Resveratrol Productivity of Wild Grapes Native to Japan: Vitis Ficifolia Var
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Research Note Resveratrol Productivity of Wild Grapes Native to Japan: Vitis ficifolia var. lobata and Vitis ficifoliavar. ganebu Shuji Shiozaki,1* Taiji Nakamura,1 and Tsuneo Ogata2 Abstract: Resveratrol production potential was determined in the leaves and berries of Vitis ficifolia Bunge var. lobata (Ebizuru) and V. ficifolia Bunge var. ganebu (Ryuukyuuganebu), wild grapes native to Japan. Ultraviolet-C (UV-C) irradiation was used to stimulate resveratrol production. Resveratrol levels in the nonirradiated leaf discs were 3.6 times higher in Ryuukyuuganebu than in Ebizuru, and levels in the Ryuukyuuganebu leaf discs were 4.4 times higher than Ebizru after UV-C irradiation. Resveratrol levels in the nonirradiated berries differed little be- tween the varieties. The resveratrol level in immature berries of both varieties increased significantly 24 hr after 15 min of UV-C irradiation. However, the resveratrol production stimulated by UV-C had a different pattern. Resve- ratrol production in Ebizuru declined during berry development and maturation, whereas that of Ryuukyuuganebu declined until veraison before it increased to almost the same level as that found during the most immature stage at harvest. Increased resveratrol in the mature berries of Ryuukyuuganebu was also detected 48 hr after UV-C ir- radiation. UV-C irradiation had no effect on the piceid level of either variety. Ryuukyuuganebu is a wild grape with a distinctive resveratrol production pattern, especially in the berry. Key words: wild grape, leaf, berry, resveratrol, piceid Japan has seven species and eight varieties of wild grapes. infections in grapes. In addition to biotic stresses, resveratrol The main species are Vitis coignetiae Pulliat, V. f lexuosa synthesis is highly stimulated by abiotic stresses such as UV Thunb., and V. ficifolia Bunge var. lobata (Regel) Nakai irradiation, ozone, wounding, and chemicals (Bavaresco and (common name, Ebizuru) (Nakagawa et al. 1991). Vitis fici- Fregoni 2001). Numerous studies have demonstrated the ben- folia var. lobata is endemic to low-altitude areas throughout eficial health effects of resveratrol, including its antioxidant Japan except for the northern part of Hokkaido and Okina- (Fremont 2000), antiatherogenic (Ramprasath and Jones 2010), wa, while V. ficifolia Bunge var. ganebu Hatusima (common anticancer (Aggarwal et al. 2004), antiplatelet aggregation name, Ryuukyuuganebu) is endemic to coastal areas of the (Wang et al. 2002), and anti-inflammatory effects (Fremont southwest islands. The leaf blades of Ebizuru and Ryuukyuu- 2000). Both piceid and resveratrol can inhibit platelet aggrega- ganebu are thick, and their abaxial side is pubescent. Ebizuru tion and eicosanoid synthesis (Kimura et al. 1985, Shan et al. and Ryuukyuuganebu have no endodormancy and can bear 1990). Piceid is effective on its own and is also hydrolyzed by fruit continuously. Their berries are very small, but the skin β-glucosidase in the intestine to produce resveratrol (Hackett accumulates high levels of anthocyanin. The anthocyanin 1986). These important physiological and biological activi- components of the skin are considerably more than those ties of resveratrol have led to many studies on the evaluation, found in some table-grape cultivars (V. labruscana) from Ja- control, and health benefits of resveratrol concentrations in pan or in wild grapes distributed in northern Japan, such as grapes and wine. Food, nutraceutical, and cosmetic indus- V. coignetiae (Mochioka et al. 1995). tries have also shown a great interest in grapes as a source In addition to anthocyanin, another characteristic poly- of resveratrol. Wine, especially red wine, and grape juice are phenol found in grapes is resveratrol (3,5,4′-trihydroxy stil- important sources of resveratrol in food. Health benefits of bene), which is present in the berry skin, seeds, and leaves moderate drinking of red wine (Guilford and Pezzuto 2011) as free (mainly trans- form) and bound forms (piceid is a and high resveratrol bioavailability from red wine and grape 3-β-glucoside of resveratrol) (Bavaresco and Fregoni 2001). juice (Stockley et al. 2012) have stimulated consumer interest. Resveratrol acts as a phytoalexin during the response to fungal Resveratrol derived from grapes has been used in nutritional supplements and cosmetics. Resveratrol levels in berry skin were determined for 32 1Graduate School of Life & Environmental Sciences, Osaka Prefecture table-grape and winegrape cultivars grown in Japan, as well University, 1-1, Gakuen-cho, naka-ku, Sakai, Osaka, 599-8531, Japan; and as for wines made from some of these grapes (Okuda and 2Faculty of Agriculture, Kochi University, Otsu 200, Monobe, Nankoku, Kochi, 783-8502, Japan. Yokotsuka 1996). Resveratrol production in the berry skins *Corresponding author (email: [email protected]) in response to UV irradiation has also been investigated in Manuscript submitted Apr 2012, revised Aug 2012, accepted Sept 2012 the leading white and red winegrape cultivars grown in Ja- Copyright © 2013 by the American Society for Enology and Viticulture. All pan (Takayanagi et al. 2004). However, little is known about rights reserved. resveratrol levels and production in the leaves and berries of doi: 10.5344/ajev.2012.12066 wild grapes native to Japan. 163 Am. J. Enol. Vitic. 64:1 (2013) 164 – Shiozaki et al. The objective of this study was to evaluate the resveratrol pan) were measured in the leaves (n = 10) of Ebizuru and productivity of the wild Japanese grape varieties Ebizuru and Ryuukyuuganebu. Leaf discs were prepared from 15 leaves of Ryuukyuuganebu. We investigated their resveratrol levels and each variety. The leaf was folded into two along the midvein their potential for its production in response to UV-C irra- and discs with a diameter of 12 mm were punched out using diation of their leaves and berries. We discuss the potential a cork borer. The two discs from each punch were assigned to for resveratrol production by Ryuukyuuganebu berries in re- a different treatment: the control or UV-C irradiation. Thirty sponse to UV-C irradiation during berry development and leaf discs were floated on sterile distilled water in a Petri dish maturation. (90 mm diam) with the abaxial side facing up. Ninety discs were placed in a total of three Petri dishes. The fresh weight Materials and Methods of a leaf disc was measured for 10 discs. Chemicals. The trans-resveratrol standard was purchased Berry materials. Experiments with berries were con- from Sigma-Aldrich (St. Louis, MO). Water, acetic acid, and ducted during 2005 and 2006. In 2005, berries with pedicels acetonitrile were HPLC grade, whereas other reagents were of Ebizuru and Ryuukyuuganebu were randomly sampled analytical grade. Standard piceid, a glucoside of resvera- from five bunches at 20-day intervals from 20 days after full trol, was prepared using a modified version of a published bloom (DAB) to harvest. In 2006, the berries were sampled method (Waterhouse and Lamuela-Raventos 1994). Briefly, at 40 and 80 DAB in each variety. The samples were placed the piceid standard was produced by methanol (MeOH) ex- in a plastic container on wetted river sand that had been dry- traction from dry roots of Polygonum cuspidatum Sieb. et sterilized at 100°C. The pedicel was inserted into the sand. Zucc. (Kojo-kon). The piceid extract was partially purified Forty berries were used in each treatment. The fresh weight by C18 reversed-phase column chromatography and fraction- of the berries (n = 15) was measured without the pedicel at ated by reversed-phase HPLC. The HPLC conditions were each sampling. as follows: flow rate, 1 mL/min; column, Nucleosil 120-5 UV-C irradiation. The UV-C light source was a Nation- C18 (Macherey-Nagel, Düren, Germany) (4.0 × 250 mm); al GL-10 (Panasonic Corp., Kadoma, Japan) (10 W) with a solvents, water adjusted to pH 2.4 with acetic acid (A) and maximum emission wavelength of 260 nm. Leaf and berry 80% v/v acetonitrile in A (B); elution program starting with samples were irradiated using the UV light at a distance of 0% solvent B at 0 min, followed by 15% at 20 min, 22% at 13 cm (420 µW/cm2) for 15 min at 25°C. Samples were then 35 min, 63% at 65 min, and 100% at 66 min; detector, UV incubated in the dark at 25°C for 24 hr (in 2005 and 2006) at 306 nm. Purity was tested through hydrolysis of the frac- or for 48 hr in 2006 for berries only. Nonirradiated controls tionated sample (major peak with a retention time of 38 min) were incubated in the same way. After incubation, the leaf using HCl or β-glucosidase; each hydrolysate was extracted discs and berries (without their pedicels) were immediately with ethyl acetate and analyzed by reversed-phase HPLC. frozen in liquid nitrogen and stored at -25°C until analysis. The retention time of the hydrolysate was compared with trans-Resveratrol and trans-piceid extraction and pu- that of trans-resveratrol. Further confirmation of piceid was rification. Frozen leaf samples were ground to a fine pow- obtained by HPLC-MS (APCI+) analysis of the fractionated der in liquid nitrogen with a mortar and pestle. trans-Piceid sample and a sample that had been UV-C irradiated (420 µW/ was only analyzed in the berry sample from 2006. A 0.5 cm2) for 15 min (the cis form). A mass-to-charge ratio of m/z g sample of leaf powder was homogenized in 20 mL 90% 229 was detected for each sample, indicating the molecular MeOH using a glass homogenizer for 1 min and then filtered. resveratrol ion. Thus, a peak with a retention time of 38 min The residue was washed with 20 mL 90% MeOH and 60 mL under these HPLC conditions was identified as trans-piceid, 100% MeOH. The berry samples including seeds (~3 g) were and this peak fraction was used as the trans-piceid standard homogenized in 40 mL 90% MeOH using a bio-mixer for 2 during analyses.