Research Note Resveratrol Productivity of Wild Grapes Native to Japan: Vitis ficifolia var. lobata and Vitis ficifolia var. ganebu

Transcription

1 Research Note Resveratrol Productivity of Wild Grapes Native to Japan: Vitis ficifolia var. lobata and Vitis ficifolia var. ganebu Shuji Shiozaki, 1 * Taiji Nakamura, 1 and Tsuneo Ogata 2 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 between 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. Resveratrol 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 irradiation. 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. The main species are Vitis coignetiae Pulliat, V. f lexuosa Thunb., and V. ficifolia Bunge var. lobata (Regel) Nakai (common name, Ebizuru) (Nakagawa et al. 1991). Vitis ficifolia var. lobata is endemic to low-altitude areas throughout Japan except for the northern part of Hokkaido and Okinawa, while V. ficifolia Bunge var. ganebu Hatusima (common name, Ryuukyuuganebu) is endemic to coastal areas of the southwest islands. The leaf blades of Ebizuru and Ryuukyuuganebu are thick, and their abaxial side is pubescent. Ebizuru and Ryuukyuuganebu have no endodormancy and can bear fruit continuously. Their berries are very small, but the skin accumulates high levels of anthocyanin. The anthocyanin components of the skin are considerably more than those found in some table-grape cultivars (V. labruscana) from Japan or in wild grapes distributed in northern Japan, such as V. coignetiae (Mochioka et al. 1995). In addition to anthocyanin, another characteristic polyphenol found in grapes is resveratrol (3,5,4 -trihydroxy stilbene), which is present in the berry skin, seeds, and leaves as free (mainly trans- form) and bound forms (piceid is a 3-β-glucoside of resveratrol) (Bavaresco and Fregoni 2001). Resveratrol acts as a phytoalexin during the response to fungal 1 Graduate School of Life & Environmental Sciences, Osaka Prefecture University, 1-1, Gakuen-cho, naka-ku, Sakai, Osaka, , Japan; and 2 Faculty of Agriculture, Kochi University, Otsu 200, Monobe, Nankoku, Kochi, , Japan. *Corresponding author ( ssgvo67@plant.osakafu-u.ac.jp) Manuscript submitted Apr 2012, revised Aug 2012, accepted Sept 2012 Copyright 2013 by the American Society for Enology and Viticulture. All rights reserved. doi: /ajev infections in grapes. In addition to biotic stresses, resveratrol synthesis is highly stimulated by abiotic stresses such as UV irradiation, ozone, wounding, and chemicals (Bavaresco and Fregoni 2001). Numerous studies have demonstrated the beneficial health effects of resveratrol, including its antioxidant (Fremont 2000), antiatherogenic (Ramprasath and Jones 2010), anticancer (Aggarwal et al. 2004), antiplatelet aggregation (Wang et al. 2002), and anti-inflammatory effects (Fremont 2000). Both piceid and resveratrol can inhibit platelet aggregation and eicosanoid synthesis (Kimura et al. 1985, Shan et al. 1990). Piceid is effective on its own and is also hydrolyzed by β-glucosidase in the intestine to produce resveratrol (Hackett 1986). These important physiological and biological activities of resveratrol have led to many studies on the evaluation, control, and health benefits of resveratrol concentrations in grapes and wine. Food, nutraceutical, and cosmetic industries have also shown a great interest in grapes as a source of resveratrol. Wine, especially red wine, and grape juice are important sources of resveratrol in food. Health benefits of moderate drinking of red wine (Guilford and Pezzuto 2011) and high resveratrol bioavailability from red wine and grape juice (Stockley et al. 2012) have stimulated consumer interest. Resveratrol derived from grapes has been used in nutritional supplements and cosmetics. Resveratrol levels in berry skin were determined for 32 table-grape and winegrape cultivars grown in Japan, as well as for wines made from some of these grapes (Okuda and Yokotsuka 1996). Resveratrol production in the berry skins in response to UV irradiation has also been investigated in the leading white and red winegrape cultivars grown in Japan (Takayanagi et al. 2004). However, little is known about resveratrol levels and production in the leaves and berries of wild grapes native to Japan. 163

2 164 Shiozaki et al. The objective of this study was to evaluate the resveratrol productivity of the wild Japanese grape varieties Ebizuru and Ryuukyuuganebu. We investigated their resveratrol levels and their potential for its production in response to UV-C irradiation of their leaves and berries. We discuss the potential for resveratrol production by Ryuukyuuganebu berries in response to UV-C irradiation during berry development and maturation. Materials and Methods Chemicals. The trans-resveratrol standard was purchased from Sigma-Aldrich (St. Louis, MO). Water, acetic acid, and acetonitrile were HPLC grade, whereas other reagents were analytical grade. Standard piceid, a glucoside of resveratrol, was prepared using a modified version of a published method (Waterhouse and Lamuela-Raventos 1994). Briefly, the piceid standard was produced by methanol (MeOH) extraction from dry roots of Polygonum cuspidatum Sieb. et Zucc. (Kojo-kon). The piceid extract was partially purified by C 18 reversed-phase column chromatography and fractionated by reversed-phase HPLC. The HPLC conditions were as follows: flow rate, 1 ml/min; column, Nucleosil C18 (Macherey-Nagel, Düren, Germany) ( mm); solvents, water adjusted to ph 2.4 with acetic acid (A) and 80% v/v acetonitrile in A (B); elution program starting with 0% solvent B at 0 min, followed by 15% at 20 min, 22% at 35 min, 63% at 65 min, and 100% at 66 min; detector, UV at 306 nm. Purity was tested through hydrolysis of the fractionated sample (major peak with a retention time of 38 min) using HCl or β-glucosidase; each hydrolysate was extracted with ethyl acetate and analyzed by reversed-phase HPLC. The retention time of the hydrolysate was compared with that of trans-resveratrol. Further confirmation of piceid was obtained by HPLC-MS (APCI+) analysis of the fractionated sample and a sample that had been UV-C irradiated (420 µw/ cm 2 ) for 15 min (the cis form). A mass-to-charge ratio of m/z 229 was detected for each sample, indicating the molecular resveratrol ion. Thus, a peak with a retention time of 38 min under these HPLC conditions was identified as trans-piceid, and this peak fraction was used as the trans-piceid standard during analyses. Grapes. The experiments were carried out in 2005 and 2006 with three 11-year-old vines of Vitis ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) grown in a research field at Osaka Prefecture University (lat: N, 30 m asl). The soil was a gray lowland, ph 5.5. Climate conditions of growing periods of the vines in 2005 and 2006 (from April until October) were as follows: average temperature, 22.6 C and 22.2 C; rainfall, 686 mm and 916 mm; and sunshine duration, 1,208 hr and 1,180 hr, respectively. During both years, full bloom of Ebizuru and Ryuukyuuganebu occurred in early June and mid-june, respectively. Leaf materials. In 2005, leaves that were free from visual defects were sampled from the fifth to the tenth nodes, counting up from the base of two-month-old shoots. Values of the chlorophyll (the maximum value in the rate of leaf green) (Chlorophylltester CT 102; Fujihira Industry Co., Tokyo, Japan) were measured in the leaves (n = 10) of Ebizuru and Ryuukyuuganebu. Leaf discs were prepared from 15 leaves of each variety. The leaf was folded into two along the midvein and discs with a diameter of 12 mm were punched out using a cork borer. The two discs from each punch were assigned to a different treatment: the control or UV-C irradiation. Thirty leaf discs were floated on sterile distilled water in a Petri dish (90 mm diam) with the abaxial side facing up. Ninety discs were placed in a total of three Petri dishes. The fresh weight of a leaf disc was measured for 10 discs. Berry materials. Experiments with berries were conducted during 2005 and In 2005, berries with pedicels of Ebizuru and Ryuukyuuganebu were randomly sampled from five bunches at 20-day intervals from 20 days after full bloom (DAB) to harvest. In 2006, the berries were sampled at 40 and 80 DAB in each variety. The samples were placed in a plastic container on wetted river sand that had been drysterilized at 100 C. The pedicel was inserted into the sand. Forty berries were used in each treatment. The fresh weight of the berries (n = 15) was measured without the pedicel at each sampling. UV-C irradiation. The UV-C light source was a National GL-10 (Panasonic Corp., Kadoma, Japan) (10 W) with a maximum emission wavelength of 260 nm. Leaf and berry samples were irradiated using the UV light at a distance of 13 cm (420 µw/cm 2 ) for 15 min at 25 C. Samples were then incubated in the dark at 25 C for 24 hr (in 2005 and 2006) or for 48 hr in 2006 for berries only. Nonirradiated controls were incubated in the same way. After incubation, the leaf discs and berries (without their pedicels) were immediately frozen in liquid nitrogen and stored at -25 C until analysis. trans-resveratrol and trans-piceid extraction and purification. Frozen leaf samples were ground to a fine powder in liquid nitrogen with a mortar and pestle. trans-piceid was only analyzed in the berry sample from A 0.5 g sample of leaf powder was homogenized in 20 ml 90% MeOH using a glass homogenizer for 1 min and then filtered. The residue was washed with 20 ml 90% MeOH and 60 ml 100% MeOH. The berry samples including seeds (~3 g) were homogenized in 40 ml 90% MeOH using a bio-mixer for 2 min and then filtered. The residue from the berry sample was washed with 20 ml 90% MeOH and 40 ml 100% MeOH. All filtrates from each sample were combined and concentrated to produce an aqueous phase, which was then mixed with 20 ml distilled water. The aqueous phase was then partitioned using an equal volume of hexane to remove lipids and chlorophyll before it was extracted three times using ethyl acetate at ph 8.0. The combined ethyl acetate extract containing transresveratrol was reduced to dryness in vacuo. The aqueous phase containing piceid was evaporated to remove acetic acid and adjusted to a ph of 7.0 using 1 N NaOH before its volume was adjusted to 50 ml with distilled water. The trans-resveratrol extract was partially purified with a Sep-Pak C 18 -ENV cartridge (Waters Corp., Milford, MA) as described (Jeandet et al. 1991) with modification. The extract was redissolved in 1 ml 100% MeOH, and 4 ml 33 mm phosphate buffer (ph 7.0) was added before centrifugation

3 V. ficifolia Resveratrol Productivity 165 at 12,500 g for 10 min. The supernatant was loaded onto the cartridge, which had been previously activated with 6 ml 100% MeOH and washed with 10 ml 33 mm phosphate buffer (ph 7.0). The pellet was washed with 5 ml 33 mm phosphate buffer and centrifuged in the same way before the supernatant was applied to the cartridge. The sample flow rate was <1 ml/min. The cartridge was purged with N 2 for 1 min to eliminate the buffer solution after the sample had been applied. trans-resveratrol was eluted in a 10 ml test tube with 5 ml ethyl acetate. The eluate was concentrated to dryness with a centrifugal concentrator (model VC-96N; TAITEC Corp., Koshigaya, Japan) and stored at -20 C until HPLC analysis. trans-piceid was also purified using a Waters Sep-Pak C 18 - ENV cartridge. The aqueous 50 ml sample was centrifuged at 12,500 g for 10 min and the supernatant was loaded onto the cartridge, activated, and washed as described above for resveratrol. The cartridge was then washed with 20 ml 33 mm phosphate buffer (ph 7.0) and 20 ml 0.1% HCl before the residual aqueous solution was eliminated from the cartridge by suction for 15 min. Piceid was then eluted using 20 ml 16% acetonitrile containing 0.1% HCl. HPLC analysis. trans-resveratrol and trans-piceid were analyzed using the same HPLC conditions. The dried samples containing trans-resveratrol were redissolved in 500 µl ethyl acetate and centrifuged at 1,800 g for 3 min. A 20 µl aliquot of the supernatant was then analyzed by HPLC. For trans-piceid, a 100 µl aliquot of 16% acetonitrile containing 0.1% HCl was injected directly into the HPLC. Resveratrol and piceid were detected by UV at 306 nm (detection limit of trans-resveratrol: 0.5 ng/20 µl). The samples were eluted at a flow rate of 1.0 ml/min from a reversed-phase column (Nucleosil C18; mm) with a guard column (Nucleosil C18; mm). The solvent system consisted of water adjusted to ph 2.4 with acetic acid (A) and 80% v/v acetonitrile in A (B). Separation was carried out using a multistep linear gradient with an increasing concentration of solvent B as follows: 0% solvent B at 0 min, 15% at 10 min, 22% at 25 min, 42.5% at 45 min, and 100% at 55 min. Stilbene compounds were identified based on their retention time when compared with authentic or prepared standards. The concentration of trans-resveratrol was quantified based on an external standard, whereas that of trans-piceid was determined as trans-resveratrol equivalents. Results trans-resveratrol accumulation in leaf discs after irradiation. There were no significant differences between varieties in fresh weight of the discs or chlorophyll content (Table 1). The trans-resveratrol content of the nonirradiated control of Ryuukyuuganebu was 3.6 times higher than that of Ebizuru. After UV-C irradiation, the trans-resveratrol content of the Ebizuru leaf discs was almost 24 times that of the control, while that of the Ryuukyuuganebu leaf discs was almost 29 times the control level. The trans-resveratrol content of UV- C-irradiated Ryuukyuuganebu leaf discs was 96.3 µg/g (fresh weight), which was 4.4 times greater than that of Ebizuru. trans-resveratrol accumulation in berries 24 hr after irradiation (2005). Berry development based on fresh weight was superior in Ryuukyuuganebu. Berry weight of Ebizuru was 0.15 g at 20 days after full bloom (DAB), 0.21 g at 40 DAB, 0.27 g at 60 DAB, and 0.33 g at 80 DAB. Berry weight of Ryuukyuuganebu was 0.17 g at 20 DAB, 0.29 g at 40 DAB, 0.36 g at 60 DAB, 0.47 g at 80 DAB, and 0.62 g at 100 DAB. Ripening occurred earlier in Ebizuru than in Ryuukyuuganebu. Veraison of Ebizuru and Ryuukyuuganebu occurred about 50 and 60 DAB, respectively. trans-resveratrol levels in Ebizuru and Ryuukyuuganebu berries increased after UV-C irradiation (Figure 1). Levels in the nonirradiated control of each variety increased slightly during berry development. In immature berries, trans-resveratrol accumulation was significantly induced by UV-C irradiation in both varieties. At 20 DAB, there was no significant difference in trans-resveratrol in response to UV-C. The accumulation of resveratrol in Ebizuru berries treated with UV-C declined as the berries developed until it reached an ineffective level at harvest (80 DAB). In contrast, the accumulation of trans-resveratrol in Ryuukyuuganebu berries treated with Table 1 trans-resveratrol in the leaf discs of V. ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) 24 hr after UV-C irradiation (2005). Variety Leaf disc characterization a Fresh wt (g) trans-resveratrol (µg/g fw) Chlorophyll tester value Control UV-C Ebizuru Ryuukyuuganebu Signif b ns ns ** ** a Values are the means of 10 leaf discs for fresh weight and 10 leaves for the chlorophyll tester value. b ns and ** indicate not significant and significant at 0.01 level by Student t-test, respectively. Figure 1 Changes in trans-resveratrol content in berries of V. ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) during development upon UV-C irradiation (2005). Berries were sampled 24 hr after UV-C irradiation.

4 166 Shiozaki et al. UV-C followed a distinct pattern during berry development. The content decreased at 60 DAB (veraison), and then at harvest (100 DAB) increased to a similar level as at 20 DAB. At harvest, trans-resveratrol in UV-C-treated Ryuukyuuganebu berries was ~17 times higher than the control. trans-resveratrol and piceid in berries 24 and 48 hr after irradiation (2006). The berry fresh weights at 40 and 80 DAB were higher in Ryuukyuuganebu (0.27 and 0.47 g, respectively) than in Ebizuru (0.21 and 0.31 g, respectively). The timing of veraison of both varieties in 2006 was almost the same as in trans-resveratrol accumulation was determined 24 hr and 48 hr after UV-C irradiation for each variety (Table 2). There were no significant differences in the controls for each variety on each sampling date. Leaving berries for an increased period on wet sand in the dark reduced the trans-resveratrol in the control with each variety, irrespective of the berry development stage. No significant differences were found in the levels of trans-resveratrol in response to UV-C irradiation in the immature berries of either variety at 40 DAB, irrespective of the length of incubation after irradiation. trans-resveratrol accumulated only in Ryuukyuuganebu berries 24 hr after UV-C irradiation at 80 DAB (Figure 1). Accumulation occurred in Ebizuru berries 48 hr after irradiation, and there was a significant difference in the levels in the UV-C treatment and the control (p < 0.01). However, the level in the Ryuukyuuganebu berries was about five times greater than that in Ebizuru berries. The piceid levels (as trans-resveratrol equivalents) were one or two orders of magnitude lower than trans-resveratrol in the control (Table 3). UV-C irradiation did not induce piceid accumulation in Ebizuru berries at each developmental stage. At 40 DAB, no piceid was detected in the control or UV-C-irradiated Ryuukyuuganebu berries. Piceid was detected in Ryuukyuuganebu berries at 80 DAB, although the increase in response to UV-C irradiation was negligible compared with the change in trans-resveratrol. Discussion The berries and leaves of Ebizuru and Ryuukyuuganebu produced resveratrol in response to UV-C irradiation. The trans-resveratrol content in both nonirradiated and irradiated leaves was higher in Ryuukyuuganebu than in Ebizuru. A strong UV-C response was found in the leaves of V. rupestris prepared from cuttings or in vitro plantlets: trans-resveratrol increased ~800 times over the nonirradiated control at 24 hr after UV-C irradiation (Bonomelli et al. 2004, Borie et al. 2004). The leaves of Ryuukyuuganebu were less responsive to UV-C than those of V. rupestris, but the levels of transresveratrol after UV-C irradiation agreed with those reported for V. vinifera (Borie et al. 2004, Jeandet et al. 1991). Only the abaxial side of the grape leaf responds strongly to UV light, resulting in a significant accumulation of resveratrol (Pool et al. 1981). Ebizuru and Ryuukyuuganebu both possess pubescence on the abaxial surfaces of their leaves. Therefore, the UV-C response of their leaves might be weaker than in other Vitis species lacking pubescence on the abaxial surface of their leaves. Resveratrol production potential in grapes is inversely correlated to susceptibility to fungal pathogens, such as Botrytis cinerea (Langcake and McCarthy 1979, Sbaghi et al. 1995, Shiraishi et al. 2010), Erysiphe necator (Shiraishi et al. 2010), Oidium tuckeri (Bavaresco and Eibach 1987), and Plasmopara viticola (Barlass et al. 1987, Bavaresco and Eibach 1987, Dercks and Creasy 1989). The leaves of a disease-resistant species responded more intensely to UV irradiation than those of a susceptible species, resulting in high resveratrol accumulation (Bonomelli et al. 2004, Borie et al. 2004). The leaves of Ryuukyuuganebu showed more trans-resveratrol production, suggesting that this variety has greater disease resistance than Ebizuru. Resveratrol accumulation in grape berries occurs in the skin and seeds, but not in the flesh (Creasy and Coffee 1988, Bavaresco and Fregoni 2001). An analysis of resveratrol in whole vinifera red grape berries 16 hr after UV irradiation showed that resveratrol production was higher in immature berries, while it decreased during berry development and ripening, whereas labrusca red grapes had high production in immature berries and decreased dramatically at veraison (Jeandet et al. 1991). Lower resveratrol production in the skin of grape berries after veraison was found in red and white Table 2 trans-resveratrol accumulation in the berries of V. ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) 24 hr and 48 hr after UV-C irradiation (2006). trans-resveratrol (µg/g fw) 24 hr 48 hr DAB a /variety Control UV-C Control UV-C 40 Ebizuru Ryuukyuuganebu Signif b ns b ns * ns 80 Ebizuru Ryuukyuuganebu Signif b ns ** ns ** a DAB: days after full bloom. b ns, *, and ** indicate not significant and significant at 0.05 and 0.01 levels by Student t-test, respectively. Table 3 Piceid levels in berries of V. ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) 24 hr and 48 hr after UV-C irradiation (2006). Piceid (trans-resveratrol equivalent µg/g fw) 24 hr 48 hr DAB a /variety Control UV-C Control UV-C 40 Ebizuru Ryuukyuuganebu nd b nd nd nd 80 Ebizuru Ryuukyuuganebu Signif c ns ns ns ns a DAB: days after full bloom. b nd: not detected. c ns indicates not significant by Student t-test.

5 V. ficifolia Resveratrol Productivity 167 grapes (Creasy and Coffee 1988). The change in resveratrol production in the whole Ebizuru berry agreed with previous findings in V. vinifera (Jeandet et al. 1991) (Figure 1). However, the change in resveratrol production of Ryuukyuuganebu during berry development and maturation was unique: it decreased at veraison, and then increased to a level equivalent to the most immature stage. Jeandet et al. (1995) suggested that resveratrol production in the skin was inversely related to anthocyanin accumulation in red grapes, possibly due to competition between stilbene synthase producing resveratrol and chalcone synthase producing anthocyanin from a common substrate. The anthocyanin content of Ryuukyuuganebu skins at harvest was 26.9 mg/g fw malvidin-3-glucoside equivalent and about nine times higher than that of Merlot (S. Shiozaki, author s unpublished data, 2011). In Ryuukyuuganebu berries, resveratrol production may not be affected by anthocyanin synthesis in the skins, unlike other species or cultivars. Resveratrol production in the seeds should be considered when the resveratrol productivity of the whole Ryuukyuuganebu berry is evaluated. The level of trans-resveratrol in grape seeds depends on the species or cultivars, ranging from 1 to 62 µg/g (fresh weight) (Bavaresco and Fregoni 2001). We have no useful information on changes in the resveratrol levels in grape seeds during seed development, not to mention the effect of UV irradiation to berries on the resveratrol levels in seeds. The differences in the resveratrol levels of Ebizuru and Ryuukyuuganebu may not result from the number and size of seeds in the berries, because no significant differences were found in these factors, i.e., the seed numbers per berry in Ebizuru and Ryuukyuuganebu were 3.4 and 3.5, respectively, while the average fresh seed weights were 16.2 and 15.0 mg, respectively (data not shown). A study of resveratrol levels in the parts of mature berries in response to biotic stress (infection by Botrytis cinerea) showed that the level remained constant in seeds, even under stress (Jeandet et al. 1995). In grapes, stilbenes are present in lignified organs, such as seeds, roots, and stems, where they may play a role in wood resistance to decay (Bavaresco and Fregoni 2001). The lignification of the grape seed testa occurs after the end of size enlargement several days before veraison (27 to 45 DAB) (Pratt 1971). Therefore, the high resveratrol level found only in the mature Ryuukyuuganebu berries is probably due to that produced in the skin rather than the seeds. In 2006, the trans-resveratrol in the berries increased until 48 hr after UV irradiation, especially in mature Ryuukyuuganebu berries (Table 2). Bais et al. (2000) reported that resveratrol levels only increased in the fully mature berry skin of winegrapes (V. vinifera) following a sufficiently long delay after UV irradiation (48 hr and 72 hr after the treatment) in specific cultivars, although the levels were much lower than those in the immature skin. Resveratrol production in the skins of fully mature table grapes was also examined in a series of studies aimed at obtaining grapes with enhanced health-promoting properties based on the high resveratrol content. Postharvest treatment of V. vinifera Napoleon grapes with UV-C light ( µw/cm 2, for 30 min) increased resveratrol in the skins by ~100 µg/g fw (~three-fold of the untreated control) after refrigerated storage at 0 C for 10 days, followed by 15 C for 5 days (Cantos et al. 2000). In the same cultivar, resveratrol in the skins reached 115 µg/g fw, more than 10-fold higher than the control, with UV-C irradiation by 510 W lamps for 30 sec from 40 cm distance after storage at 22 C for 3 days (Cantos et al. 2001). In berry skins of three white and four red table varieties (V. vinifera and V. labruscana), the resveratrol induction kinetics and the maximum content during storage at 22 C after UV-C irradiation depended on the cultivar; the peak occurred between the third and seventh day, and the maximum levels ranged from ~10 to 23 µg/g fw (Cantos et al. 2002). Resveratrol content per unit fresh weight of skins becomes very low when expressed as per unit fresh weight of berries (Jeandet et al 1991). Therefore, the high resveratrol content found here in whole berries after UV irradiation and its increase up to 48 hr after UV irradiation (by 49.2 µg/g fw berries, more than 27-fold of the unirradiated control) suggest that mature Ryuukyuuganebu berries have a greater resveratrol production potential than Ebizuru and other Vitis species/cultivars. The small Ryuukyuuganebu berries are unsuitable as table grapes. However, the mature berries containing artificially induced resveratrol could be used for red wine or juice with high health-promoting effects or in the nutraceutical and cosmetic industries. The optimal UV exposure conditions and time lapse until maximum response of fully mature Ryuukyuuganebu berries should be examined in future studies. Finally, the piceid levels in the berries were lower than the resveratrol levels in both species (Table 3). UV irradiation had little effect on the piceid levels. Consistantly low piceid levels were also reported in grape leaves irradiated with UV-C (Douillet-Breuil et al. 1999). The turnover of piceid may be unrelated to changes in the resveratrol levels in grapes, even after UV irradiation. Conclusion Ebizuru and Ryuukyuuganebu are wild grapes native to Japan that produce resveratrol in their leaves and berries in response to UV-C irradiation. A comparison of resveratrol accumulation in leaves in response to UV-C irradiation showed that Ryuukyuuganebu had a higher resveratrol production potential than Ebizuru. Data on resveratrol accumulation in the berries in response to UV-C irradiation revealed the unique resveratrol production potential of the mature Ryuukyuuganebu berry. In Ebizuru, the production potential decreased throughout development and maturation. In contrast, the production potential of Ryuukyuuganebu decreased at veraison and then increased until maturity, resulting in a resveratrol production potential at maturation equivalent to that of the immature green stage. Thus, Ryuukyuuganebu may be a useful wild grape resource for resveratrol production. Literature Cited Aggarwal, B.B., A. Bhardwaj, R.S. Aggarwal, N.P. Seeram, S. Shishodia, and Y. Takada Role of resveratrol in prevention and therapy of cancer: Preclinical and clinical studies. Anticancer Res. 24:

6 168 Shiozaki et al. Bais, A.J., P.J. Murphy, and I.B. Dry The molecular regulation of stilbene phytoalexin biosynthesis in Vitis vinifera during grape berry development. Aust. J. Plant Physiol. 27: Barlass, M., R.M. Miller, and T.J. Douglas Development of methods for screening grapevines for resistance to infection by downy mildew. II. Resveratrol production. Am. J. Enol. Vitic. 38: Bavaresco, L., and R. Eibach Investigations on the influence of N fertilizer on resistance to powdery mildew (Oidium tuckeri) downy mildew (Plasmopara viticola) and on phytoalexin synthesis in different grapevine varieties. Vitis 26: Bavaresco, L., and C. Fregoni Physiological role and molecular aspects of grapevine stilbenic compounds. In Molecular Biology & Biotechnology of the Grapevine. K. Roubelakis-Angelakis (ed.), Kluwer, Dordrecht. Bonomelli, A., L. Mercier, J. Franchel, F. Baillieul, E. Benizri, and M.C. Mauro Response of grapevine defenses to UV-C exposure. Am. J. Enol. Vitic. 55: Borie, B., P. Jeandet, A. Parize, R. Bessis, and M. Adrian Resveratrol and stilbene synthase mrna production in grapevine leaves treated with biotic and abiotic phytoalexin elicitors. Am. J. Enol. Vitic. 55: Cantos, E., J.C. Espin, and F.A. Tomas-Barberan Postharvest induction modeling method using UV irradiation pulses for obtaining resveratrol-enriched table grapes: A new functional fruit? J. Agric. Food. Chem. 49: Cantos, E., J.C. Espin, and F.A. Tomas-Barberan Postharvest stilbene-enrichment of red and white table grape varieties using UV-C irradiation pulses. J. Agric. Food. Chem. 50: Cantos, E., C. Garcia-Viguera, S. de Pascual-Teresa, and F.A. Tomas- Barberan Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of cv. Napoleon table grapes. J. Agric. Food. Chem. 48: Creasy, L.L., and M. Coffee Phytoalexin production potential of grape berries. J. Amer. Soc. Hort. Sci. 113: Dercks, W., and L.L. Creasy The significance of stilbene phytoalexins in the Plasmopara viticola-grapevine interaction. Physiol. and Mol. Plant Pathology 34: Douillet-Breuil, A.C., P. Jeandet, M. Adrian, and R. Bessis Changes in the phytoalexin content of various vitis spp. in response to ultraviolet C elicitation. J. Agric. Food. Chem. 47: Fremont, L Biological effects of resveratrol. Life Sci. 66: Guilford, J.M., and J.M. Pezzuto Wine and health: A review. Am. J. Enol. Vitic. 62: Hackett, A.M The metabolism of flavonoid compounds in mammals. In Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological and Structure-Activity Relationships. V. Cody et al. (eds.), pp Wiley & Sons, New York. Jeandet, P., R. Bessis, and B. Gautheron The production of resveratrol (3,5,4 -trihydroxystilbene) by grape berries in different developmental stages. Am. J. Enol. Vitic. 42: Jeandet, P., R. Bessis, M. Sbaghi, and P. Meunier Production of the phytoalexin resveratrol by grapes as a response to Botrytis attack under natural conditions. J. Phytopathol. 143: Kimura, Y., H. Okuda, and S. Arichi Effects of stilbenes on arachidonate metabolism in leukocytes. Biochim. Biophy. Acta. 834: Langcake, P., and W.V. McCarthy The relationship of resveratrol production to infection of grapevine leaves by Botrytis cinerea. Vitis 18: Mochioka, R., M. Yamaguchi, S. Horiuchi, H. Matsui, and H. Kurooka Chemotaxonomic classification of native wild grapes by anthocyanins in berry skin. J. Japan. Soc. Hort. Sci. 64: Nakagawa, S., S. Horiuchi, H. Matsui, E. Yuda, S. Yamada, Y. Murai, and H. Komatsu Distribution and leaf morphology of wild grapes native to Japan. J. Japan. Soc. Hort. Sci. 60: Okuda, T., and K. Yokotsuka Trans-resveratrol concentrations in berry skins and wines from grapes grown in Japan. Am. J. Enol. Vitic. 47: Pool, R.M., L.L. Creasy, and A.S. Frackelton Resveratrol and the viniferins, their application to screening for disease resistance in grape breeding programs. Vitis 20: Pratt, C Reproductive anatomy in cultivated grapes A review. Am. J. Enol. Vitic. 22: Ramprasath, V.R., and P.J.H. Jones Anti-atherogenic effects of resveratrol. Eur. J. Clin. Nutri. 64: Sbaghi, M., P. Jeandet, B. Faivre, R. Bessis, and J.C. Fournioux Development of methods using phytoalexin (resveratrol) assessment as a selection criterion to screen grapevine in vitro cultures for resistance to grey mould (Botrytis cinerea). Euphytica 86: Shan, C.W., S.Q. Yang, H.D. He, S.L. Shao, and P.W. Zhang Influence of 3,4,5-trihydroxystilbene-3-β-mono-d-glucoside on rabbits platelet aggregation and thromboxane B 2 production in vitro. Acta Pharmacol. Sin. 11: Shiraishi, M., H. Chijiwa, H. Fujishima, and K. Muramoto Resveratrol production potential of grape flowers and green berries to screen genotypes for gray mold and powdery mildew resistance. Euphytica 176: Stockley, C., P.L. Teissedre, M. Boban, C. Di Lorenzo, and P. Restani Bioavailability of wine-derived phenolic compounds in humans: A review. Food Funct. 3: Takayanagi, T., T. Okuda, Y. Mine, and K. Yokotsuka Induction of resveratrol biosynthesis in skins of three grape cultivars by ultraviolet irradiation. J. Japan. Soc. Hort. Sci. 73: Wang, Z., J. Zou, Y. Huang, K. Cao, Y. Xu, and J.M. Wu Effect of resveratrol on platelet aggregation in vivo and in vitro. Chin. Med. J. 115: Waterhouse, A.L., and R.M. Lamuela-Raventos The occurrence of piceid, a stilbene glucoside, in grape berries. Phytochemistry 37: