Occurrence and Estimation of trans-Resveratrol in One-Year-Old Canes from Seven Major Chinese Grape Producing Regions

The concentration of trans-resveratrol in 165 grape cane samples from three major grape production regions and four large distribution centers of Chinese wild Vitis species were determined by reversed-phase high-performance liquid chromatography (HPLC). Among the different genotype groups and purpose of uses, cultivars of V. vinifera had much higher amounts of trans-resveratrol than did the cultivars of both V. labrusca or V. labrusca and V. vinifera hybrids, and within the V. vinifera species, significantly higher amounts of trans-resveratrol were found in wine grapes compared to table ones. No significant differences were observed between V. labrusca and its hybrids from crosses with V. vinifera, and between red cultivars and white ones (P < 0.05 or P < 0.01). The contents of trans-resveratrol, as a normal constituent occurring in grape canes, in Chinese wild species of V. amurensis, V. pentagona, and V. davidii from their native habitats were also relatively high.

Grapes and wine are the primary dietary sources of resveratrol in the human diet. In the past 20 years, China achieved much success in the development of its grape and wine industry. All the cultivated grape cultivars in China are mainly grown in three major regions, indicated as C1, C2, and C3 ( Figure 1). Most Chinese wild Vitis species are distributed in four major eco-geographic centers, labeled W1, W2, W3, and W4 ( Figure 1).

Figure 1. Geographical location of the sampling sites (black points).
Provinces with a sizeable amount of grape cultivation in China are Xinjiang, Hebei, Shandong, Liaoning and Henan [18]. Additionally, China is one of the most important centers of origin of Vitis species and several wild Chinese Vitis species that occur widely in their native habitats, such as V. amurensis in the north-east regions of China, V. davidii and V. pentagona in the southern regions, have been developed to produce wine [19,20]. Large quantities of grape canes are produced as solid wastes during the annual pruning campaigns of the viticulture industry. Compared with other solid byproducts from wineries and vineyards such as grape pomace [21], grape stems [22], grape seeds [23], and so on, people have paid little attention to grape cane wastes with respect to the possible recovery of high value-added compounds. These pruning wastes represent a great potential source of natural antioxidants, and after suitable treatment these low-cost residues could contribute to the sustainable development of related industries. Currently, these grape cane wastes are disposed of in landfills, burned in situ or used as fuels, indicating low-value utilizations [24]. We know of only a few previous studies that have addressed the resveratrol content in a single sample of Pinot Noir (V. vinifera) grape cane [25,26]. The diversity of the health-promoting properties of trans-resveratrol has sparked intense research interest devoted to developing resveratrol-enriched foodstuffs or plants and exploiting other new potential sources to meet the increasing demand [27][28][29].
The main objectives of the present work were: (i) to investigate and evaluate the trans-resveratrol content in one-year-old canes at the grape germplasm level in order to acquire information for the future utilization of these wastes; (ii) to compare grape canes with other main known sources of transresveratrol; (iii) to estimate the annual yield and the potential economic value of trans-resveratrol in grape cane wastes from seven large grape-concentrating areas of China.

Genotypic variation of trans-resveratrol contents in grape canes from two germplasm repertoires
A total of 118 grape cultivars (51 from Yangling and 67 from Yantai), mainly belonging to three important commercial grape groups: V. vinifera, V. labrusca, and V. labrusca and V. vinifera hybrids, were analyzed for trans-resveratrol content. The frequency distribution and median of amounts of trans-resveratrol in one-year-old canes were similar in the two repositories (Figures 2 and 3).
The wine grape cultivars of V. vinifera had higher trans-resveratrol contents than other grape genotypic groups in this study in the two repositories. trans-Resveratrol content in wine grape cultivars of V. vinifera ranged from 664.7 to 1,751.6 mg kg −1 of cane fresh weight (FW) with a median of 906.6 mg kg −1 of cane FW in Yangling ( Figure 2) and from 570.8 to 1,452.9 mg kg −1 of cane FW with a median of 780.9 mg kg −1 of cane FW in Yantai ( Figure 3). The trans-resveratrol concentrations posted by wine grape cultivars of V. vinifera varied significantly with their genetic backgrounds. The highest value was found in 'Pinot Noir', one of the most famous wine grape cultivars, reaching 1,751.6 and 1,452.9 mg kg −1 of cane FW in Yangling and Yantai, respectively. It is somewhat similar to the previous reports that the highest trans-resveratrol concentrations were also found in red wines from cv. 'Pinot Noir' [30,31].

Figure 2.
Box and whisker plots of Yangling Grape Germplasm Repository. The median and mean value are indicated by the horizontal bar and the plus sign inside the box, respectively; the height of the box represents the interquartile range (IQR), which is the difference between the third quartile and the first quartile of the data. The whisker (the vertical lines from the top and bottom of the box) extends to a distance of 1.5 times IQR. Data outside these whiskers are marked by the black dots. The numbers in front of the black dots represent the accession numbers (Table 1). V-T, table grape cultivars of V. vinifera; V-W, wine grape cultivars of V. vinifera; LV-T, table grape cultivars of hybrids between V. labrusca and V. vinifera; AL-T, table grape of hybrid between V. aestivalis and V. labrusca; R-T, table grape of V. rotundifolia.  (Table 1). V-T, table grape cultivars of V. vinifera; V-W, wine grape cultivars of V. vinifera; LV-T, table grape cultivars of hybrids between V. labrusca and V. vinifera; L-J, juice grape cultivars of V. labrusca; VR-W, wine grape cultivar of hybrid between V.vinifera and V. riparia. LR-Rs, rootstock grape cultivar of hybrid between V. labrusca and V. riparia.
For all grape cultivars of three species in this study the average trans-resveratrol content in grapes of V. vinifera was 937.9 mg kg −1 of cane FW, and significantly higher than that (571.3 mg kg −1 of cane FW) of the V. labrusca and V. vinifera hybrids in Yangling (P < 0.05 and P < 0.01). The cultivars of V. vinifera from Yantai had significantly higher amounts of trans-resveratrol than did both V. labrusca and V. labrusca and V. vinifera hybrids (P < 0.05 and P < 0.01). However, there were no significant differences between cultivars of V. labrusca and those of V. labrusca and V. vinifera hybrids in Yantai. With regard to grape cultivars' purpose of uses both originating from V. vinifera and V. labrusca and V. vinifera hybrids, wine grapes had significantly higher trans-resveratrol than did table grapes in both Yangling and Yantai (P < 0.05 and P < 0.01), whereas there were no significant differences between juice cultivars of V. labrusca and table cultivars of V. vinifera and V. labrusca and V. vinifera hybrids in Yantai. In addition, no significant differences of trans-resveratrol contents were observed between the red and green cultivars among different species in both Yangling and Yantai ( Table 2).
The results of the study on the two grape germplasm repositories indicated that the amounts of trans-resveratrol in one-year-old canes of various cultivars were strongly influenced by their genetic background. trans-Resveratrol synthesis in plants can usually be triggered by exogenous stress factors, such as UV irradiation and the presence of pathogenic fungi, as discussed previously, but its synthesis in grape canes during the dormant season with low fungal infection conditions and weak UV irradiation might be a non-specific response to those stresses. Therefore, trans-resveratrol can be considered as a normal constituent of grape canes, as the similar results reported by Langcake and Pryce [32] in the lignified stems of cv. Müller-Thurgau. Table 2. Comparison of trans-resveratrol contents (mean value ± S.D., mg kg −1 of FW) in grape cane among different genotypic groups or purpose of uses of two grape germplasm repositories. The lowercases and uppercases mean significant variation of average trans-resveratrol in grape cane of Yantai Grape Germplasm Repository among different genotypic groups or purpose of uses at P < 0.05 and P < 0.01 levels (ANOVA), respectively; and different letters indicate significant variation; a V, V. vinifera; b LV, hybrid of V. labrusca and V. vinifera; c L, V. labrusca; d Not available; T * and *** indicate significant difference of average trans-resveratrol in grape cane in the same grape germplasm repository between different genotypic groups or purpose of uses at P < 0.05 and P < 0.01 levels (Student's t-test), respectively.

Occurrence and prediction of trans-resveratrol in main grape cultivars from China and comparison with other known sources
The concentration ranges and averages with standard deviations of trans-resveratrol in the main cultivars from three major grape production areas and four important distribution centers of Chinese wild Vitis species are shown in Table 3. Occurrences of trans-resveratrol in the main known sources and transgenic plants were summarized in Table 4.  The lowercases and uppercases mean significant variation at 0.05 and 0.01 level, respectively; and the values in any two groups with different letters indicate significant variation (P < 0.05 or P < 0.01); a The numbers in the following blanks represent the same accession numbers to Tab    The observed trans-resveratrol contents in grape canes were higher than the reported amounts in most of the other known sources, with a few exceptions (Tables 3, 4). Furthermore, trans-resveratrol occurs in Polygonum cuspidatum and peanut roots in the range from a few tens to a few thousands mg kg −1 dry weight, showing a marked fluctuations appearing to be related to various factors [10,37,29]. In contrast, grape canes represent a novel and stable source of trans-resveratrol.
In this study, 165 grape cane samples were used to estimate the annual yields and the economic outputs of trans-resveratrol from grape cane wastes of seven major grape production regions of China (Table 3). Predicted annual yields of trans-resveratrol from each region were calculated with the mean and range values shown in Table 3, with an approximate annual grape cane production rate of 1 t ha −1 [38] and cultivated area of each major production region [39]. Estimated annually economic outputs of each region were generated from the average yields values of trans-resveratrol, at a commercial price of food-grade product ranging from US$ 2,000 to US$ 3,000 kg −1 reported by Baur and Sinclair when they evaluated the costs of daily or yearly trans-resveratrol intake for a human [40]. The total value of trans-resveratrol from grape cane wastes in China could reach up to US$ 543.8-815.7 million year −1 based on cultivation data of 2006. Actually, the vineyard area in China, with a steady increase year after year, reached approximately 475 mha in 2009 [41]. Recovery and utilization of trans-resveratrol from these agricultural pruning wastes, as a sideline production, indicates a huge economic potential.

Plant material
One hundred and sixty five (165) grape cane samples used in this study (Table 1), including seven raisin grapes of V. vinifera from Turpan, Xinjiang (C1), eight wine grapes of V. vinifera from Helan, Ningxia (C2), 51 grape cultivars from the experimental vineyard of grape germplasm repository of College of Enology, Northwest A&F University at Yangling, Shaanxi (C2), 67 grape cultivars from the grape germplasm collection of Changyu Pioneer Wine Co. Ltd in Yantai, Shandong Province (C3), one wine grape hybrid of V. labrusca and V. vinifera, five wine grapes of V. amurensis, and four wine grape hybrids of V. vinifera and V. amurensis from Tonghua, Jilin (W1), 11 genotypes of V. pentagona from Lantian, Shaanxi, Danfeng, Shaanxi, and Yunxi, Hubei, and Douan, Guangxi (W2, W4), 11 grape cultivars or genotypes of V. davidii from Zhijiang, Hunan, Hongjiang, Huan, Chongyi, Jiangxi, and Yushan, Jiangxi (W3). The ideal one-year-old canes with moderate growth vigor (approximately 0.8-1.0 cm diameter) were collected from the above locations during the 2008 pruning practice. All cane samples were frozen in liquid nitrogen, ground through a 0.5 mm sieve using an electrical grinder (final particle size < 0.5 mm), stored under vacuum in labeled plastic containers, and then stored at −20 °C in a freezer until extraction.

Chemicals
trans-Resveratrol standard (purity >97%) was purchased from Sigma-Aldrich Chemical Co. (Shanghai, China). Methanol and acetonitrile were HPLC grade solvents from Tianjin Kermel Chemical Reagent Co. Ltd. (Tianjin, China). Analytical grade acetic acid was from Xi'an Chemistry Factory (Xi'an, China). Water was purified using the Milli-Q system (Millipore, Bedford, MA, USA).

Extraction of trans-resveratrol from grape canes
Triplicate samples of ground grape cane (5 g, fresh weight) were extracted three times with acidified methanol solution (40 mL, 1 N HCl/methanol/water, 1/80/19, v/v/v), and extraction was performed under continuous stirring (600 rpm) at 20 °C for 1h in an external water bath. The extracts were centrifuged at 8,000 g for 15 min at 4 °C using a Sorvall RC-5C Plus centrifuge (Kendro Laboratory Products, Newton, CT, USA). All the supernatants were combined in a 250 mL flask and concentrated in a Büchi RE-111 evaporator (Switzerland) at 35 °C to a volume of 10 mL. The final concentrate solution was filtered through a 0.22 µm nylon micro-membrane and stored at −40 °C until analysis.

Chromatographic analysis
The chromatographic analyses were carried out on a Shimadzu liquid chromatograph system (Shimadzu Corp, Kyoto, Japan) equipped with a quaternary pump coupled with a photodiode array detector and a UV-Vis detector.
Samples were injected onto a Shim-Pack VP-ODS C 18 column (250 mm × 4.6 mm, 5 μm) at room temperature. The mobile phase was acidified water containing 3% acetic acid (A) and acetonitrile (B). The gradient program is as follows: B: 0.00-5.00 min, 0-8.5%; 5.00-16.50 min, 8.5-2.0%; 16.50-35.00 min, 2.0-18%; 35.00-50.00 min, 18-20%; 50.00-65.00 min, 20-30%; 65.00-70.00 min, 30-0%. The column held at 30 °C was flushed at a flow rate of 0.8 mL/min. The DAD detector was applied to ascertain the maximum absorbance wavelength of trans-resveratrol within a range of 200 to 400 nm. The UV-vis detector was conducted at 306 nm for quantitation of trans-resveratrol in grape cane extracts with the external standard. Chromatographic identification and confirmation of transresveratrol were based on comparing retention times with the authentic standard and on-line UV absorption spectrum data. Results were acquired and processed by the Shimadzu Workstation CLASS-VP 6.12 software (Shimadzu Corp, Kyoto, Japan). Typical HPLC chromatographies of grape cane extracts and trans-resveratrol standard are shown in Figure 4.

Statistical analysis
Mean values of each grape cane sample were from three replicates, and were employed for further analysis. For the data of two grape germplasm repositories, the cultivar variations of trans-resveratrol contents in one-year-old canes were evaluated at the germplasm level, and the box-and-whisker plots, which are helpful in interpreting the distribution of data, were generated by the software of Sigmaplot version 10.0 (Systat Software Inc., US) to display range, median, and distribution density of variables in sample size. The Student's independent-sample t-test and one-way analysis of variance (ANOVA) were carried out by SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA)

Conclusions
Grape canes are generated in a substantial quantity as agricultural pruning wastes in the grape industry. trans-Resveratrol has been proven to be a normal constituent of grape canes and its content varies largely with different genotypic groups as well as their purpose of uses. Compared with other known sources, the trans-resveratrol content in grape cane wastes is high enough to indicate they are a potentially new and stable commercial source. Occurrence of trans-resveratrol in main grape cultivars cultivated in seven major grape production regions in China shows a great potential economic value.