Biological Activities of Flavonoid Glycosides Isolated from Angelica keiskei

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 37, No. 1, pp. 78~83 (2005). ©The Korean Society of Food Science and Technology. F.öB ªÒ* flavonoid glycosides~ ÒW

(2) Ò+ÁBß{ÁB+*ÁBãÎ ()¦Zö 8F\². Biological Activities of Flavonoid Glycosides Isolated from Angelica keiskei Jae-Seok Shim, Seung-Deok Kim, Tae-Seok Kim*, and Kyung-Nam Kim R&D Center for Food Technology, Pulmuone Co., Ltd. Recently, much attention has been focused on plant antioxidants, because they are expected to protect against oxidative damage, possibly preserving biological functions of cells. Antioxidant compounds were isolated from Angelica keiskei through extraction with 80% EtOH, and fractionations were carried out sequentially with nhexane, chloroform, ethyl acetate, n-butanol, and water. Two active compounds were isolated from ethyl acetate fraction by silica gel column chromatography, and were identified as isoquercitrin (quercetin-3-O-β-D-glucose) and hyperoside (quercetin-3-O-β-D-galactose). Isoquercitrin and hyperoside showed strong antioxidative potency, as revealed by evaluation of their ABTS, DPPH, OH, and H2O2 radical-scavenging activities, and ex vivo DNA damage-protecting effects. Key words: antioxidant activity, Angelica keiskei, isoquercitrin, hyperoside, DNA damage protection. B. . *W ~ ê¢ ~ "ö '« £Ò¾ "¢, j² f ?f Âb F¾~ ÖzB¢ BB~ Ï~Jº ãË b æz> ® (4). &' Âb F¾~ ÖzBº αtocopherol, vitamin, carotenoids, flavonoids, tannins, pycnogenol, thiazineê zb > ®b, V^ ® N ~ö V epigallocatechin gallatef ?f bî~ Öz W rJ^ ® (5-8). $ "öº ¿ S& î÷ ®º ãÖ öò jî¢ ¢>ö²ê '·~ ®;j ²~ ;ö êæ >º Â Wª j /~ î÷j .O~ '~ ;j Fæ ~º ©b ²B>B j² ¿~ Ïö & & ² Ã& >î (9). &¢, F., öÒ, ò¾Ò, " ~ ï j²º jæ", ZVî j F~ ®, 7 ß® F.º flavonoids ~ FÏ phytochemical j ï F ~ ®Ú ~ VËW «" Î"ö & B ~² ê¯> ® (10,11). F.(Angelica keiskei)º j& æOöB ¶~º ¾Ò "~ &; j .ÏbB «¢#, Fâ. $º ã.¢ ®Ò(12), ÒWj ¾æÚº '« FVÖ, flavonoids, coumarin, saponin" '« ZVî ß® FV ²îª Ú ®. >Ú ® (13). F.~ ÎËf .{, *Ë÷, ãÛ ö "*º»b ÒÏ>Ú z *æ ®, " öº æ.Ã BF 5 *ëW BF ö & ÒW >Ú ®b¾(14,15) · Ò' ÎËö & & jç ç . V¢B öº F.~ ÒWj «~º ·ë~. " ² Ã& >&~ Ëçb ®~ '·' GöB ÚOÚ, î÷~ Oæ 5 ², zÛB ~ ; VËWö & & Ã&> ® (1). *~ z*ç¾ zB ~ '« î÷f Ú ÚöB oxidative stressö ~ pªì Ö>º W Ö²«ö V~º ©b rJ^ ®º, superoxide, nitric oxide, nitrogen dioxide, hydroxyl, peroxyl, alkoxyl, hydroperoxy " ?f W Ö²«. f *~ &Ò"; 7ö pªì B>Ú zf f & NB î÷~ "º ¶ ·Ï~ ® (2). W Ö²« ö ~ oxidative stress~ Ã&º ^ Ú Wª~ Öz' ¶ç 5 DNA æ j Û~ ^ Ú çW~ 2Z¢ .¾b &æ ¯W î÷j ¢b Ê z*ç~ /ê" pf &W ®º ©b rJæº oxidative stress ö & ôf & ®Ú z, ö &w~ .O 5 ~ò > ®º ÖzB~ BB ê¯> ® (3). z 5 '« î÷j .O 5 ~ò~V * Ï'b .Vö BHT, BHAf ?f W ÖzB < ®º n *Corresponding author: Tae-Seok Kim, R&D Center for Food Technology, Pulmuone Co., Ltd, Seodaemun Post Office P.O. Box 146, Seodaemun-Ku, Seoul 120-600, Korea Tel: 82-2-3277-8580 Fax: 82-2-3277-8503 E-mail: [email protected] 78.

(3) F.öB ªÒB flavonoid glycosides~ ÒW. ¢~b Öz Wj 7b Öz æ Wªj ªÒ ~&, æWª~ Öz W 5 DNA ¶ç ÛB ¦¢ G;~& .. Òò 5 O» Òò. þ Òò F.º Z¦Zö¿öB FV³ öò / Aj ÒÏ~&, öò¢ ÿÖ~ ªê ê ªöj ïÿö &~B ºÂÏ ò ÒÏ~& . F.~ ºÂ 5 ª³ B ªê ò 100 g" öêR~ ³êê ºÂ >N jv' ¸~~ 80%(v/v) öêR 400 mL¢ b~, çN öB æÿn >ºÂ ~& . -² ºÂB ò¢ Whatman filter paper(No.2)¢ Ï~ "~, ºÂj rotary vacuum evaporator(EYELA NE1001S, Japan) ³»~ Ï ¢ j *® B ê ÿÖ~ öêR ºÂb(12.7 g)j B~ & . ºÂ~ ª³f Ï ~ Wj Ï êÛ ª³j Ï~ & . ¯, öêR ºÂbj ³»~ Ï ¢ B b[ö £ 2V~ n-hexanej &~ n-hexane ª³b(1.81 g)j á, ÿ¢ O»b chloroform(2.45 g), ethyl acetate(1.23 g), n-butanol (3.12 g)j &~ ''~ ª³bj áîb îæïb Îf b[(1.05 g)j áî . r ''~ ª³f 2² >~&, ' ª³bf rotary vacuum evaporator Ï ¢ j*® B ê 10% öêRö Öz þö ÒÏ~& . Öz bî~ ªÒ 5 ;B ª³ê Öz W 7öB &Ë W ¸f ª³j Ï ~ silica gel column chromatography»b ¢ bîj ªÒ ~& (Fig. 1). W ¸f ª³j silica gel(Merck, 70-230 mesh) chloroform : acetone : methanol : water(5 : 3 : 2 : 0.5)~ Ï. b ÏêB columnöB ÏÂB C 6B~ ²ª³b j á, 7 W ¸f ª³j chloroform : acetone : methanol : water(6 : 3 : 2 : 0.5)~ öB ÏÂB C 3B~ ² ª³bj áî . C 3B~ ª³bö & Öz Wj G; ~ &Ë W ¸f ª³ö & acetylation(16) >w j ê¯Ê ¢ n-hexane : chloroform : methanol(6.5 : 3 : 1)~ Ï b ÏÂ ê deacetylation(16)j ê¯~ « v B~ ¢ zbj áî . z> I: Mp 235oC; UVλmax(MeOH) 206, 255, 357 nm; (+NaOH) 210, 270, 325, 411 nm; (+AlCl3) 210, 267, 398 nm; (+NaOAc) 209, 270, 364 nm; (+NaOAc/H3BO3) 208, 261, 296, 375 nm; 1H-NMR(500 MHz, CD3OD) δ: 7.69(1H, d, J = 2Hz, H-2'), 7.59(1H, dd, J = 2.0Hz, J = 8.5Hz, H-6'), 6.88(1H, d, J = 8.5Hz, H-5'), 6.40(1H, d, J = 2.0Hz, H-8), 6.20(1H, d, J = 2.0Hz, H-6), 5.22(1H, d, J = 7.5Hz, H-1''); 13C-NMR(125 MHz, CD3OD): 178.6(C-4), 165.7(C-7), 162.4(C-5), 158.3(C-9), 157.8(C-2), 149.2(C-3'), 145.2(C-4'), 134.9(C-3), 122.3(C-1'), 121.9(C-6'), 116.7(C-5'), 115.3(C-2'), 104.8(C-10), 103.1(C-1''), 99.3(C-6), 94.1(C-8), 77.4(C-5''), 76.8(C-3''), 74.5(C-2''), 70.3(C4''), 61.5(C-6'').. 79. z> II: Mp 230oC; UVλmax(MeOH) 258, 271, 300, 359 nm; (+NaOH) 217, 272, 333, 411 nm; (+AlCl3) 211, 275, 416 nm; (+NaOAc) 211, 261, 362 nm; (+NaOAc/H3BO3) 210, 263, 297, 380 nm; 1H-NMR(500 MHz, CD3OD) δ: 7.66(1H, dd, J = 2Hz, J = 8.5Hz, H-6'), 7.50(1H, d, J = 2.0Hz, H-6'), 6.81(1H, d, J = 8.5Hz, H-5'), 6.40(1H, d, J = 2.0Hz, H-8), 6.20 (1H, d, J = 2.0Hz, H-6), 5.34(1H, d, J = 8Hz, H-1''); 13C-NMR (125 MHz, CD3OD): 178.3(C-4), 165.4(C-7), 162.3(C-5), 157.1 (C-9), 157.3(C-2), 149.2(C-3ä), 145.9(C-4'), 134.5(C-3), 123.1 (C-1'), 122.2(C-6'), 117.1(C-5'), 116.3(C-2'), 105.0(C-10), 102.9 (C-1''), 99.8(C-6), 94.6(C-8), 76.0(C-5''), 73.8(C-3''), 72.1(C-2''), 68.7(C-4''), 61.2(C-6''). 88ªC zb~ ª¶ Cj * VVªCj ~& . 1HNMR, 13C-NMR, HMQC 5 HMBC¢ G;~&b ÒÏ VV º BrukerÒ~ Avance 500 Îj Ï~& . Öz W~ /; DPPH radical ²`»: DPPH radical ²Wf öêRö ³êê C ò 10 µLö 200 µM DPPH/EtOH 190 µL¢ & ê 37oCöB 30ª* >wÎ r 517 nmöB 7ê¢ G;~&b, ò & öêRj & &ö & ò¢ Iîj r~ 7ê 6² ;ê¢ Ò~& (17). ABTS radical ²`»: ABTS radical ² Wf 2 mM ABTS(2,2'-azino-bis-(3-ethyl benthiazoline-6-sulfonic acid), 15 µM H2O2 Ò 0.25 µM HRP¢ F 50 mM Na-phosphate buffer(pH 7.5) 1 mLj 25oCöB 5ª* ;~Î r 730 nm öB 7ê& ¢;~² Fæ> öêRö ÏÎ ò 10 µL¢ &~ 5ª ê 730 nmöB 7ê 6²¢ G;~&. (18). Hydroxyl radical ²`»: Hydroxyl radical ² Wf Fenton reactionö ~ OH radicalj BB ö & ² Wj G;~& . 2.8 mM deoxyribose, 25 µM FeCl3, 100 µM EDTA, 2.8 mM H2O2¢ 10 mM KH2PO4/KOH buffer (pH 7.4)ö ³êê BB ò 100 µL¢ &~ ¾ b~ reaction volumn 1.2 mL& >² ~& . ê reaction mixture~ ascorbate~ ³ê& 100 µM >ê ascorbate¢ Î & ê 37oCöB * ÿn 200 rpm~ ³ê ²*~º shaking incubatoröB >w Î r 1% TBA 1 mL" 2.8% TCA 1 mLj &~ OHf~ >wö ~ WB deoxyribose ~ malondialdehyde(MDA) FÒbî"~ >wj *~ 80oCö B 20ª* &Ê çNb ï'V . n-Butanol 3.2 mL j &~ 30.* ;~² z ÚB ¾ b ê 3,000 rpmö B 20ª* öªÒB >wö ~ WB ï²bîj ªÒ ~&b ï²bîj F *[ 200 µL¢ ~ 532 nmö B ELISA reader 7ê¢ G;~& (19). Hydrogen peroxide ²`»: 2 mM H2O2¢ F phosphate buffer saline(pH 7.4) 600 µLö ¢;³ê~ ò 300 µL¢ &~ 5ª êö 230 nmöB 7ê¢ G;~& (20)..

(4) ®"²æ B 37 ² B 1 ^ (2005). 80. Table 1. Antioxidant activity of different solvent fractions of Angelica keiskei ethanol extract Sample concentrations (%) Samples. 0.05. 0.01. 0.005. Inhibition (%) EtOH extract n-Hexane fraction Chloroform fraction Ethyl acetate fraction BuOH fraction Water fraction. 95 55 83 97 92 90. 31 26 29 95 83 54. 16 12 15 51 50 5. Ex vivo DNA ¶ç 6² Î /; Ú ª2 ^º j. * ² 8* çf rbj _ æ pf ; &ç¶¦V £ 5 mL~ .j haparinated sterile tubeö Aj *. 100 µL¢ 1 mL~ 10% fetal bovine serumj F RPMI 1640ö Df ê Histopaque 1077j Ï ª2òj ªÒ~& . Ú ª2 ^ö ò¢ ÿÖ ê PBSö Ï~ 10-1,000 µg/mLÒ~ ³ê cell ö ¾Ò~ ïË(30ª, 4oC)öB >wÊ >w Â ª 2 ^ö oxidative stress(100 µM H2O2)¢ 4oCöB 5ª ÿn & Ê oxidative stressö ~ DNA ¶çj FBÎ ê comet assay¢ ~& (21).. Fig. 1. Isolation of isoquercitrin and hyperoside.. Ö 5 8 Öz bî~ ªÒ 5 ;B ºÂbö & ª³ê Öz Wj G; Ö" ethyl acetate ª³ ò ³ê 0.01%öB 95% ç~ ¸f Öz Wj ¾æÚ 0.005%~ &³êöBê 50% ç~ Öz Wj ¾æÚÚ(Table 1) silica gel column chromatography»j Ï~ ethyl acetate ª³b¦V Öz bîj ªÒ~& . b& ethyl acetate ª³j silica gel(Merck, 70-230 mesh)" OB Ï ¢ j*® B ê silica gel chloroform : acetone : methanol : water(5 : 3 : 2 : 0.5)~ Ï b ÏêB column(5 Ü45 cm)öB ÏÂ~ TLC(thin layer chromatography, Merck, 60F254) plate çöB *~& jÝ zbj Îj C 6B~ ª³(Fr.I-VI)j áî . 6B~ ª³bö & Öz W ¦Ã Ö" W &Ë ¸² G;B Fr.IIIj chloroform : acetone : methanol : water(6 : 3 : 2 : 0.5)~ Ï b ÏÂ~ Fr.III-Af Fr.III-B~ vB~ ª³bj áî vB~ ª³b 7 Öz W ¸f Fr.III-Bö & ªÒ ·ëj ê¯~& . Fr.III-Bº zb B "7~ ßö®V r^ö acetylation >wj ~& . ¯ Fr.III-B¢ pyridine 5 mLö ê acetic anhydride 5 mL¢ ï~öB '~~& . NöB 15* ç v> Î ê >wj >f ethyl acetate ª V ºÂ~& . Ethyl acetate [j 5% HCl, z NaHCO3 5 NaCl ^¿ ê, MgSO4 anhydrous î> ~ " 6{.

(5) F.öB ªÒB flavonoid glycosides~ ÒW. Fig. 2. Chemical structure of isoquercitrin and hyperoside. (R)=Glucose: Isoquercitrin, (R)=Galactose: Hyperoside.. ³»~& . áÚê bîj silica gel column chromatography¢. ~ n-hexane : chloroform : methanol(6.5 : 3 : 1)~ Ï b ÏêB columnöB ÏÂB acetylationB 3B~ ¢ b î Fr.III-B(a), Fr.III-B(b) 5 Fr.III-B(c)¢ áî acetylationB 3B~ ª³bö & Öz W G;j * deacetylation V . ¯, 3B~ ª³bj '' 20 mL~ methanolö Vö 2 mL~ 5% KOH¢ IÚ v>~B 2* ç >w Î ê ¢;ï~ DOWEX 50WX4-400 ·N v~>æ¢ IÚ 7z Ê " 6{ ³»~ Öz W ¦Ã ê « ;BB ¢ bî zb I" zb II¢ áî . zb If ï ªöçb 1H-NMR Ê¿Þ"öB δ5.22(J = 7.5Hz, d)öB~ anomeric proton" 13C-NMR Ê¿Þ" öB δ103.1, 77.4, 76.8, 74.5, 70.3 5 61.5öB glucoseö ~ ê² b¢ &V > ®î, NaOH, AlCl3, NaOAc 5 NaOAc/H3BO3 shift reagent Î& UV Ê¿Þ" band I,II~ bathochromic shift~º ©b j flavonoid Ï~ C-5, C-7, C-3' 5 C-4'ö free ç~ OH& Ò~º zbªj r > ®î . ç~ Vf ^ò¶ò(22)~ jv¢ ÛB z b If C21H20O12~ ª¶j &æ ª¶ï 464.4 isoquercit-. Fig. 3. Antioxidant activities of isoquercitrin. (A): DPPH radical, (B): ABTS radical, (C): OH radical, (D): H2O2.. 81. rin(quercetin-3-O-β-D-glucose)ªj « {~& (Fig. 2). Isoquercitrinf ¢>'b Âbö Ò~º flavonoids~ ¢ «b böB ªÒ> ®æò þöB ÒÏB Ò ò F.öB isoquercitrin~ ªÒö & º *Ò ~ rJ^ ®æ pf çæ ÒWö & Ï & j º ©b 'B . zb II ï ªöçb UV, 1H-NMR 5 13C-NMR Ê¿Þ" zb I" Ö FÒ~&, N6f 1H-NMR Ê ¿Þ"öB ~ δ5.34(J = 8.0Hz, d)öB~ anomeric proton" 13 C-NMR Ê¿Þ"~ δ102.9, 76.0, 73.8, 72.1, 68.7 5 61.2b ¦ª galatose ~~Nj º; > ®î . ç~ Vf ^ò¶ò(23)~ jv¢ ÛB zb IIº C21H20O12~ ª¶j &æ ª¶ï 464.4 hyperoside(quercetin-3-O-β-Dgalactose)ªj « {~& (Fig. 2). Hyperosideº F.öB ªÒ>î º (10)& ®æ ò bîö & Öz W º jç ç . V¢B ¢ êV ~ Özf &NB ÒWö & ê®º & jº ©b 'B . Isoquercitrin hyperoside~ Öz W F.¦V ªÒB isoquercitrin" hyperosideö & Ö z Wj DPPH radical ²», ABTS radical ²», OH radical ²» 5 H2O2 ²»b G;~& . 4&æ assay» öB Îv isoquercitrin" hyperosideö & Öz Wf ³ ê~'b Ã&>º ãËj &, ß® &³êöB 90% ç~ radical ² Wj ¾æÚÚ ;K Öz Wj & . Isoquercitrin~ Öz W(Fig. 3)öB DPPH radical ²» f 5 µg/mL ³êöB £ 60% ;ê~ ¸f Öz Wj ¾ æî, $ ABTS radical ²»öBê 3 µg/mL &³êöB £ 55% ;ê~ ¸f Öz Wj ¾æî . Ò OH radical ²»" H2O2 ²»öBê '' 25 µg/mL" 10 µg/ mL~ ³êöB 50% ç~ Öz Wj ¾æî . *~ Ö "öB OH radical ²»" H2O2 ²»~ ãÖöº DPPH.

(6) 82. ®"²æ B 37 ² B 1 ^ (2005). Fig. 4. Antioxidant activities of hyperoside. (A): DPPH radical, (B): ABTS radical, (C): OH radical, (D): H2O2.. Fig. 5. Percentage inhibitory effect of isoquercitrin and hyperoside on H2O2-induced oxidative DNA damage in human lymphocyte cell. (A): DNA protecting effect of isoquercitrin, (B): DNA protecting effect of hyperoside, N: Negative control, P: Positive control.. radical ²»" ABTS radical ²»ö j~ £* ¸f ³ êöB Öz Wj ¾æÚº ãË ®æò, OH radical" H2O2& î' Ú Ú~ Öz ÊöB DNAf ?f ª¶ .ÎöB~ Öz' ¶çö &7 &N ® º (24, 25)¢ J jv' &³êöB OH radical" H2O2ö & B Öz Wj ¾æÞ º ©f 7º ~¢ Ú. " > ® . V¢B &æ assay»ö ~~æ p DPPH radical ² »" ABTS radical ²»" ?f V.' W G;" þ DNA ¶ç" ?f ÚÚ ª¶.ÎöB~ Öz' ¶ç" &7 &N ®º OH radical" H2O2 ² W ¸ º ©f b isoquercitrinj j FÒ flavonoidsö & [ ¢ ÛB ²Ò¢ Öë'b FÏ~² 'Ï > ® º &ËWj Ò " > ® . Hyperoside~ Öz W(Fig. 4)j *~ 4&æ assay»ö ~ B G; Ö" isoquercitrin" ~ ÿ Öz Wj ¾æî . f ?f Ö"º quercetin¾ quercetin VÚ~ ãÖ Wö 7º 'Ëj ~º © hydroxyl group~ > ¾ *~ö ~ ©¢º (26)ö V¢ hyperosidef isoquer-. citrin~ ª¶ öB ~ «~ò ¢ ö %?f quercetin Ïj &æ ®V r^ö ÿ¢ Ïö ÖB ~ «~º Öz Wö 'Ëj ~æ pV r^ ©b 6>Ú ê . Isoquercitrin hyperoside~ DNA ¶ç 6² Î /; Isoquercitrin" hyperosideö & DNA ¶ç 6² Î"¢ G ; Ö" Fig. 5öB º :f ? negative control(N)" positive control(P)j V&b isoquercitrin" hyperosideº ³ê ê Öz' ¶çj «f lymphocytes~ DNA ¶çö & ^ Î"¢ ¾æÚº ©j r > ® . Isoquercitrin" hyperoside~ ³êê Wj ÚÚ 100 µg/mL~ ³êöB 50% ç~ DNA ¶ç ^ Î"& ¾æÎj { > ®º, comet assayº ªC þ" Ò >~B ò~ .&' 8 j * > ì, ò ³ê ~'b positive controlö j DNA ¶ç ^ Î"& ¾æÎj * > ® . ê *~ þöB DNA ¶çö & ^ Î"& {B isoquercitrin" hyperoside ö FÒ ¢ &æ ®º flavonoids~ Wj B jv~ ~ Nö V W~.

(7) F.öB ªÒB flavonoid glycosides~ ÒW. æz¢ G;~ f W*~ ç&Wj Ò~º & jº ©b 6 B .. º. £. F.öB Öz W bîj ªÒ~V * F.¢ 80% öêR ºÂ~ ºÂbö & êÛ ª³j ~ n-hexane, chloroform, ethyl acetate, n-butanol 5 water~ C 5&æ ª³ b ¾*Ú Öz Wj G; Ö" ethyl acetate ª³~ W &Ë ¸j ethyl acetate ª³b¦V Öz W b îj ªÒ ;B~& . Silica gel column chromatography¢ Û ~ chloroform : acetone : methanol : water~ Ï j 6N 'b æã~B ²ª³b ¾* Öz W &Ë ¸ f ²ª³ö &B acetylation >wj ~ ¢ n-hexane : ethyl acetate : methanol~ Ï b ¢ bî ªÒ ê deacetylation >wj ö « vB~ ¢ zbj ³ ® . vB~ ¢ zbö & Cj VVªCj Û B ê¯ Ö" v zbj '' isoquercitrin" hyperoside « ÿ;~& . Isoquercitrin" hyperosideö & Öz Wj DPPH radical ²», ABTS radical ²», OH radical ²» 5 H2O2 ²»b G; Ö" 4&æ assay» ö &B &³êöB Öz Wj ¾æî, $ DNA ¶çö & ^ Î"¢ G; Ö" isoquercitrin" hyperoside Îv ¶çB DNAö & ^ Î"¢ ¾æÚº ©j { > ®î . 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