Research Note Resveratrol Productivity of Wild Grapes Native to : 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: ssgvo67@.osakafu-u.ac.jp) 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. min and then filtered. The residue from the berry sample was Grapes. The experiments were carried out in 2005 and washed with 20 mL 90% MeOH and 40 mL 100% MeOH. All 2006 with three 11-year-old vines of Vitis ficifolia var. lo- filtrates from each sample were combined and concentrated bata (Ebizuru) and V. ficifoliavar. ganebu (Ryuukyuuganebu) to produce an aqueous phase, which was then mixed with 20 grown in a research field at Osaka Prefecture University (lat: mL distilled water. The aqueous phase was then partitioned 34°32’N, 30 m asl). The soil was a gray lowland, pH 5.5. Cli- using an equal volume of hexane to remove lipids and chloro- mate conditions of growing periods of the vines in 2005 and phyll before it was extracted three times using ethyl acetate at 2006 (from April until October) were as follows: average tem- pH 8.0. The combined ethyl acetate extract containing trans- perature, 22.6°C and 22.2°C; rainfall, 686 mm and 916 mm; resveratrol was reduced to dryness in vacuo. The aqueous and sunshine duration, 1,208 hr and 1,180 hr, respectively. phase containing piceid was evaporated to remove acetic acid During both years, full bloom of Ebizuru and Ryuukyuu- and adjusted to a pH of 7.0 using 1 N NaOH before its volume ganebu occurred in early June and mid-June, respectively. was adjusted to 50 mL with distilled water. Leaf materials. In 2005, leaves that were free from vi- The trans-resveratrol extract was partially purified with a sual defects were sampled from the fifth to the tenth nodes, Sep-Pak C18-ENV cartridge (Waters Corp., Milford, MA) as counting up from the base of two-month-old shoots. Values of described (Jeandet et al. 1991) with modification. The extract the chlorophyll (the maximum value in the rate of leaf green) was redissolved in 1 mL 100% MeOH, and 4 mL 33 mM (Chlorophylltester CT 102; Fujihira Industry Co., Tokyo, Ja- phosphate buffer (pH 7.0) was added before centrifugation

Am. J. Enol. Vitic. 64:1 (2013) V. ficifolia Resveratrol Productivity – 165 at 12,500 × g for 10 min. The supernatant was loaded onto trans-Resveratrol accumulation in berries 24 hr after the cartridge, which had been previously activated with 6 irradiation (2005). Berry development based on fresh weight mL 100% MeOH and washed with 10 mL 33 mM phosphate was superior in Ryuukyuuganebu. Berry weight of Ebizuru buffer (pH 7.0). The pellet was washed with 5 mL 33 mM was 0.15 g at 20 days after full bloom (DAB), 0.21 g at 40 phosphate buffer and centrifuged in the same way before the DAB, 0.27 g at 60 DAB, and 0.33 g at 80 DAB. Berry weight supernatant was applied to the cartridge. The sample flow of Ryuukyuuganebu was 0.17 g at 20 DAB, 0.29 g at 40 DAB, rate was <1 mL/min. The cartridge was purged with N2 for 0.36 g at 60 DAB, 0.47 g at 80 DAB, and 0.62 g at 100 DAB. 1 min to eliminate the buffer solution after the sample had Ripening occurred earlier in Ebizuru than in Ryuukyuugane- been applied. trans-Resveratrol was eluted in a 10 mL test bu. Veraison of Ebizuru and Ryuukyuuganebu occurred about tube with 5 mL ethyl acetate. The eluate was concentrated 50 and 60 DAB, respectively. to dryness with a centrifugal concentrator (model VC-96N; trans-Resveratrol levels in Ebizuru and Ryuukyuuganebu TAITEC Corp., Koshigaya, Japan) and stored at -20°C until berries increased after UV-C irradiation (Figure 1). Levels in HPLC analysis. the nonirradiated control of each variety increased slightly

trans-Piceid was also purified using a Waters Sep-Pak 18C - during berry development. In immature berries, trans-resve- ENV cartridge. The aqueous 50 mL sample was centrifuged ratrol accumulation was significantly induced by UV-C irra- at 12,500 × g for 10 min and the supernatant was loaded onto diation in both varieties. At 20 DAB, there was no significant the cartridge, activated, and washed as described above for difference in trans-resveratrol in response to UV-C. The accu- resveratrol. The cartridge was then washed with 20 mL 33 mulation of resveratrol in Ebizuru berries treated with UV-C mM phosphate buffer (pH 7.0) and 20 mL 0.1% HCl before declined as the berries developed until it reached an ineffec- the residual aqueous solution was eliminated from the car- tive level at harvest (80 DAB). In contrast, the accumulation tridge by suction for 15 min. Piceid was then eluted using 20 of trans-resveratrol in Ryuukyuuganebu berries treated with mL 16% acetonitrile containing 0.1% HCl. HPLC analysis. trans-Resveratrol and trans-piceid were analyzed using the same HPLC conditions. The dried samples Table 1 trans-Resveratrol in the leaf discs of V. ficifolia containing trans-resveratrol were redissolved in 500 µL ethyl var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) acetate and centrifuged at 1,800 × g for 3 min. A 20 µL 24 hr after UV-C irradiation (2005). Leaf disc trans-Resveratrol aliquot of the supernatant was then analyzed by HPLC. For a characterization (µg/g fw) trans-piceid, a 100 µL aliquot of 16% acetonitrile containing Fresh wt Chlorophyll 0.1% HCl was injected directly into the HPLC. Resveratrol Variety (g) tester value Control UV-C and piceid were detected by UV at 306 nm (detection limit Ebizuru 26.4 1.20 0.9 21.8 of trans-resveratrol: 0.5 ng/20 µL). The samples were eluted Ryuukyuuganebu 30.1 1.13 3.3 96.3 at a flow rate of 1.0 mL/min from a reversed-phase column Signifb ns ns ** ** (Nucleosil 120-5 C18; 4.0 × 250 mm) with a guard column aValues are the means of 10 leaf discs for fresh weight and 10 leaves (Nucleosil 120-5 C18; 4.0 × 10 mm). The solvent system con- for the chlorophyll tester value. sisted of water adjusted to pH 2.4 with acetic acid (A) and bns and ** indicate not significant and significant at 0.01 level by 80% v/v acetonitrile in A (B). Separation was carried out Student t-test, respectively. using a multistep linear gradient with an increasing concen- tration 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 irra- diation. There were no significant differences between variet- ies 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 Ebi- zuru. 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- Figure 1 Changes in trans-resveratrol content in berries of V. ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu (Ryuukyuuganebu) during C-irradiated Ryuukyuuganebu leaf discs was 96.3 µg/g (fresh development upon UV-C irradiation (2005). Berries were sampled 24 hr weight), which was 4.4 times greater than that of Ebizuru. after UV-C irradiation.

Am. J. Enol. Vitic. 64:1 (2013) 166 – Shiozaki et al.

UV-C followed a distinct pattern during berry development. trans-resveratrol content in both nonirradiated and irradiated The content decreased at 60 DAB (veraison), and then at har- leaves was higher in Ryuukyuuganebu than in Ebizuru. A vest (100 DAB) increased to a similar level as at 20 DAB. At strong UV-C response was found in the leaves of V. rupestris harvest, trans-resveratrol in UV-C-treated Ryuukyuuganebu prepared from cuttings or in vitro plantlets: trans-resveratrol berries was ~17 times higher than the control. increased ~800 times over the nonirradiated control at 24 hr trans-Resveratrol and piceid in berries 24 and 48 hr after UV-C irradiation (Bonomelli et al. 2004, Borie et al. after irradiation (2006). The berry fresh weights at 40 and 2004). The leaves of Ryuukyuuganebu were less responsive 80 DAB were higher in Ryuukyuuganebu (0.27 and 0.47 g, to UV-C than those of V. rupestris, but the levels of trans- respectively) than in Ebizuru (0.21 and 0.31 g, respectively). resveratrol after UV-C irradiation agreed with those reported The timing of veraison of both varieties in 2006 was almost for V. vinifera (Borie et al. 2004, Jeandet et al. 1991). Only the same as in 2005. trans-Resveratrol accumulation was the abaxial side of the grape leaf responds strongly to UV determined 24 hr and 48 hr after UV-C irradiation for each light, resulting in a significant accumulation of resveratrol variety (Table 2). There were no significant differences in (Pool et al. 1981). Ebizuru and Ryuukyuuganebu both possess the controls for each variety on each sampling date. Leav- pubescence on the abaxial surfaces of their leaves. Therefore, ing berries for an increased period on wet sand in the dark the UV-C response of their leaves might be weaker than in reduced the trans-resveratrol in the control with each variety, other Vitis species lacking pubescence on the abaxial surface irrespective of the berry development stage. No significant of their leaves. differences were found in the levels of trans-resveratrol in Resveratrol production potential in grapes is inversely cor- response to UV-C irradiation in the immature berries of ei- related to susceptibility to fungal pathogens, such as Botrytis ther variety at 40 DAB, irrespective of the length of incuba- cinerea (Langcake and McCarthy 1979, Sbaghi et al. 1995, tion after irradiation. trans-Resveratrol accumulated only in Shiraishi et al. 2010), Erysiphe necator (Shiraishi et al. 2010), Ryuukyuuganebu berries 24 hr after UV-C irradiation at 80 Oidium tuckeri (Bavaresco and Eibach 1987), and Plasmo- DAB (Figure 1). Accumulation occurred in Ebizuru berries para viticola (Barlass et al. 1987, Bavaresco and Eibach 1987, 48 hr after irradiation, and there was a significant difference Dercks and Creasy 1989). The leaves of a disease-resistant in the levels in the UV-C treatment and the control (p < 0.01). species responded more intensely to UV irradiation than However, the level in the Ryuukyuuganebu berries was about those of a susceptible species, resulting in high resveratrol five times greater than that in Ebizuru berries. accumulation (Bonomelli et al. 2004, Borie et al. 2004). The The piceid levels (as trans-resveratrol equivalents) were leaves of Ryuukyuuganebu showed more trans-resveratrol one or two orders of magnitude lower than trans-resveratrol production, suggesting that this variety has greater disease in the control (Table 3). UV-C irradiation did not induce pi- resistance than Ebizuru. ceid accumulation in Ebizuru berries at each developmental Resveratrol accumulation in grape berries occurs in the stage. At 40 DAB, no piceid was detected in the control or skin and seeds, but not in the flesh (Creasy and Coffee 1988, UV-C-irradiated Ryuukyuuganebu berries. Piceid was de- Bavaresco and Fregoni 2001). An analysis of resveratrol in tected in Ryuukyuuganebu berries at 80 DAB, although the whole vinifera red grape berries 16 hr after UV irradiation increase in response to UV-C irradiation was negligible com- showed that resveratrol production was higher in immature pared with the change in trans-resveratrol. berries, while it decreased during berry development and ripening, whereas labrusca red grapes had high production Discussion in immature berries and decreased dramatically at veraison The berries and leaves of Ebizuru and Ryuukyuuganebu (Jeandet et al. 1991). Lower resveratrol production in the skin produced resveratrol in response to UV-C irradiation. The of grape berries after veraison was found in red and white

Table 2 trans-Resveratrol accumulation in the berries of Table 3 Piceid levels in berries of V. ficifolia var. lobata (Ebizuru) V. ficifolia var. lobata (Ebizuru) and V. ficifolia var. ganebu and V. ficifolia var. ganebu (Ryuukyuuganebu) 24 hr and 48 hr (Ryuukyuuganebu) 24 hr and 48 hr after UV-C irradiation (2006). after UV-C irradiation (2006). trans-Resveratrol (µg/g fw) Piceid (trans-resveratrol equivalent 24 hr 48 hr µg/g fw) DABa/variety Control UV-C Control UV-C 24 hr 48 hr DABa/variety Control UV-C Control UV-C 40 Ebizuru 0.6 31.3 0.4 27.3 40 Ryuukyuuganebu 0.8 43.8 0.2 31.9 Ebizuru 0.02 0.03 0.01 0.02 b Signifb nsb ns * ns Ryuukyuuganebu nd nd nd nd 80 80 Ebizuru 3.7 3.7 1.3 9.4 Ebizuru 0.17 0.13 0.10 0.04 Ryuukyuuganebu 5.3 23.8 1.8 49.2 Ryuukyuuganebu 0.07 0.29 0.12 0.37 Signifb ns ** ns ** Signifc ns ns ns ns aDAB: days after full bloom. aDAB: days after full bloom. bns, *, and ** indicate not significant and significant at 0.05 and 0.01 bnd: not detected. levels by Student t-test, respectively. cns indicates not significant by Student t-test.

Am. J. Enol. Vitic. 64:1 (2013) V. ficifolia Resveratrol Productivity – 167 grapes (Creasy and Coffee 1988). The change in resveratrol untreated control) after refrigerated storage at 0°C for 10 production in the whole Ebizuru berry agreed with previous days, followed by 15°C for 5 days (Cantos et al. 2000). In the findings in V. vinifera (Jeandet et al. 1991) (Figure 1). How- same cultivar, resveratrol in the skins reached 115 µg/g fw, ever, the change in resveratrol production of Ryuukyuuganebu more than 10-fold higher than the control, with UV-C irra- during berry development and maturation was unique: it de- diation by 510 W lamps for 30 sec from 40 cm distance after creased at veraison, and then increased to a level equivalent storage at 22°C for 3 days (Cantos et al. 2001). In berry skins to the most immature stage. Jeandet et al. (1995) suggested of three white and four red table varieties (V. vinifera and V. that resveratrol production in the skin was inversely related labruscana), the resveratrol induction kinetics and the maxi- to anthocyanin accumulation in red grapes, possibly due to mum content during storage at 22°C after UV-C irradiation competition between stilbene synthase producing resveratrol depended on the cultivar; the peak occurred between the third and chalcone synthase producing anthocyanin from a common and seventh day, and the maximum levels ranged from ~10 to substrate. The anthocyanin content of Ryuukyuuganebu skins 23 µg/g fw (Cantos et al. 2002). Resveratrol content per unit at harvest was 26.9 mg/g fw malvidin-3-glucoside equivalent fresh weight of skins becomes very low when expressed as and about nine times higher than that of Merlot (S. Shiozaki, per unit fresh weight of berries (Jeandet et al 1991). Therefore, author’s unpublished data, 2011). In Ryuukyuuganebu berries, the high resveratrol content found here in whole berries after resveratrol production may not be affected by anthocyanin UV irradiation and its increase up to 48 hr after UV irradia- synthesis in the skins, unlike other species or cultivars. tion (by 49.2 µg/g fw berries, more than 27-fold of the unirra- Resveratrol production in the seeds should be considered diated control) suggest that mature Ryuukyuuganebu berries when the resveratrol productivity of the whole Ryuukyuu- have a greater resveratrol production potential than Ebizuru ganebu berry is evaluated. The level of trans-resveratrol in and other Vitis species/cultivars. The small Ryuukyuuganebu grape seeds depends on the species or cultivars, ranging from berries are unsuitable as table grapes. However, the mature 1 to 62 µg/g (fresh weight) (Bavaresco and Fregoni 2001). berries containing artificially induced resveratrol could be We have no useful information on changes in the resveratrol used for red wine or juice with high health-promoting effects levels in grape seeds during seed development, not to men- or in the nutraceutical and cosmetic industries. The optimal tion the effect of UV irradiation to berries on the resveratrol UV exposure conditions and time lapse until maximum re- levels in seeds. The differences in the resveratrol levels of sponse of fully mature Ryuukyuuganebu berries should be Ebizuru and Ryuukyuuganebu may not result from the num- examined in future studies. ber and size of seeds in the berries, because no significant Finally, the piceid levels in the berries were lower than the differences were found in these factors, i.e., the seed numbers resveratrol levels in both species (Table 3). UV irradiation had per berry in Ebizuru and Ryuukyuuganebu were 3.4 and 3.5, little effect on the piceid levels. Consistantly low piceid lev- respectively, while the average fresh seed weights were 16.2 els were also reported in grape leaves irradiated with UV-C and 15.0 mg, respectively (data not shown). A study of res- (Douillet-Breuil et al. 1999). The turnover of piceid may be veratrol levels in the parts of mature berries in response to unrelated to changes in the resveratrol levels in grapes, even biotic stress (infection by Botrytis cinerea) showed that the after UV irradiation. level remained constant in seeds, even under stress (Jeandet et al. 1995). In grapes, stilbenes are present in lignified organs, Conclusion such as seeds, roots, and stems, where they may play a role Ebizuru and Ryuukyuuganebu are wild grapes native to in wood resistance to decay (Bavaresco and Fregoni 2001). Japan that produce resveratrol in their leaves and berries in The lignification of the grape seed testa occurs after the end response to UV-C irradiation. A comparison of resveratrol ac- of size enlargement several days before veraison (27 to 45 cumulation in leaves in response to UV-C irradiation showed DAB) (Pratt 1971). Therefore, the high resveratrol level found that Ryuukyuuganebu had a higher resveratrol production po- only in the mature Ryuukyuuganebu berries is probably due tential than Ebizuru. Data on resveratrol accumulation in the to that produced in the skin rather than the seeds. berries in response to UV-C irradiation revealed the unique In 2006, the trans-resveratrol in the berries increased until resveratrol production potential of the mature Ryuukyuu- 48 hr after UV irradiation, especially in mature Ryuukyuu- ganebu berry. In Ebizuru, the production potential decreased ganebu berries (Table 2). Bais et al. (2000) reported that res- throughout development and maturation. In contrast, the pro- veratrol levels only increased in the fully mature berry skin duction potential of Ryuukyuuganebu decreased at veraison of winegrapes (V. vinifera) following a sufficiently long delay and then increased until maturity, resulting in a resveratrol after UV irradiation (48 hr and 72 hr after the treatment) in production potential at maturation equivalent to that of the specific cultivars, although the levels were much lower than immature green stage. Thus, Ryuukyuuganebu may be a use- those in the immature skin. Resveratrol production in the ful wild grape resource for resveratrol production. skins of fully mature table grapes was also examined in a series of studies aimed at obtaining grapes with enhanced Literature Cited health-promoting properties based on the high resveratrol Aggarwal, B.B., A. Bhardwaj, R.S. Aggarwal, N.P. Seeram, S. content. Postharvest treatment of V. vinifera Napoleon grapes Shishodia, and Y. Takada. 2004. Role of resveratrol in prevention 2 with UV-C light (1780–2300 µW/cm , for 30 min) increased and therapy of cancer: Preclinical and clinical studies. Anticancer resveratrol in the skins by ~100 µg/g fw (~three-fold of the Res. 24:2783-2840.

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