갈근으로부터분리한암세포증식저해물질. Anti-proliferative Components in the Roots Extract from. z w (Pueraria thunbergiana)

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1 w z Kor. J. Pharmacogn. 40() : 46 Ê 50 (2009) 갈근으로부터분리한암세포증식저해물질,2 Á,2 Á Á½ Á Áy Á½ Á½ ³ 3 Á 2 Á, * w yw, 2 ³ w w w, 3 w w w Anti-proliferative Components in the Roots Extract from Pueraria thunbergiana Mok Ryeon Baek,2, Yeon Hee Choi,2, Dae Seok Yoo, Mi Ri Kim, Sang Un Choi, Kyung Sik Hong, Young Sup Kim, Young-Kyoon Kim 3, Kang Ro Lee 2 and Shi Yong Ryu, * Korea Research Institute of Chemical Technology, Daejeon College of Pharmacy, Sungkyunkwan University, Suwon College of Forest Science, Kookmin University, Seoul , Korea Abstract The antitumor activity of the roots extract of Pueraria thunbergiana was investigated on the basis of cytotoxicity upon the cultured human tumor cell lines, in vitro. The purification of methylene chloride (MC) soluble part and ethylacetate (EA) soluble part of extract by column chromatography furnished seven isoflavonoids, two triterpenoids, one but-2-enolide. The structures of them were established by chemical and spectroscopic means to be lupeol (), β-sitosterol (2), biochanin A (3), (-)-tuberosin (4), calycosin (5), daidzein (6), puerarin (7), daidzin (8), (+)-puerol-b 2-O-β-glucopyranoside (9), formononetin- 7-O-β-glucopyranoside (0). Each isolates (~0) were evaluated for inhibitory activities on the proliferation of cultured human tumor cell lines such as A549, SK-OV-3, HCT-5 and SK-MEL-2, respectively. Key words Pueraria thunbergiana, Leguminosae, Isoflavonoid, Cytotoxic activity. (~ƒ, Pueraria thunbergiana Benth) g (Leguminosae) w w ù e w w, w. isoflavonoid daidzin, daidzein, puerarin, genistein, formononetin triterpenoid soyasapogenol A kudzusapogenol B, š polysaccharide, coumarin š š.,2) flavonoid y,, x q w š. 3) y w helicobacter pylori³ wy, ü ³ y y 4) z, w y z 5), g y 6) z, 7) allelopathy, w 8), z. A549 ( ss ) 4 s w x ü (in vitro) s &NBJM TZSZV!LSJDUSFLS 5FM wz t 300 w z w (Pueraria thunbergiana) methanol (MeOH) yw w z š. MeOH z w ƒ z ƒ chromatography mw w 0 w. 0 yw»» mw yw ³ ƒ yw s w s wz šwš w. x x w w ½ ³ z w tt w yw (KR ).»» H 3 C-NMR spectra Brucker 46

2 Vol. 40, No., AM-300 AMX 500 d w. s x sulforhodamine B (SRB) bioassay w ww x w s A549 (non small cell. 7) lung carcinoma), SK-OV-3 (ademoncarcinoma, ovary malignant ascites), SK-MEL-2 (malignant melanoma, metastasis to skin of thigh) HCT-5 (colon adenocarcinoma) human origin tumor cell line, (NCI) l w yw w. 2.0 kg MeOH 7 2z þew., w MeOH 2.0 kg, xk k z methylene chloride (MC) ethylacetate (EA), n-butanol (BuOH) zw MC z 93.0 g EA z 66.0 g, BuOH z g. MC z 77.0 g silica gel column chromatography (MC : MeOH = 00 :, 20 :, 5 :, : ) w 4 z (Fr.~Fr.4) ù, Fr.2 (32.9 g) silica gel column chromatography (n-hexane : EA = 5 :, 7 :, 3 : ) w 8 z (Fr.2~Fr.28) ù. Fr.22 Fr.23 ƒƒ (yw ) 3.5 g x (yw 2).7 g. Fr.25 (4.9 g) silica gel column chromatography (n-hexane : EA = 7 :, 5 :, 4 : ) RP-8 column chromatography (60~80% MeOH) w y (yw 3) mg w. Fr.26 (2.6 g) RP-8 column chromatography (20~60% MeOH) w y (yw 4) mg. Fr.4 (3.0 g) silica gel column chromatography (MC : MeOH = 50 :, 40 :, 30 :, 20 : ) w y (yw 5) mg. wr EA z l y z EA w (y w 6) 3.0 g. yw 6 w ù 25.0 g silica gel column chromatography (MC : MeOH = 50 :, 40 : ) w 9 z (Fr.E~ Fr.E9) ù š, Fr.E2 y (yw 7).8 g, Fr.E4 (yw 8) 4.4 g, Fr.E6 (yw 9) mg Fr.E8 y (yw 0) 0.0 mg ƒƒ. Lupeol () 3 C-NMR (25 MHz, CDCl (C-), 27.4 (C-2), 79.0 (C-3), 38.8 (C-4), 55.3 (C-5), 8.3 (C-6), 34.2 (C-7), 40.8 (C-8), 50.4 (C-9), 37. (C-0), 20.9 (C-), 25. (C-2), 38.0 (C-3), 42.8 (C-4), 27.4 (C-5), 35.5 (C-6), 43.0 (C-7), 48.3 (C-8), 47.9 (C- 9), 50.9 (C-20), 29.8 (C-2), 40.0 (C-22), 27.9 (C-23), 5.3 (C-24), 6. (C-25), 5.9 C-26), 4.5 (C-27), 8.0 (C-28), 09.3 (C-29), 9.3 (C-30) Biochanin A (3) H-NMR (300 MHz, DMSO-d (3H, s, -OMe), 6.20 (H, d, J = 2. Hz, H-6), 6.36 (H, d, J = 2. Hz, H-8), 6.96 (2H, d, J = 6.9 Hz, H-3', 5'), 7.46 (2H, d, J = 6.8 Hz, H-2', 6'), 8.32 (H, s, H-2) (-)-Tuberosin (4) H-NMR (300 MHz, DMSO-d 6.30 (3H, s, -CH 3 ),.33 (3H, s, -CH 3 ), 3.95 (H. d, J =.4 Hz, H-6), 3.99 (H, d, J =.4 Hz, H-6), 5.9 (H, s, H-a) 5.56 (H, d, J = 9.8 Hz, H-3'), 5.98 (H, s, OH-6a), 6.9 (H, s, H-0), 6.20 (H, d, J = 2.2 Hz, H- 4), 6.35 (H, d, J = 9.8 Hz, H-4'), 6.44 (H, dd, J = 8.4, 2.2 Hz, H-2), 7.07 (H, s, H-7), 7.22 (H, d, J = 8.4 Hz, H-), 9.66 (H, d, J = 2.2 Hz, H-4); 3 C-NMR (25 MHz, DMSO-d (C-),.4 (C-a), 09.9 (C-2), 58.7 (C-3), 02.6 (C-4), 55.6 (C-4a), 69.3 (C-6), 75.0 (C- 6a), 2.7 (C-7), 22.0 (C-7a), 4.4 (C-8), 54.7 (C-9), 98.4 (C-0), 59.8 (C-0a), 84.5 (C-a), 27.6 (-(CH 3 ) 2 -), 27.7 (-(CH 3 ) 2 -), 76.3 (C-2'), 27.6 (C-3'), 2.8 (C-4') Calycosin (5) H-NMR (300 MHz, DMSO-d (3H, s, -OMe), 6.86 (H, d, J = 8.2 Hz, H-5'), 6.9 (H, d, J = 2.2 Hz, H-8), 6.99 (H, dd, J = 8.8, 2.2 Hz, H-6), 7.04 (H, dd, J = 8.2,.9 Hz, H-6'), 7.2 (H, d, J =.9 Hz, H-2'), 8.02 (H, d, J = 8.8 Hz, H-5), 8.38 (H, s, H-2), 9.05 (H, s, OH-3') Daidzein (6) H-NMR (300 MHz, DMSO-d (2H, d, J = 8.6 Hz, H-3', 5'), 6.85 ( H, d, J = 2. Hz, H-8), 6.93 (H, dd, J = 8.7, 2. Hz, H-6), 7.37 (2H, d, J = 8.6 Hz, H-2', 6'), 7.96 (H, d, J = 8.7 Hz, H- 5), 8.27 (H, s, H-2); 3 C-NMR (25 MHz, DMSO-d (C-2), 24.2 (C-3), 75.4 (C-4), 27.9 (C-5), 5.8 (C-6), 63. (C-7), 02.7 (C-8), 57.8 (C-9), 7.3 (C-0), 23.2 (C-'), 30.7 (C-2'), 5.6 (C-3'), 58. (C-4'), 5.6 (C-5'), 30.7 (C-6') Puerarin (7) H-NMR (300 MHz, DMSO-d (H, d, J = 9.8 Hz, H-Glc-), 6.79 (2H, d, J = 8.6 Hz, H-3', 5'), 6.98 (H, d, J = 8.8 Hz, H-6), 7.39 (2H, d, J = 8.6 Hz, H-2', 6'), 7.93 (H, d, J = 8.8 Hz, H-5), 8.34 (H, s, H-2) Daidzin (8) H-NMR (300 MHz, DMSO-d 6 +D 2 O 5.08 (H, d, J = 7.4 Hz, H-Glc-), 6.8 (2H, d, J = 8.6 Hz, H-3', 5'), 7.4 (H, dd, J = 8.9, 2.2 Hz, H-6), 7.23 (H, d, J = 2.2 Hz, H-8), 7.40 (2H, d, J = 8.6 Hz, H-2', 6'), 8.04 (H, d, J = 8.9 Hz, H-5), 8.39 (H, s, H-2); 3 C- NMR (25 MHz, DMSO-d (C-2), 23.7 (C-3), 74.7 (C-4), 26.9 (C-5), 5.5 (C-6), 6.4 (C-7), 03.4 (C-8), 57.2 (C-9), 8.4 (C-0), 22.3 (C-'), 30.0 (C-

3 48 Kor. J. Pharmacogn. Fig.. Structures of compounds ~0 2'), 4.9 (C-3'), 56.9 (C-4'), 4.9 (C-5'), 30.0 (C-6'), 99.9 (Glc C-), 73. (Glc C-2), 76.5 (Glc C-3), 69.6 (Glc C-4), 77.2 (Glc C-5), 60.6 (Glc C-6) (+)-Puerol-B 2-O-β-glucopyranoside (9) H-NMR (300 MHz, DMSO-d (H, dd, J = 7.8, 4.5 Hz, H-4a), 3.03 (H, dd, J = 4.5, 2.6 Hz, H'-4a), 3.82 (H, s, -OMe), 5.0 (H, d, J = 5.2 Hz, H-Glc-), 6.00 (H, m, H-4), 6.33 (H, d, J =.4 Hz, H-2), 6.6 (2H, d, J = 8.5 Hz, H-3', 5'), 6.7 (H, dd, J = 8.7, 2.4 Hz, H-5"), 6.88 (H, d, J= 2.4 Hz, H-3"), 6.94 (2H, d, J = 8.5 Hz, H-2', 6'), 7.56 (H, d, J = 8.7 Hz, H-6"); 3 C-NMR (25 MHz, DMSOd (C-), 3.3 (C-2), 65.9 (C-3), 83.6 (C-4), 38.2 (C-4a), 26.6 (C-'), 30.3 (C-2'), 4.8 (C-3'), 55.9 (C-4'), 4.8 (C-5'), 30.3 (C-6'), 2. (C-"), 56.3 (C- 2"), 0.2 (C-3"), 62.9 (C-4"), 08.5 (C-5"), 3.3 (C- 6"), 55.4 (OMe), 00.2 (Glc C-), 73. (Glc C-2), 76.5 (Glc C-3), 69.9 (Glc C-4), 77.4 (Glc C-5), 60.8 (Glc C-6) Formononetin-7-O-β-glucopyranoside (0) H-NMR (300 MHz, DMSO-d (3H, s, -OMe), 5.08 (H, d, J = 4.7 Hz, H-Glc-), 6.98 (2H, d, J = 8.4 Hz, H-3', 5'), 7.4 (H, d, J = 8.8 Hz, H-6), 7.23 (H, s, H-8), 7.52 (2H, d, J = 8.4 Hz, H-2', 6'), 8.04 (H, d, J = 8.8 Hz, H-5), 8.43 (H, s, H-2); 3 C-NMR (25 MHz, DMSOd (C-2), 23.3 (C-3), 74.6 (C-4), 26.9 (C-5), 5.6 (C-6), 6.4 C-7), 03.4 (C-8), 57.0 (C-9), 8.4 (C-0), 23.9 (C-'), 30.0 (C-2'), 3.6 (C-3'), 58.9 (C- 4'), 3.6 (C-5'), 30.0 (C-6'), 55. (OMe), 99.9 (Glc C- ), 73. (Glc C-2), 77.2 (Glc C-3), 69.6 (Glc C-4), 76.4 (Glc C-5), 60.6 (Glc C-6) s wy (in vitro cytotoxicity) d x w s A-549 (non small cell lungcarcinoma), SK-OV-3 (adenocarcinoma, ovary malignant ascites), SK-MEL-2 (malignant melanoma, metastasis to skin of thigh), HCT 5 (colon adenocarcinoma) s

4 Vol. 40, No., human origin tumor cell line, (NCI) l w yw w s wy NCI protocol d w. 9) š yw H-NMR spectrum 3 C-NMR spectrum mwš tt w lupeol w. yw 2 x 0) H-NMR Ÿ w data x) w β-sitosterol y. yw 3 y H-NMR 3 C-NMR data x2) w biochanin A w. yw 4 y H-NMR spectrum 3 C-NMR spectrum mwš tt w (-)- tuberosin w. wr yw 5 y 3) x4) w calycosin ew. yw 6 H-NMR 3 C-NMR data tt w daidzein y w. yw 7 2,5) spectral data x6)e w, daidzein (yw 6) 8 carbon glucoseƒ w C- puerarin y w. yw 8 H-NMR 3 C- NMR data tt w daidzein (yw 6) 7 hydroxy» glucoseƒ w daidzin y w yw 9 H-NMR spectrum isoflavonoid. 3) H-NMR spectrum w ù, isoflavonoid H-NMR spectrum p 2 proton signal (8.32 ppm). 3 C-NMR data w aromatic ring w furan ring, w glucosideƒ w d w, x7) w yw 9 (+)-puerol-b 2-O-β-glucopyranoside y w. yw 0 H-NMR 3 C-NMR data mw daidzin (yw 8) 4 e hydroxy»ƒ methoxy» ey š, tt w formononetin-7-o-βglucopyranoside y w. 8,9) wr, ƒ yw (~0) ƒƒ SRB (Sulfrhodamine B) bioassay w A549 ( ss ), SK-OV-3 (ù ), SK-MEL-2 (v ) HCT-5 ( ) 4 human tumor cells w x ü s w z w yw, 3~6 ƒƒ s w IC 50 (50% s w z ùkü )e doxorubicin w x w û w s wz (Table I). Table I. Inhibition of tumor cell proliferation by components isolated from Pueraria thunbergiana. Compound IC 50 (µg/ml) a A549 SK-OV-3 SK-MEL-2 HCT >30 >30 >30 > >30 >30 >30 >30 8 >30 >30 >30 >30 9 >30 >30 >30 >30 0 >30 >30 >30 >30 doxorubicin a IC 50 value of compounds against each cancer cell line, which was defined as a concentration (µg/ml) that caused 50 % inhibition of cell proliferation in vitro. (~ƒ, Pueraria thunbergiana Benth) MeOH z z chromatography w 7 isoflavonoid yw, 2 triterpenoid yw, but-2-enolide yw w. yw ƒƒ yw»» ( H- NMR, 3 C-NMR) data lupeol (), β-sitosterol (2), biochanin A (3), (-)-tuberosin (4), calycosin (5), daidzein (6), puerarin (7), daidzin (8), (+)-puerol-b 2-O-β-glucopyranoside (9), formononetin-7-o-β-glucopyranoside (0) y w. yw SRB bioassay mw human tumor cell w s w yw, 3~6 w s w z ùkþ.» z» x w» ( y : S20808L004) ww. x. Jun, M., Fu, H. Y., Hong, J., Wan, X., Yang, C. S. and Ho, C. T. (2003) Comparison of antioxidant activities of isoflavones from kudzu root (Pueraria lobata Ohwi). Journal of Food

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