Phenolic Constituents of Althaea rosea Canival.

Transcription

1 w z Kor. J. Pharmacogn. 38(3) : 222 Ê 226 (2007) r ½ zá Á ³wÁ½»xÁ * ³ w w w tyw Phenolic Constituents of Althaea rosea Canival. Do Hoon Kim, Min Cheol Yang, Kyu Ha Lee, Ki Hyun Kim and Kang Ro Lee* Natural Pruducts Laboratory, College of Pharmacy, Sungkyunkwan University, Suwon , Korea Abstract Seven compounds, scopoletin (1), p-hydroxyphenethyl trans-ferulate (2), 1-(α-L-rhamnosyl(1 6)-β-D-glucopyranosyloxy)-3,4,5-trimethoxybenzene (3), benzyl α-l-rhamnopyranosyl(1 6)-β-D-glucopyranoside (4), suberic acid (5), sebacic acid (6) and scopolin (7) were isolated from the methanol extract of the roots of Althaea rosea Canival. Compounds 1 ~ 7 were first isolated from this plant. The isolated compounds were tested for their cytotoxicity against human cancer cell lines using a SRB method in vitro. Key words Malvaceae, Althaea rosea, Phenolic constituent, Cytotoxicity (Althaea rosea Canival) ù ƒ sw (Malvaceae) 2 ³ ( ƒƒ) š w, x,, z š,, w, x, mx, e. 1-3) (A. rosea) phenolic, 4) sterols š 5) antiviral activity, 6) antiinflammatory activity 7). ù w A. rosea w w k. ü l y, p s ww, y (A. rosea) methanol w column chromatography w 5 r 2 suberic acids w yw»» ³ w. w y w 7 w SRB 4 s, A549 ( s s ), SK-OV-3 (ù ), SK-MEL-2 (v ) HCT-15 ( ) w s ww. &NBJMLSMFF!TLLVBDLS '"9 x x x (A. rosea) w y w z w š, t ³ w w w t (SKKU ).»» NMR Varian Unity-Inova-500 spectrophotometer w. EI-MS spectrum JMS700(Jeol, Japan) FAB-MS VG70-VSEG(VG Analytical, UK) w š, nitrobenzyl alcohol matrix w. LPLC column Lobar F -A LiChroprep Si 60 (Merck) column Lobar F -A LiChroprep RP C-18 (Merck) column w š, pump FMI (QSY-0) w. HPLC Gilson 306 pump Econosil F RP C u (length : 250 mm, I.D 10 mm) column Shodex refractive index detecter(ri-101) w w. Column chromatography silica gel Kiesel gel 60 (Merck) LiChroprep C-18(Merck) w š, molecular sieve column chromatography packing Sephadex LH-20(Pharmacia) w. TLC plate Kiesel gel 60 F254 precoated plate(art.552, Merck) w š, TLC 10% H 2 w, column chromatography 1,»k 222

2 Vol. 38, No. 3, Fig. 1. Structures of compounds 1-7 isolated from the roots of Althaea rosea Canival. 1 p ƒƒ w. 3.0 kg methanol 3z w. w MeOH» 250 g. Methanol (1.6 L) ƒw xk k z n-hexane (55 g), chloroform (13 g), n-buoh (40 g) zw. Chloroform z (13 g) chloroform :methanol (8:1) silica gel column chromatography w 4 z (C1-C4) ù, C2 z (0.5 g) n- hexane :chloroform :ethyl acetate (15:13:1) silica gel column chromatography z, Econosil F RP C-18 HPLC (70% MeOH in water) w yw 1 (3 mg). C4 z (2.9 g) n-hexane: chloroform : methanol (3 : 8 : 1) silica gel column chromatography w z methylene chloride: methanol (1:1) w Sephadex LH-20 column chromatography w z Econosil F RP C-18 HPLC(40% acetonitrile) w w oil yw 2 (10 mg). n-butanol z (40 g) 20% methanol~100% methanol w gradient RP C-18 silica gel column chromatography w 4 z (B1- B4) ù. B1 z (20 g) chloroform:methanol :water (7:5 :1) xk k z B1 (5 g) 80% methanol w Sephadex LH-20 column chromatography w š, chloroform:mathanol:water (7:5:0.5) w silica gel column chromatography z, Econosil F RP C-18 HPLC (15% acetonitrile) w oil yw 3 (6 mg). B2 z (1.7 g) chloroform : methanol:water (12:5:1) w silica gel column chromatographyz 80% methanol w Sephadex LH-20 column chromatography wš, chloroform : methanol : water (12:5:1) w Lobar F -A column chromatography w z, Econosil F RP C-18 HPLC (22% acetonitrile) w oil yw 4 (5 mg), yw 5 (30 mg) ƒƒ. B3 z (1.6 g) 80% methanol w Sephadex LH-20 column chromatography w 6 z (B31-B36) ù z, B35 (150 mg) Econosil F RP C-18 HPLC (24% acetonitrile) w yw 6 (12 mg) š, B34 (200 mg) acetonitrile (23%) w RP C-18 silica Lobar F -A column chromatography w z, Econosil F RP C-18 HPLC (23% acetonitrile) w oil yw 7 (11 mg). Scopoletin (1) White powder; m.p. : 203 ~ 205 o C; FAB-MS m/z:215 [M+Na] + ; 1 H-NMR (500 MHz, CD 3 OD) δ: 7.87 (1H, d, J = 9.5 Hz, H-4), 7.12 (1H, s, H-5), 6.78 (1H, s, H-8), 6.21 (1H, d, J = 9.5 Hz, H-3), 3.92 (3H, s, OCH 3 ); 13C-NMR (125 MHz, CD (C-2), (C-7), (C-8a), (C-4), (C-6), 111.4

3 224 Kor. J. Pharmacogn. (C-3), (C-4a), (C-5), (C-8), 55.7 (OCH 3 ). p-hydroxyphenethyl trans-ferulate (2) Yellow oil; EI-MS m/z :314 [M] + ; 1 H-NMR (500 MHz, CD (1H, d, J=16.0 Hz, H-7), 7.12 (1H, d, J=2.0 Hz, H- 2), 7.07 (2H, d, J=8.0 Hz, H-2', 6'), 7.04 (1H, dd, J=8.0, 2.0 Hz, H-6), 6.81 (1H, d, J=8.0 Hz, H-5), 6.72 (2H, d, J = 8.0 Hz, H-3', 5'), 6.40 (1H, d, J = 16.0 Hz, H-8), 3.89 (3H, s, OCH 3 ), 3.47 (2H, t, J=7.0 Hz, H-1"), 2.76 (2H, t, J = 7.0 Hz, H-2"); 13 C-NMR (125MHz, CD (C-9), (C-4'), (C-4), (C-3), (C-7), (C-1'), (C-2'), (C-6'), (C-1), (C-6), (C-8), (C-3'), (C-5), (C-5'), (C-2), 55.2 (C, OCH3), 41.3 (C-1"), 34.6 (C-2"). 1-(α-L-Rhamnosyl(1 6)-β-D-glucopyranosyloxy)-3, 4,5-trimethoxybenzene (3) Colorless oil ; EI-MS m/z :492 [M] + ; 1 H-NMR (500 MHz, CD (2H, s, H-2, 6), 4.82 (1H, d, J=7.0 Hz, H-1'), 4.71 (1H, d, J=2.0 Hz, H-1"), 4.01 (1H, d, J=9.0 Hz, H -6'a), 3.83 (6H, s, 2 OCH 3 ), 3.72 (3H, s, OCH 3 ), 3.65 ~ 3.55 (4H, m), 3.60 (1H, d, J = 9.0 Hz, H-6'b), 3.49 ~ 3.38 (4H, m), 1.22 (3H, d, J = 9.0 Hz, H-6"); 13 C-NMR (125 MHz, CD (C-1), (C-3), (C-5), (C-4), (C-1'), (C-1"), 96.4 (C-2), 96.4 (C-6), 78.0 (C-3'), 77.0 (C-5'), 75.0 (C-2'), 74.1 (C-4"), 72.5 (C-3"), 72.2 (C- 2"), 71.6 (C-4'), 69.9 (C-5"), 67.9 (C-6'), 61.4 (OCH 3 ), 56.8 (2 OCH 3 ), 18.0 (C-6"). Benzyl-O-α-L-rhamnopyranosyl (1 6)-β-Dglucopyranoside (4) Yellow oil; FAB-MS m/z :417 [M+H] + ; 1 H-NMR (500 MHz, CD ~7.28 (5H, m, H-2,3,4,5,6), 4.87 (1H, d, J=12.0 Hz, H-7a), 4.79 (1H, d, J = 1.5 Hz, H-1"), 4.66 (1H, d, J = 12.0 Hz, H-7b), 4.33 (1H, d, J = 7.5 Hz, H-1'), 3.71 (1H, m, H-6'a), 3.65 (1H, dd, J = 12.0, 6.0 Hz, H-6'b), 3.74 ~ 3.63 (4H, m), 3.42 ~ 3.23 (4H, m), 1.28 (3H, d, J = 6.5 Hz, H-6"); 13 C- NMR (125 MHz, CD (C-1), (C-2), (C-6), (C-3), (C-5), (C-4), (C-1'), (C-1"), 76.9 (C-3'), 75.7 (C-5'), 73.9 (C-2'), 72.8 (C-4"), 71.2(C-4'), 71.0 (C-3"), 70.6 (C-7), 70.5 (C- 2"), 68.6 (C-6'), 66.9 (C-5"), 16.9 (C-6"). Suberic acid (5) White powder; m.p. : 144 ~ 146 o C; EI-MS : m/z 174 [M] + ; 1 H-NMR (500 MHz, CD (4H, t, J = 8.0 Hz, H-2, 2'), 1.63 (4H, m, H-3, 3'), 1.38 (4H, m, H-4, 4'); 13 C-NMR(125 MHz, CD (2C-1, 1'), 33.6 (2C, C-2, 2'), 28.6 (2C, C-3, 3'),24.7 (2C, C-4, 4'). Sebacic acid (6) White powder; m.p. : 133 ~ 137 o C; EI-MS m/z : 202 [M] + ; 1 H-NMR (500 MHz, CD (4H, t, J = 7.0 Hz, H-2, 2'), 1.61 (4H, m, H-3, 3'), 1.36 (8H, m, H-4, 4', 5, 5'); 13 C-NMR (125 MHz, CD (2C-1, 1'), 33.7 (2C, C-2, 2'), 28.8 (2C, C-3, 3'), 24.8 (4C, C-4, 4', 5, 5'). Scopolin (7) Colorless oil; FAB-MS m/z : 355 [M+H] + ; 1 H-NMR (500 MHz, DMSO-d 6 ) δ: 7.96 (1H, d, J = 9.5 Hz, H-4), 6.95(1H, s, H-5), 6.81(1H, s, H-8), 6.34 (1H, d, J = 9.5 Hz, H-3), 5.03 (1H, d, J = 8.0 Hz, H-1'), 4.40 ~ 3.06 (6H, m), 3.84 (3H, s, OCH 3 ) ; 13 C-NMR (125 MHz, DMSO-d 6 ) δ: (C-2), (C-7), (C-8a), (C-4), (C-6), (C-5), (C- 4a), (C-3), (C-8), (C-1'), 77.5 (C-5'), 76.5 (C-3'), 74.1 (C-2'), 69.8 (C-4'), 60.8 (C-6'), 56.5 (OCH 3 ). s x s x Sulforhodamine-B (SRB) bioassay 8) w w yw ww. x s A549 (non small cell lung carcinoma), SK-OV-3 (adenocarcinoma, ovay malignant ascites), SK-MEL-2 (malignant melanoma, metastasis to skin of thigh) HCT-15 (colon adenocarcinoma) w s x w w. š yw 1, 5, 6, 7 ù š. 1 H-NMR, 13 C-NMR MS rp x w w., yw 1 7- hydroxy-6-methoxy coumarin (scopoletin) 9-11), yw 5 suberic acid 12,13), yw 6 sebacic acid 14), yw 7 15,16) scopolin ƒƒ y w. yw 2 oil 10% H 2 (EtOH) w. EI-MS rp molecular ion peakƒ m/z 314, 1 H- 13 C-NMR rp ww molecular formula C 18 H 18 O 5 w š, l sy 10 w. 1 H-NMR spectrum δ 7.44 (1H, d, J= 16.0 Hz, H-7) δ 6.40 (1H, d, J = 16.0 Hz, H-8) olefinic proton trans w y w š, δ 7.12 (1H, d, J = 2.0 Hz, H-2), 7.04 (1H, dd, J =8.0, 2.0Hz, H- 6) 6.81 (1H, d, J=8.0 Hz, H-5)vj w y proton 1,3,4 ey wš, δ 7.07 (2H, d, J=8.0 Hz, H-2', 6'), δ 6.72 (2H, d, J=8.0 Hz, H-3', 5')vj ƒ d 1,4 ey w y w.

4 Vol. 38, No. 3, δ 3.47 (2H, t, J=7.0 Hz, H-1") δ 2.76 (2H, t, J=7.0Hz, H-2") aliphatic proton vj mw phenylethyl w w w š, δ 3.89 (3H, s, OCH 3 ) mw methoxy group y w. 13 C-NMR spectrum 12 aromatic carbon vj sww, 1 ketone (δ 168) vj 2 olefinic carbon (δ 140.8, 117.6) vj w, 2 oxygenated carbon (δ 55.2, 41.3) vj d w. x 17,18) mw yw 2 p- hydroxyphenethyl trans-ferulate y w. yw 3 oil 10% H 2 (EtOH) y. EI-MS rp mw molecular ion peakƒ m/z 492, 1 H- 13 C-NMR rp ww molecular formula C 21 H 32 O 13 w š, l sy 6 w. 1 H-NMR spectrum δ 6.45 (2H, s, H-2, 6)vj w y proton w š, anomeric proton peak vjƒ δ 4.82 (1H, d, J=7.0 Hz, H-1'), δ 4.71(1H, d, J=2.0 Hz, H-1") 2 w w š, ƒƒ coupling constant (J=7.0, 2.0Hz) l β-form α-form d w. δ 3.83 (6H, s, 2 OCH 3 ) δ 3.72 (3H, s, OCH 3 ) peaks 3 methoxyl groups w. proton peaksƒ overlapping δ 4.01~3.38 d š, aliphatic proton δ 1.22 (3H, d, J=9.0 Hz, H-6") α-rhamnosyl methyl proton d w. 13 C-NMR rp aromatic carbon δ (C-1), (2C, C-3, 5), (C-4) w š, chemical shift δ (C-1'), (C-1"), 78.0 (C-3'), 77.0 (C-5'), 75.0 (C-2'), 74.1 (C-4"), 72.5 (C-3"), 72.2 (C-2"), 71.6 (C-4'), 69.9 (C-5"), 67.9 (C-6') w. š aliphatic carbon δ 18.0 (C-6") 1 H- 13 C-NMR rp mw α-rhamnosyl w. x 19) mw yw 3 1-(α-L-rhamnosyl(1 6)-β-D-glucopyranosyloxy)-3,4,5-trimethoxybenzene w š, x 19) data w y w. yw 4 oil 10% H 2 (EtOH) w y. FAB-MS rp molecular ion peak ƒ + m/z 417[M+H], 1 H- 13 C-NMR rp ww molecular formula C 19 H 28 O 10 w š, l sy 6 w. 1 H-NMR rp δ 7.44~ 7.28 (5H, m) w y proton vj d w š, 4.87 (1H, d, J = 12.0 Hz, H-7a), 4.66 (1H, d, J =12.0 Hz, H-7b) AB-system vj d w. δ 4.79 (1H, d, J = 1.5 Hz, H-1"), δ 4.33 (1H, d, J =7.5Hz, H-1') anomeric proton peak, coupling constantƒ J=7.5 Hz, J=1.5Hz β-form α-form w d w. Aliphatic proton 1.28 (3H, d, J = 6.5 Hz, H-6") α- rhamnosyl methyl proton d w. 13 C-NMR rp 2 vj w y carbon δ (C-1), (C-2), (C-6), (C-3), (C-5), (C-4) d w. x 20) mw yw 4 benzyl-o-α-lrhamnopyranosyl(1 6)-β-D-glucopyranoside w š, x 21) data w yw 4 y w. (A. rosea) ƒ w w t w wš w w š p w ƒ x. MeOH l 7 w š yw»» w, yw 1 7-hydroxy-6-methoxycoumarin (scopoletin), yw 2 p-hydroxyphenethyl trans-ferulate, yw 3 1-(α-L-rhamnosyl(1 6)-β-D-glucopyranosyloxy)-3,4,5-trimethoxybenzene, yw 4 benzyl-oα-l-rhamnopyranosyl(1 6)-β-D-glucopyranoside, yw 5 suberic acid, yw 6 sebacic acid, yw 7 7-Oβ-D-glucopyranosyl-6-methoxycoumarin (scopolin) ƒƒ y w. 1-7 š yw. w w A549 ( ss ), SK-OV-3 (ù ), SK-MEL-2 (v ) HCT-15 ( ) 4 human tumor cells w x ü s w z w yw s w ùkû (ED 50 values>30.0 µg/ml).»» NMR ³ w œ»» z, FAB-MS EI-MS w» w. x 1. (2003) w, 712, w,.

5 226 Kor. J. Pharmacogn. 2. (1996) œ ªƒ Œ ~, 705, e,. 3.»y(2000) w, 332, w,. 4. Feng, Y., Xu, L., Yang, S. and Zou, Z. (2005) Chemical constituents isolated from flowers of Althaea rosea (I). Zhongcaoyo., 36: Matlawska, I. K. Z., Akad, M. I. K. M. and Poznan, P. (1992) Flavonoids in the flowers of Althaea rosea Cav. var. nigra (Malvaceae). Herba Polonica., 38: Asrea, K., Bucar, F., Witvrouw, M., and Pannecouque, C. (2001) Antiviral activity against human immunodeficiency virus type 1(HIV-1) and type 2 (HIV-2) of ethnobotanically selected Ethiopian medicinal plants. Phytother. Res. 15: Wang, D., Shang, J., Yu, Q. H. and Zhongguo, Z. Y. Z. Z. (1989) Analgesic and anti-inflammatory effects of the flower of Althaea rosea (L.) Cav. Zhongguo Zhongyao Zazhi, 14: Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMabon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S. and Boyd, M. R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst., 82: Kwon, Y. S., Shin, S. J., Kim, M. J. and Kim, C. M.(2001) A new coumarin from stem of Angelica dahurica. Arch. Pharm. Res., 25: Kim, J. S., Kim, J. C., Shin, S. H., Lee, E. J., Jin, W. Y., Bae, K. W., Son, K. H., Kim, H. P., Kang, S. S. and Chang, H. W. (2006) Chemical constituents of the root of Dystaenia takeshimana and their anti-inflammatory activity. Arch. Pharm. Res., 29: Razdan, T. K., Qadri, B., Harkar, S. and Waight, E. S.(1987) Chromones and coumarins from Skimmia laureola. Phytochemistry, 26: Junichi, K., Akiko, M., Yasuko and M., Yasuko, T. (1989) Studies on the constituents of Trichosanthes root. III. Constituents of roots of Trichosanthes bracteata Vougt. Yakugaku Zasshi, 109: Junichi, K. and Yasuko, T. (1989) Studies on the constituents of Trichosanthes root. I. Constituents of roots of Trichosanthes kirilowii Maxim. var. japonicum Kitam. Yakugaku Zasshi, 109: Awasthi, K. K., Misra and M. K. (1977) Chemical constituents of Caesalpinia pulcherrimas stem bark and flowers. J. Indian Chem Soc., 54: Kwon, Y. S. and Kim, C. M. (1996) A Study on the chemical constituents from Leaves of Fraxinus rhynchophylla. Kor. J. Pharmacogn., 27: Chang, B. S. and Kim, C. M. (2004) The Chemical structures and their antioxidant activity of the components isolated from the Heartwood of Hemiptelea davidii. Kor. J. Pharmacogn., 35: Hiroyuki, K., Yutaka, S., and Hiroko, S. (1982) A new phenolic compound from Heracleum lanatum Michx. var. nippinicum Hara. Chem. Pharm. Bull., 30: Faten M, M. D. and Manfred, G. R. (2003) Ecdysteroids and other constituents from Sida spinosa L. Phytochemistry, 62: Jaconnet, O. A., Christian, T., Sevser, S., Jocelyne, R. and Kurt, H. (1999) A phenolic glycoside and N-(p-coumaroyl)- tryptamine from Ravensara anisata. Phytochemistry, 52: Toshio, M., Akira, U., Nobuo, T., Hiromi, K. and Hiroko, K. (1987) Studies on the glycosides of Epimedium grandiflorum MORR. var. thunbergianum (MIQ.) Nakai. I. Chem. Pharm. Bull., 35: Nunziatina, D. T., Luca, R., Jativa, C., Giovanna and S., Cosimo, P. (1996) Aryl and triterpenenic glycosides from Margyricarpusetosus. Phytochemistry, 42: ( )