Antioxidant Capacity and Polyphenol Content of Extracts from Crops Cultivated in Japan, and the Effect of Cultivation Environmen

Home , Arctium, Myoga, Perilla frutescens

Food Sci. Technol. Res., 19 (1), 69–79, 2013

Antioxidant Capacity and Polyphenol Content of Extracts from Crops Cultivated in

Japan, and the Effect of Cultivation Environment

1* 2 2 1 1 Ichiho Mikami-Konishide , Shiro Murakami , Keijiro Nakanishi , Yumiko Takahashi , Minako Yamaguchi , 3 1 1 Tetsuo Shioya , Jun Watanabe and Akihiro Hino

1 National Food Research Institute (NFRI), National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 2 Nakanishi Incorporated, 2-11-21 Takezono, Tsukuba, Ibaraki 305-0032, Japan 3 2-10-22 Ottominami, Tsuchiura, Ibaraki 300-0845, Japan

Received October 29, 2011; Accepted October 14, 2012

Oxygen radical absorbance capacity (ORAC) value and polyphenol contents of extracts from 71 crops cultivated in Japan were determined. Hydrophilic ORAC values (H-ORAC) ranged from 194 − 29,830 μmol TE/100 g fresh weight (FW), whereas lipophilic ORAC values (L-ORAC) ranged from 6 − 2,076 μmol TE/100 g FW. High total ORAC value was indicated in the following vegetables and fruits: mulukhiya (Corchorus olitorius), perilla (Perilla frutescens), water spinach (Ipomoea aquatica), edible burdock (Arctium lappa), blueberries (Vaccinium corymbosum) and hardy kiwifruit (Actinidia arguta) (6,562 − 31,802 μmol TE/100 g FW). These extracts showed a strong positive correlation between ORAC value and polyphenol content (r = 0.98). In addition, to gain a better understanding of ORAC variability in Jap- anese crops, we also discussed the influence of environmental factors on the ORAC value of stem mustard (Brassica juncea) “Umino” grown in three different regions. Regional variations of ORAC in stem mustard likely result from different levels of ultraviolet radiation and fertilizer application regimens specific to those regions.

Keywords: ORAC, polyphenol content, stem mustard (Brassica juncea) “Umino”, environmental effects

Introduction many Japanese consumers are seeking information about the There is increasing evidence that oxidative stress is as- effects of agricultural products on their health. Therefore, the sociated with the development of many chronic and degen- government has established several programs, “local produc- erative diseases, as well as the aging process (Awika et al., tion for local consumption” and “information which is useful 2003; Bacchiocca et al., 2006; Cho et al., 2005; Miller et al., for food selection and food safety” (ii), aimed at improving 2008; Samaniego Sánchez et al., 2007). Although the human the health of the Japanese through promoting the production body has the ability to eliminate free radicals, such ability of high-quality foods by local farmers. These programs also is commonly insufficient (Seeram et al., 2008). Therefore, contribute to the economic development of local agriculture dietary antioxidants, such as in fruits and vegetables, can in Japan by revitalizing various high- quality agricultural help to decrease the adverse effects of reactive oxygen spe- products. cies in humans. With the aging of Japanese society, food, Determination of the antioxidant capacity of locally pro- especially fruits and vegetables, is increasingly viewed as a duced food is important for assessing its ability to induce means of disease prevention and good health maintenance. effects on people’s health. The ORAC assay is a commonly According to studies of food consumption performed by the adopted and relatively standardized method to determine Ministry of Agriculture, Forestry, and Fishery in 2004 (i), antioxidant capacity in foods (Cao et al., 1993; Huang et al., 2002; Prior et al., 2003; Wu et al., 2004a). The ORAC meth- *To whom correspondence should be addressed. od measures scavenging capacity against the peroxyl radical, E-mail: [email protected] which is the predominant free radical involved in lipid oxi- 70 I. Mikami-Konishide et al. dation in foods and biological systems (Mikami et al., 2009; Each farm’s growing period was as follows: (A) Ibaraki; 24 Takebayashi et al., 2010; Wu et al., 2004b). In 2010, the Sep. 2009 − 14 Dec. 2009, 17 Sep. 2010 − 5 Jan. 2011, (B) USDA released the second database of the antioxidant capac- Yamagata; 12 Aug. 2009 − 12 Dec. 2009, 12 Sep. 2010 − 24 ity of 326 selected foods determined using ORAC method- Jan. 2011, (C) Kanagawa; 5 Oct. 2009 − 10 Feb. 2010, 12 ology (iii). Furthermore, drinks and supplements marketed Sep. 2010 − 24 Jan. 2011. The basal (I) and additional (II) in the USA are labeled with the ORAC value. However, in fertilizer used in this study were as follows: Tsukuba farm [(I) Japan, various antioxidant assay methods have been used for (II); Silver Almighty (composted animal manure; 8-10-10, a prolonged period. Therefore, unification and standardiza- N-P-K, Fukuei Hiryo Co., Hyogo, Japan)], Takahata farm [(I); tion of the antioxidant assay is needed and is currently being Sun-ace (12-8-10, N-P-K, Taiyo fertilizer Co., Ibaraki, Ja- developed (Watanabe et al., 2010). Several studies on ORAC pan), (II); Shikishima-6 (6-8.5-6, N-P-K, Taki chemical Co., values of local farm produce have recently been performed Hyogo, Japan)], Miura farm [(I) (II); Ainakasei (14-16-10, (Kitazume et al., 2011; Oki et al., 2009; Sato et al., 2010). N-P-K, Katakura Chikkarin Co., Tokyo, Japan)]. And at Tsu- The term “Functional foods” is an established food products kuba farm, (I) and (II) fertilizer were applied at 80 and 48 kg category in Japan (Arai, 1996), and has strong consumer ac- N ha-1, respectively. At Takahata farm, (I) and (II) were ap- ceptance (iv). In addition, a program to ensure the quality of plied at 240 and 120 kg N ha-1, respectively. At Miura farm, (I) local health-related products is currently being carried out and (II) were applied at 42 and 112 kg N ha-1, respectively. in Japan (Tsushida, 2009). However, ORAC values for most After harvesting, 50 − 100 g of bulk sample from of the three Japanese farm products have not yet been determined. There- different plants was used for lyophilization. Each part of the fore, constructing a database of various food products, each plants was separately lyophilized (leaf, petiole, stem, flower labeled with its corresponding ORAC value, will be required. bud, and leaf of flower bud), powdered using a mill, and The objective of this study is to further explore the stored at −30℃ until used. ORAC values of various crops produced in Japan. We ex- Reagents 6-Hydroxy-2,5,7,8-tetramethyl chroman-2- amined 71 samples of locally grown agricultural products in carboxylic acid (Trolox), fluorescein (sodium salt) (FL), and Ibaraki prefecture in Japan. In addition we investigated the gallic acid were purchased from Sigma-Aldrich (St. Louis, role of various factors, such as growing conditions, which MO). Methyl-β-cyclodextrin (MCD) was obtained from affect the antioxidant capacity of the food, in order to gain a Junsei Chemical Co Ltd (Tokyo, Japan). Folin-Ciocalteu’s better understanding of ORAC variability. In particular, we phenol reagent was purchased from MP Biomedicals, LLC focused on the influence of environmental factors, ultraviolet (Illkirch, France). 2,2’-Azobis (2-amidinopropane) dihydro- radiation, and fertilizer application, on ORAC values. For chloride (AAPH, extra pure reagent), sodium carbonate and this study, we employed stem mustard (Brassica juncea), other reagents were purchased from Wako Pure Chemical which has a comparably high ORAC value, and was grown Industries (Osaka, Japan). Except for AAPH, special grade in three different regions. chemicals were used in this experiment.

Materials and Methods Plant materials Seventy samples of different crops were purchased from retail stores in Ibaraki prefecture, Japan in 2006-2011 (Table 1). All samples (except for samples No. 60, 61, 65 in Table 1) were separated into three 50 − 100 g units of edible parts, lyophilized, then powdered using a mill and stored at −30℃ until used. Rice (No. 60 and 61) and Kinako (No. 65, roasted and ground green soybeans) were kept at −30℃ in their original form until analysis. As shown in Fig. 1, stem mustard (Brassica juncea) “Umino” was cultivated by six farmers in three prefectures: Ibaraki [36° 5’ N, 140° 6’ E, Tsukuba region, two farmers (A-1, 2), open field plot, direct-seeded], Yamagata [37° 58’ N, 140° 10’ E, Takahata region, one farmer (B), tunnel plot, Fig. 1. Distribution of the three regions where stem planted 2 – 5 week transplants] and Kanagawa [35° 9’ N, mustard “Umino” was cultivated in this study. 139° 39’ E, Miura region, three farmers (C-1, 2, 3), open Takahata city is about 30 km away from Yamagata city. Miura city field plot, direct-seeded], and were harvested in 2009 − 2011. in Kanagawa prefecture is about 30 km away from Yokohama city. Antioxidant Capacity of Extracts from Crops Cultivated in Japan 71

Table 1. ORAC and TP values of selected crops in Japan.

Moisture HTP LTP (mg of TP H-ORAC L-ORAC Total-ORAC No. English name, Description Japanese name Scientific name Date of N*1 Content (mg of GAE/ GAE/100 g (mg of GAE/ (μmol TE/ (μmol TE/ (μmol TE/ purchase (%) 100 g FW) FW) 100 g FW) 100 g FW) 100 g FW) 100 g FW)

【Fruit】 1 Blueberry Buruberi Vaccinium corymbosum 2008/6/27 3 86.2 ± 0.1 196.9 ± 58.6 1.4 ± 0.2 198.3 ± 58.6 6543 ± 327 19 ± 6 6562 ± 332 2 Fig Ichijiku Ficus carica 2008/8/9 3 86.5 ± 1.5 22.4 ± 8.3 2.3 ± 0.5 24.7 ± 8.6 968 ± 271 130 ± 27 1098 ± 262 3 Hardy kiwifruit Sarunashi Actinidia arguta 2008/8/9 3 87.0 ± 0.2 407.6 ± 14.9 4.3 ± 0.2 411.9 ± 14.8 9420 ± 552 56 ± 18 9476 ± 534 4 Kiwifruit Kiui Actinidia chinensis 2008/8/9 3 86.4 ± 0.2 16.0 ± 2.0 2.4 ± 0.1 18.4 ± 2.0 659 ± 53 40 ± 7 699 ± 58 5 Japanese pear, “Kousui” Nashi Pyrus pyrifolia 2008/8/9 3 88.0 ± 0.6 6.2 ± 2.0 0.2 ± 0.0 6.5 ± 2.0 218 ± 25 14 ± 1 232 ± 26 6 Pomegranate Zakuro Punica granatum 2008/10/22 3 75.9 ± 0.4 61.6 ± 7.4 8.2 ± 0.6 69.8 ± 7.1 1378 ± 93 53 ± 4 1431 ± 97

【Fruit vegetable】 7 Bell pepper, Yellow-green Banana piman Capsicum annuum 2008/7/27 3 93.1 ± 0.0 58.6 ± 5.7 1.5 ± 0.2 60.1 ± 5.9 1161 ± 51 74 ± 4 1235 ± 49 8 Bell pepper, Green Piman Capsicum annuum 2008/6/27 3 93.2 ± 0.9 59.9 ± 17.4 4.0 ± 1.4 63.9 ± 18.8 1305 ± 385 107 ± 33 1411 ± 418 9 Cucumber Kyuri Cucumis sativus 2008/6/27 3 93.5 ± 0.2 9.2 ± 1.1 3.8 ± 0.3 13.0 ± 0.9 194 ± 30 78 ± 8 272 ± 37 10 Cucumis melo Kin makuwauri Cucumis melo 2008/7/27 3 93.8 ± 0.2 18.5 ± 2.2 0.3 ± 0.0 18.7 ± 2.2 249 ± 16 10 ± 4 258 ± 20 11 Eggplant Mizu nasu Solanum melongena 2008/6/30 3 92.9 ± 0.2 61.7 ± 7.5 0.6 ± 0.0 62.3 ± 7.5 2110 ± 245 64 ± 4 2174 ± 249 12 Eggplant Nasu Solanum melongena 2008/6/30 3 93.3 ± 0.1 52.0 ± 1.8 0.5 ± 0.1 52.5 ± 1.8 1796 ± 150 50 ± 1 1846 ± 149 13 Green pepper Shishito Capsicum annuum 2008/6/30 3 92.2 ± 0.4 71.9 ± 2.1 2.7 ± 0.1 74.6 ± 2.3 1631 ± 117 132 ± 11 1762 ± 125 14 Hot pepper, Large Togarashi Capsicum annuum 2008/6/30 3 92.7 ± 0.4 82.3 ± 8.5 2.7 ± 0.2 85.0 ± 8.5 1754 ± 220 120 ± 1 1874 ± 220 15 Hot pepper, Small Togarashi Capsicum annuum 2008/6/30 3 89.7 ± 0.4 118.0 ± 3.8 10.0 ± 1.9 128.0 ± 2.9 3290 ± 222 558 ± 50 3848 ± 199 16 Melon, Ahrusu, “Asahi” Ahrusu meron Cucumis melo 2008/8/9 1 85.8 13.1 NT 13.4 268 6 274 17 Melon, Ahrusu, “Nadarou” Ahrusu meron Cucumis melo 2008/8/9 1 86.3 14.4 NT 14.8 283 7 290 18 Melon, Andesu Ahrusu meron Cucumis melo 2008/6/27 3 85.0 ± 0.2 16.6 ± 2.0 0.6 ± 0.0 17.2 ± 2.1 287 ± 17 15 ± 0 302 ± 17 19 Melon, Kinsho Kinsho meron Cucumis melo 2008/6/27 3 89.2 ± 0.9 14.0 ± 1.8 0.4 ± 0.0 14.4 ± 1.8 259 ± 44 10 ± 2 270 ± 45 20 Melon, Quincy Kinsho meron Cucumis melo 2008/6/27 3 87.3 ± 0.2 18.6 ± 1.6 0.6 ± 0.2 19.2 ± 1.4 337 ± 68 17 ± 2 354 ± 70 21 Melon, Takami Takami meron Cucumis melo 2008/6/27 3 88.0 ± 1.5 13.4 ± 3.1 0.4 ± 0.2 13.8 ± 3.2 211 ± 38 11 ± 3 222 ± 41 22 Momordica charantia Nigauri Momordica 2008/7/27 3 93.5 ± 0.5 30.9 ± 6.2 3.0 ± 0.2 33.9 ± 6.4 581 ± 82 110 ± 11 690 ± 93 23 Myoga Myoga Zingiber mioga 2008/7/23 3 95.4 ± 0.1 16.0 ± 1.9 8.6 ± 0.7 24.6 ± 1.2 580 ± 59 97 ± 10 678 ± 50 24 Myoga-take Myogatake Zingiber mioga 2008/7/27 3 96.9 ± 0.1 8.6 ± 0.4 1.2 ± 0.1 9.8 ± 0.5 301 ± 19 71 ± 2 373 ± 21 25 Okra Okura Abelmoschus esculentus 2008/7/27 3 89.9 ± 0.2 124.3 ± 2.6 0.5 ± 0.1 124.8 ± 2.7 1812 ± 192 77 ± 8 1889 ± 200 26 Paprika, Purple Papurika Capsicum annuum 2008/7/27 3 93.5 ± 0.3 55.1 ± 0.6 2.0 ± 0.1 57.1 ± 0.6 1429 ± 174 66 ± 3 1495 ± 173 27 Pepo,Oriental pickling melon Ao uri Cucumis melo 2008/7/15 3 96.0 ± 0.1 8.6 ± 0.4 0.6 ± 0.1 9.3 ± 0.3 175 ± 7 21 ± 4 196 ± 11 28 Pumpkin Edosaki kabocha Cucurbita maxima 2008/7/15 3 69.3 ± 0.7 22.2 ± 1.4 9.1 ± 0.4 31.4 ± 1.3 595 ± 24 280 ± 10 875 ± 14 29 Tomato Tomato Lycopersicum esculentum 2008/6/27 3 93.6 ± 0.5 13.1 ± 1.6 1.6 ± 1.3 14.7 ± 1.5 391 ± 31 27 ± 10 418 ± 40 30 Zucchini, Yellow Zukkini Cucurbita pepo 2008/7/23 3 94.2 ± 0.3 9.7 ± 0.8 1.6 ± 0.3 11.3 ± 0.9 207 ± 55 95 ± 44 302 ± 72 31 Zucchini, Green Zukkini Cucurbita pepo 2008/7/27 3 92.7 ± 0.8 17.3 ± 8.2 1.6 ± 0.5 18.9 ± 8.3 414 ± 121 56 ± 15 470 ± 127

【Leaf and stem vegetable】 32 Welsh onion, Blanched Negi Allium fistulosum 2008/7/15 3 91.7 ± 0.4 17.3 ± 3.7 3.2 ± 0.6 20.6 ± 4.3 333 ± 59 90 ± 20 423 ± 79 Welsh onion, Leaves Negi Allium fistulosum 2008/7/15 3 92.8 ± 0.5 16.9 ± 3.7 5.6 ± 1.4 22.5 ± 5.0 509 ± 128 191 ± 50 700 ± 178 33 Asparagus Asuparagasu Asparagus 2008/8/9 3 92.6 ± 0.6 22.7 ± 0.9 2.9 ± 0.5 25.6 ± 1.3 1062 ± 150 83 ± 19 1145 ± 135 34 Cabbage, Purple Murasaki kyabetsu Brassica oleracea 2008/7/27 3 90.7 ± 0.4 110.2 ± 23.1 1.2 ± 0.2 111.5 ± 23.2 3412 ± 673 57 ± 13 3470 ± 685 35 Garden rocket Rukkora Eruca vesicaria 2008/7/27 3 92.3 ± 0.0 90.0 ± 5.6 2.4 ± 0.1 92.4 ± 5.6 2845 ± 144 422 ± 44 3267 ± 173 36 Garlic Ninniku Allium sativum 2008/6/30 3 73.3 ± 1.2 33.1 ± 0.9 1.4 ± 0.2 34.5 ± 0.8 2284 ± 295 26 ± 3 2310 ± 297 37 Garlic, Odorless Mushu ninniku Allium sativum 2008/6/30 3 75.9 ± 0.7 19.4 ± 5.3 0.8 ± 0.1 20.2 ± 5.3 2504 ± 91 17 ± 2 2521 ± 92 38 Komatsuna Komatsuna Brassica rapa 2008/7/27 3 94.4 ± 0.3 27.5 ± 2.6 3.3 ± 0.3 30.8 ± 2.8 897 ± 88 199 ± 27 1096 ± 95 39 Lettuce Retasu Lactuca sativa 2008/7/15 3 95.8 ± 0.1 10.9 ± 1.1 1.1 ± 0.1 12.0 ± 1.1 449 ± 64 41 ± 4 491 ± 68 40 Mulukhiya Moroheiya Corchorus olitorius 2008/7/23 3 80.3 ± 0.4 251.1 ± 23.0 15.1 ± 1.4 266.1 ± 22.0 9351 ± 888 817 ± 98 10168 ± 849 41 Onion, Red Aka tamanegi Allium cepa 2008/6/27 3 89.9 ± 0.8 35.4 ± 8.5 1.6 ± 0.5 36.9 ± 8.9 927 ± 181 38 ± 5 965 ± 180 42 Onion, Organic Yuki tamanegi Allium cepa 2008/6/27 3 93.2 ± 0.1 21.6 ± 1.8 0.9 ± 0.1 22.5 ± 1.8 476 ± 70 23 ± 2 500 ± 69 43 Perilla Egoma Perilla frutescens 2008/7/27 3 84.1 ± 1.3 691.4 ± 98.0 34.1 ± 5.1 725.5 ± 97.7 27400 ± 5656 1218 ± 154 28618 ±5805 44 Perilla,Green Ao shiso Perilla frutescens 2008/7/23 3 80.4 ± 1.9 857.9 ± 54.2 92.7 ± 7.2 950.6 ± 60.7 29830 ± 1827 1972 ± 209 31802 ±1850 45 Perilla, Red Aka shiso Perilla frutescens 2008/7/27 3 84.7 ± 0.4 586.5 ± 60.3 64.4 ± 7.5 650.8 ± 59.4 24797 ± 2582 1420 ± 157 26217 ±2685 46 Rhubarb Rubabu Rheum rhaponticum 2008/7/27 3 93.9 ± 1.4 30.7 ± 8.4 2.3 ± 0.2 32.9 ± 8.6 1196 ± 365 121 ± 20 1317 ± 360 47 Saltwort Okahijiki Salsola komarovii 2008/7/27 3 89.5 ± 0.3 130.0 ± 15.4 2.6 ± 0.4 132.6 ± 15.8 4317 ± 315 269 ± 16 4586 ± 327 48 Sunny lettuce Saniretasu Lactuca sativa 2008/7/27 3 93.4 ± 0.4 36.4 ± 3.5 6.6 ± 0.8 43.0 ± 4.2 1355 ± 247 185 ± 20 1540 ± 265 49 Water spinach-1 Entsuai Ipomoea aquatica 2008/7/27 3 88.4 ± 0.4 215.3 ± 24.7 18.4 ± 1.6 233.7 ± 26.2 6410 ± 545 437 ± 55 6847 ± 594 Water spinach-2 Kushinsai Ipomoea aquatica 2008/7/27 3 90.9 ± 1.0 88.4 ± 7.5 6.2 ± 0.9 94.7 ± 8.3 3508 ± 687 390 ± 42 3898 ± 700

【Root crop】 50 Carrot Ninjin Daucus carota 2008/7/15 3 87.7 ± 0.6 11.3 ± 0.4 3.6 ± 0.7 14.8 ± 0.8 326 ± 21 81 ± 11 407 ± 32 51 Edible burdock Gobou Arctium lappa 2010/3/26 3 86.2 ± 0.1 4.6 ± 0.4 1.5 ± 0.4 6.1 ± 0.4 6711 ± 675 35 ± 3 6747 ± 678 52 Ginger, Rhizome Shoga Zingiber officinale 2008/6/27 3 96.0 ± 0.4 15.7 ± 1.4 41.2 ± 4.8 56.9 ± 5.0 698 ± 50 1498 ± 109 2196 ± 59 53 Ginger, Rhizome *2 Hashoga Zingiber officinale 2008/7/23 3 94.4 ± 0.4 18.2 ± 2.1 53.3 ± 2.8 71.5 ± 4.8 716 ± 69 2076 ± 152 2792 ± 218 54 Japanese radish Karami daikon Raphanus sativus 2008/7/27 3 93.7 ± 0.2 19.3 ± 1.5 1.6 ± 0.2 20.8 ± 1.5 710 ± 33 67 ± 13 777 ± 21 55 Lotus root Renkon Nelumbo nucifera 2008/7/27 3 91.4 ± 0.7 35.3 ± 11.1 0.8 ± 0.1 36.1 ± 11.1 1175 ± 247 42 ± 11 1217 ± 238 56 Radish, Root-1 Hatsuka daikon Raphanus sativus 2008/7/27 3 93.8 ± 0.1 49.4 ± 1.2 1.4 ± 0.0 50.9 ± 1.2 2113 ± 113 61 ± 20 2175 ± 93 Radish, Root-2 Radeisshu Raphanus sativus 2008/7/27 3 94.5 ± 0.4 44.0 ± 4.7 1.3 ± 0.0 45.3 ± 4.7 2453 ± 424 57 ± 9 2511 ± 415 Radish, Leaf-1 Hatsuka daikon Raphanus sativus 2008/7/27 3 89.8 ± 0.3 85.4 ± 0.0 12.0 ± 0.4 97.5 ± 0.3 4578 ± 202 216 ± 21 4794 ± 192 Radish, Leaf-2 Radeisshu Raphanus sativus 2008/7/27 3 92.7 ± 0.8 37.3 ± 6.8 5.5 ± 0.8 42.9 ± 7.2 2326 ± 611 144 ± 13 2470 ± 624 57 Turnip Kabu Brassica rapa 2008/7/27 3 93.7 ± 0.2 14.2 ± 0.2 0.8 ± 0.1 15.0 ± 0.3 430 ± 60 31 ± 3 461 ± 63 Turnip, Leaf Kabu Brassica rapa 2008/7/27 3 93.8 ± 0.5 34.6 ± 5.2 5.9 ± 2.0 40.5 ± 6.7 1017 ± 189 160 ± 35 1177 ± 211

【Cereal 】 58 Corn, “Mirai” Tomorokoshi Zea mays 2008/7/23 3 76.9 ± 0.5 46.8 ± 2.7 7.1 ± 1.4 53.9 ± 3.7 686 ± 46 229 ± 98 915 ± 142 59 Corn, “Pure white” Tomorokoshi Zea mays 2008/8/9 4 76.1 ± 0.7 38.5 ± 3.8 4.1 ± 0.6 42.5 ± 4.1 612 ± 122 150 ± 34 761 ± 146 60 Rice, Brown rice, “Koshihikari” Kome Oryza sativa 2011/2/25 1 NT NT NT NT 1235 514 1749 61 Rice, Brown rice, “Milky queen” Kome Oryza sativa 2006/8/2 1 NT 44.0 NT 44.0 1010 425 1435 【Tubers and roots】 62 Potato Jagaimo Solanum tuberosum 2008/7/15 3 79.7 ± 0.4 36.2 ± 5.8 0.9 ± 0.3 37.0 ± 5.5 583 ± 85 45 ± 3 628 ± 82 63 Potato, “Andesu Red” Jagaimo Solanum tuberosum 2008/7/27 3 83.1 ± 0.6 27.0 ± 3.0 1.4 ± 0.3 28.4 ± 2.8 834 ± 93 71 ± 11 905 ± 104 64 Sweet potato, “Beniazuma” Satsumaimo Ipomoea batatas 2008/8/9 3 65.6 ± 0.8 24.3 ± 3.8 4.5 ± 0.9 28.8 ± 4.7 782 ± 116 90 ± 29 872 ± 88

【Pulse】 65 Kinako *3 Uguisu kinako Pisum sativum 2010/11/24 1 NT NT NT NT 8583 1094 9677 66 Soybean sprout Daizu moyashi Glycine max 2008/7/15 3 91.9 ± 0.1 40.0 ± 1.7 3.1 ± 0.7 43.1 ± 1.3 1479 ± 74 88 ± 4 1567 ± 76 67 Soybean, Immature Edamame Pisum sativum 2008/7/27 3 73.0 ± 0.1 43.0 ± 1.0 6.4 ± 1.4 49.4 ± 1.0 1334 ± 66 87 ± 18 1421 ± 85

【Mushroom】 68 Brown shimeji Bunashimeji Hypsizigus marmoreus 2008/7/15 3 90.8 ± 0.9 23.4 ± 0.4 8.5 ± 1.1 31.9 ± 0.7 693 ± 37 524 ± 24 1217 ± 61 69 King trumpet mushroom Eringi Pleurotus eryngii 2008/7/15 3 90.4 ± 0.4 29.0 ± 2.5 2.2 ± 0.3 31.2 ± 2.6 642 ± 47 116 ± 8 759 ± 43 70 Oyster mushroom Hiratake Pleurotus ostreatus 2008/7/15 3 90.0 ± 0.2 75.3 ± 3.2 2.8 ± 0.6 78.0 ± 2.9 1774 ± 314 144 ± 15 1919 ± 328

Total ORAC value was calculated by combining L-ORAC and H-ORAC values. TP value was calculated by combining polyphenol content from hexane/dichloromethane (1:1) extracts (LTP) and MWA extracts (HTP). Data expressed as mean values represent analysis of samples (means ± standard deviations). *1 N: the number of samples *2: Young rhizome with leaves *3: Roasted and ground green soybeans NT: not tested 72 I. Mikami-Konishide et al.

Sample preparation Approximately 0.5 or 1 g of freeze 15 μL of diluted Folin-Ciocalteu reagent was mixed and dried sample, milled rice (No. 60, 61) and Kinako (No. 65) incubated for 5 min at room temperature and 75 μL of 2% were applied to the extraction by hexane/dichloromethane sodium carbonate solution was added. After incubation for (1:1) using an extraction system (ASE200, Dionex Co., Osa- 15 min at room temperature, absorbance of the solution was ka, Japan) as described by Wu et al. (2004b) and Oki et al. determined at 750 nm using a plate reader (TERMO max, (2008). Then, the hexane/dichloromethane was evaporated Molecular Devices, Sunny vale, CA). Gallic acid was used using nitrogen gas and the remaining sample was dissolved to prepare the standard curve. Results were expressed as mg in 2 to 4 mL of acetone. This solution was then used for the gallic acid equivalents (GAE) per 100 g of FW. TP value was lipophilic ORAC (L-ORAC) assay after being appropriately calculated by combining polyphenol content from hexane/di- diluted with 7% MCD solvent (w/v) in a 50% acetone-water chloromethane (1:1) extracts (LTP) and MWA extracts (HTP). mixture (v/v) (Huang et al., 2002). Methanol/water/acetic acid (90:9.5:0.5; MWA) extracts were obtained from the re- Results and Discussion maining residue after obtaining hexane/dichloromethane (1:1) ORAC and TP values of selected crops in Japan As extracts using ASE200 (Kitazume et al., 2011). The volume shown in Table 1, the extracts from 70 specimens of crops of the MWA extracts was fixed at 25 mL and used for hy- cultivated in Ibaraki prefecture in Japan were prepared by se- drophilic ORAC (H-ORAC) assay. For the Folin-Ciocalteu quential extraction with hexane/dichloromethane (1:1) and assay, hexane/ dichloromethane (1:1) extracts and MWA MWA using freeze-dried samples, and their ORAC value and extracts prepared by ASE200 were used (Kitazume et al., TP content were measured. Total ORAC value was calcu- 2011). Hexane and dichloromethane solvent was substituted lated by combining L-ORAC and H-ORAC values. TP value with dimethyl sulfoxide (DMSO) by evaporation with nitro- was calculated by combining polyphenol content from hex- gen gas before the assay (Suda et al., 2005). ane/dichloromethane (1:1) extracts (LTP) and MWA extracts ORAC assay The ORAC value of the extracts was (HTP). evaluated according to the method of Oki et al. (2008) and H-ORAC values ranged from 194 to 29,830 μmol TE/100 Huang et al. (2002) with slight modifications. In summary, g FW, whereas L-ORAC values ranged from 6 to 2,076 each 35 μL of diluted sample or Trolox standard, was mixed μmol TE/100 g FW. TP values varied in the range of 7 to with 115 μL of fluorescein solution [(110.7 nM/assay buffer 951 mg GAE/100 g FW. High total ORAC value was mainly

(75 mM KH2PO4-K2HPO4 at pH 7.4)] in a clear, 96-well mi- indicated in leaf and stem vegetables: mulukhiya (No. 40 in croplate (Falcon #353072) and incubated at 37℃ for 10 min. Table 1, Corchorus olitorius, 10,168 μmol TE/100g FW), Next, a reading was taken using a plate reader (Powerscan perilla (No. 43 − 45, Perilla frutescens, 28,879 ± 2,287 μmol HT, DS Pharma Biomedical Co., Osaka, Japan) before the TE/100 g FW), water spinach (No. 49, Ipomoea aquatica, addition of AAPH solution. Then, 50 μL of AAPH solution 5,373 ± 1,474 μmol TE/100 g FW), purple cabbage (No. 34, (31.7 mM/assay buffer) was rapidly added to each well using Brassica oleracea, 3,470 μmol TE/100 g FW), saltwort (No. a multi-channel pipette. Immediately following the addition 47, Salsola komarovii, 4,586 μmol TE/100 g FW), and radish of AAPH, the plate was agitated for 5 s prior to the first read- leaf (No. 56, Raphanus sativus, 3,632 ± 1,142 μmol TE/100 ing and for 3 s before each subsequent reading. Readings g FW). High total ORAC values were also indicated in the were done at 2 min intervals for 90 min for H-ORAC assay, following: edible burdock (No. 51, Arctium lappa, 6,747 and for 120 min for L-ORAC assay. Excitation and emission μmol TE/100 g FW) in the root crop, blueberry (No. 1, Vac- filter wavelengths were set at 485 nm and 528 nm, respec- cinium corymbosum, 6,562 μmol TE/100 g FW) and hardy tively. Data were expressed as μmol Trolox equivalents (TE) kiwifruit (No. 3, Actinidia arguta, 9,476 μmol TE/100 g per 100 g of fresh weight (FW). Instead of using AAPH solu- FW) in the fruit, small hot pepper (No. 15, Capsicum annu- tion (31.7 mM/assay buffer) for H-ORAC assay, AAPH solu- um, 3,848 μmol TE/100 g FW) in the fruit vegetable, uguisu- tion (63.4 mM/assay buffer) was used for L-ORAC assay. kinako (No. 65, roasted and ground green beans, Pisum sati- Total ORAC value was calculated by combining L-ORAC vum, 9,677 μmol TE/100 g FW) in pulse (Table 1). A rather and H-ORAC values. high L-ORAC value was found in the following plants: Total phenolics (TP) analysis The TP analysis was mulukhiya (No. 40, Corchorus olitorius, 817 μmol TE/100 based on the Folin-Ciocalteu method (Sun et al., 2005), g FW), perilla (No. 43 − 45, Perilla frutescens, 1,537 ± 318 slightly modified by Kitazume et al. (2011) and Tsuruta et μmol TE/100 g FW), ginger (No. 52, 53, Zingiber officinale, al. (2007). Concentrated Folin-Ciocalteu reagent was diluted 1,787 ± 289 μmol TE/100 g FW) and uguisu- kinako (No. two-fold with water. Water (60 μL) and 10 μL of sample 65, roasted and ground green soybeans, Pisum sativum, 1,094 extracts were mixed in a clear, 96-well microplate. Next, μmol TE/100g FW). H-ORAC values were much higher than Antioxidant Capacity of Extracts from Crops Cultivated in Japan 73 L-ORAC values, with the exception of ginger. ORAC values of leafy vegetables at different development The USDA database containing antioxidant capacities of stages, grown in different environments and with different 326 selected foods measured using ORAC methodology (iii) fertilization regimes. They indicated that differences in cul- demonstrates high ORAC values in herb, berry and cereal tivation practices result in significant changes in the antioxi- grain. However, in this study, leafy vegetables (such as mu- dant properties of each plant. lukhiya and perilla), edible burdock and hardy kiwi showed For a better understanding of ORAC variability, we ex- higher ORAC values. amined several factors that may affect ORAC values. The term “functional foods” indicates the “tertiary” function of foods, and differs from the conventional “primary” and “secondary” functions that are related to nutrition and preference, respectively (Arai, 1996). Food has various functions, and consumption of each agricultural product differs in terms of its effects on the human body. The products from the six farms examined constitute a large part of agricultural output of Ibaraki prefecture (v), and present a good source of calories, dietary fiber or minerals. However, their ORAC value per gram is at best moderate: brown rice (No. 60, 61, Oryza sativa, 1,592 ± 157 μmol TE/100 g FW), sweet potato (No. 64, Ipomoea batatas, 872 μmol TE/100 g FW), melon (No. 16 − 21, Cucumis melo, 285 ± 40 μmol TE/100 g FW), lettuce (No. 39, Lactuca sativa, 491 μmol TE/100 g FW), tomato (No. 29, Lycopersicum esculentum, 418 μmol TE/100 g FW), and welsh onion (No. 32, Allium fistulosum, 561 ± 138 μmol TE/100 g FW). Although examination of food intake is not being carried out at this time, a balanced intake of various agricultural products is important. Figure 2 shows the correlation between ORAC value and polyphenol content of the crop extracts assessed in this study. The results demonstrate that the ORAC value of these extracts has a strong positive correlation with polyphenol content, suggesting that antioxidant capacity of these crops appear to result mainly from the polyphenol content. In this study, we performed multiple measurements of ORAC level in samples of the same products purchased from different farmers: water spinach (No. 49 − 1 and 2: 6,847 and 3,898 μmol TE/100 g FW) and radish (No. 56 − 1 and 2: root; 2,175 and 2,511, leaf; 4,794 and 2,470 (μmol TE/100 g FW)). These variations in ORAC value may be explained by variations in the strains, growth stages or the environment. Genotype is the principal factor contributing to variations in antioxidant capacity of fruits and vegetables (How- ard et al., 2002; Pandjaitan et al., 2005; Reyes-Carmona et al., 2005). However, the cultivation environment is also an important factor that determines variability of antioxidant capacity (Zhao et al., 2007; Piergiovanni et al., 2010). Antioxi- dant capacity of fruits and vegetables grown organically ver- Fig. 2. Correlation between ORAC value and poly-phenol content of crop extracts from Ibaraki prefecture. sus that of conventionally grown produce has also been the ORAC value and total polyphenol content of crop extracts corre- subject of multiple studies, due to an increased demand for spond to Table 1. A; MWA (methanol/water/acetic acid (90:9.5:0.5) information on organic produce (Mitchell et al., 2007; Wang extracts, B; hexane/dichloro-methane (1:1) extracts, C; total value et al., 2008; Zhao et al., 2007). Zhao et al. (2007) compared of two solutions (A and B extracts). 74 I. Mikami-Konishide et al.

ORAC value of stem mustard ”Umino” extract The ORAC value of stem mustard leaf (5,475 ± 2,055 μmol TE/100 g FW) was almost the same as that of water spinach (No. 49). Water spinach had the third highest ORAC value of the examined vegetables (Table 1) after perilla (No. 43 − 45) and mulukhiya (No. 40). A part of expanded stem of stem mustard is utilized as Zha cai; however, stem mustard leaf is currently an underutilized resource, at least in Japan. However, by measuring ORAC values of each part of stem mustard, stem mustard leaf was found to have a high ORAC value and can be considered a good-quality material. For these reasons, here we used each part of the stem mustard, cultivated in three different regions, for the following evaluation of ORAC variation. Stem mustard (Brassica juncea (Linn.) Czern. et Coss. Fig. 4. Correlation between ORAC and TP values of stem mustard extracts. var. tumida Tsen et Lee) “Umino” is a stable cold resistant The correlation was calculated using the data of season II (2010 − inbred strain bred by Nakanishi and Murakami. It was reg- 2011) (see Fig. 3.). istered in 2007 (vi) and is one of the special local crops in Ibaraki prefecture. However, there are no reports about its wide leaf and thick petiole. Its expanded stem is ideal for antioxidative capacity. This strain has an expanded stem, lightly pickled vegetable, which is well known as Zha cai. It has been cultivated only in Ibaraki prefecture, but was recently adapted to grow in three different regions (Ibaraki, Yamagata and Kanagawa prefectures). Therefore, interest in the evaluation of the strain against various cultivation conditions has increased. First, we compared ORAC values of each part of the stem mustard plant [leaf (L), petiole (P), stem (S), flower bud (F) and leaf of flower bud (LF)]. As shown in Fig. 3, remarkable differences in ORAC value were observed in different parts of the stem mustard in all three prefectures; ORAC value of the leaf was 7 − 12, 10 − 30 times higher than that of stem and petiole, respectively. In addition, ORAC value showed a strong positive correlation with polyphenol content (Fig. 4), suggesting that antioxidative capacity of the stem mustard results mainly from the polyphenol content. Second, we focused on the “growth stage”, and compared ORAC values of stem mustard extracts (Fig. 3). As a result, ORAC value of the leaf harvested at the “flower bud (LF) differentiation stage” tended to be higher than that of “vegetative growth (L) stage”. This suggests that variations in the Fig. 3. ORAC values of stem mustard extracts. phenolic level and antioxidant capacity of stem mustard are ORAC assay results are expressed as the means of more than two experiments (μmol TE/100 g of FW) of bulk samples from three dependent on its growth stage. different plants (means ± standard deviations). Stem mustard “Umi- With respect to “growth stage”, Pandjaitan et al. (2005) no” was harvested from the three regions: Ibaraki (A), Yamagata (B) and Zhao et al. (2007) showed that the influence of matu- and Kanagawa (C) prefectures in 2009 − 2011. A-1, 2, B, C-1, 2, 3 indicate the different farmers in the three prefectures. Each stem ration on hydrophilic and total antioxidant capacities was mustard part was separately prepared for lyophilization (see Materi- dependent upon the vegetable type and production environ- als and Methods): L (leaf), P (petiole), S (stem), F (flower bud), LF ment. For example, significant elevated values at the mature (leaf of flower bud). From top to bottom: growing season I (2009 head stage were only observed in pac choi (Brassica rapa) − 2010) and II (2010 − 2011). Vegetative periods in each region are indicated in Fig. 5-I and Materials and Methods. Black squares from high tunnel fields and spinach from open fields. But in indicate the moisture content (%). other types of vegetables (Red leaf lettuce and Romaine let- Antioxidant Capacity of Extracts from Crops Cultivated in Japan 75 tuce), production environment did not affect the difference in tard “Umino” As shown in Figs. 3 and 4, the ORAC and ORAC value according to harvest stage (Zhao et al., 2007). TP values of the stem mustard extracts from Ibaraki were At the same time, mid-maturity spinach (Spinacia oleracea) higher than those of the other two regions. Therefore, we ex- leaves had higher levels of total phenolics, total flavonoids, amined effects of cultivation environment by comparing the and antioxidant capacity than immature and mature leaves following data of the three regions: UV index, day length, (Pandjaitan et al., 2005). In this case, spinach genotypes vegetative periods, rainfall, temperature and fertilizer (Figs. 5 should be harvested at the mid-maturity stage for consumers and 6). We also investigated several hypotheses to elucidate to benefit from the elevated levels of health-promoting flavo- the factors that affect ORAC values in stem mustard in these noids present in the leaves. regions. As a result, we paid special attention to UV radiation Third, we examined differences in cultivation areas and (Fig. 5) and fertilizer (Fig. 6) as the main cultivation factors compared ORAC values of the stem mustard extracts from affecting ORAC value of stem mustard in these regions. three prefectures. As a result, variability of ORAC value was In plants, the biosynthesis of flavonoids (polyphenolic observed (Fig. 3); ORAC value of leaf, petiole and stem in compounds) produced through the phenylpropanoid pathway Tsukuba (Ibaraki) was two times, 1.5 − 3.1 times, 1.7 − 3 is known to be activated by environmental stresses, such as times higher than those of other regions, respectively, in sea- nutrient deficiency, wounding, pathogens, and UV radiation son I (2009 − 2010). Although differences in ORAC value (Dixon and Paiva, 1995; Mitchell et al., 2007). Connor et al. among season II (2010 − 2011) samples were smaller than (2002) showed that differences in antioxidant capacity may those of season I, the tendency for high ORAC values in reflect differences in cultivation practices among locations, samples grown in Ibaraki was observed. including differences in water stress, mineral nutrient avail- Effects of cultivation environment on ORAC of stem mus- ability, and UV radiation.

Fig. 5. Graphic representation of UV index, day length (I), rainfall, maximum and minimum temperatures (II) during cultivation of stem mustard “Umino” in 2009 − 2010 (Left of I and II), and 2010 − 2011 (Right of I and II). From top to bottom: observation points A (Tsukuba city in Ibaraki prefecture), B (Yamagata city in Yamagata prefecture), C (Yokohama city in Kanagawa prefecture). Dotted lines and black squares in (I) indicate the vegetative periods and hours of day length in a month, respectively. Yamagata and Yokohama city (observation points) is about 30 km away from Takahata and Miura city (cultivation regions), respectively. Ultraviolet (UV) index: an international standard measurement of the strength of the UV radiation from the sun. Meteorological data containing UV index, day length, rainfall, and temperature in the three observation points were from Japan Meteorological Agency (vii). 76 I. Mikami-Konishide et al.

Fig. 6. Graphic representation of the amount of fertilizer employed on each farm. I and II indicate the amount of fertilizer used as basal fertilizer and additional fertilization (kg ha-1), respectively. Basal fertilizer was added before seeding. Additional fertilizer was added at the fourth or fifth leaf term. Tsukuba (Ibaraki) and Takahata (Yamagata) farmers used chemical fertilizer containing organic manure; on the other hand, purely chemical fertilizers were used in Miura (Kanagawa) farm. The rates of fertilizer application are indicated in the Materials and Methods.

UV index (vii) is an international standard measurement anthocyanin contents among a range of blackberry genotypes of the strength of the UV radiation from the sun, and is cat- produced in different climatic regions, and showed that wild egorized into five exposure levels: minimal (1 − 2), low (3 blackberry exhibited the highest values. They concluded that − 4), moderate (5 − 6), high (7 − 9), and very high (10 +). high concentrations of protective polyphenolic compounds As shown in Fig. 5-I, when we take account of each grow- had resulted from greater exposure of the unsheltered wild ing period [Tsukuba (Ibaraki); 24 Sep. 2009 − 14 Dec. 2009, plants to extreme conditions. However, Josuttis et al. (2010) 17 Sep. 2010 − 5 Jan. 2011, Takahata (Yamagata); 12 Aug. reported that effects of UV radiation differed depending upon 2009 − 12 Dec. 2009, 12 Sep. 2010 − 24 Jan. 2011, Miura the types of phenolics; the contents of anthocyanin (cyani- (Kanagawa); 5 Oct. 2009 − 10 Feb. 2010, 12 Sep. 2010 − 24 din 3-glucoside) and flavonols (quercetin 3-glucuronide and Jan. 2011)], the effect of the UV radiation in Ibaraki was the kaempferol 3-glucoside) were decreased in the fruits grown strongest among the three regions. The averages of monthly under UV blocking film compared to open-field grown UV index during each growing period (I) 2009-2010 and (II) strawberries. At the same time, other phenolics were not 2010-2011 in the three regions were as follows: Ibaraki; (I) consistently affected by UV radiation. Therefore, in future, 8.25, (II) 8.76, Kanagawa; (I) 7.02, (II) 6.60, Yamagata; (I) further analysis using high-performance liquid chromatogra- 7.15, (II) 5.88. The plants in Yamagata were grown in a tun- phy (HPLC) is required to clarify not only the contributors to nel plot, which may reduce the amount of UV radiation and stem mustard antioxidant capacity, but also to explain ORAC sunshine compared to plants grown in the open field. The variations among plants grown in different regions. day length in Ibaraki was 9 − 10% shorter than that of Kana- Another stress factor is temperature regime. It is known gawa; however, the effect of UV radiation on ORAC value that stem mustard is ruined by frost. Therefore, Kanagawa, was unclear. where temperatures do not fall below the freezing point, is Solar UV-B radiation elicits a variety of acclimation considered to be favorable for its cultivation (Fig. 5-II). In responses, which typically include increased activity of an- Ibaraki, temperatures drop to minus five degrees or less dur- tioxidant enzymes, increased DNA repair capacity, and ac- ing late January and plants are normally harvested before cumulation of phenolic compounds that serve as “sunscreens” that period. Temperatures in Yamagata did not greatly affect or UV filters (Caldwell et al., 2007). Exposure to increased the plants, since they were grown in tunnel plots. Accord- levels of UV radiation during cultivation caused the leaves ing to the literature (Josuttis et al., 2010; Wang and Zheng, of red leaf lettuce to redden and an increase in ORAC values 2001), the content of kaempferol (3-glucoside- malonate) and concentrations of total phenols and main flavonoids, could be sensitive to temperature and UV, similar to the UV such as quercetin and cyanidin glycosides, as well as luteolin sensitivity of anthocyanin levels in strawberries. Therefore, conjugates and phenolic acids (García-Macías et al., 2007). significant differences in temperature regime may affect stem Reyes-Carmona et al. (2005) compared values of ORAC, mustard antioxidant capacity. Precipitation does not affect ferric reducing antioxidant power (FRAP), total phenolic and stem mustard strongly, as it is a winter crop. Antioxidant Capacity of Extracts from Crops Cultivated in Japan 77 Figure 6 displays the amount of fertilizer used at each and nutrient availability are important factors that determine farm. Ibaraki and Yamagata farmers relied on chemical ORAC values. In future work, comparison on the same back- fertilizer containing organic manure while purely chemical ground will be necessary to address the issues of nutrient fertilizers were used at the Kanagawa farm. Depending on availability. the farm, the amounts and the sources of the basal and addi- In this study, we showed the wide range of ORAC and tional fertilizers differed greatly. As previously reported, the TP values of 71 types of farm products in Japan. In addi- increase in total phenolic content seems to be stimulated by tion, we discussed growing conditions as factors that affect low nitrogen supply; the amount of nitrogen fertilizer used in the antioxidant capacity of crops. We expect that our data Ibaraki was the lowest among the three farms. The amount on ORAC values will help familiarize the Japanese popula- of nitrogen fertilizer used in Kanagawa (154 kg N ha-1) and tion with indicators of crop quality that are connected with Yamagata (360 kg N ha-1) farms was 1.2 and 2.8 times larger health outcomes. In addition, elucidating the causes behind than in Ibaraki (128 kg N ha-1), respectively. high ORAC values in crops will help local farmers ensure a Dixon and Paiva (1995) attributed the accumulation of steady supply of high-quality farm produce to the market. phenolic compounds to nitrogen deficiency. 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Oxygen-radical increased, especially under conventional fertilization. They absorbance capacity assay for antioxidants. Free Radical Biol. also reported an interesting tendency demonstrated by ORAC Med., 14, 303-311. values in response to fertilizer rates; in general, H-ORAC Caldwell, M.M., Bornman, J.F., Ballaré, C.L., Flint, S.D. and Ku- and total ORAC values of pac choi tended to decrease at landaivelu, G. (2007). Terrestrial ecosystems, increased solar higher fertilization, while L-ORAC reached the highest lev- ultraviolet radiation, and interactions with other climate change els at a medium fertilizer rate. Meanwhile, Toor et al. (2006) factors. Photochem. Photobiol. Sci., 6, 252-266. reported that different nutrient sources (mineral nutrient solu- Cho, M.J., Howard, L.R., Prior, R.L. and Clark, J.R. (2005). 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