Food Sci. Technol. Res., +, (-), +20ῌ+3-, ,**0

Characteristics of Flavonoids in Niihime Fruit - a New Sour Citrus Fruit

ῌ Yoshiaki MIYAKE

Faculty of Human Wellness, Tokaigakuen University, Nagoya, .02ῌ2/+., Japan

Received November ,+, ,**/; Accepted June ,, ,**0

Niihime fruit, produced in the coastal area of the Sea of Kumano in Mie prefecture, is a new sour citrus fruit. This is the first study to examine the characteristics of the flavonoids in niihime fruit. The content of flavonoids in the juice and peel of niihime fruit was determined by HPLC and their distribution was examined in comparison with seven other species of sour citrus fruits. Niihime fruit was found to contain a comparatively high quantity of bioactive flavonoids of the flavanone with rutinose sugar chain such as and as well as the polymethoxyflavones such as nobiletin and tangeretin. The peel extract of niihime fruit exhibited comparatively high antioxidative activity among sour citrus fruits using the DPPH radical scavenging assay. Furthermore, the flavonoids eluted from niihime fruit by over time in hot and cold solutions of water, /ῌ ethanol, and ,/ῌ ethanol, which are commonly used in the field of food processing and cooking, were examined. The flavonoids eluted from niihime fruit in hot ,/ῌ ethanol solution was highest content in solutions, and the content of flavonoids eluted in / min was an approximate half of content eluted in 0* min. The flavanone glycosides, eriocitrin and hesperidin, were eluted in higher contents in hot solutions than in cold solutions. The polymethoxyflavones, nobiletin and tangeretin, were eluted to some extent in hot /ῌ ethanol but were found in low contents in cold solutions. The highest contents were eluted in hot ,/ῌ ethanol. The di#erence in the elution properties between flavanone glycosides and polymethoxyflavones is considered to be a result of their hydrophobic properties. The scavenging activity of DPPH radical for eluates was shown to increase over time, and the activity was suggested to be related to the elution content of eriocitrin, which is an antioxidant in niihime fruit. This study showed that niihime fruit contains a comparative abundance of bioactive flavonoids and these flavonoids are able to be eluted using hot solutions of water and ethanol.

Keywords: flavonoid, niihime fruit, sour citrus fruit

Introduction tivities (Attaway, +33.: Benavente-Garcia et al., +331). Citrus fruit contains many physiologically bioactive Eriocitrin, which is a flavanone and is abundant substances such as ascorbic acid, carotenoids, flavonoids, in lemon and lime fruits, has been reported to have anti- and coumarins, which have antimutagenic, antitumor, oxidative activity on lipid peroxidation and a suppressive antioxidative, and anticarinogenic activities (Attaway, e#ect on oxidative stress in vivo (Miyake et al., +332b; +33.: Middleton and Kandaswami, +33.; Higashimoto et Minato et al., ,**-). Nobiletin and tangeretin, which are al., +332). Sour citrus fruit is a group of citrus fruit with polymethoxyflavones abundant in shiikuwasha fruit, the characteristics of high acidity and a strong citrus have been reported as having anti-tumor and anti- flavor, and includes citrus fruits such as lemon, lime, carcinogenic activity (Kawaii et al., +333a; Murakami et yuzu, sudachi, kabosu, daidai, and shiikuwasha. Sour al., ,***; Suzuki et al., ,**.). Given these findings, citrus citrus fruit is used widely in food seasoning and bever- flavonoids have gained a lot of attention for their many ages, including alcohol beverages, because they possess a physiology characteristics linked to the prevention of desirable citrus flavor and sour taste. Recently, the lifestyle diseases, and their distribution in citrus fruit has physiological activity of sour citrus fruit has been re- been widely examined (Kawaii et al., +333b). ported in experimental animals. A suppressive e#ect for Niihime fruit, which is produced in Mie prefecture high blood pressure is provided by the intake of lemon along the coast of the Sea of Kumano, is a new sour citrus juice (Miyake et al., +332a), a hypocholesterolaemic e#ect fruit. It is thought to be an autogenous citrus consisting is provided by the intake of kabosu juice (Ogawa and of a mandarin and tachibana (Citrus tachibana) cross hy- Mochizuki, ,**-) and an increase in calcium bio- bridization, and was registered as a new nursery plant in availability is provided by the intake of sudachi juice (Nii +331 (Japan the plant variety protection, No. /13+, ++/+./ et al., ,**.). Flavonoids in citrus fruit have been reported +331). People of the region have eaten green niihime to have many biological functions in antioxidative, anti- fruit and ripe niihime fruit and used it for food seasoning carcinogenic, cardiovascular, and anti-inflammatory ac- fried and roasted fish and for adding a sour flavor to distilled alcohol. However, the flavonoid characteristics ῌ To whom correspondence should be addressed. of niihime fruit have yet to be reported. In this study, E-mail: [email protected] the distribution of flavanone glycosides and poly- Characteristics of Flavonoids in Niihime Fruit 187 methoxyflavones in niihime fruit was examined in com- in sour citrus fruit was carried out in accordance with the parison with other sour citrus fruits for first time. Fur- retention time of standard flavonoids. Standard flavonoids thermore, the elution properties of flavonoids from were used for the determination of flavonoids in sour niihime fruit were examined using di#erent solutions in citrus fruit. The content of flavonoids in juice and peel order to evaluate the characteristics of flavonoids in was shown as meanῌSD (N῎.)inmg/+** mL juice and niihime fruit for food processing and cooking. mg/+** g peel. The trace level (*.*+ῌ*.+ mg/+** mL juice or +** g peel) was shown as trace, and the level of less than Materials and Methods *.*+ mg/+** mL juice or +** g peel was shown as nd (no Samples and Reagents Citrus fruits, consisting of detect). lemon (produced in California), lime (produced in Mexico), Extraction of niihime fruit with water-ethanol solution yuzu (produced in Miyazaki), sudachi (produced in Toku- The peel of ripe niihime fruit was extracted with water- shima), kabosu (produced in Ohita), and shiikuwasha (pro- ethanol solutions and the content of flavonoids in extract duced in Okinawa) were purchased from a supermarket. was determined by HPLC. The peel (+.* g) of niihime Fruits were obtained at approximately the same time and fruit, which was crushed using a food processor, was from the same store. Daidai and niihime (green and ripe extracted with +* mL of water, /῍ ethanol, ,/῍ ethanol, fruits) were obtained courtesy of the Mie Prefectural Sci- /*῍ ethanol, 1/῍ ethanol, and +**῍ ethanol over night at ence and Technology Promotion Center, Agricultural Re- room temperature (N῎-). The content of flavonoids in search Division. The green niihime fruit, which has a peel extract was determined by HPLC according to the green peel, was collected on Nov. +, ,**. and the ripe fruit, described method. The extraction e$ciency (῍)of which has a yellow peel, was collected on Jan. +/, ,**.. flavonoids in each water-ethanol solution was calculated Eriocitrin was obtained by purification from lemon peel based on the highest content of flavonoids in the solution. (Miyake et al., +331a). Nobiletin and tangeretin were re- Data are presented as meanῌSD (N῎-). ceived as a gift courtesy of Dr. M. Yano and Dr. M. DPPH radical scavenging activity The ninety-six-well Sugiura of the Department of Citriculture, Okitsu, Nation- microplate was used for the assay of DPPH radical al Institute of Fruit Tree Science, Shizuoka. Other flavo- scavenging assay. Regarding the assay of peel extract of noids of neoeriocitrin, , naringin, hesperidin, and sour citrus fruit (N῎.), the reaction mixture contained neohesperidin (HPLC grade) and reagents were purchased +** mLof*.+- mg/mL DPPH (+,+-diphenyl-,-picrylhydra- from Funakoshi, Ltd., Tokyo, Japan and Wako Pure zyl) dissolved in ethanol, 3* mLof*.+ M TrisῌHCl bu#er Chemical Industries, Ltd., Osaka, Japan, respectively. (pH 1..), ..* mL of test sample for peel extract, and 0.* mL Determination of flavonoids in sour citrus fruit The water. For the assay of niihime fruit elution (N῎.), the content of flavonoids in the juice and peel of four fruits reaction mixture contained +** mLof*.+- mg/mL DPPH each of lemon, lime, yuzu, kabosu, sudachi, daidai, shi- dissolved in ethanol, +0 mLof*./ M TrisῌHCl bu#er (pH ikuwasha, green niihime, and ripe niihime was deter- 1..), and 2..* mL of eluate. After +-h incubation at room mined by HPLC (JASCO Co., Ltd., Tokyo, Japan). The temperature, the absorbance was recorded at /+1 nm juice of sour citrus fruit was obtained by hand squeezing using a plate reader (Sunrise Rainbow, Tecan, Ltd.). after cutting the fruit with a knife. The pulp was re- Results were expressed as percent decrease with respect moved by filtration using gauze. After the flavonoids to control values (Chen and Ho et al., +33/; Kikuzaki et al., content in the juice was analyzed by HPLC, the juice of all ,**,). The control sample contained solvent (ethanol or four sour fruits was mixed. The pH and acidity levels of water of sample solvent) in place of the test sample. the juice were measured using a Metrohm Herisau appa- Ascorbic acid was used as the reference sample. Data are ratus (1+3S, Metrohm-Shibata Co., Ltd., Tokyo, Japan). presented as meanῌSD (N῎.). The peel from the squeezed residue was crushed using a Elution of flavonoids from niihime fruit with solution food processor, and a peel sample (+.* g) was extracted Niihime green fruit (+2..ῌ,*.0 g, +3.0ῌ*.0/ meanῌSD) was with +* mL of 1/῍ ethanol, which has the highest level of cut in half using a knife. All cut fruit was soaked in ,** extraction e$ciency in water-ethanol solutions (Table ,), mL of a cold solution at .῏ and a hot solution of 3*῏, over night at room temperature. The flavonoids con- which consisted of water, /῍ ethanol solution, and ,/῍ tents in the juice and the peel extract (N῎.) were deter- ethanol solution (N῎.). The fruit was left to stand at mined by HPLC using a YMC-Pack ODS column (YMC Co., room temperature and stirred occasionally with a grass Ltd., Kyoto, Japan, column size; f..0῍,/* mm, particle rod. The eluate was taken from + mL of solution at +, -, /, size; / mm) and ,2* nm UV detection. The mobile phase +*, ,*, .*,and0* min. The initial temperatures of the cold consisted of a linear gradient of methanol in distilled solution (.῏) and hot solution (3*῏) were changed to +/῏ water with /῍ acetic acid as follows: +/῍ (*-+* min), +/ῌ and -/ ῏, respectively, after examination for 0* min at /*῍ (+*ῌ-* min), /*ῌ3*῍ (-*ῌ./ min), +**῍ (./ῌ/* min). room temperature (,,῏). The quantity of flavonoids in The column temperature was maintained at .*῏, and the the sampling solutions was determined by HPLC in ac- flow rate was +.* mL/min. The retention time of stand- cordance with the above method, and the elution content ard flavonoids was ,-.* min (eriocitrin), ,... min (neo- from niihime fruit (g) was calculated. The sampling so- eriocitrin), ,1.* min (narirutin), ,2.* min (naringin), ,3., min lutions were assayed using the DPPH radical scavenging (hesperidin), -*.* min (neohesperidin), .-.2 min (nobiletin), assay in accordance with the above method. Data are and ././ min (tangeretin). The identification of flavonoids presented as meanῌSD (N῎.). 188 Y. MIYAKE

Results and Discussion Regarding green and ripe niiheme fruit, eriocitrin, Distribution of Flavonoids in sour citrus fruits The narirutin, naringin, hesperidin, nobiletin, and tangeretin pH level of juice from sour citrus fruits including niihime were detected in the juice and peel; however, no neo- was between ,.. to ,.2, and the acidity level was between eriocitrin or neohesperidin were detected. Niihime fruit -.0 to 0.+ (Table +). Niihime juice from green and ripe was shown to have flavanone glycosides with rutinose fruits was shown to have characteristics including low sugar chain, such as eriocitrin, narirutin, and hesperidin. pH and high acidity, identical to other sour citrus fruits. The content of flavanone glycosides with rutinose sugar Flavonoids are found in a variety of vegetables and fruits. chain was found to be higher than that with neohes- In particular, flavanone glycosides and polymethoxy- peridiose sugar chain in sour citrus fruits, with the excep- flavones are reported to be particularly abundant in tion of daidai. Naringin, neoeriocitrin, and neohesperi- citrus fruit (Miyake et al., +332c; Kawaii et al., +333b). din with neohesperidose sugar chain are reported to taste Flavanones in citrus fruits exist mainly as glycosides bitterer than narirutin, eriocitrin, and hesperidin, which with rutinose sugar chain (0-O-a-L-rhamnosyl-b-D-glu- have rutinose sugar chain (Rouse#, +32*; Miyake et al., cose) such as eriocitrin, narirutin, and hesperidin, and +332c). Daidai juice was shown to taste very bitter be- with neohesperidose sugar chain (,-O-a-L-rhamnosyl-b-D- cause of a high content of neoeriocitrin, naringin, and ) such as neoeriocitrin, naringin, and neohesperi- neohesperidin of flavanone glycoside with neohesperi- din (Fig. +). Polymethoxyflavones, such as nobiletin and dose sugar chain. However, niihime juice was shown to tangeretin, are also particularly abundant in citrus fruits taste less bitter because it contained only a low content of (Fig. +). In this study, the quantity of these flavonoids in naringin, which is a flavanone glycoside with a neohes- the juice and peel of sour citrus fruits, including niihime, peridose sugar chain. In Table +, green niihime (green was determined for the purpose of examining the distri- peel) and ripe niihime (yellow peel) were shown to contain bution characteristics of flavonoids in niihime fruit (Table the bioactive flavonoids hesperidin and eriocitrin. The +). The content of flavonoids in the peel of all sour citrus juice of green niihime had a higher content of eriocitrin, fruits was shown to be higher than that in the juice. narirutin, and hesperidin than that of ripe niihime. The

Fig. +. of flavonoids in sour citrus fruit. Characteristics of Flavonoids in Niihime Fruit 189

Table +. Determination of flavonoids in juice and peel of sour citrus fruit.

Table ,. Flavonoid content in extract of niihime peel extracted by ethanol-water solution.

peel of green niihime contained a higher content of ported to be the highest among several citrus fruits eriocitrin, hesperidin, nobiletin, and tangeretin than ripe (Kawaii et al., +333b). In this study, it was shown that the niihime fruit. In addition, the green niihime fruit was bioactive flavonoids nobiletin and tangeretin contain in found to contain an abundance of flavonoids in compari- the juice and peel of niihime fruit. The content of nobil- son with the ripe niihime fruit. Hesperidin is reported to etin in the peel of green niihime was shown to be higher have antihypertensive activity (Galati et al., +330) and is than that of shiikuwasha. widely distributed in citrus fruits (Kawaii et al., +333b). Extraction of flavonoids from niihime fruit in water- Eriocitrin is reported to exhibit the highest antioxidative ethanol solution The extraction e$ciency of flavonoids activity among all citrus flavonoids and is especially ab- from niihime peel (ripe fruit) was examined as shown in undant in lemon and lime (Miyake et al., +332c). Eri- Table ,. In this study, water and ethanol were used for ocitrin has also been identified in sudachi fruit (Kawaii et extraction solutions, as they are common solutions in the al., +333b). In the present study, eriocitrin was shown to field of food processing and cooking. Solutions of water, also be found in niihime fruit, although the content in /ῌ ethanol, ,/ῌ ethanol, /*ῌ ethanol, 1/ῌ ethanol, and niihime fruit was lower than that in lemon fruit. Poly- +**ῌ ethanol were used for the extraction of flavonoids methoxyflavones, nobiletin and tangeretin, were shown from niihime peel. The content of flavonoids in water to be more abundant in shiikuwasha and niihime fruits and low-content ethanol solutions was generally lower than in other sour citrus fruits (Table +). The content of than in high-content ethanol solutions. The extraction nobiletin and tangeretin in shiikuwasha fruit was re- e$ciency of eriocitrin, narirutin, hesperidin, nobiletin, and 190 Y. MIYAKE

Fig. ,. Antioxidative activity of peel extract of sour citrus fruits. The DPPH radical scavenging activity of the peel extract of sour citrus fruits was measured.

tangeretin was to shown to be highest with 1/ῌ ethanol citrus fruits, including niihime fruit. The peel extract of solution. That of naringin was highest with /*ῌ ethanol sour citrus fruit was examined for antioxidative activity solution but was at 31ῌ with 1/ῌ ethanol solution. using DPPH radical scavenging (Fig. ,). Niihime ex- Based on these results, 1/ῌ ethanol solution was shown hibited high antioxidative activity in the assay of sour to be a suitable extraction solution using water and etha- citrus fruits. Among citrus flavonoids, eriocitrin and nol. A 1/ῌ ethanol solution was used for the extraction neoeriocitrin are reported to exhibit high antioxidative solution in the determination of flavonoids in sour citrus activity because of their structure containing a catechol peel in Table +. In Table ,, the flavanone glycosides group at the -῍ and .῍ position, which has radical-scav- eriocitrin, narirutin, naringin, and hesperidin were eluted enging ability (Miyake et al., +331b). The antioxidant in to some extent in water or low-content ethanol solutions, the peel extract of lemon is reported to have been isolated and their contents in +**ῌ ethanol were lower than that as eriocitrin (Miyake, et al., +331a). Furthermore, we in /*ῌ and 1/ῌ ethanol solutions. However, the content attempted to fractionate the antioxidative substances of polymethoxyflavones, nobiletin and tangeretin, was from the peel extract of daidai, and one was identified as very low in water and was high in 1/ῌ and +**ῌ ethanol neoeriocitrin (data not shown). Eriocitrin and neo- solutions. The di#erence in the elution properties of eriocitrin were identified in the lemon, daidai, lime, flavonoids was suggested to be related to the hydrophobic sudachi, and niihime (Table +). The antioxidative activi- nature of flavonoids. ty of these fruits was suggested to be related to the Antioxidative activity of sour citrus fruits Aging and presence of eriocitrin and neoeriocitrin being antioxida- carcinogenesis is suspected to be strongly associated with tive flavonoids. The green niihime was suggested to excess oxidative stress in the body. Antioxidants in exhibit a higher activity than ripe niihime because of its food, which have been used for the prevention of lipid high content of eriocitrin in comparison with ripe nii- peroxidation in food storage, have been shown to have a hime. However, the high antioxidative activity of shi- suppressive e#ect on oxidative stress in vivo, and are ikuwasha could not be explained by these flavonoids. thought to play a role in the prevention of atherosclerosis Shiikuwasha fruit was suggested to contain antioxidants and complications from diabetes (Yagi, +321; Osawa et al., other than these, which will require further research. +33*). Citrus flavonoid is also thought to play a role in Elution property flavonoids from niihime fruit to solu- the prevention of disease because it has been reported to tion Niihime fruit was shown to contain bioactive fla- have antioxidative activity and a suppressive e#ect on vonoids such as eriocitrin, hesperidin, nobiletin, and oxidative stress in vivo (Middleton and Kandaswami, +33.; tangeretin (Table +). In this study, the elution properties Miyake et al., +332b). Based on this background, the of these flavonoids from niihime fruit were investigated present study evaluated the antioxidative activity of sour using hot and cold solutions of water, /ῌ ethanol, and Characteristics of Flavonoids in Niihime Fruit 191

Fig. -. Elution content of flavanone glycosides from nii- Fig. .. Elution content of polymethoxyflavones from nii- hime fruit in hot and cold solutions of water and ethanol. hime fruit in hot and cold solutions of water and ethanol. (A) is elution content of eriocitrin from niihime fruit. (B) (A) is elution content of nobiletin from niihime fruit. (B) is is elution content of hesperidin from niihime fruit. elution content of tangeretin from niihime fruit.

,/ῌ ethanol solutions, which are commonly used in food peridin. The contents of eriocitrin and hesperidin were processing and cooking. In Table ,, the extraction e$- higher in hot solutions than in cold solutions (Fig. -). ciency of 1/ῌ ethanol solution was the highest in water- The content of flavonoids eluted in / min was an approx- ethanol solutions; however, this solution is seldom used imate half of content eluted in 0* min. The dissolution in food processing or cooking because of the high ethanol property of purified eriocitrin to solution is di#erent from content. /ῌ and ,/ῌ ethanol solutions are used com- that of purified hesperidin showing that eriocitrin dis- monly because these solutions are available in commer- solves easily in water while hesperidin does not dissolve cial products, such as alcoholic beverages. Based on this easily in water. However, the changes over time in background, the water, /ῌ ethanol, and ,/ῌ ethanol eriocitrin contents eluted from niihime fruit were shown solutions were used for determining elution properties in to be similar to that of hesperidin. Based on the present this study. Moreover, this study attempted to gain basic results, hot ,/ῌ ethanol solution is suitable for eluting data for the application of the bioactive flavonoids in the bioactive flavanone glycosides eriocitrin and hes- niihime fruit to food science. Niihime fruits were cut peridin from niihime fruit. Eriocitrin and hesperidin using a knife and the cut fruit was soaked in hot or cold were shown to be eluted to some extent from niihime fruit solutions of water and ethanol for up to 0* min. The in a hot solution of water and /ῌ ethanol. content of flavonoids in the eluate was determined by As for the polymethoxyflavones nobiletin and tangere- HPLC (Fig. -, Fig. .). In comparison with other solutions, tin, the changes over time in the contents of nobiletin hot ,/ῌ ethanol solution exhibited the highest elution eluted from niihime fruit were similar to that of tan- content of the flavanone glycosides eriocitrin and hes- geretin (Fig. .). The eluted contents of nobiletin and 192 Y. MIYAKE

xidative activity in comparison with other sour citrus fruits. These bioactive flavonoids in niihime fruit were eluted e#ectively from niihime fruit in hot solution, and hot ,/ῌ ethanol was shown to be the preferable solution for the elution of nobiletin and tangeretin. These results identified the characteristics of the flavonoids in niihime and demonstrate their possible use and e#ective applica- tion in food processing and cooking.

Acknowledgements I would like to thank Mr. T. Maegawa and Mr. H. Ichinokiyaka of the Mie Prefectural Science and Technol- ogy Promotion Center, Agricultural Research Division, for the gift of niihime and daidai fruits. I would also like to thank Dr. M. Yano and Dr. M. Sugiura of the Department of Citriculture, Okitsu, National Institute of Fruit Tree Science, Shizuoka for the gift of the nobiletin and tangeretin. I am also grateful to Mr. M. Hiramitsu of Pokka Corporation, Ltd for the experimental Fig. /. DPPH radical scavenging activity of eluates of nii- advice. hime fruit. References Attaway, J.A. (+33.) Citrus Juice Flavonoids with Anticarcino- tangeretin were highest in hot ,/ῌ ethanol solution. genic and Antitumor Properties, in Food phytochemicals for cancer prevention. I Fruits and vegetables (Huang, M.T., The content of flavonoids eluted in / min for hot ethanol Osawa, T., Ho, C.T., Rosen, R.T., eds.) pp. ,.*ῌ,.2, ACS Wash- 0* solutions was an approximate half of content eluted in ington, D.C. min. The flavonoids were eluted to some extent in hot Benavente-Garcia, O., Castillo, J., Marin, F.R., Ortuno, A. and Del solutions of /ῌ ethanol, but only to a very low content in Rio, J.A. (+331) Uses and properties of citrus flavonoids. J. other solutions. The elution of nobiletin and tangeretin Agric. Food Chem., ./, ./*/ῌ./+/. was shown to require a hot ethanol solution with a con- Chen, C.-W. and Ho, C.-T. (+33/) Antioxidant properties of poly- , centration of more than /ῌ. Nobiletin and tangeretin phenols extracted from green and black tea. J. Food Lipids, , -/ῌ.0. are di$cult to dissolve in water and ethanol solution of Emmons, C.L, Peterson, D.M. and Paul, G.L. (+333) Antioxidant low concentration in comparison with eriocitrin and capacity of oat (Avena sativa L.) extracts. ,. In vitro antioxida- hesperidin because of their strong hydrophobic property. tive activity and content of phenolic and tocol antioxidants. J. The strong hydrophobic property of nobiletin and Agric. Food Chem., .1, .23.ῌ.232. tangeretin was suggested to a#ect the elution property; Higashimoto, M., Yamato, H., Kinouchi, T. and Ohnishi, Y. (+332) therefore, they are eluted from niihime fruit to some Inhibitory e#ects of citrus fruits on the mutagenicity of +- + , - . - extent in hot solutions of /ῌ ethanol, but are eluted to the methyl- , , , -tetrahydro-beta-carboline- -carboxylic acid treat- ed with nitrite in the presence of ethanol. Mutat Res. .+/, ,+3ῌ highest content in hot ,/ῌ ethanol. In food processing ,,0. and cooking for niihime fruit, hot solution, preferably hot Galati, E.M., Trovato, A., Kirjavainen, S., Forestieri, A.M., Ros- ,/ῌ ethanol was identified as a suitable solution for sitto, A. and Monforte, M.T. (+330) Biological e#ects of hes- eluting the bioactive polymethoxyflavones nobiletin and peridin, a citrus flavonoid. (Note III): antihypertensive and tangeretin. diuretic activity in rat. Farmaco., /+, ,+3ῌ,,+. The antioxidative activity of eluates, which were eluted Kikuzaki, H., Hisamoto, M., Hirose, K., Akiyama, K. and Tani- ,**, in hot and cold solutions of water and ethanol, were guchi, H. ( ) Antioxidant properties of ferulic acid and its related compounds. J. Agric. Food Chem., /*, ,+0+ῌ,+02. examined for DPPH radical scavenging activity (Fig. /). Kawaii, S., Tomono, Y., Katase, E., Ogawa, K. and Yano, M. (+333 The antioxidative activity of the eluates in hot solution a) Antiproliferative activity of flavonoids on several cancer was higher than in cold solution. The increasing change cell lines. Biosci Biotechnol Biochem., 0-, 230ῌ233. of antioxidative activity observed until 0* min (Fig. /) Kawaii, S., Tomono, Y., Katase, E., Ogawa, K. and Yano, M. (+333 was similar to that of the eluted content of eriocitrin and b) Quantitation of flavonoid constituents in citrus fruits. J. hesperidin (Fig. -). Eriocitrin has been reported to have Agric Food Chem., .1, -/0/ῌ-/1+. +33. the highest antioxidative activity among citrus flavon- Middleton, E. and Kandaswami, C. ( ). Potential Health- Promoting Properties of Citrus Flavonoids. Food Tecnol., .2, oids (Miyake et al., +331b; +332c). Therefore, the anti- ++/ῌ++3. oxidative activity of the eluate was suggested to be relat- Minato, K., Miyake, Y., Fukumoto, S., Yamamoto, K., Kato, Y., ed to the elution content of eriocitrin in the solution. Hot Shimomura, Y. and Osawa, T. (,**-) Lemon flavonoid, eri- solutions, such as water, /ῌ ethanol, and ,/ῌ ethanol, octrin, suppresses exercise-induced oxidative damage in rat were suggested to be the most suitable solutions for liver. Life Sciences, 1,, +0*3ῌ+0+0. eluting antioxidative flavonoids from niihime. Miyake, Y., Yamamoto, K. and Osawa, T. (+331a) Isolation of 1 In the present study, the sour citrus fruit niihime was eriocitrin (eriodictyol -rutinoside) from lemon fruit (Citrus limon BURM. f.) and its antioxidative activity. Food Sci. shown, for the first time, to contain an abundance of Technol. Int. Tokyo, -, 2.ῌ23. bioactive flavonoids, such as eriocitrin, hesperidin, nobile- Miyake, Y., Yamamoto, K., Morimitsu, Y. and Osawa, T. (+331b) tin, and tangeretin. Niihime fruit exhibited high antio- Characteristics of Flavonoids in Niihime Fruit 193

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