Potential of Grape Wastes as a Natural Source of Bioactive Compounds

Grapes are widely used in the wine and juice industries, which can lead to massive amounts of waste, mostly grape peels and seeds. The antioxidant capacities, total phenolic and flavonoid contents and phenolic profiles of peels and seeds from 30 grape varieties were systemically assessed. The antioxidant activities of fat-soluble, water-soluble and insoluble-bound fractions of grape peels and seeds were evaluated using ferric-reducing antioxidant power and Trolox equivalent antioxidant capacity assays, and their total phenolic contents and total flavonoid contents were determined by the Folin-Ciocalteu method and AlCl3 colorimetry, respectively. It was found that the antioxidant capacities were diverse among different grape peels and seeds. Moreover, several phenolic compounds were identified and quantified, including gallic acid, cyanidin-3-glucoside, epicatechin, catechin gallate, ferulaic acid, rutin and resveratrol, which could contribute to the antioxidant capacities of these grape peels and seeds. Several grape wastes with strong antioxidant activity could be abundant sources of natural bioactive compounds, and have the potential for development into functional foods, food additives and pharmaceuticals.


Introduction
Grape is a famous fruit all over the world, and is widely used in wine and juice industries, which can lead to massive amounts of wastes, including grape peels and seeds [1]. It is reported that these wastes contain a variety of phytochemicals, especially phenols and flavonoids like anthocyanins, resveratrol, tannin and quercetin [2][3][4][5][6][7][8]. These bioactive components possess various outstanding bioactivities, such as antibacterial, anticancer, antioxidant, anti-inflammation and hepatic and cardiovascular protection effects [9][10][11][12][13], and have great safety and effectiveness advantages in preventing chronic diseases [14][15][16][17]. They can be used as raw materials to produce functional Molecules 2018, 23, 2598; doi:10.3390/molecules23102598 www.mdpi.com/journal/molecules foods, food additives and pharmaceuticals [18][19][20][21][22][23]. Many factors can influence the composition and contents of bioactive compounds in fruits, like genotype, growth environment (soil, water, sunlight, etc.) and maturity, among which genotype usually has the greatest impact [24][25][26]. Thus, we could hypothesize that grapes with diverse genotypes should have different composition and contents of bioactive compounds, so it is worthwhile to assess the antioxidant capacities while determining the phenolic and flavonoid contents of peels and seeds from different grape varieties. In the present study, the antioxidant capacities of peels and seeds from 30 grape varieties were measured, and their total phenolic contents and total flavonoid contents were evaluated. In addition, the phenolic and flavonoid constituents were identified and quantified using HPLC analysis. This should prove helpful for the full utilization of grape peels and seeds.

Ferric Reducing Antioxidant Power (FRAP) of the Grape Peels and Seeds
The FRAP was used as one of the indices to assess antioxidant capacities of these grape peels and seeds. The FRAP assay is established on the basis of the ability that antioxidants reduce ferric ions to ferrous ions [27], which is a simple and commonly employed method to evaluate antioxidant capacity [28][29][30]. The FRAP values of these grape peels and seeds are presented in Table 1.
For the 10 grape seeds, the total FRAP values varied from 312.429 ± 11.760 to 858.121 ± 35.507 µmol Fe(II)/g FW with a 3-fold difference. Pearl Black Grape (Xinjiang, China), Black Grape (Yunnan, China), Red Grape (Yunnan, China), Red Grape (Xinjiang, China) and Red Grape (California, CA, USA) possessed the top-five antioxidant capacities, which were 858.121 ± 35.507, 837.242 ± 21.578, 616.485 ± 29.629, 562.018 ± 19.437 and 520.390 ± 19.974, respectively. Kyoho Grape (Guangxi, China) possessed the lowest antioxidant capacity, which was 312.429 ± 11.760 µmol Fe(II)/g FW. In addition, the ranges of FRAP values for three fractions were in a decreasing order: fat-soluble (250.876 ± 8.208 to 726.495 ± 23.487 µmol Fe(II)/g FW) > water-soluble (44.148 ± 3.996 to 144.767 ± 3.348 µmol Fe(II)/g FW) > insoluble-bound (0.383 ± 0.033 to 1.881 ± 0.184 µmol Fe(II)/g FW) (p < 0.001, p = 0.038, respectively). According to the results described above, the FRAP values of fat-soluble fractions were generally higher than those of water-soluble fractions, which were distinctly higher than those of insoluble-bound fractions. These results indicated that the antioxidants responsible for the reducing power of grape peels and seeds were most fat-soluble compounds with some water-soluble and a little insoluble-bound ones. When evaluating total antioxidant capacities of grape peels and seeds, all of the three fractions should be taken into consideration. In addition, the FRAP values of grape seeds were apparently higher than those of grape peels (p < 0.001). As compared to other materials, the FRAP values of the tested grape peels were higher than those of most edible macro-fungi, vegetables, fruits and fruit wastes (peels and seeds) [31][32][33][34], and also higher than those of some wild fruits and edible and wild flowers [35,36]. Moreover, the FRAP values of the tested grape seeds were higher than those of most edible macro-fungi, vegetables, wild fruits, edible and wild flowers, fruits and fruit wastes (peels and seeds) [31][32][33][34][35][36], and higher than those of some medicinal plants [37]. So grape peels and seeds could be abundant resources of natural antioxidants with great potential to produce functional foods, food additives and pharmaceuticals.

Trolox Equivalent Antioxidant Capacity (TEAC) of the Grape Peels and Seeds
Most natural antioxidants are multifunctional, and the antioxidant capacities of plant samples are generally impacted by multiple factors, such as the extraction solvent, extraction method and measurement method, leading to difficulty to completely demonstrate antioxidant capacities using a single method. Therefore, an authentic antioxidant assessing system requires evaluations of multiple aspects, and it is essential to conduct different experiments to assess the antioxidant activity which might be associated with diverse mechanisms of action [38]. The TEAC assay is a simple, fast, repeatable and widely used method for the evaluation of antioxidant capacity [39,40]. The TEAC assay is on the basis of the capability of antioxidants to scavenge the ABTS •+ radical, and can be used for measuring antioxidant capacities of fat-soluble, water-soluble and insoluble-bound components in the same sample [41]. As reported, vitamin C, vitamin E, butylated hydroxytoluene, butylated hydroxyanisole and Trolox were often applied as reference standards [42,43]. Here, Trolox was employed. The TEAC values of the peels and seeds from 30 grape varieties are displayed in Table 2. water-soluble (24.555 ± 1.907 to 64.105 ± 0.106 µmol trolox/g FW) > insoluble-bound (0.159 ± 0.003 to 1.320 ± 0.071 µmol trolox/g FW) (p < 0.001, p = 0.023, respectively).
As seen from the description before, the TEAC values of fat-soluble fractions were generally higher than those of water-soluble fractions, which were distinctly higher than those of insoluble-bound fractions. It meant that the antioxidants, which were responsible for the free radical scavenging activities of grape peels and seeds, were most fat-soluble compounds with some water soluble and a little insoluble-bound ones. When the total antioxidant capacities of grape peels and seeds are about to be assessed, three fractions should all be counted in. In addition, the TEAC values of the grape seeds were extremely higher than those of the grape peels (p < 0.001). Besides, the TEAC values of the grape peels were higher than those of most edible macro-fungi, vegetables, fruits and fruit waste (peels and seeds) [31][32][33][34], and higher than those of some wild fruits and edible and wild flowers [35,36]. Furthermore, the TEAC values of the grape seeds were higher than those of most edible macro-fungi, vegetables, edible and wild flowers, fruits and fruit waste (peels and seeds) [31][32][33][34][35][36], and higher than those of some wild fruits and medicinal plants [37], so grape peels and seeds could be developed into functional foods, food additives and pharmaceuticals regarding antioxidants.

Total Phenolic Contents (TPC) of 30 Grape Peels and 10 Grape Seeds
The TPC values of these grape peels and seeds were determined by the Folin-Ciocalteu method, which is based on the reaction that electrons are transferred from phenolic compounds to the Folin-Ciocalteu reagent in alkaline medium, and is a simple, rapid and reproducible method [44].
The TPC values of these grape peels and seeds are given in Table 3.
Based on the above demonstration, the TPC values of the grape seeds were drastically higher than those of the grape peels (p < 0.001). Besides, the TPC values of the grape peels were higher than those of most edible macro-fungi, vegetables, fruits and fruit waste (peels and seeds) [31][32][33][34], and higher than those of some wild fruits and edible and wild flowers [35,36]. Moreover, the TPC values of the grape seeds were higher than those of most edible macro-fungi, vegetables, edible and wild flowers, wild fruits, fruits and fruit wastes (peels and seeds) [31][32][33][34][35][36], and higher than those of some medicinal plants [37], so grape peels and seeds could be used to extract phenols with further applications in the functional food, pharmaceutical, food additive and cosmetic industries. Furthermore, it should be pointed out that some non-phenolic components such as organic acids and sugars, which also possess reducing capacity, could affect the measurement of total phenolic contents determined by the Folin-Ciocalteu method, leading to overestimated total phenolic contents [45,46]. In addition, varied phenols might response to the Folin-Ciocalteu reagent differently and several flavonoids present low responses, which might cause an underestimate of total phenolic contents [47][48][49].

Total Flavonoid Contents (TFC) of the Grape Peels and Seeds
The TFC values of these grape peels and seeds were estimated by the AlCl 3 colorimetry method according to the literature reported by Kalia et al., which is based on the reaction that the 3-hydroxy-4-hydroxyl or 5-hydroxy-4-carbonyl or o-2-phenolic hydroxyl of flavonoids is combined with Al 3+ to form a red complex under an alkaline condition, and is a simple, rapid and repeatable method [50]. The TFC values of these grape peels and seeds are given in Table 4.
As illustrated before, the TFC values of the grape seeds were higher than those of the grape peels (p < 0.001). Both of the TFC values of the grape peels and seeds were lower than those of most medicinal plants and some common plant/tree waste [51,52]. Moreover, extracts with higher TPC values did not always have higher TFC values, different extracts contained different levels of TFC as a portion of phenols [51][52][53]. So it should be pointed out that grape peels and seeds were valuable resources of natural phenols but not flavonoids.

Correlations between Total FRAP, TEAC, TPC and TFC Values
The correlations between FRAP, TEAC, TPC and TFC values (based on the total values of three fractions) were detected using a simple linear regression model, and the results were displayed in Figures 1 and 2.   For grape seeds, according to Figure 2, FRAP values and TEAC values were intensely correlated to TPC values (R² = 0.993, p < 0.001 and R² = 0.945, p < 0.001, respectively), but not correlated to TFC values (R² = 0.007, p = 0.825 and R² = 0.041, p = 0.574, respectively). The outcomes suggested that phenolic components could be the main contributors to the antioxidant capacities of the grape seeds, but flavonoid compounds had little influence on the antioxidant capacities of grape seeds. For grape peels, as seen from Figure 1, FRAP values and TEAC values were highly correlated to TPC values (R 2 = 0.865, p < 0.001 and R 2 = 0.892, p < 0.001, respectively), and moderately correlated to TFC values (R 2 = 0.760, p < 0.001 and R 2 = 0.732, p < 0.001, respectively). The outcomes revealed that phenolic components could be the main ingredients responsible for the antioxidant capacities of the grape peels, and flavonoid compounds might contribute to the antioxidant capacities of grape peels but were not the main contributors. In addition, TPC values were weakly correlated with TFC values (R 2 = 0.596, p < 0.001). It suggested that flavonoids comprised only a small part of phenolic components of the grape peels. Furthermore, FRAP values were significantly correlated with TEAC values (R 2 = 0.970, p < 0.001), so the antioxidant ingredients in the grape peels could reduce oxidants (like Fe(III)) and scavenge free radicals (like ABTS •+ ).
For grape seeds, according to Figure 2, FRAP values and TEAC values were intensely correlated to TPC values (R 2 = 0.993, p < 0.001 and R 2 = 0.945, p < 0.001, respectively), but not correlated to TFC values (R 2 = 0.007, p = 0.825 and R 2 = 0.041, p = 0.574, respectively). The outcomes suggested that phenolic components could be the main contributors to the antioxidant capacities of the grape seeds, but flavonoid compounds had little influence on the antioxidant capacities of grape seeds. Additionally, there was no linear correlation between TPC values and TFC values (R 2 = 0.010, p = 0.779), which suggested that phenolic components of the grape seeds were rarely flavonoids. Moreover, the correlation between FRAP values and TEAC values was remarkable (R 2 = 0.935, p < 0.001), so the antioxidant components in these grape seeds could also reduce oxidants (like Fe(III)) and scavenge free radicals (like ABTS •+ ).
The results illustrated above are consistent with many previous studies, which have reported that phenolic components were the main contributors responsible for the antioxidant capacities, and could reduce oxidants and scavenge free radicals [31,[33][34][35][36]. On the contrary, these results were quite different from some other studies that reported a very weak correlation (R 2 = 0.0337) between the FRAP values and TEAC values [32,37], indicating that the ingredients possessing reducing activities and those possessing free radicals scavenging activities in the 62 fruits were not the same, and a very weak correlation (R 2 = 0.0404) between the TEAC values and TPC values, suggesting that phenolic components could not be the main contributors to the free radicals scavenging abilities of the 62 fruits. Li et al. [37] also reported a very weak correlation between the TEAC values and the FRAP values (R 2 = 0.1563) as well as the FRAP values and the TPC values (R 2 = 0.1966), which suggested that phenolic components could not be the main contributors to activities of the 223 medicinal plants to reduce oxidants.
As for the 10 grape seeds, four phenols including gallic acid, cyanidin-3-glucoside, epicatechin and catechin gallate were found in all of them, and the content ranges were as follows, respectively: 0.022 ± 0.001 to 0.236 ± 0.009 mg/g FW with a 10-fold difference; 0.058 ± 0.003 to 0.840 ± 0.052 mg/g FW with a 14-fold difference; 0.877 ± 0.065 to 2.156 ± 0.156 mg/g FW with a 2-fold difference; 0.028 ± 0.002 to 0.176 ± 0.008 mg/g FW with a 7-fold difference, respectively. The seeds of Red Grape (Yunnan, China), Ito Kyoho Grape (Yunnan, China), Red Grape (Yunnan, China) and Red Grape (Yunnan, China) possessed the highest level of gallic acid, cyanidin-3-glucoside, epicatechin and catechin gallate, respectively. These results also prove our hypothesis that grapes with diverse genotypes have different composition and contents of bioactive compounds. Furthermore, it was reported that phenols like cyanidin-3-glucoside, resveratrol and rutin possessed varies bioactivities, such as antibacterium, antioxidant, anti-inflammation and hepatic and cardiovascular protection, so grape peels and seeds from juice and wine industries could be valuable resources to extract phenols with further use in producing functional foods, food additives and pharmaceuticals.
The above results were expressed on the weight of fresh material. In addition, the moisture contents of the grape peels and seeds are displayed in Table 6, which could be used to express the results on the weight of dry material. Table 6. Moisture contents of the tested grape peels and seeds.

Name of Grapes
Place of Production Part of Grapes Moisture Contents (%)

Sample Preparation
Grapes from 30 varieties produced in China, USA and Australia ( Figure 4) were obtained from local shops in Guangzhou, China. Grapes were washed with double distilled water and dried at room temperature. The grapes were separated into peels, pulps and seeds, and then the peels and seeds were respectively ground into particles using a special grinder for food processing. After that, accurate 2.000 g of the samples were weighed and extracted with 10 mL tetrahydrofuran at 30 • C for 30 min in a shaking water bath [55]. The samples were centrifuged at 4200 g for 10 min, and the supernatants were gathered. The extraction was repeated twice, and the supernatants were collected as the fat-soluble fractions. Subsequently, the residues were extracted with 10 mL methanol-acetic acid-water (50:3.7:46.3, v/v/v) mixture at 30 • C for 30 min in a shaking water bath, which was also repeated twice, and the supernatants were gathered up as the water-soluble fractions. Furthermore, the residues were hydrolyzed with 5 mL sodium hydroxide solution (2 mol/L NaOH, 10 mmol/L EDTA, 1% ascorbic acid) at 37 • C for 30 min in a shaking water bath, and then acidified to pH = 2 with 6 mol/L hydrochloric acid solution [56]. The mixtures were extracted twice with 5 mL n-hexane to eliminate fatty acids, which might be released during alkaline hydrolysis. Immediately, the mixtures were extracted twice with 5 mL diethyl ether and ethyl acetate mixture (1:1, v/v), and the organic phases were collected. The extracts were dried out at room temperature under a stream of N 2 using an evaporator and reconstituted in ethanol as the insoluble-bound fractions. All extracts were preserved at −20 • C until tested.

FRAP Assay
The FRAP assay was conducted referring to the literature with minor alterations [27]. Briefly, the FRAP reagent was a mixture of 300 mmol/L sodium acetate-acetic acid buffer (pH = 3.6), 10 mmol/L TPTZ solution (40 mmol/L hydrochloric acid solution as solvent) and 20 mmol/L FeCl3 solution (10:1:1, v/v/v), and it was prepared freshly and warmed to 37 °C in a water bath before used. The 0.1 mL properly diluted sample was combined with 3 mL FRAP reagent. After incubated at room temperature for 4 min, a CANY 722 visible spectrophotometer (Shanghai, China) was used to measure the absorbance of the mixtures at 593 nm. The size and volume of cuvette were 1 cm × 1 cm × 4.5 cm and 4.5 mL, respectively. The assay volumes were 1/2 to 2/3 of the volume of cuvette. The results were expressed as μmol Fe (II)/g FW of the grape peels or seeds.

TEAC Assay
The TEAC assay was carried out according to the literature with minor alterations [41]. Accordingly, the ABTS •+ stock solution was a mixture of 7 mmol/L ABTS solution and 2.45 mmol/L potassium persulphate solution (1:1, v/v), which was incubated in the dark for at least 16 h at room temperature and used within 2 days. The ABTS •+ working solution was obtained by diluting the stock solution with ethanol to an absorbance of 0.710 ± 0.05 at 734 nm. The samples were diluted approximately until they can inhibit 20-80% blank absorbance. Subsequently, the 0.1 mL properly diluted sample was mixed with 3.8 mL ABTS •+ working solution and measured at 734 nm after incubated at room temperature for 6 min. The percent of inhibition of absorbance was calculated to evaluate of the antioxidant capacity. The results were expressed as μmol Trolox/g FW of the grape peels or seeds.

FRAP Assay
The FRAP assay was conducted referring to the literature with minor alterations [27]. Briefly, the FRAP reagent was a mixture of 300 mmol/L sodium acetate-acetic acid buffer (pH = 3.6), 10 mmol/L TPTZ solution (40 mmol/L hydrochloric acid solution as solvent) and 20 mmol/L FeCl 3 solution (10:1:1, v/v/v), and it was prepared freshly and warmed to 37 • C in a water bath before used. The 0.1 mL properly diluted sample was combined with 3 mL FRAP reagent. After incubated at room temperature for 4 min, a CANY 722 visible spectrophotometer (Shanghai, China) was used to measure the absorbance of the mixtures at 593 nm. The size and volume of cuvette were 1 cm × 1 cm × 4.5 cm and 4.5 mL, respectively. The assay volumes were 1/2 to 2/3 of the volume of cuvette. The results were expressed as µmol Fe (II)/g FW of the grape peels or seeds.

TEAC Assay
The TEAC assay was carried out according to the literature with minor alterations [41]. Accordingly, the ABTS •+ stock solution was a mixture of 7 mmol/L ABTS solution and 2.45 mmol/L potassium persulphate solution (1:1, v/v), which was incubated in the dark for at least 16 h at room temperature and used within 2 days. The ABTS •+ working solution was obtained by diluting the stock solution with ethanol to an absorbance of 0.710 ± 0.05 at 734 nm. The samples were diluted approximately until they can inhibit 20-80% blank absorbance. Subsequently, the 0.1 mL properly diluted sample was mixed with 3.8 mL ABTS •+ working solution and measured at 734 nm after incubated at room temperature for 6 min. The percent of inhibition of absorbance was calculated to evaluate of the antioxidant capacity. The results were expressed as µmol Trolox/g FW of the grape peels or seeds.

Determination of TPC
The TPC values were determined based on procedures reported by Singleton, Orthofer and Lamuela-Raventos [49]. Briefly, a properly diluted sample (0.5 mL) was added to Folin-Ciocalteu reagent (0.2 mol/L, 2.5 mL). After 4 min, saturated sodium carbonate solution (about 75 g/L, 2 mL) was added to the mixture. The mixture was incubated at room temperature for 2 h, and then the absorbance was measured at 760 nm. The results were expressed as milligram gallic acid equivalent (mg GAE)/g FW of the grape peels or seeds.

Determination of TFC
The TFC values were determined according to the literature reported by Kalia et al. [50]. Accordingly, a properly diluted sample (0.5 mL) was mixed with ethanol solution (95%, v/v, 1.5 mL), AlCl 3 solution (10%, w/v, 0.1 mL), potassium acetate solution (1 mol/L, 0.1 mL) and double distilled water (2.8 mL). The mixture was incubated for 30 min at room temperature, and then the absorbance was measured at 415 nm. The results were expressed as mg quercetin equivalent (mg QE)/g FW of the grape peels or seeds.

HPLC Analysis
The phenolic and flavonoid components in the samples were detected by HPLC-PDAD (photodiode array detector) based on the method reported by Cai et al. with small modifcations [54]. In detail, the HPLC system included a Waters (Milford, MA, USA) 1525 binary HPLC pump separation module with an auto-injector and employed a Waters 2996 PDAD. Separation was carried out with an Agilent Zorbax Extend-C18 column (250 × 4.6 mm, 5 µm) at 40 • C with a gradient elution solution A, composed of formic acid solution (0.1%, v/v), and solution B, methanol, which were routinely delivered at a flow rate of 0.8 mL/min according to the procedure: 0 min, 95% (A); 15 min, 80% (A); 20 min, 70% (A); 25 min, 63% (A); 40 min, 60% (A); 60 min, 50% (A); 65 min, 50% (A); 65.1 min, 95% (A); and 70 min, 95% (A). Fat-soluble, water-soluble and insoluble-bound fractions were combined together before sampling. The spectra were recorded between 200 and 600 nm to characterize the peak patterns. Phenolic and flavonoid components were identified by the retention time and UV-Vis spectra comparing with standards and quantified by the peak area under maximum absorption wavelength, and the results were expressed as mg/g FW of the grape peels or seeds.

Data Analysis
All tests were conducted in triplicate and the values were expressed as mean ± SD (standard deviation). Data analysis was performed using SPSS 22 (International Business Machines Corporation, Armonk, NY, USA) and Excel 2007 (Microsoft Corporation, Redmond, WA, USA).

Conclusions
In this study, the antioxidant capacities and total phenolic and flavonoid contents of peels and seeds from 30 grape varieties were systematically evaluated. The antioxidant capacities and phenolic and flavonoid contents of the grape peels and seeds were greatly different, and those of the three fractions were, in decreasing order: fat-soluble fractions > water-soluble fractions > insoluble-bound fractions. Antioxidant components in these grape peels and seeds could reduce oxidants and scavenge free radicals, and phenols were the main contributors to the antioxidant capacities, and flavonoids were not major contributors to these activities. Several phenolic compounds such as gallic acid, cyanidin-3-glucoside, epicatechin, catechin gallate, ferulaic acid, rutin and resveratrol were identified and quantified in these grape peels and seeds. These grape wastes could be abundant sources of natural bioactive compounds for developing functional foods, food additives and pharmaceuticals.