Potentials of representative heirloom vegetables on Shonai region of Yamagata, Japan

*Corresponding author: Takeshi Nagai,Graduate School of Agricultural Sciences, Yamagata University, Yamagata 9978555, Japan. E-mail: nagatakenagatake@yahoo.co.jp, tnagai@tds1.tr.yamagata-u.ac.jp Received: 20 December 2018 ; Accepted: 12 February 2019 R E S E A R C H A R T I C L E


INTRODUCTION
Vegetables plays important roles as not only resources for supplying micronutrients such as vitamins and minerals that are essential for normal nutrition and metabolisms but also as low-caloric and low-fatty foods. In addition, many kinds of functional ingredients contain in it. Phenolics have multiple biological functions such as antioxidant and antibacterial properties (Fawole et al., 2012) and synergistic effects and protective properties against life style-related diseases such as arteriosclerosis, arthritis, brain dysfunction, cardiovascular diseases, cancer, hypertension, and inflammation (Verma et al., 2018). Reactive oxygen species as superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals, which cause oxidative damage of human body, are factors of onset of many diseases containing cancer. Therefore, consumption of vegetables, which are rich in fibers, minerals, phenolics, and vitamins, help to prevent oxidative stress and to reduce incidence of these diseases (Fidrianny et al., 2018). Moreover, it is reported that intake of antioxidants such as phenolics and vitamins retard ageing (Ross and Kasum, 2002).
Heirloom vegetables are crops that seeds are maintained and conservation of species is continued by growers in limited areas over generations. It is considered that these species have values as cultural properties because these are genetic resources for breeding materials and are in close relations with traditional eating habits in a region. However, traditional varieties rapidly disappear worldwide, therefore, it has been discussing the way of conservation of varieties (Tomiyoshi and Ueno, 2016). Recently, there is a nationwide trend to revalue existence and benefits of heirloom vegetables. Representative heirloom vegetables in Japan are as follows: Shishigatani pumpkin, Kujonegi, and Fushimi pepper in Kyoto (JA-Kyoto), Aizumaru eggplant, Arakudakukitachi, and Tachikawa burdock in Aizu region of Fukushima (Association to protect traditional vegetables in Aizu; Mizuno and Sumino, 2008), and Nakajimana, Futatsukakarashina, and Kaga Glycine soja in Kanazawa region of Ishikawa (Kanazawa City Agricultural Products Branding Association). Particularly, there are many types of heirloom vegetables in Yamagata, Japan (179 varieties in 2018). Among them, 87 varieties of vegetables are cultivated in Shonai region of Yamagata, Japan (Tsuruoka Creative City of Gastronomy Promotion Committee, 2018). However, few studies have explored even proximate composition and nutritional properties of these vegetables. It is expected that many vegetables are more nutritious than commonly available vegetables, as these has not undergo selective breeding aimed at good appearance and convenience for broad area distribution. Consumers nowadays tend to require foods with high nutritional values and health functionalities. This work was performed to evaluate proximate composition and physicochemical and nutritional properties of main heirloom vegetables on Shonai region of Yamagata, Japan, and furthermore to elucidate health functionalities of these vegetables.

Materials
Fourteen fresh heirloom vegetables (Atsumikabu, Chijimina, Fujisawakabu, Hirataakanegi, Karatori, Kirariboshi, Makomodake, Mindennasu, Mosodake, Natsuna, Okitanasu, Tagawakabu, Tomoebuki, and Tonojimakyuri) were purchased from produce stands on Shonai region of Yamagata, Japan (Fig. 1), and were used in the study. These are representative heirloom vegetables on this region. In addition, these are eaten frequently on the region. The information about these genetic materials is summarized in the book (Yamagata Forum for the Indigenous Crops, 2012). All chemicals were of regent grade.

Determination of proximate composition
Moisture was measured using a Moisture Determination Balance (FD-600; Kett Electric Laboratory, Tokyo, Japan). Crude proteins were determined by the Kjeldahl method using a conversion factor of 6.25. Crude lipids were analyzed by ether extraction. Measurements of crude ashes were used an electric furnace (AMI-II; Nitto Kagaku Co. Ltd., Aichi, Japan). Carbohydrates were calculated by difference. Crude fibers were measured as described by Nakamura et al. (1998). Salts were determined using a digital salinity concentration meter (EB-158P, EISHIN Co., Ltd., Hiroshima, Japan). Calories were calculated using the FAO conversion factors (Isobe et al., 2011).

Physicochemical properties
Total soluble solids (TSS) were determined using a digital refractometer (PAL-Pâtissier, Atago Co. Ltd., Tokyo, Japan). The pH values were measured using a digital pH meter (PHL-40, DKK-TOA Co., Tokyo, Japan). Alkalinity was estimated as described by Miura et al. (2006). Free amino acid contents were determined by the TNBS method (Sugawara and Soejima, 1996) using L-leucine as standard. Vitamins B1 and B2 were measured by the p-aminoacetophenone method and by the lumiflavin fluorescence method, respectively (Nakamura et al., 1998). Vitamin C was determined by α,α'-dipyridyl method (The Vitamin Society of Japan, 1990). β-Carotene, lycopene, and chlorophyll (a, b) were measured using acetone-hexane extraction method (Nagata and Yamashita, 1992). Phenols and flavonoids were determined as described by Slinkard and Singleton (1977) and by Kim et al. (2003), respectively, using quercetin dihydrate as standard.

Functional properties
Antioxidative activities of extracts from vegetables were evaluated as described by Nagai et al. (2018). Ascorbic acid (AA), tert-butyl-4-hydroxyanisole (BHA), 2,6-di-t-butyl-4methylphenol (BHT), α-tocopherol (TP), and trolox (TL) were used as positive controls, and distilled water or 80% methanol were used as negative one. Superoxide anion radicals, hydroxyl radicals, and DPPH radicals scavenging activities were determined as described by Nagai et al. (2018). Activities [TL equivalents scavenging capacity (TESC); mM TE/kg FW] were also expressed as millimoles of TL equivalents per kg of fresh weight of vegetables. ACE and hyaluronidase inhibitory activities were measured as described by Nagai et al. (2018).

Statistical analysis
Each assay was repeated 3 times independently and results were reported as means ± standard deviation (SD).

Proximate composition
Proximate composition per 100g of vegetables is shown in Table 1. Moisture contents ranged from 90.1-96.0 g except for Karatori tubers and Chijimina. Content in Karatori tubers was similar to eddoe fresh bulbs, but was fairly lower than taro fresh bulbs (Kagawa, 2018

Physicochemical properties
Vegetables were cut into small pieces, ground in a mortar, and then TSS and pH at 20ºC were measured. Highest TSS value was detected in Chijimina, followed by in Hirataakanegi, Atsumikabu, and Okitanasu, whereas Karatori tubers were lowest because of existence of great quantities of starches (Table 1). Correlation between carbohydrate contents and TSS contents was with R 2 = 0.648 except for Karatori tubers and Tomoefuki and was with R 2 = 0.833 except for Fujisawakabu. The pH values ranged from 5.4-6.4. High alkarinity was shown in Chijimina, followed by Makomodake, Kirariboshi, and Fujisawakabu, whereas Tonojimakyuri, Mindennasu, and Okitanasu were low. There were varietal differences on alkarinity among turnip roots and leafy vegetables. Generally, grains, meats, and fish and shellfishes are classified as acid foods, whereas vegetables, fruits, mushrooms, and seaweeds are categorized as alkaline ones. Highest free amino acid contents were shown in Mosodake, followed by in Chijimina, Kirariboshi, and Makomodake, whereas Karatori stems were low.

Functional components
Kirariboshi showed high vitamin B1 value, followed by Chijimina and Natsuna, whereas turnip roots as Fujisawakabu, Karatori tubers, Tomoefuki, and Makomodake were low ( Next, vegetables were cut into small pieces, and were homogenized with 2 volumes of distilled water or 80% methanol. Suspension was centrifuged at 30,000 x g for 30 min at 4ºC, and supernatants were filtered with glass wool (distilled water extracts: WE; methanol extracts: ME). Phenols and flavonoids contents of these extracts are shown in

Hydroxyl radical scavenging activity
In WEs, Kirariboshi and Karatori stems showed remarkably high hydroxyl radical scavenging activities about 85.7 and 85.3%, respectively, although activities did not reach that of 0.1 mM TL (Table 3). Activity on Natsuna was similar to that in 0.01 mM BHT. On the contrary, Tonojimakyuri and Mindennasu were low. TESC ranged from 0.13-1.54 x 10 3 mM TE/kg FW. In MEs, Tomoefuki exhibited significantly high activity about 88.2%, followed by Karatori stems, whereas Mosodake was low about 34.3%. TESC ranged from 0.39-1.59 x 10 3 mM TE/ kg FW. Correlation was not observed between activities on WEs or MEs and phenols or flavonoids contents.

Hyaluronidase inhibitory activity
Sodium cromoglicate (SC) is used as one of commercially available anti-allergic drug against atopic dermatitis based on food allergies, allergic rhinitis, and allergic conjunctivitis. Hyaluronidase inhibitory activities were also expressed as millimoles of SC equivalents per kg of fresh weight of vegetables [SC equivalents inhibitory capacity (SCEIC); mM SCE/kg FW]. In WEs, Atsumikabu and Karatori stems showed extremely high activities about 83.3 and 81.7%, respectively, followed by Fujisawakabu and Kirariboshi (Table 4). Meanwhile, Okitanasu, Makomodake, and Mosodake were low about 14.0-18.9%. SCEIC was estimated to 0.20-1.43 x 10 2 mM SCE/kg FW. In MEs, Karatori stems possessed significantly high activity about 92.7%, followed by Okitanasu, Karatori tubers, and Kirariboshi. Fujisawakabu, Mindennasu, Chijimina, and Makomodake showed middle activities ranging from 48.1-57.6%, whereas Tonojimakyuri was lowest. SCEIC ranged from 0.21-1.58 x 10 2 mM SCE/ kg FW. Correlation was not observed between activities and phenols or flavonoids contents. Ippoushi et al. (2000) investigated hyaluronidase inhibitory activities on 46 vegetables and herb extracts. Extracts prepared from 8 Labiatae plants and borage showed activities. Moreover, they isolated and identified rosmarinic acid as inhibitor from lemon balm extract. Kimura et al. (2007) reported that activities on 7 heirloom vegetables in Yamanashi, Japan were low or not detected. Hirata (2010) elucidated that none of activities were low, although 32 vegetables exhibited activities among 43 vegetables in Yamaguchi, Japan.
In this study, it was revealed for the first time the nutritional and health-promoting properties of representative heirloom vegetables on Shonai region of Yamagata, Japan. Further research is going to investigate the properties of other vegetables because of existence of many native varieties of vegetables in this area. It is also necessary to research the nutrients and functionalities on each part of vegetable. Cooking may affect the contents of useful components in vegetables. Detailed analysis of nutritional and functional properties on vegetables before and after cooking is in progress to develop novel processing methods of these vegetables.

CONCLUSIONS
These findings demonstrated that heirloom vegetables used in this study served as good sources of vitamins, phenolics, and antioxidants compared to corresponding commercially available vegetables. Positively eating of these vegetables can probably contribute to health promotion to prevent life style-related diseases such as cancer, hypertension, and inflammation. Furthermore, it also may have potentials for preservation of species and for promotion of sustainable cultivation of heirloom vegetables.