In Vitro Effect of Yuza (Citrus junos SIEB ex TANAKA) Extracts on Proliferation of Human Prostate Cancer Cells and Antioxidant Activity

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 2, pp. 339~344 (2004). ©The Korean Society of Food Science and Technology. In vitro çöB F¶(Citrus junos SIEB ex TANAKA) Ï. ºÂb~ z^

(2) ÛBÎ 5 ÖzW FãÁã* *ÞL ®'·. In Vitro Effect of Yuza (Citrus junos SIEB ex TANAKA) Extracts on Proliferation of Human Prostate Cancer Cells and Antioxidant Activity Kyung-Mi Yoo and In-Kyeong Hwang* Department of Food and Nutrition, College of Human Ecology, Seoul National University Effects of 18 kinds of yuza extracts on viability of prostate cancer cells, DU 145 and LN-CaP, were investigated. Chloroform and methanolic extracts of yuza peel exhibited moderate cytotoxicity against both cancer cell lines dose-dependently and also showed antioxidant activity matching on inhibition of cell viability (author’s intension not clear). Chloroform extract of yuza peel exhibited highest radical-scavenging activity and cytotoxicity against prostate cancer cells in vitro. Key words: Citrus junos SIEB ex TANAKA, polyphenol, prostate cancer cell, antioxidant activity, cytotoxicity. B. . Ò ß® æO ô FB Òf þ z 5 BOzB W ^z¢ "º öb ~b " Ã&º& /Ï Ã &~º î÷¢ ~& (9). ê BzB " Ã &>º ÊZÞ® b *ãFzö ~ Ò Ã& > ® . ¾ òWî~b Îæº zf z~ ê¯ ô ê¿>Ú¢ò r > ®º zb * B ê ~ò ô f Û FB >º î÷ 7~ ~¾ . V¢B *ãFzf ~ò .O z 7º~ > ® . F¶(Citrus junos SIEB ex TANAKA)º ¢, ÖÒ¾¢öB ò ÒV> ®º "¢ . ¢öBº yuzu, $º sumikan ¢º ªb çë'b Ï> ® "bf "Gj &æ ®Ò 5 Ï», ÏÏ®ö Î&~ ® (10-12). ß ® F¶~ ëß ËV r^ö j Î&~º r Ò . $º .Ò z æ~² ÒÏ> ® . ÖÒ¾¢öBê .¦V F¶º ³¦ ËV 5 j æf æ~² ÒÏ>Ú z b 6V ~òö ±f "¢ ;>Ú z . F¶º " F ¶Ób ò Ú F¶N î¾ $º *Û Ç, ÷"ö Î &~ ® . ¾ " ô Ö>º F¶º 70% ç æ F¶Ó~ öòò ÒÏ>Ú · F¶ Ï» n s>Ú¢ 6ö f ® . "öº F¶ Ê~ Ïb F¶ ÒÏ £* Ã&> ®b¾ *® 6& Ï ª~æ pj F¶ ³&ö ÚJæj " ® . z× F¶ö & º ¢>Wª" F¶ Ê O¿O» ö & (14-16)& " Ú^z . ®&GöB " >Ú z~ F¶ö & Ò Wö & ¢ Þ &® ~ ; VËWj C® Ëê ¦&&~ VËW ®öò ÏF > ®j © . $ " 6 ~~ VËW. òW ÊÞ.Ê~ Ã&, ' ~ãJ"" ^z~ ÊZÞz *& f &æ òW ¯W î~~ B * þ Ã&> ® . zj j .{, Ë÷ òW ¯W î÷f Wê" ¾¢ ®^~ Bº& Ã&> ® . î~~ .O Ï'b " well-being~ j º ~º ãËb ÒWj ô F ®ö & & Ã& >îb ö & & ê¯> ® . ß® ôf Ò öB "¢ S~ Ã&º Ë&N î~" &æ z, òãï î~" ?f ¯W î~~ B" >jf' &ê¢ &æ ®º ©b >î (1-3). "¢ î÷j ÛB ~º F 7~ ~¾º &æ '·Wª7~ ~¾ vitamin Cf E& ®V r^ ©b rJ^ zb¾ "~ ¢ ^öB Öz j ~º ©f jæ"ö jî¢ "¢ö ô Ú®º polyphenol, ß® flavonoids ¢º © C&æ ® (4-8). Â ®f · VËWö 'Ë j "º Ö> '·Wªj F~ ®Ú ~£® ²Ò 5 V ËW ®b &ËWj ;A ®b ¦&&~ ®b ÏF > ® . ~ Bö ~~ *ãFzf Î ¶ z Ò B*öB 3*¢ Næ~º zb G *"~ *Corresponding author : In-Kyeong Hwang, Department of Food and Nutrition, College of Human Ecology, Seoul National University, San 56-1, Shillim-dong, Kwanak-gu, Seoul 151-742, Korea Tel: 82-2-880-6837 Fax: 82-2-884-0305 E-mail: [email protected] 339.

(3) ®"²æ B 36 ² B 2 ^ (2004). 340. & B® ê¯>Ú "¢ 7~ ß; '·Wª z^ Û B"; ö 'Ëj º Ö"(17-19)& ¾J ®b ¾ F¶ö & VËW º Ö j ç . V¢B öBº ÖÒ¾¢f ¢öB " Ö>º ãçK ®º "¢ F¶ö & ÒW ® ²ÒB~ & ËWj ¦Æ~V * &æ Ï ºÂb~ Öz W j ÚÚ *ãF z^~ ^ÒÎ"ö & &ËWj B~¶ ~& .. ~ 10.* b~, çNöB 10ª* O~ ê 517 nmöB 7ê¢ G;~& . &f THF 0.2 mLö DPPHÏ 0.8 mL¢ & ê çNöB 10ª* O~ ê 517 nmöB 7ê ¢ G; ©b ~& .. Òò 5 O». ^

(4) ëW /; DU 145, LN-CaP~ *ãFz ^º ^" f¯ (KCLB)öB ª· Aj 10% fetal bovine serum(FBS: Sigma Chemical Co., St. Louis, MO, USA)f 1% penicillin 5 streptomycin(Sigma Chemical Co., St. Louis, MO, USA) B RPMI-1640(GIBCOTM, Invitrogen, CA, USA)j Ï~ V· ~& . F¶ ºÂb~ ^ëW þf Nagao (21)~ O»ö V¢ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT: Sigma Chemical Co., St. Louis, MO, USA) £j Ò Ï~ ~& . ¯ 5Ü104 cell/well ³ê 96 well plateö ª" ê 37oC, 5% CO2 incubatoröB 72* V· ê, V· ö ÒÏB Væ¢ B~ îÚ Væ 180 µLf F¶ ºÂ b 20 µL¢ Î&~& . Î& F¶ ºÂb~ ³êº '' 0, 2.5, 12.5, 25.0 µg/mLî . 24* V· ê Væ¢ B ~ 0.5 mg/mL ³ê~ MTT£j well 200 µLO ª". r 4* ê ELISA microplate reader(Bio-rad, Benchmark, USA)¢ Ï~ 595 nmöB 7ê¢ G;~& .. ò þö ÒÏB F¶(Citrus junos SIEB ex TANAKA)º *¢Îê jê æöB 2001j 10¢ 15¢, 11ú 15¢, 12ú 15¢ö «~ ÒÏ~& . F¶º ?W ;êö V¢ 10ú 15¢ Öbj ?F¶(immature yuza), 11ú 15¢ Öbj F¶(intermediate yuza), 12ú 15¢ Öbj "? F¶ (mature yuza) ª~&b ''f "b, "G" N ª ~& . F¶~ "bf "Gf ÿÖV(freeze dryer, FD 550g Il-Sin Engineering, Seoul, Korea) ÿÖ Î ê ªêV (FM-700W food mixer, Han Il, Korea) ªê~ −20oCö & ~ þ~& . b, G Ï ºÂ 5 >N êÖ >{Vö V F¶ "G, "b ªöf '' Ö, ª, zêR Bb Wj Ò~B ºÂ~& . ¯, F¶ 50 gö Ö 50 mLj I 4oCöB 30ª* N >(Bio Shaker, BR-30LF, TAITEC, Japan)öB êû ê Ö Ïj ²>~& . Îf ºÒö ª 50 mLj IÚ ?f O»b ºÂj ê ²>~ Îf ºÒö zêR 50 mLj IÚ ?f O»b BN'b ºÂ~& . ''~ Ï f ³»V(Büchi, 461, Germany) 6{~B ³», ~ Z²¢ G;~&b tetrahydrofuran(THF)Ï 3 mLö IÚ −20oCö & ~B ÒÏ~& . BB Ï ºÂ òº F ¶ê 3«~, "b, "G, ºÂÏ 3«~ æ C 18&æ& .. DPPH radical scavenging activity (%) = sample absorbance (1− ----------------------------------------------------- )Ü100 control absorbance. Photomicrography ^~ æ; ;ê¢ ÚÚV * ~ O»ö V¢ DU 145 *ãF z^ö & ^ ëW þj ~& . j? F¶ ª ºÂb ³êº 0, 12.5, 25 µg/mL ºÂbj Î& ê 24*ÿn V·~B þ" &~ ^ Î · æz¢ *ãb &V~ êã*ã(CK 2, Olympus, Japan)j Ï~&b Vö ËOB z¢(Olympus, Japan) ¢ Ï~ 100VN Òêj 3Ú jv ~& .. C ¾¦ ï C ¾¦ïf Folin ciocalteu»(6)j ¢¦ æ;~ G;~ & . CB 1 mL~ ò¾ &bî(chlorogenic acid: Sigma Chemical Co., St. Louis, MO, USA)j THFÏ 1 mLö Ã~> 9 mLj If ê v> ~ 3ª ê Folin ciocalteu phenol reagent(Sigma Chemical Co., St. Louis, MO, USA) 1 mL j Î&~ v>~& . 5ª ê, 7% Na2CO3 Ï 10 mLj & ~ v> ~ Ã~> 25 mL C ê 23oCöB 90ª ÿ n ;~8 . ;~ ê ª77êê(DU 530 spectrophotometer, Beckman, 4300N, Fullerton, USA)¢ Ï~ 750 nmöB 7ê¢ G;~& . C ¾¦ïf chlorogenic acid(Sigma Chemical Co., St. Louis, MO, USA) & ¦ïFj ·W~ êÖ~&b 100 g Û7ïö & mg chlorogenic acid (CAE)b ¾æÚî .. Ûê ªC Î þÖ"º SAS package programj Ï~ ANOVA ¾Ò~&b F~W ¦;f F~ >& p < 0.05, p < 0.01öB Duncan’s multiple range test¢ Ï~ ªC~& .. 1,1-Diphenyl-2-picrylhydrazy(DPPH) ¶F8 ²Ë /; Chu (17)~ O»ö V¢ F¶ Ï ºÂb~ ³êê C Ï 0.2 mLö 4Ü10−4 M 1,1-diphenyl-2-picrylhydrazy(DPPH: Sigma Chemical Co., St. Louis, MO, USA)Ï 0.8 mL¢ &. C¾¦ ï DPPH ¶F8 ²Ë /; >{Vö V¢ F¶~ "b 5 "G ºÂb ÖzWö N& ®ºæ rjV *~ C¾¦ ï 5 ¶FV ² Wj ªC~& (Table 1). Vê ÚÚ W? F¶ö. Ö 5 8 F¶¦*ê, Ï ê ºÂb >N W? F¶, W?F¶, "W? F¶~ "b 5 "Gj Ö, ª, zêR ºÂ~ áf ºÂb ïf Table 1" ? ?W;êö V ºÂ >Nj ÚÚ j? F¶öB º Â >N &Ë ¸~ . FV Ï ê ºÂ >Nj ÚÚ "b, "G Îv zêR ºÂb~ >N FV Ï ºÂ bö j~ z ¸f ãËj & ..

(5) F¶ ºÂb~ z^ ÛBÎ" 5 ÖzW. 341. Table 1. Recovered weight, the total phenolics contents, and scavenging activity of DPPH radicals of yuza peel extracts Peel extract. Weight recovered (mg/mL). Total phenolics2). Scavenging activity3) (%). Immature. Hexane Chloroform MeOH. 5.98 11.33 20.44. 50.2c 82.1b 90.2b. 60.6b 79.7a 46.7c. Intermediate. Hexane Chloroform MeOH. 6.01 4.36 16.2. 65.5c 92.5a 99.2a. 71.5a 79.8a 66.1b. Mature. Hexane Chloroform MeOH. 10.44 11.96 40.28. 66.5c 116.5a 112.9a. 65.5b 83.4a 76.1a. Maturity1). 1). All mean values are triplicate determinations. Values in the same column (total phenolics and scavenging activity) that are followed by a different letter are significantly different (p<0.01) by Duncan’s multiple range test. 1)Immature green, intermediate mature yellow, and mature deep-yellow yuzas were harvested at Wando on October 15, November 15, and December 15, respectively. 2)Total phenolics content, expressed in milligrams of chlorogenic acid equivalents per 100 g of fresh fruits. 3)Means of DPPH radical scavenging activity on 1 mg/mL of each extract.. Fig. 1. Effects of yuza peel extracts on the viability of DU 145 human prostate cancer cells. DU 145 cells were seeded in 96-well plates and cultured for 72 hr, and then treated with various yuza extracts. After culturing for 72 hr, cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay, and was expressed as the percentage of the value obtained in the control culture treated with the vehicle alone THF(tetrahydrofuran). **p<0.01, *p<0.05.. B W? F¶& F > C¾¦ ï Ã& >º ãËj & . "W? F¶ "b~ C¾¦ ïf W? F¶ "bö j~ 70 mg% ;ê ¸~ . ¾ "W? F¶ "G~ C¾ ¦ ïf W? F¶ "Gö j 50 mg% ;ê ¸~ . " bf "G~ ^ &æ Ï ºÂb Îv "¢ W?> C ¾¦ ï Ã&~º ãËj & . ¶FV ² Wêº ^ Ï ºÂb Îv "W? F¶öB &Ë ¸² ¾æ¾ "W ? F¶~ ÖzW ¸f ©b . ¾ C¾¦ ï ¸j> DPPH ¶FV ²Wê ¢'b ¸² ¾ æ¾æ p~ . W? F¶ "b~ ãÖ ª ºÂb~. Fig. 2. Effects of yuza flesh extracts on the viability of DU 145 human prostate cancer cells. DU 145 cells were seeded in 96-well plates and cultured for 72 hr, and then treated with various yuza extracts. After culturing for 72 hr, cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay, and was expressed as the percentage of the value obtained in the control culture treated with the vehicle alone THF(tetrahydrofuran). **p<0.01, *p<0.05.. C¾¦ ïf zêR ºÂb Ôæò DPPH ¶FV ² Wf z ¸f ©b ¾æÒ . "W? "G~ ãÖ ª ºÂb~ C¾¦ ïf 45.1 mg% DPPH ¶FV ² Wf 67.3 mg%¢ ¾æîb¾ zêR ºÂb~ C¾¦ ïf ª ºÂb~ 2V ;ê >º 89.4 mg% DPPH ¶FV ² Wf £* ¸f 72.5%¢ ¾æî . V¢B DPPH ¶FV ² W zêRöB ô ºÂ>º W bî ªöB ºÂ>º jW bî ©b ÒòB . ¯, F¶& F~º ÖzßWf WÏ öB ºÂ>º W Wª" þ jW Ï öB ºÂ>º $. jW bî þ z ² ·Ï ©b Îê ..

(6) ®"²æ B 36 ² B 2 ^ (2004). 342. Fig. 3. Effects of yuza peel extracts on the viability of LN-CaP human prostate cancer cells. LN-CaP human prostate normal cells were seeded in 96-well plates and cultured for 72 hr, and then treated with various yuza extracts. After culturing for 72 hr, cell viability was evaluated by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and was expressed as the percentage of the value obtained in the control culture treated with the vehicle alone THF (tetrahydrofuran). **p<0.01, *p<0.05.. Fig. 4. Effects of yuza flesh extracts on the viability of LN-CaP human prostate cancer cells. LN-CaP human prostate normal cells were seeded in 96-well plates and cultured for 72 hr, and then treated with various yuza extracts. After culturing for 72 hr, cell viability was evaluated by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and was expressed as the percentage of the value obtained in the control culture treated with the vehicle alone THF(tetrahydrofuran). **p<0.01, *p<0.05.. z^

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(8) Ò Î F¶ "b, "G" >{Vö V 3&æ Ï ºÂb~ * ãF z^ ÛB Î"¢ ÚÚV *~ DU 145, LN-CaP~ *ãF z^ö & ^ Ò þj ~& (Fig. 1, 2, 3, 4). þö ÒÏ DU 145, LN-CaP ^º ?f *ãF z ^æò ^ nöB~ ÿW DU 145º androgenö j ~' LN-CaPº androgenö ~' . androgen ^Òf Î¶~ ÎWWj &~º ^Òb ^Ò~ "ï ªjº *ãF z^~ WËj ûº (9). V ¢B 2&æ ^ö ºÂbj ¾ÒB ^ ëWj ÚÚ*b B ^Ò ~' ";" j ~' "; 7 Ú¶ ";. j z Î"'b ÛB ~ºæ r > ®î . F¶ Ï ºÂb 7 ª Ï ºÂbf v &æ z^ö ÛB Î"& Îv ±f ©b ¾æÒ (Fig. 1, 2). ¯, "b, "G ª ºÂb Îv DU-145, LN-CaP ^öB &ö j~ 50%;ê~ ¸f ÛBNj ¾æî . $ "W? F¶ö ®Ú B ªÏ ºÂb" zêR ºÂbf ^Ò ~' *ãF z^f -æ pf z^ Îv ³ê ~' ÛBNj ¾æÚî . ¾ W? F¶ ºÂb v ^" Îv ³êö V¢ 60-90%~ ^ Nj ¾æÚ ^ Ò Î"& Ô~ . æ ^ ëW ãËWf W?öB W? F¶& F > W Ã&>îb ãËf "b, ". Table 2. Recovered weight, the total phenolics contents and scavenging activity of DPPH radicals of yuza flesh extracts Flesh extract. Weight recovered (mg/mL). Total phenolics2). Scavenging activity3) (%). Immature. Hexane Chloroform MeOH. 3.46 5.80 19.160. 45.2b 34.3c 60.8b. 58.9c 63.2b 62.9b. Intermediate. Hexane Chloroform MeOH. 23.740 5.61 27.40. 56.9b 40.1c 69.7a. 59.0c 65.6 b 63.7b. Mature. Hexane Chloroform MeOH. 7.88 7.77 36.050. 60.1b 45.1b 89.4a. 73.1a 67.3a 72.5a. Maturity1). 1). All mean values are triplicate determinations. Values in the same column (total phenolics and scavenging activity) that are followed by a different letter are significantly different (p<0.01) by Duncan's multiple range test. 1)Immature green, intermediate mature yellow, and mature deep-yellow yuzas were harvested at Wando on October 15, November 15, and December 15, respectively. 2)Total phenolics content, expressed in milligrams of chlorogenic acid equivalents per 100 g of fresh fruits. 3)Means of DPPH radical scavenging activity on 1 mg/mL of each extract..

(9) F¶ ºÂb~ z^ ÛBÎ" 5 ÖzW. 343. "'b z^¢ ÛB ® . $ Kawaii (19)" Johnson (20)f "¢ö FB >ÏW VËW bî ¯, flavonoidf epigallocatechin gallate _f quercetinö ~ z^ ÛB Î"¢ in vitroöB « : ® . ¢^ öB ÚÚ ©" ? F¶~ ^ÒÎ"ê F¶& F æÏ W, >ÏW bî þ ·Ï~ Úæº ©b Òò> ß® "b¢ ªÒæ p S > ®º F¶º "¢ö j ãçK ®º "¢ F © . ¾ F¶~ *ãF z ^ ÛB Î"¢ ¦ z { ® ~V *~ ^ç~ z î¾ « { ~ & z B® Ú^¢ .. º. £. F¶º W?> "bf "G~ C¾¦ ïf ôjæ ¶FV ²Ë ¸jæº ãËj & . F¶¦V ºÂ 18&æ Ï ºÂb~ Öz W" *ãF z^ DU 145, LN-CaPö & ^ëW þj Ö" "bf "G Î v W? F¶ W? ê >{ F¶& W ¸ ² ¾æî . Ï ê ÚÚ "W? F¶ "b, "G~ ª, zêR ºÂbf *ãF z^ DU 145ö &~ 60% ç~ ^Ò Î"¢ ¾æÒb zêR ºÂb~ Î "ê ³êö V¢ ç® ¸f ©b ¾æÒ . $ ^ çj *ãb ÚÚ Ö" ºÂb~ ¾Ò ³ê& Ã&> ^ Ò £² Úæº ©j { > ®î . ^ Ò~ ãËf ºÂb~ ¶FV ² W ¸j> ¸² ¾æÒ .. 6Ò~ Fig. 5. Photomicrograph of DU 145 cell treated which 24 hr cytotoxicity of addition with different concentrations of chloroform extract of mature yuza peel. (a) Control, (b) Treatment of 12.5 µg/mL chloroform extracts of mature yuza peel, (c) Treatment of 25 µg/mL chloroform extracts of mature yuza peel.. G, Ï ê Îv jÝ ãËj ¾æî . Ö" " r F¶~ Wª 7 ªö ¾ º bî" zêRö ¾ º bî ^ëWj z ÛB > ® . $ Table 2, 3ö ¾æÂ Ö"f ? ¶FV ²W ¸f "W? F ¶ ºÂb ^Ò Î"ê ¸bæ ¶FV ²W bî ^ ÒÎ"ö ±f Î"¢ ¾æÚº ©b ÒòB . z^

(10) ~ *ã 8 *ãF z^ DU 145~ ^ æz¢ ÚÚV *~ * ã&Vj ~& (Fig. 5). '' 0, 12.5, 25 µg/mL ¾Ò ê 24* Êö ¾æÂ Ö"¢ ÚÚ ¾Ò ³ê& ¸j î> ç~ f>º ;ê& æ ^>& &ö j ç® 6² ©j " > ®Ú ^ëWö ~ ^ Ò ";j Ð ® ÒòB . MTT £j Ï ^ë W þ" ? ºÂb~ ¾Ò ³ê& Ã& > ^ ç ô vâò æ apoptotic bodies& ;W>º ©j Ú j ©f ^ cc*ç ¢Ú¾ Ò>º necrosis"; ¢V º apoptosis Ò ";j Ð ®º ©b ÒòB. . Nara (21)ö ~~ ®7~ carotenoidsº *ãFz^ PC-3j Ïª® ÛB~ β-carotenef 20 µM/mL ³êöB Î. ¢^f 2003jê ³âVF&ÒbV(ARPC)~ æöö ~ ê¯>îb ö 6Òãî .. ^. ò. 1. Ames BM, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. 90: 7915-7922 (1993) 2. Willet CW. Diet and health: What should we eat? Science 264: 532-537 (1994) 3. Wang H, Cao G, Prior RL. Total antioxidant capacity of fruits. J. Agric. Food Chem. 44: 701-705 (1996) 4. Dabrowska-Ufniarz E, Dzieniszewski J, Jarosz M, Wartanowicz M. Vitamin C concentration in gastric juice in patients with precancerous lesions of the stomach and gastric cancer. Med. Sci. Monit. 8: 96-103 (2002) 5. Drake IM, Davies MJ, Mapstone NP, Dixon MF, Schorah CJ, White KL, Chalmers DM, Axon AT. Ascorbic acid may protect against human gastric cancer by scavenging mucosal oxygen radicals. Carcinogenesis 17: 559-562 (1996) 6. Lee KW, Lee HJ, Kang KS, Lee CY. Preventive effects of vitamin C on carcinogenesis. Lancet 359:172-176 (2002) 7. Vinson JA, Su X, Zubik, Bose P. Phenol antioxidant quantity and quality in food: Fruits. J. Agric. Food Chem. 49: 5315-5321 (2001) 8. Hertog MG, Feskeens JM, Hollman CH, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease. Lancet 342: 1007-1011 (1993) 9. Jane M. Dairy food, dairy fat, and cancer: A review of epidemiological evidence. Nutr. Res. 18: 905-937 (1998) 10. Nakanishi M, Hisatake M. Studies on the using of yuza (Citrus junos) juice in food industry field. New Food Ind. 17: 39-45.

(11) 344. ®"²æ B 36 ² B 2 ^ (2004). (1988) 11. Yamasaki Y, Hisatake M, Nakanishi M. Long-term frozen storage of yuza (Citrus junos) juice produced in business container. Kochi Food Ind. 20: 34-45 (1989) 12. Yamasaki Y, Hisatake M. On the quality of yuza (citrus junos) juice produced in kochi prefecture. Kochi Food Ind. 2: 44-51 (1990) 13. Lee HY, Kim YM, Shin DH, Sun BK. Aroma Components in Korean citron (Citrus medica). Korean J. Food Sci. Technol. 19: 361-365 (1987) 14. Lee YC, Kim IH, Jeoung JW, Kim HK, Park MH. Chemical characteristics of citron (Citrus junos) juice. Korean J. Food Sci. Technol. 26: 552-556 (1994) 15. Jeoung JW, Kwon DJ, Hwang JB, Jo YJ. Influence of the extraction method on quality of citron juice. Korean J. Food Sci. Technol. 26: 704-708 (1994) 16. Jeoung JW, Lee YC, Kim JH, Kim OW, Nahmgung B. Cooling properties and quality changes during storage of citron (Citrus. junos). Korean J. Food Sci. Technol. 28: 1071-1077 (1996) 17. Chu YH, Chan CL, Hsu HF. Flavonid content of several vegetables and their antioxidant mushrooms (Agricus bisporus). J. Sci. Food Agric. 80: 561-566 (2000) 18. Lee CY, Smith NL. Apples: An important source of antioxidants in the American diet. N.Y. Fruit Q. 8: 15-17 (2000) 19. Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M. Quantitation of flavonoid constituents in citrus fruits. J. Agric. Food Chem. 47: 3565-3571 (1999) 20. Johnson MK, Loo G. Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. Mutat. Res. 459: 211-218 (2000) 21. Kotake-Nara E, Kuchiro M, Zhang H, Sugawara T, Miyashita, Nagao A. Carotenoids affect proliferation of human prostate cancer cells. J. Nutr. 131: 3303-3306 (2001) (2004j 4ú 8¢ %>; 2004j 4ú 17¢ j).

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