60 °C and then 10 min at room temperature. The absorbance was measured at 750 nm by the ultraviolet/visible (UV/vis) spectrophotometer (UV- 9200, BioTECH Engineering Management, U.K.). The calibration curve was drawn with GA. PC was expressed as milligrams of gallic acid equivalent (GAE) per gram of DM. Antioxidant Capacity (AC). The AC of vine shoot extracts was measured by means of the DPPH assay.12 A total of 50 μL of vine shoot extracts or standard (Trolox) was added to 1.45 mL of DPPH radical (0.06 mM). After an incubation of 30 min at room temperature, the absorbance was measured at 515 nm. The DPPH value of the extracts was expressed in micromolar Trolox equivalent per milliliter (μMTE). HPLC. The HPLC analyses were conducted for the identification and quantification of polyphenols from vine shoot extracts.13 The HPLC−diode array detection (DAD) analyses of polyphenols were carried out with a HPLC system (Waters Alliance, Milford, MA) equipped with a quaternary Waters e2695 pump. A UV−vis photodiode array spectrophotometer Waters 2998 was used, with the control system and data collection Empower 3 software. HPLC analyses of polyphenols were carried out on a Discovery C18, 5 μm, 250 × 4.6 mm, column (Supleco, Bellefonte, PA) with a C18, Supelguard Discovery 18, 20 × 4 mm, 5 μm, precolumn (Supelco, Bellefonte, PA) at 30 °C. Separation of 100 μL of cane extract was performed at a flow rate of 0.5 mL min−1. Mobile phase A consisting of
3
Y = a0 +
3
2
3
∑ anXn+ ∑ annXn2+ ∑ ∑ n=1
n=1
n=1 m=n+1
anmX nX m
(1)
where Y is the response, a0, an, am, and anm are the constant, linear, quadratic, and two factor interaction coefficients, respectively. Statistical Analysis. Each extraction process was repeated on three different vine shoot samples. Means and standard deviations (error bars in the figures) of data were calculated. Variance analyses (ANOVA) and least significant difference (LSD) test were performed by STATGRAPHICS Centurion XV (StatPoint Technologies, Inc.).
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RESULTS AND DISCUSSION Experimental Design and Statistics. The effect of time, temperature, and β-CD concentration (β) on polyphenol extraction from vine shoots was studied. The RSM design was conceived taking into consideration the solubility limitation of β-CD at concentrations above 50 mg/mL10 and temperature limitation because of the possible heat degradation/oxidation of polyphenols. RSM is a statistical and mathematical method employed for the development, improvement, and optimization of extraction processes.14 Table 1 shows the experimental design and combinations of the coded variables with their responses (PC and AC). Total PC ranged from 0.36 to 2.48 mg 3388
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
Article
− 4.893βt
(3)
Comparing the significance of the studied parameters, it can be noticed that the effect of time was more significant for extracting higher PCs but had less effect on their AC. This might be due to the oxidation phenomena that is likely to occur when subjecting polyphenols to high temperatures (>50 °C) for long durations. In contrast, the β-CD concentration played a more important role for conserving the AC of polyphenols than enhancing their quantity (PC) (Figures 1a and 2a). Multiple Response Optimization. Figure 3 shows the overlay plots for PC and AC at 66.6 °C (optimal temperature). As marked by the black dot, the optimal conditions for the maximization of PC are 46.8 mg/mL β-CD for 6.2 h, while those for AC are 46.8 mg/mL β-CD for 0.7 h. These are the quantitative and qualitative optimums, respectively. However, for industrial applications of vine-shoot-derived polyphenols, it 3389
2
94.6 88.7
AC = −812.072 + 25.3102β + 198.619t + 24.1344T
PC = 0.161426 − 0.0072592β − 0.0819191t − 0.0149214T − 0.000469537β + 0.00206658βt + 0.000806414βT + 0.0122602t + 0.00273404tT + 0.000280171T AC = −48.9255 + 12.2366β + 262.684t − 2.86155T − 0.106453β2 − 4.893βt + 0.389216βT − 0.959765t2 − 1.15653tT + 0.190782T2
The positive effect of t, T, and β was expected. The temperature augments molecular diffusion; it decreases solvent viscosity, enhancing the solute solubilization and diminishing surface tension.15 CDs are likely to form, within their lipophilic cavities and through non-covalent forces, inclusion complexes with hydrophobic polyphenols, increasing thus their water solubility.16,17 Time comes immediately after the temperature factor in terms of its significance (Figure 1a). It plays an important role in the diffusion process, which is a relatively slow operation of three steps: (1) internal diffusion of the solvent, (2) solubilization of the target molecule, and (3) diffusion of the solubilized molecules to the surface of the particle.18 The significant positive effect of time is therefore expected for polyphenol extraction from vine shoots. Effect of the Time, Temperature, and β-CD Concentration on AC. Similar to PC, AC is positively affected by the time, temperature, and β-CD concentration (Figure 2a). As shown in Figure 2b, the increase in the β-CD concentration and time of the extraction process linearly augments AC up to 1740.37 μMTE. The temporary locking of polyphenols within CD cavities stabilizes host molecules from oxidation, light, and heat degradation,16 which probably gave linear positive effects for t, T, and β. Excluding insignificant parameters, the regression equation becomes
regression coefficient, R2 (%)
(2)
2
PC = −2.0886 + 0.0116057β + 0.196472t + 0.0461738T
2
Table 2. Second-Order Polynomial Equations for PC and AC, Including Insignificant Parameters, with R2 Also Shown for the Equations
of GAE/g of DM, while AC was between 800.96 and 1740.37 μMTE. The relation between the responses and the experimental parameters (β, t, and T) was demonstrated by statistical analyses to fit second-order polynomial equations shown in Table 2. The analyses of the predicted values, calculated from the regression equations, showed a high correlation with experimental values and resulted in the calculation of the coefficients of determination (R2), which were shown to have high levels of adequacy (Table 2). Effect of the Time, Temperature, and β-CD Concentration on PC. According to the pareto chart (Figure 1a) and the response surface plot (Figure 1b), time, temperature and βCD concentration have linear positive effects on polyphenol extraction from vine shoots. No quadratic effects were observed nor interaction between the parameters. Excluding insignificant parameters, the regression equation becomes
regression equation (including insignificant parameters)
Journal of Agricultural and Food Chemistry
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
Article
Journal of Agricultural and Food Chemistry
Figure 1. (a) Standardized pareto chart and (b) estimated response surface for PC.
Figure 2. (a) Standardized pareto chart and (b) estimated response surface for AC.
Experimental results were compared to those predicted by the model. As shown in Table 3, the difference of the values Table 3. Predicted and Experimental Results of PC and AC at the Multiple Optimum Conditions (66.6 °C, 4.4 h, and 37.7 mg/mL β-CD) multiple optimum
predicted
experimental
mean absolute error
PC (mg of GAE/g of DM) AC (μMTE)
2.5 1881.3
2.45 1864
0.14 48.7
obtained by experimentation and model prediction are within the range of the mean absolute error, suggesting a good agreement of the model with experimental results. Consequently, 66.6 °C and 37.7 mg/mL aqueous CD seem to be adequate optimal conditions for the maximization of both PC and AC. In the studied domain, all of the quadratatic effects (t2, T2, and β2) of the parameters were insignificant. Time, temperature, and β-CD concentration had positive linear impacts on PC and AC (Figures 1a and 2a). The β-CD concentration in water is an experimental condition limited by its solubility and economic impact. Similarly, the temperature elevation is governed by the oxidation and/or degradation phenomena that it causes to polyphenols and also by its energy consumption that affects the overall cost of the process. Because of all of the aforementioned reasons, the linear extrapolation of the time parameter was possible because it was suspected to further increase PC and AC. Figure 4 shows PC and AC as a function of long periods of time (from 5 to 54 h) at 66.6 °C and 37.7 mg/mL β-CD. The highest PC and AC, 5.8 mg of GAE/g of DM and 3147 μMTE, respectively, were reached after 48 h of extraction. Beyond this time, PC
Figure 3. Superposition plots of PC and AC. The optimums for (●) PC, (▲) AC and (×) both responses are also shown.
is important to maintain the AC of the molecules through the extraction process. The simultaneous maximization of the polyphenol quantity (PC) and their quality (AC) is therefore primordial. Moreover, the optimization of the process is likely to diminish its cost. The simultaneous optimum for both responses was obtained with 37.7 mg/mL β-CD for 4.4 h at 66.6 °C. It permitted the decrease in β-CD consumption (from 46.8 to 37.7 mg/mL), thus reducing the overall cost of the process. The suggested multiple optimum is therefore an economic choice for industrial purposes that decreases the time and β-CD consumption, maintaining a high AC of concentrated polyphenol extracts. To verify the exactitude of the model, the optimal conditions for the optimization of both of the responses (PC and AC) were repeated. The extraction process of polyphenols from Syrah vine shoots was conducted at 66.6 °C for 4.4 h with a 37.7 mg/mL aqueous β-CD solvent. 3390
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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Journal of Agricultural and Food Chemistry
Ethanol is likely to enhance the polyphenol extraction process because it acts on both the solvent properties and the matrix. It influences the polarity of the solvent and its capacity to solubilize lipophilic polyphenols. Moreover, ethanol can chemically and biophysically alter biological membranes by affecting cell permeability.19 The faster kinetics of polyphenol extraction with ethanol is therefore expected because it affects membrane permeability and, thus, facilitates molecular diffusion. With regard to the AC of the extracted molecules, a comparison was performed between polyphenols obtained by ethanol extraction and those by β-CD (37.7 mg/mL). For the same polyphenol yield (2.6 mg of GAE/g of DM), AC of 1000 μMTE was found with water, AC of 1100 μMTE was found with ethanol, and AC of 1864 μMTE was obtained with β-CD. The oxidation or degradation phenomena affecting polyphenol AC was more likely to occure in water and ethanol/water mixtures than with β-CD-containing solvent. This observation was maintained with higher polyphenol yields. For 5.8 mg of GAE/g of DM, AC of 2000 μMTE was found with ethanol compared to AC of 3146 μMTE obtained with β-CD. After 48 h of extraction at 66.6 °C with 50% ethanol/water mixture, PC of 24 mg of GAE/g of DM and AC of 6000 μMTE were obtained (data not shown). In comparison to β-CD, AC was only ameliorated 1.9 times, even when PC was 4.13 times higher. The negative effect of the ethanol and temperature combination on the polyphenol AC was highlighted within 48 h of extraction. Better antioxidant power and stability were reported for polyphenols encapsuled with CD compared to methanol extract.20 The difference of AC among ethanol and βCD extracts might result from (1) the difference in the composition and diversity of the extracted polyphenols and (2) the encapsulation role of β-CD in the protection of polyphenol from possible degradation by oxidation, heat, and UV light.21 The identification and quantification of polyphenols from βCD- and hydroethanolic-assisted extractions were conducted by HPLC. Both extracts had the same initial total PC of 5.8 mg of GAE/g of DM but different antioxidant activities (Table 4). As shown in Table 4, GA, HBA, FA, and TCA were identified in the β-CD extract obtained at the following conditions (37.7 mg/mL, 48 h, and 66.6 °C). Higher concentrations of polyphenols were identified in β-CD compared to the hydroethanolic extract. For instance, FA and HBA were by far better extracted with β-CD-containing solvent compared to 50% ethanol (Table 4). The higher yield of polyphenols and their improved diversity could be the reason for the enhanced AC activity observed with the β-CD extracts (3146 μMTE) compared to the hydroethanolic extracts (2000 μMTE). With regard to the lower price of β-CD compared to ethanol and the higher quantity of ethanol used per experiment (7.8 g of ethanol equivalent to 50% ethanol/water mixture versus 0.754 g of β-CD equivalent to 37.7 mg/mL taken at the optimal conditions), it is more economic for industrial purposes to use β-CD. The importance of this latter is not limited to the extraction process because polyphenol encapsulation by β-CD
Figure 4. PC and AC as a function of time at 66.6 °C and 37.7 mg/mL β-CD.
diminishes, probably because of polyphenol degradation caused by exposure to a relatively high temperature for a long period of time. The linear extrapolation of time permitted the amelioration of PC and AC by 2.3 and 1.7 times, respectively, enabling the obtainment of the highest concentration of bioactive polyphenols (5.8 mg of GAE/g of DM) with β-CD-assisted extraction (37.7 mg/mL) after 48 h at 66.6 °C. β-CD and Organic Solvent Extraction. The efficiency of β-CD in the extraction of antioxidant polyphenols has been evaluated in comparison to organic solvent extraction with 50% ethanol/water, shown to be the optimal ethanolic mixture compared to 0, 25, and 75% ethanol/water in terms of polyphenol extraction from vine shoots.8 As shown in Figure 5, a PC of 2.6 mg of GAE/g of DM was reached after 0.5 h of extraction with 50% ethanol/water, while
Figure 5. PC and AC as a function of time at 66.6 °C and 50% ethanol/water.
it took 5 h with β-CD (37.7 mg/mL) (Figure 4) and 10 h with pure water (data not shown) to attain the same phenolic yield.
Table 4. PC of β-CD- and Ethanol-Assisted Extracts from Syrah Vine Shootsa β-CD 50% ethanol
GA (μg/g of DM)
HBA (μg/g of DM)
FA (μg/g of DM)
TCA (μg/g of DM)
AC (μMTE)
PC (mg of GAE/g of DM)
2100 a 1958 b
6.5 a 1.7 b
17.6 nd
72.5 a 60.2 b
3146 a 2000 b
5.8 5.8
a
nd corresponds to non-detectable. GA, gallic acid; HBA, 4-hydroxybenzoic acid; FA, ferulic acid; TCA, trans-cinnamic acid; AC, antioxydant capacity; and PC, polyphenol content. Different letters (a, b, and c) indicate significant statistical difference between means. 3391
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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Journal of Agricultural and Food Chemistry Table 5. PC of Syrah, Cabernet Sauvignon, Cabernet Franc, and Tempranillo Vine Shoot Extractsa
Syrah Cabernet Sauvignon Cabernet Franc Tempranillo
GA (μg/g of DM)
HBA (μg/g of DM)
FA (μg/g of DM)
TCA (μg/g of DM)
PA (μg/g of DM)
R (μg/g of DM)
AC (μMTE)
2100 a 1792 b 2162 c 1463 d
6.5
17.6 a 4.43 b 7c
7.25
nd 64.1 a 73.2 a 66 a
nd 1929.7 a 2230.8 b 2300.2 c
3146 a 4800 b 3700 c 7000 d
PC (mg of GAE/g of DM) 5.8 7.5 7.5 7.5
a b b b
a
nd corresponds to non-detectable. GA, gallic acid; HBA, 4-hydroxybenzoic acid; FA, ferulic acid; TCA, trans-cinnamic acid; PA, protocatechuic acid; R, resveratrol; AC, antioxydant capacity; and PC, polyphenol content. Different letters (a, b, and c) indicate significant statistical difference between means.
Notes
heightens their stability, masks their unpleasant taste, and improves their bioavailabity and half-life.21 Therefore, their removal is unnecessary, as is the case for organic solvents, after the extraction process, especially when they will be expected to be used in industrial applications. In comparison to organic solvent (50% ethanol), β-CD-assisted extraction was shown to be an efficient method in terms of antioxidant polyphenol extraction from vine shoots. Comparison of Vine Shoot Cultivars. β-CD-assisted extraction of polyphenols was conducted on three other vine shoot cultivars proposed by a local viticulture for the valorization of their shoots: Cabernet Sauvignon, Cabernet Franc, and Tempranillo. The comparison was realized using the optimal conditions found with Syrah (β-CD, 37.7 mg/mL; T, 66.6 °C; and t, 48 h). Different diversities and quantities of polyphenols were identified among the extracts (Table 5). For example, in Tempranillo cultivar, three polyphenols were identified: GA, PA, and R. Among all of the studied vine shoots, Tempranillo had the highest concentration of R (2300.2 μg/g of DM), Cabernet Franc had the highest concentrations of GA (2162 μg/g of DM), and Syrah had the highest concentrations of FA (17.6 μg/g of DM) and HBA (6.5 μg/g of DM). All cultivars had the same PA content (73.2 μg/g of DM). No correlation was shown between the identified molecules and the antiradical capacities of the extract AC. However, all vine shoot cultivars were shown to be a rich source of highly antioxidant polyphenols. GA, PA, FA, TCA, HBA, and R were found by many authors in vine shoot extracts.1,22,23 Our study focused on the differences between Syrah, Cabernet Sauvignon, Cabernet Franc, and Tempranillo cultivars in terms of their AC, PC, and diversity. All in all, we have clearly shown that β-CD-assisted extraction is an environmentally friendly process for the extraction of polyphenols with a high radical-scavenging capacity. At an industrial scale and compared to organic solvent, β-CDs are cheaper and safer for several applications and processes. This type of natural antioxidant molecule extraction is quite efficient in terms of quantity and quality of the polyphenols extracted from vine shoots. However, a potential co-extraction of phytochemicals is likely to occur when using β-CDs. Future studies on the identification and characterization of these phytochemicals is essential to consider this process as GRAS.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Château Kefraya, Bekaa, Lebanon, for logistic support. ABBREVIATIONS USED β-CD, β-cyclodextrin; PC, polyphenol content; AC, antioxidant capacity; t, time; T, temperature; GA, gallic acid; HBA, 4hydroxybenzoic acid; FA, ferulic acid; TCA, trans-cinnamic acid; PA, protocatechuic acid; R, resveratrol; GRAS, generally recognized as safe; RSM, response surface methodology; DPPH, 2,2-diphenyl-picrylhydrazyl; Trolox, (±)-6-hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid; HPLC, highperformance liquid chromatography; DM, dry matter; μMTE, micromolar trolox equivalent per milliliter; GAE, gallic acid equivalent; DAD, diode array detection; UV, ultraviolet
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +96178821568 or +330781419515. E-mail: [email protected]. Funding
The authors acknowledge the Research Council of Saint Joseph University of Beirut for financial support through Project FS54. 3392
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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Journal of Agricultural and Food Chemistry (11) Singleton, V. L.; Orthofer, R.; Lamuela-Raventós, R. M.; Lester, P. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin−Ciocalteu reagent. Methods Enzymol. 1999, 299, 152−178. (12) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWTFood Sci.Technol. 1995, 28, 25−30. (13) Vergara, C.; von Baer, D.; Mardones, C.; Wilkens, A.; Wernekinck, K.; Damm, A.; Macke, S.; Gorena, T.; Winterhalter, P. Stilbene levels in grape cane of different cultivars in southern Chile: Determination by HPLC−DAD−MS/MS method. J. Agric. Food Chem. 2012, 60, 929−933. (14) Myers, R. H.; Montgomery, D. C.; Anderson-Cook, C. M. Response Surface Methodology: Process and Product Optimization Using Designed Experiments, 3rd ed.; John Wiley & Sons: Hoboken, NJ, 2009; Vol. 705. (15) Ibañez, E.; Herrero, M.; Mendiola, J. A.; Castro-Puyana, M. Extraction and characterization of bioactive compounds with health benefits from marine resources: Macro and micro algae, cyanobacteria, and invertebrates. In Marine Bioactive Compounds; Hayes, M., Ed.; Springer: New York, 2012; pp 55−98. (16) Del Valle, E. M. Cyclodextrins and their uses: A review. Process Biochem. 2004, 39, 1033−1046. (17) Loftsson, T.; Brewster, M. E. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J. Pharm. Sci. 1996, 85, 1017−1025. (18) Mazza, G. Functional Foods: Biochemical and Processing Aspects; CRC Press: Boca Raton, FL, 1998; Vol. 1. (19) Goldstein, D.; Chin, J. Interaction of ethanol with biological membranes. Fed. Proc. 1981, 40 (7), 2073−2076. (20) Mantegna, S.; Binello, A.; Boffa, L.; Giorgis, M.; Cena, C.; Cravotto, G. A one-pot ultrasound-assisted water extraction/cyclodextrin encapsulation of resveratrol from Polygonum cuspidatum. Food Chem. 2012, 130, 746−750. (21) Fang, Z.; Bhandari, B. Encapsulation of polyphenolsA review. Trends Food Sci. Technol. 2010, 21, 510−523. (22) Delgado-Torre, M. P.; Ferreiro-Vera, C.; Priego-Capote, F.; Pérez-Juan, P. M.; Luque de Castro, M. D. Comparison of accelerated methods for the extraction of phenolic compounds from different vineshoot cultivars. J. Agric. Food Chem. 2012, 60, 3051−3060. (23) Karacabey, E.; Mazza, G. Optimisation of antioxidant activity of grape cane extracts using response surface methodology. Food Chem. 2010, 119, 343−348.
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β‑Cyclodextrin-Assisted Extraction of Polyphenols from Vine Shoot Cultivars Hiba N. Rajha,*,†,‡ Stephanie Chacar,† Charbel Afif,† Eugene Vorobiev,‡ Nicolas Louka,† and Richard G. Maroun† †
Centre d’Analyses et de Recherche, Unité de Recherche Technologie et Valorisation Alimentaire (UR TVA), Faculté des Sciences, Université Saint-Joseph, B.P. 11-514 Riad El Solh, Beirut 1107 2050, Lebanon ‡ Sorbonne Universités, Université de Technologie de Compiègne, Laboratoire Transformations Intégrées de la Matière Renouvelable (UTC/ESCOM, EA 4297 TIMR), Centre de Recherche Royallieu, CS 60 319, Compiègne 60 203 CEDEX, France ABSTRACT: This work optimized the β-cyclodextrin (β-CD)-assisted extraction process of polyphenols from vine shoots. The efficiency of β-CD was compared to that of ethanol in terms of the quantity and antioxidant capacity (AC) of the extracted polyphenols. Response surface methodology permitted the optimization of the β-CD concentration, time, and temperature. The optimal polyphenol content (PC) [5.8 mg of gallic acid equivalent (GAE)/g of dry matter (DM)] and AC [3146 micromolar trolox equivalent per milliliter (μMTE)] were initially obtained with Syrah cultivar after an extraction of 48 h at 66.6 °C with a 37.7 mg/mL aqueous β-CD solvent. The same PC (5.8 mg of GAE/g of DM) was reached with 50% ethanol/water solvent after 1.65 h. However, a lower AC was found with ethanol (2000 μMTE) compared to β-CD. A comparison of the PC and AC of four different vine shoot cultivars was realized. Our results clearly show the capacity of β-CD to amplify polyphenol extraction from vine shoots. KEYWORDS: polyphenols, β-cyclodextrin, response surface methodology, high-performance liquid chromatography, vine shoots
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conducted on the β-CD-assisted extraction of polyphenols from vine shoots. The current study was carried out with the aim of defining the optimal β-CD concentration, time, and temperature for the recovery of polyphenols from vine shoots, comparing the efficiency of β-CD to organic solvent extraction, studying polyphenol diversity and antioxidant activity of the extracts, and comparing polyphenol contents of four diffirent vine shoot cultivars.
INTRODUCTION Viticulture is an agricultural activity important worldwide because of its valuable economic and health benefits.1 For highquality grape production, cultural practices are annually performed in vineyards, such as pruning. The latter affects vine form, shape, and size. It also influences the production of grapes in terms of quantity and quality. Vine shoots are pruning wastes thrown or burned in the field for their disposal, thus causing environmental problems.2 Vine shoots are value-added industrial products regarding their content in high-value phytochemicals, such as polyphenols.3 The extraction and purification processes are primordial steps for the extraction of biomolecules from natural sources. They permit the utilization of bioactive compounds in pharmaceuticals, nutraceuticals, cosmetics, etc.4 Organic solvents are commonly used in the conventional extraction processes for polyphenol recovery from plants.5 Alternative environmentally friendly extraction processes, such as high-voltage electrical discharges, supercritical fluid extraction, pulsed electric fields, ultrasounds, etc., were conceived to diminish organic solvent use and their toxicological and safety concerns.6−8 However, the availability of these methods is limited by their requirement of advanced and costly equipment. Cyclodextrins (CDs) are cyclic oligosaccharides generally recognized as safe (GRAS), widely used in food industries for their capacity to form inclusion complexes with bioactive compounds, increasing thus their stability, solubility, and bioavailability.9 The use of aqueous βcyclodextrin (β-CD) was studied for the recovery of polyphenols from grapes and their pomace.10 Extraction parameters, such as time, temperature, and solvent mixture, are likely to influence the process, having possible interactions among the variables.3,4 To our knowledge, no research has been © 2015 American Chemical Society
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MATERIALS AND METHODS
Reagents. All reagents were of analytical grade. Sodium carbonate (Fluka, Buchs, Switzerland), Folin’s phenol reagent (Scott Science, U.K.), and gallic acid (GA, Sigma Chemical Co., St. Louis, MO) were used in the Folin−Ciocalteu assay. 2,2-Diphenyl-picrylhydrazyl (DPPH) radical and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (Trolox) were purchased from Sigma Chemical Co. (St. Louis, MO). The high-performance liquid chromatography (HPLC) solvents were acetonitrile (Merck, Darmstadt, Germany), formic acid (Scharlau, Barcelona, Spain), and water (Scharlau, Barcelona, Spain) of HPLC ultra gradient grade. Phenolic compound standards of GA, protocatechuic acid (PA), ferulic acid (FA), transcinnamic acid (TCA), 4-hydroxybenzoic acid (HBA), and resveratrol (R) were from Sigma Chemical Co. (St. Louis, MO). Raw Material. Vitis vinifera var. Syrah vine shoots were used for the optimization process of polyphenol extraction by response surface methodology (RSM). However, other varieties were also studied: Tempranillo, Cabernet Sauvignon, and Cabernet Franc. Vine shoots were pruned in 2013 and stored at room temperature until use. Vine Received: Revised: Accepted: Published: 3387
February 4, 2015 March 18, 2015 March 18, 2015 March 18, 2015 DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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Journal of Agricultural and Food Chemistry
Table 1. RSM Central Composite Design of Three Factors and Five Levels and the Experimental Responses (PC and AC) of βCD-Assisted Extraction from Vine Shoots run factorial design
center points
star points
β-CD concentration, β (mg/mL) 20 40 20 40 20 40 20 40 30 30 30 30 30 30 13.82 46.82 30 30 30 30
(−1) (+1) (−1) (+1) (−1) (+1) (−1) (+1) (0) (0) (0) (0) (0) (0) (−α) (+α) (0) (0) (0) (0)
time, t (h) 1.5 1.5 5 5 1.5 1.5 5 5 3.25 3.25 3.25 3.25 3.25 3.25 3.25 3.25 0.30 6.19 3.25 3.25
temperature, T (°C)
(−1) (−1) (+1) (+1) (−1) (−1) (+1) (+1) (0) (0) (0) (0) (0) (0) (0) (0) (−α) (+α) (0) (0)
40 40 40 40 60 60 60 60 50 50 50 50 50 50 50 50 50 50 33.18 66.82
PC (mg of GAE/g of DM)
AC (μMTE)
0.43 0.69 0.93 0.96 1.43 1.65 1.75 2.48 1.04 1.29 1.14 1.38 1.20 1.15 0.85 1.05 0.36 2.02 0.56 1.76
800.96 1190.57 1250.79 1130.34 1340.51 1710.88 1540.44 1740.37 1220.06 1390.49 1370.00 1270.04 1240.55 1410.99 1120.72 1390.49 1090.60 1460.97 940.66 1740.37
(−1) (−1) (−1) (−1) (+1) (+1) (+1) (+1) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (−α) (+α)
0.1% (v/v) formic acid in water and mobile phase B consisting of 100% acetonitrile were used. The elution profile had the following proportions (v/v) of solvent B: 5 min, 15−20%; 45 min, 20−44.5%; 1 min, 100%; kept at 100% of B for 9 min; and followed by 5 min of stabilization at 15%. Spectrophotometric detection was performed at 280 nm. Experimental Design. The optimization of the β-CD-assisted extraction process of polyphenol from vine shoots was conducted by RSM. The independent variables were β-CD concentration, time, and temperature. They were coded at five levels (−α, −1, 0, 1, and +α), in a central composite design (23 + star) of 20 experiments, including six center points. Table 1 (first four columns) shows the central composite design with the range of variables and their coded values. Two responses were studied: PC and AC. Data analyses were carried out using STATGRAPHICS Plus 4.0 for Windows and were fitted to a second-degree regression equation of the form
shoots were cut into cylinders of 1 cm height and a mean diameter of 5 mm. Dry Matter (DM) Content. Vine shoots were dried in a convictive oven at 105 °C for 48 h for the measurement of the DM content. The DM of Syrah, Cabernet Sauvignon, Cabernet Franc, and Tempranillo vine shoots was found to be 60, 65, 57, and 69%, respectively. Solid−Liquid Extraction. The solid−liquid extraction process of polyphenols from cylindrical vine shoots was performed with a solid/ liquid ratio of 1:20 (w/v).8 After β-CD aqueous extraction at different concentration, time, and temperature combinations, extracts were centrifuged at 5000 rpm for 15 min. β-CD aqueous concentrations were prepared by dissolving the required weight of β-CD in the specific water volume in a 50 °C water bath under stirring. Total Polyphenol Content (PC). The total PC was determined by the Folin−Ciocalteu colorimetric assay.11 A total of 1 mL of Folin− Ciocalteu reagent (10-fold diluted) was mixed to 200 μL of extract. Then, 800 μL of sodium carbonate (75 g/L) was added. The mixture was incubated for 10 min at 60 °C and then 10 min at room temperature. The absorbance was measured at 750 nm by the ultraviolet/visible (UV/vis) spectrophotometer (UV- 9200, BioTECH Engineering Management, U.K.). The calibration curve was drawn with GA. PC was expressed as milligrams of gallic acid equivalent (GAE) per gram of DM. Antioxidant Capacity (AC). The AC of vine shoot extracts was measured by means of the DPPH assay.12 A total of 50 μL of vine shoot extracts or standard (Trolox) was added to 1.45 mL of DPPH radical (0.06 mM). After an incubation of 30 min at room temperature, the absorbance was measured at 515 nm. The DPPH value of the extracts was expressed in micromolar Trolox equivalent per milliliter (μMTE). HPLC. The HPLC analyses were conducted for the identification and quantification of polyphenols from vine shoot extracts.13 The HPLC−diode array detection (DAD) analyses of polyphenols were carried out with a HPLC system (Waters Alliance, Milford, MA) equipped with a quaternary Waters e2695 pump. A UV−vis photodiode array spectrophotometer Waters 2998 was used, with the control system and data collection Empower 3 software. HPLC analyses of polyphenols were carried out on a Discovery C18, 5 μm, 250 × 4.6 mm, column (Supleco, Bellefonte, PA) with a C18, Supelguard Discovery 18, 20 × 4 mm, 5 μm, precolumn (Supelco, Bellefonte, PA) at 30 °C. Separation of 100 μL of cane extract was performed at a flow rate of 0.5 mL min−1. Mobile phase A consisting of
3
Y = a0 +
3
2
3
∑ anXn+ ∑ annXn2+ ∑ ∑ n=1
n=1
n=1 m=n+1
anmX nX m
(1)
where Y is the response, a0, an, am, and anm are the constant, linear, quadratic, and two factor interaction coefficients, respectively. Statistical Analysis. Each extraction process was repeated on three different vine shoot samples. Means and standard deviations (error bars in the figures) of data were calculated. Variance analyses (ANOVA) and least significant difference (LSD) test were performed by STATGRAPHICS Centurion XV (StatPoint Technologies, Inc.).
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RESULTS AND DISCUSSION Experimental Design and Statistics. The effect of time, temperature, and β-CD concentration (β) on polyphenol extraction from vine shoots was studied. The RSM design was conceived taking into consideration the solubility limitation of β-CD at concentrations above 50 mg/mL10 and temperature limitation because of the possible heat degradation/oxidation of polyphenols. RSM is a statistical and mathematical method employed for the development, improvement, and optimization of extraction processes.14 Table 1 shows the experimental design and combinations of the coded variables with their responses (PC and AC). Total PC ranged from 0.36 to 2.48 mg 3388
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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− 4.893βt
(3)
Comparing the significance of the studied parameters, it can be noticed that the effect of time was more significant for extracting higher PCs but had less effect on their AC. This might be due to the oxidation phenomena that is likely to occur when subjecting polyphenols to high temperatures (>50 °C) for long durations. In contrast, the β-CD concentration played a more important role for conserving the AC of polyphenols than enhancing their quantity (PC) (Figures 1a and 2a). Multiple Response Optimization. Figure 3 shows the overlay plots for PC and AC at 66.6 °C (optimal temperature). As marked by the black dot, the optimal conditions for the maximization of PC are 46.8 mg/mL β-CD for 6.2 h, while those for AC are 46.8 mg/mL β-CD for 0.7 h. These are the quantitative and qualitative optimums, respectively. However, for industrial applications of vine-shoot-derived polyphenols, it 3389
2
94.6 88.7
AC = −812.072 + 25.3102β + 198.619t + 24.1344T
PC = 0.161426 − 0.0072592β − 0.0819191t − 0.0149214T − 0.000469537β + 0.00206658βt + 0.000806414βT + 0.0122602t + 0.00273404tT + 0.000280171T AC = −48.9255 + 12.2366β + 262.684t − 2.86155T − 0.106453β2 − 4.893βt + 0.389216βT − 0.959765t2 − 1.15653tT + 0.190782T2
The positive effect of t, T, and β was expected. The temperature augments molecular diffusion; it decreases solvent viscosity, enhancing the solute solubilization and diminishing surface tension.15 CDs are likely to form, within their lipophilic cavities and through non-covalent forces, inclusion complexes with hydrophobic polyphenols, increasing thus their water solubility.16,17 Time comes immediately after the temperature factor in terms of its significance (Figure 1a). It plays an important role in the diffusion process, which is a relatively slow operation of three steps: (1) internal diffusion of the solvent, (2) solubilization of the target molecule, and (3) diffusion of the solubilized molecules to the surface of the particle.18 The significant positive effect of time is therefore expected for polyphenol extraction from vine shoots. Effect of the Time, Temperature, and β-CD Concentration on AC. Similar to PC, AC is positively affected by the time, temperature, and β-CD concentration (Figure 2a). As shown in Figure 2b, the increase in the β-CD concentration and time of the extraction process linearly augments AC up to 1740.37 μMTE. The temporary locking of polyphenols within CD cavities stabilizes host molecules from oxidation, light, and heat degradation,16 which probably gave linear positive effects for t, T, and β. Excluding insignificant parameters, the regression equation becomes
regression coefficient, R2 (%)
(2)
2
PC = −2.0886 + 0.0116057β + 0.196472t + 0.0461738T
2
Table 2. Second-Order Polynomial Equations for PC and AC, Including Insignificant Parameters, with R2 Also Shown for the Equations
of GAE/g of DM, while AC was between 800.96 and 1740.37 μMTE. The relation between the responses and the experimental parameters (β, t, and T) was demonstrated by statistical analyses to fit second-order polynomial equations shown in Table 2. The analyses of the predicted values, calculated from the regression equations, showed a high correlation with experimental values and resulted in the calculation of the coefficients of determination (R2), which were shown to have high levels of adequacy (Table 2). Effect of the Time, Temperature, and β-CD Concentration on PC. According to the pareto chart (Figure 1a) and the response surface plot (Figure 1b), time, temperature and βCD concentration have linear positive effects on polyphenol extraction from vine shoots. No quadratic effects were observed nor interaction between the parameters. Excluding insignificant parameters, the regression equation becomes
regression equation (including insignificant parameters)
Journal of Agricultural and Food Chemistry
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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Journal of Agricultural and Food Chemistry
Figure 1. (a) Standardized pareto chart and (b) estimated response surface for PC.
Figure 2. (a) Standardized pareto chart and (b) estimated response surface for AC.
Experimental results were compared to those predicted by the model. As shown in Table 3, the difference of the values Table 3. Predicted and Experimental Results of PC and AC at the Multiple Optimum Conditions (66.6 °C, 4.4 h, and 37.7 mg/mL β-CD) multiple optimum
predicted
experimental
mean absolute error
PC (mg of GAE/g of DM) AC (μMTE)
2.5 1881.3
2.45 1864
0.14 48.7
obtained by experimentation and model prediction are within the range of the mean absolute error, suggesting a good agreement of the model with experimental results. Consequently, 66.6 °C and 37.7 mg/mL aqueous CD seem to be adequate optimal conditions for the maximization of both PC and AC. In the studied domain, all of the quadratatic effects (t2, T2, and β2) of the parameters were insignificant. Time, temperature, and β-CD concentration had positive linear impacts on PC and AC (Figures 1a and 2a). The β-CD concentration in water is an experimental condition limited by its solubility and economic impact. Similarly, the temperature elevation is governed by the oxidation and/or degradation phenomena that it causes to polyphenols and also by its energy consumption that affects the overall cost of the process. Because of all of the aforementioned reasons, the linear extrapolation of the time parameter was possible because it was suspected to further increase PC and AC. Figure 4 shows PC and AC as a function of long periods of time (from 5 to 54 h) at 66.6 °C and 37.7 mg/mL β-CD. The highest PC and AC, 5.8 mg of GAE/g of DM and 3147 μMTE, respectively, were reached after 48 h of extraction. Beyond this time, PC
Figure 3. Superposition plots of PC and AC. The optimums for (●) PC, (▲) AC and (×) both responses are also shown.
is important to maintain the AC of the molecules through the extraction process. The simultaneous maximization of the polyphenol quantity (PC) and their quality (AC) is therefore primordial. Moreover, the optimization of the process is likely to diminish its cost. The simultaneous optimum for both responses was obtained with 37.7 mg/mL β-CD for 4.4 h at 66.6 °C. It permitted the decrease in β-CD consumption (from 46.8 to 37.7 mg/mL), thus reducing the overall cost of the process. The suggested multiple optimum is therefore an economic choice for industrial purposes that decreases the time and β-CD consumption, maintaining a high AC of concentrated polyphenol extracts. To verify the exactitude of the model, the optimal conditions for the optimization of both of the responses (PC and AC) were repeated. The extraction process of polyphenols from Syrah vine shoots was conducted at 66.6 °C for 4.4 h with a 37.7 mg/mL aqueous β-CD solvent. 3390
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
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Journal of Agricultural and Food Chemistry
Ethanol is likely to enhance the polyphenol extraction process because it acts on both the solvent properties and the matrix. It influences the polarity of the solvent and its capacity to solubilize lipophilic polyphenols. Moreover, ethanol can chemically and biophysically alter biological membranes by affecting cell permeability.19 The faster kinetics of polyphenol extraction with ethanol is therefore expected because it affects membrane permeability and, thus, facilitates molecular diffusion. With regard to the AC of the extracted molecules, a comparison was performed between polyphenols obtained by ethanol extraction and those by β-CD (37.7 mg/mL). For the same polyphenol yield (2.6 mg of GAE/g of DM), AC of 1000 μMTE was found with water, AC of 1100 μMTE was found with ethanol, and AC of 1864 μMTE was obtained with β-CD. The oxidation or degradation phenomena affecting polyphenol AC was more likely to occure in water and ethanol/water mixtures than with β-CD-containing solvent. This observation was maintained with higher polyphenol yields. For 5.8 mg of GAE/g of DM, AC of 2000 μMTE was found with ethanol compared to AC of 3146 μMTE obtained with β-CD. After 48 h of extraction at 66.6 °C with 50% ethanol/water mixture, PC of 24 mg of GAE/g of DM and AC of 6000 μMTE were obtained (data not shown). In comparison to β-CD, AC was only ameliorated 1.9 times, even when PC was 4.13 times higher. The negative effect of the ethanol and temperature combination on the polyphenol AC was highlighted within 48 h of extraction. Better antioxidant power and stability were reported for polyphenols encapsuled with CD compared to methanol extract.20 The difference of AC among ethanol and βCD extracts might result from (1) the difference in the composition and diversity of the extracted polyphenols and (2) the encapsulation role of β-CD in the protection of polyphenol from possible degradation by oxidation, heat, and UV light.21 The identification and quantification of polyphenols from βCD- and hydroethanolic-assisted extractions were conducted by HPLC. Both extracts had the same initial total PC of 5.8 mg of GAE/g of DM but different antioxidant activities (Table 4). As shown in Table 4, GA, HBA, FA, and TCA were identified in the β-CD extract obtained at the following conditions (37.7 mg/mL, 48 h, and 66.6 °C). Higher concentrations of polyphenols were identified in β-CD compared to the hydroethanolic extract. For instance, FA and HBA were by far better extracted with β-CD-containing solvent compared to 50% ethanol (Table 4). The higher yield of polyphenols and their improved diversity could be the reason for the enhanced AC activity observed with the β-CD extracts (3146 μMTE) compared to the hydroethanolic extracts (2000 μMTE). With regard to the lower price of β-CD compared to ethanol and the higher quantity of ethanol used per experiment (7.8 g of ethanol equivalent to 50% ethanol/water mixture versus 0.754 g of β-CD equivalent to 37.7 mg/mL taken at the optimal conditions), it is more economic for industrial purposes to use β-CD. The importance of this latter is not limited to the extraction process because polyphenol encapsulation by β-CD
Figure 4. PC and AC as a function of time at 66.6 °C and 37.7 mg/mL β-CD.
diminishes, probably because of polyphenol degradation caused by exposure to a relatively high temperature for a long period of time. The linear extrapolation of time permitted the amelioration of PC and AC by 2.3 and 1.7 times, respectively, enabling the obtainment of the highest concentration of bioactive polyphenols (5.8 mg of GAE/g of DM) with β-CD-assisted extraction (37.7 mg/mL) after 48 h at 66.6 °C. β-CD and Organic Solvent Extraction. The efficiency of β-CD in the extraction of antioxidant polyphenols has been evaluated in comparison to organic solvent extraction with 50% ethanol/water, shown to be the optimal ethanolic mixture compared to 0, 25, and 75% ethanol/water in terms of polyphenol extraction from vine shoots.8 As shown in Figure 5, a PC of 2.6 mg of GAE/g of DM was reached after 0.5 h of extraction with 50% ethanol/water, while
Figure 5. PC and AC as a function of time at 66.6 °C and 50% ethanol/water.
it took 5 h with β-CD (37.7 mg/mL) (Figure 4) and 10 h with pure water (data not shown) to attain the same phenolic yield.
Table 4. PC of β-CD- and Ethanol-Assisted Extracts from Syrah Vine Shootsa β-CD 50% ethanol
GA (μg/g of DM)
HBA (μg/g of DM)
FA (μg/g of DM)
TCA (μg/g of DM)
AC (μMTE)
PC (mg of GAE/g of DM)
2100 a 1958 b
6.5 a 1.7 b
17.6 nd
72.5 a 60.2 b
3146 a 2000 b
5.8 5.8
a
nd corresponds to non-detectable. GA, gallic acid; HBA, 4-hydroxybenzoic acid; FA, ferulic acid; TCA, trans-cinnamic acid; AC, antioxydant capacity; and PC, polyphenol content. Different letters (a, b, and c) indicate significant statistical difference between means. 3391
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Journal of Agricultural and Food Chemistry Table 5. PC of Syrah, Cabernet Sauvignon, Cabernet Franc, and Tempranillo Vine Shoot Extractsa
Syrah Cabernet Sauvignon Cabernet Franc Tempranillo
GA (μg/g of DM)
HBA (μg/g of DM)
FA (μg/g of DM)
TCA (μg/g of DM)
PA (μg/g of DM)
R (μg/g of DM)
AC (μMTE)
2100 a 1792 b 2162 c 1463 d
6.5
17.6 a 4.43 b 7c
7.25
nd 64.1 a 73.2 a 66 a
nd 1929.7 a 2230.8 b 2300.2 c
3146 a 4800 b 3700 c 7000 d
PC (mg of GAE/g of DM) 5.8 7.5 7.5 7.5
a b b b
a
nd corresponds to non-detectable. GA, gallic acid; HBA, 4-hydroxybenzoic acid; FA, ferulic acid; TCA, trans-cinnamic acid; PA, protocatechuic acid; R, resveratrol; AC, antioxydant capacity; and PC, polyphenol content. Different letters (a, b, and c) indicate significant statistical difference between means.
Notes
heightens their stability, masks their unpleasant taste, and improves their bioavailabity and half-life.21 Therefore, their removal is unnecessary, as is the case for organic solvents, after the extraction process, especially when they will be expected to be used in industrial applications. In comparison to organic solvent (50% ethanol), β-CD-assisted extraction was shown to be an efficient method in terms of antioxidant polyphenol extraction from vine shoots. Comparison of Vine Shoot Cultivars. β-CD-assisted extraction of polyphenols was conducted on three other vine shoot cultivars proposed by a local viticulture for the valorization of their shoots: Cabernet Sauvignon, Cabernet Franc, and Tempranillo. The comparison was realized using the optimal conditions found with Syrah (β-CD, 37.7 mg/mL; T, 66.6 °C; and t, 48 h). Different diversities and quantities of polyphenols were identified among the extracts (Table 5). For example, in Tempranillo cultivar, three polyphenols were identified: GA, PA, and R. Among all of the studied vine shoots, Tempranillo had the highest concentration of R (2300.2 μg/g of DM), Cabernet Franc had the highest concentrations of GA (2162 μg/g of DM), and Syrah had the highest concentrations of FA (17.6 μg/g of DM) and HBA (6.5 μg/g of DM). All cultivars had the same PA content (73.2 μg/g of DM). No correlation was shown between the identified molecules and the antiradical capacities of the extract AC. However, all vine shoot cultivars were shown to be a rich source of highly antioxidant polyphenols. GA, PA, FA, TCA, HBA, and R were found by many authors in vine shoot extracts.1,22,23 Our study focused on the differences between Syrah, Cabernet Sauvignon, Cabernet Franc, and Tempranillo cultivars in terms of their AC, PC, and diversity. All in all, we have clearly shown that β-CD-assisted extraction is an environmentally friendly process for the extraction of polyphenols with a high radical-scavenging capacity. At an industrial scale and compared to organic solvent, β-CDs are cheaper and safer for several applications and processes. This type of natural antioxidant molecule extraction is quite efficient in terms of quantity and quality of the polyphenols extracted from vine shoots. However, a potential co-extraction of phytochemicals is likely to occur when using β-CDs. Future studies on the identification and characterization of these phytochemicals is essential to consider this process as GRAS.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Château Kefraya, Bekaa, Lebanon, for logistic support. ABBREVIATIONS USED β-CD, β-cyclodextrin; PC, polyphenol content; AC, antioxidant capacity; t, time; T, temperature; GA, gallic acid; HBA, 4hydroxybenzoic acid; FA, ferulic acid; TCA, trans-cinnamic acid; PA, protocatechuic acid; R, resveratrol; GRAS, generally recognized as safe; RSM, response surface methodology; DPPH, 2,2-diphenyl-picrylhydrazyl; Trolox, (±)-6-hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid; HPLC, highperformance liquid chromatography; DM, dry matter; μMTE, micromolar trolox equivalent per milliliter; GAE, gallic acid equivalent; DAD, diode array detection; UV, ultraviolet
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +96178821568 or +330781419515. E-mail: [email protected]. Funding
The authors acknowledge the Research Council of Saint Joseph University of Beirut for financial support through Project FS54. 3392
DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
Article
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DOI: 10.1021/acs.jafc.5b00672 J. Agric. Food Chem. 2015, 63, 3387−3393
