Vol. 47, No. 3, diammonium salt(abts), Fe 2+ antioxidant potential(frap) 를측정하였다. 재료및방법 chelating, ferric reducing 실험재료 연구에사용된배롱나무는 2012년 5월충북

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1 생약학회지 Kor. J. Pharmacogn. 47(3) : (2016) 배롱나무의항산화활성성분 우경완 1,2 심미옥 2 박종일 2 김민석 2,3 서원세 1 조현우 2 권학철 4 박종철 5 이강노 1 * 1 성균관대학교약학대학천연물약품화학연구실, 2 한약진흥재단한약재연구팀, 3 원광대학교한의학과병리학교실, 4 한국과학기술연구원천연물연구소, 5 순천대학교한약자원개발학과 Chemical Constituents from the Stems of Lagerstroemia indica and Their Anti-oxidant Effect Kyeong Wan Woo 1,2, Mi Ok Sim 2, Eel Jong Park 2, Min Suk Kim 2,3, Won Se Suh 1, Hyun Woo Cho 2, Hak Cheol Kwon 4, Jong Cheol Park 5, and Kang Ro Lee 1 * 1 Natural Product Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Korea 2 Traditional Korean Medicines Research Team, Naional Development Institute of Korea Medicine, 288, Woodlandgil, Anyangmyeon, Jangheunggun, Jeollanamdo 59338, Korea 3 Department of Pathology, College of Korean Medicine, Wonkwang University, Iksan 54538, Korea 4 Natural Product Research Center, Korea Institute of Science and Technology, Gangnueng, Gangwon-do 25451, Korea 5 Department of Oriental Medicine Resources, Sunchon National University, Suncheon 57922, Korea Abstract Phytochemical investigation of the 80% MeOH extract from the stems of Lagerstroemia indica resulted in the isolation of eighteen compounds; four norsesquiterpenes, fourteen phenolic derivatives. Their chemical structures were characterized by spectroscopic methods to be tachioside (1), isotachioside (2), 2,4,6-trimethoxyphenyl β-d-glucopyranoside (3), gallic acid 4-methyl ether (4), protocatechuic acid (5), gallic acid (6), vanillic acid (7), vanillin (8), 2-methoxy-5- hydroxymethyl-phenyl-1-o-(6''-galloyl)-β-d-glucopyranoside (9), 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside (10), 4-hydroxy-3-methoxyphenyl-1-O-(6'-O-galloyl)-β-D-glucopyranoside (11), vomifoliol (12), vomifoliol 9-O-β-Dglucopyranoside (13), 6R, 9R- 3- oxo- α- ionol- 9- O- β- D- glucopyranoside (14), dihydrophaseic acid 4'-O-β-D-glucopyranoside (15), β-hydroxypropiovanillone 3-O-β-D-glucopyranoside (16), myrciaphenone A (17), and coumaric acid (18). Compounds 1-5 and 7-18 were isolated for the first time from this plant. Compounds 1-18 were investigated for their antioxidant properties using DPPH and ABTS radical scavenging capacity assay, Fe 2+ chelating, and FRAP assay. It was found that 4, 6, and 11 possessed the highest antioxidant capacities. Key words Lythraceae, Lagerstroemia indica, Norsesquiterpene, Phenol, Anti-oxidant effect 배롱나무 (Lagerstroemia indica) 는부처꽃과 (Lythraceae) 에속하며, 낙엽교목으로원산지는중국이고, 우리나라에는관상용으로이용되고있다. 1) 주로충청도이남지역에서자생하고, 나무껍질은연한홍자색이며얇은조각으로떨어지고흰무늬가생긴다. 꽃은 7~9 월에홍색으로피고, 잎은타원형또는달걀을거꾸로세워놓은모양이다. 2) 배롱나무는한방에서자미화 ( 紫薇花 ), 자미근 ( 紫薇根 ) 으로꽃과뿌리를썼으며각각은감기, 이뇨제로사용하였다. 3) 배롱나무에서 * 교신저자 ( ):krlee@skku.edu (Tel): 연구된식물화학적성분으로는 phenol 성화합물, 4) triterpene, 5) alkaloid 6) 등이있고, 그중몇몇화합물은항염증및항암활성을나타내었다. 본연구에서는국내에자생하는천연자원으로부터활성성분연구의일환으로, 배롱나무가지추출물에서추가적으로성분연구를진행하였다. 배롱나무 80% MeOH 추출물을극성별용매분획하고, 각각의분획을 column chromatography 법을이용하여 18 종의화합물을분리하였고, 구조는 1 H, 13 C NMR 그리고 MS 를이용하여결정하였다. 분리된화합물의항산화활성을확인하기위해 2,2-,diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) 204

2 Vol. 47, No. 3, diammonium salt(abts), Fe 2+ antioxidant potential(frap) 를측정하였다. 재료및방법 chelating, ferric reducing 실험재료 연구에사용된배롱나무는 2012년 5월충북괴산에서채취하였으며, 교신저자인이강노교수가동정하여사용하였다. 음건하여세절후사용하였으며, 표본 (SKKU- NPL-1203) 은성균관대학교약학대학표본실에보관하고있다. 기기및시약 1 H NMR과 13 C NMR spectra는 Varian UNITY INOVA 500 NMR spectrometer를이용하여측정하였다. IR spectra는 Bruker IFS-66/S FT-IR spectrometer를이용하였다. FAB mass spectra는 JEOL JMS700 mass spectrometer를이용하였다. Semi-preparative HPLC는 Gilson 306 pump와 Shodex refractive index detector를함께이용하였고, column으로는 J sphere ODS-M80 column( mm I.D.) 을이용하였다. Column chromatography에이용된충진제는 silica gel 60(Merk Co., mesh), RP-C18 silica gel(ymc GEL ODS-A, 12 nm, S-75 μm) 과 Sephadex LH-20(Pharmacia Co.) 가이용되었다. TLC는 Merck precoated silica gel F254 plates를이용하였으며, RP TLC 로는 RP-C18 F254s plates가이용되었다. UV light를이용하여 254 nm와 365 nm 파장에서 1차적으로확인하고 anisaldehyde-sulfuric acid를이용하여발색확인하였다. 2,2- Diphenyl-1-picrylhydrazyl(DPPH), 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(abts), potassium persulfate, 2,4,6-tri(2-pyridyl)-striazine(TPTZ), iron(iii) chloride hexa-hydrate(fecl 3.6H 2 O), FeSO 4.7H 2 O, iron(ii) chloride, ferrozine, folin-ciocalteu reagent, sodium carbonate(na 2 CO 3 ), aluminium nitrate, potassium acetate등은 Sigma-Aldrich에서구입해서사용하였으며, ELISA microplate reader(infinite 200 pro, TECAN, Austria) 를이용하여흡광도값을측정하였다. 추출및분리 배롱나무 (L. indica) 의가지 (5 kg) 는 80% MeOH 용매를이용하여추출및여과하였다. 여과된추출액은감압농축기를이용, 농축하여 MeOH 농축액 (300 g) 을얻었다. MeOH 농축액을증류수 800 ml에녹인후에 n- hexane, chloroform, ethyl acetate, n-butanol을이용해순차적인용매분획을시행하여각각 17, 9, 8, 55 g을얻었다. Chloroform 분획 9g을 silica gel column( mesh, 360 g), chloroform/meoh(30:1~1:1) 조건으로진행하였고, 4개의소분획 (C1-C4) 을얻었다. 소분획 C1(2.2 g) 은 silica gel column(chloroform/meoh=60:1) 을이용하여 4개로 (C11- C14) 으로나누었다. C12(1.0 g) 을 RP-C 18 silica gel column (100% MeOH) 을진행하였고, 그중 C121(200 mg) 을 RP- C 18 semi-prep HPLC(35% CH 3 CN) 정제과정을거쳐화합물 8(13 mg) 을얻었다. C127(25 mg) 또한 RP-C 18 semiprep HPLC(95% CH 3 CN) 정제과정을거쳐화합물 13(9 mg) 을얻을수있었다. C14(900 mg) 을 RP-C 18 silica gel column(90% MeOH) 을진행하였고, 그중 C141을 Sephadex (80% MeOH) 와 RP-C 18 semi-prep HPLC(30% CH 3 CN) 정제과정을거쳐화합물 12(9 mg) 을얻었다. Ethyl acetate분획 8g을 RP-C 18 silica gel colum을 90% MeOH 조건으로진행하였고, 10개의소분획 (E1-E10) 을얻었다. 소분획 E1(1.3 g) 을 silica gel column(chloroform/meoh=7:1) 와 RP-C 18 semi-prep HPLC(25% CH 3 CN) 정제과정을거쳐화합물 6(9 mg) 을얻었다. 소분획 E2(500 mg) 을 Sephadex(80% MeOH) 와 RP-C 18 semi-prep HPLC(30% CH 3 CN) 정제과정을거쳐화합물 3(9 mg), 5(13 mg), 그리고 11(110 mg) 을얻었다. 소분획 E3(500 mg) 을 Sephadex(80% MeOH) 와 RP- C 18 semi-prep HPLC(40% CH 3 CN) 정제과정을거쳐화합물 4(45 mg) 과 18(3 mg) 을얻었다. 소분획 E4(400 mg) 을 Sephadex(80% MeOH) 와 RP-C 18 semi-prep HPLC(45% CH 3 CN) 정제과정을거쳐화합물 7(20 mg) 을얻었다. 소분획 E5(3.2 g) 을 RP-C 18 silica gel column(40~100% MeOH) 을진행하였고, 그중 E51을 Sephadex(80% MeOH) 와 RP- C 18 semi-prep HPLC(50% CH 3 CN) 정제과정을거쳐화합물 10(21 mg) 과 19(10 mg) 을얻었다. Butanol분획 55 g을 HP-20을진행하여당부분을제거한 B분획을 silica gel column(chloroform/meoh=5:1) 을이용하여 4개로 (B1-B4) 으로나누었다. B2(2.7 g) 을 RP-C 18 silica gel colum(90% MeOH) 조건으로진행하였고, 9개소분획 (B21-B29) 으로분리하였다. 소분획 B21(520 mg) 을 Sephadex(80% MeOH) 와 RP-C 18 semi-prep HPLC(10% CH 3 CN) 정제과정을거쳐화합물 1(20 mg), 2(15 mg) 그리고 15(5 mg) 을얻었다. 소분획 B22(500 mg) 을 Sephadex(80% MeOH) 와 RP-C 18 semiprep HPLC(15% CH 3 CN) 정제과정을거쳐화합물 16(11 mg) 을얻었다. 소분획 B27(40 mg) 을 Sephadex(80% MeOH) 와 RP-C 18 semi-prep HPLC(30% CH 3 CN) 정제과정을거쳐화합물 14(4 mg) 을얻었다. 화합물 1 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 6.80 (1H, d, J=3.0 Hz, H-2), 6.73 (1H, d, J=8.5 Hz, H-5), 6.57 (1H, dd, J=8.5, 3.0 Hz, H-6), 4.80 (1H, d, J=7.5 Hz, H-1'), 3.68 (3H, s, 3-OCH 3 ); 13 C NMR (125 MHz, CD 3 OD) δ (C-4), (C-2), (C-1), (C-6), (C-5), (C-1'), (C-3), 76.9 (C-3'), 76.6 (C-5'), 73.9 (C-2'), 70.3 (C-4'), 61.4 (C- 6'), 55.6 (3-OCH 3 ); FAB MS: m/z 303 [M+H] +. 화합물 2 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.02 (1H, d, J=9.0 Hz, H-5), 6.80 (1H, d, J=3.0 Hz, H-2), 6.28 (1H, dd, J=9.0, 3.0 Hz, H-6), 4.80

3 206 Kor. J. Pharmacogn. (1H, d, J=7.5 Hz, H-1'), 3.70 (3H, s, 3-OCH 3 ); 13 C NMR (125 MHz, CD 3 OD) δ (C-4), (C-2), (C-1), (C-6), (C-5), (C-1'), (C-3), 76.9 (C-3'), 76.6 (C-5'), 73.9 (C-2'), 70.3 (C-4'), 61.4 (C- 6'), 55.4 (3-OCH 3 ); FAB MS: m/z 303 [M+H] +. 화합물 3 Colorless gum; 1 H NMR (600 MHz, CD 3 OD) δ 6.48 (2H, s, H-3, 5), 4.83 (1H, d, J=7.5 Hz, H-1'), 3.91 (1H, dd, J=8.0, 3.0 Hz, H-2'), 3.80 (6H, s, OCH 3 ), 3.69 (3H, s, H-9), 3.65 (1H, t, J=6.5, 2.5 Hz, H- 4'), 3.44 (2H, t, J=6.5, 2.5 Hz, H-3', 5'); 13 C NMR (150 MHz, CD 3 OD) δ (C-4), (C-2, 6), (C- 1'), 96.1 (C-3, 5), 78.4 (C-5'), 78.1 (C-3'), 75.0 (C-2'), 71.7 (C-4'), 62.7 (C-6'), 61.2 (C-9), 56.5 (OCH 3 ); FAB MS: m/z 347 [M+H] +. 화합물 4 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.07 (2H, s, H-2, 6), 3.83 (3H, s, 4-OCH 3 ); 13 C NMR (125 MHz, CD 3 OD) δ (C-7), (C-3, 5), (C-4), (C-1), (C-2, 6), 52.4 (4- OCH 3 ); FAB MS: m/z 185 [M+H] +. 화합물 5 White amorphous powder; 1 H NMR (500 MHz, CD 3 OD) δ 7.43 (1H, d, J=2.0 Hz, H-2), 7.40 (1H, dd, J=8.0, 2.0 Hz, H-6), 6.79 (1H, d, J=8.0 Hz, H- 5); 13 C NMR (125 MHz, CD 3 OD) δ (C-7), (C-4), (C-3), (C-6), (C-1), (C-2), (C-5); FAB MS: m/z 155 [M+H] +. 화합물 6 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.10 (2H, s, H-2, 6); 13 C NMR (125 MHz, CD 3 OD) δ (C-7), (C-3, 5), (C-4), (C-1), (C-2, 6); FAB MS: m/z 171 [M+H] +. 화합물 7 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.60 (1H, dd, J=8.0, 1.5 Hz, H-6), 7.56 (1H, d, J=1.5 Hz, H-2), 6.92 (1H, d, J=8.0 Hz, H-5), 3.91 (3H, s, 3-OCH 3 ); 13 C NMR (125 MHz, CD 3 OD) δ (C-7), (C-3), (C-4), (C-1), (C-6), (C-2), (C-5), 56.3 (3-OCH 3 ); FAB MS: m/z 169 [M+H] +. 화합물 8 White powder; 1 H NMR (500 MHz, CDCl 3 ) δ 9.83 (1H, s, H-7), 7.44 (1H, dd, J=8.5, 2.0 Hz, H-6), 7.43 (1H, d, J=2.0 Hz, H-2), 7.05 (1H, d, J=8.5 Hz, H-5), 3.97 (3H, s, 3-OCH 3 ); 13 C NMR (125 MHz, CDCl 3 ) δ (C-7), (C-3), (C-4), (C-1), (C-6), (C-5), (C-2), 56.3 (3-OCH 3 ); FAB MS: m/z 153 [M+H] +. 화합물 9 Yellowish gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.10 (2H, s, H-2', 6'), 7.07 (1H, d, J=8.0 Hz, H-3), 7.03 (1H, d, J=2.0 Hz, H-6), 6.75 (1H, dd, J=8.0, 2.0 Hz, H-4), 4.89 (1H, d, J=7.5 Hz, H-1''), 4.59 (1H, dd, J=12.0, 3.0 Hz, H-6a''), 4.52 (1H, s, H-7), 4.42 (1H, dd, J=12.0, 5.0 Hz, H-6b''), 3.86 (3H, s, 2-OCH 3 ), 3.73 (1H, m, H-5''), 3.54 (1H, dd, J=9.5, 7.5 Hz, H-2''), 3.52 (1H, t, J=9.5 Hz, H-3''), 3.47 (1H, t, J=9.5 Hz, H-4''); 13 C NMR (125 MHz, CD 3 OD) δ (C-7'), (C-2), (C-1), (C-3', 5'), (C-4'), (C-5), (C-1'), (C-4), (C-3), (C-6), (C-2', 6'), (C-1''), 77.8 (C-3''), 75.5 (C-5''), 74.7 (C-2''), 71.8 (C-4''), 64.8 (C-7, 6''), 56.7 (2-OCH 3 ); FAB MS: m/z 469 [M+H] +. 화합물 10 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.05 (2H, s, H-2', 6'), 6.37 (2H, s, H-3, 5), 4.81 (1H, d, J=7.5 Hz, H-1''), 4.61 (1H, dd, J=12.0, 3.0 Hz, H-6a''), 4.41 (1H, dd, J=12.0, 5.0 Hz, H-6b''), 3.76 (1H, m, H-5''), 3.67 (3H, s, 2, 6-OCH 3 ), 3.62 (4-OCH 3 ), 3.50~3.33 (3H, m, glc); 13 C NMR (125 MHz, CD 3 OD) δ (C-1'), (C-2, 6), (C-4), (C-3', 5'), (C-4'), (C-1), (C-1'), (C-2', 6'), (C-1''), 94.8 (C-3, 5), 76.2 (C-3''), 74.4 (C-5''), 73.4 (C-2''), 70.3 (C-4''), 63.5 (C-6''), 59.5 (2, 6-OCH 3 ), 55.1 (4-OCH 3 ); FAB MS: m/z 499 [M+H] +. 화합물 11 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.19 (2H, s, H-2', 6'), 6.74 (1H, d, J=2.0 Hz, H-6), 6.68 (1H, d, J=8.0 Hz, H-2), 6.60 (1H, dd, J=8.0, 2.0 Hz, H-3), 4.90 (1H, d, J=8.0 Hz, H-1''), 4.70 (1H, dd, J=12.0, 3.0 Hz, H-6a''), 4.38 (1H, dd, J=12.0, 5.0 Hz, H- 6b''), 3.72 (3-OCH 3 ), 3.76~3.47 (4H, m, glc); 13 C NMR (125 MHz, CD 3 OD) δ (C-7'), (C-1), (C-3), (C-3', 5'), (C-4), (C-4'), (C-1'), (C-5), (C-2', 6'), (C-6), (C-1''), (C-2), 77.1 (C-3''), 74.6 (C-5''), 74.2 (C-2''), 71.1 (C-4''), 64.6 (C-6''), 56.2 (3-OCH 3 ); FAB MS: m/z 455 [M+H] +. 화합물 12 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 5.87 (1H, s, H-4), 5.79 (1H, dd, J=15.5, 6.5 Hz, H-8), 5.78 (1H, d, J=15.5 Hz, H-7), 4.31 (1H, m, H- 9), 2.47 (1H, d, J=16.5 Hz, H-2b), 2.15 (1H, d, J=16.5 Hz, H-2a), 1.90 (3H, s, H-13), 1.23 (3H, d, J = 6.5 Hz, H-10), 1.03 (3H, s, H-11), 1.00 (3H, s, H-12); 13 C NMR (125 MHz, CD 3 OD) δ (C-3), (C-5), (C-8), (C-7), (C-4), 79.9 (C-6), 68.7 (C-9), 49.9 (C-2), 42.2 (C-1), 24.7 (C-12), 23.8 (C-10), 23.5 (C- 11), 20.0 (C-13); FAB MS: m/z 225 [M+H] +. 화합물 13 Amorphous powder; 1 H NMR (500 MHz, CD 3 OD) δ 5.88 (1H, s, H-4), 5.79 (1H, dd, J=15.5, 6.5

4 Vol. 47, No. 3, Hz, H-8), 5.78 (1H, d, J=15.5 Hz, H-7), 4.41 (1H, d, J=7.5 Hz, H-1'), 4.31 (1H, m, H-9), 3.85 (1H, dd, J = 12.5, 2.0 Hz, H-6a'), 3.62 (1H, dd, J = 12.5, 5.0 Hz, H- 6b'), 3.36 (1H, m, H-3'), 3.28 (2H, m, H-4', 5'), 3.15 (1H, t, J=8.5 Hz, H-2'), 2.47 (1H, d, J=16.5 Hz, H-2b), 2.15 (1H, d, J=16.5 Hz, H-2a), 1.90 (3H, s, H-13), 1.23 (3H, d, J=6.5 Hz, H-10), 1.03 (3H, s, H-11), 1.01 (3H, s, H- 12); 13 C NMR (125 MHz, CD 3 OD) δ (C-3), (C-5), (C-8), (C-7), (C-4), (C-1'), 79.9 (C-6), 78.0 (C-3', 5'), 77.3 (C-9), 75.2 (C-2'), 71.6 (C-4'), 62.8 (C-6'), 50.7 (C-2), 42.4 (C-1), 24.5 (C-12), 23.8 (C-11), 21.2 (C-10), 19.6 (C-13); FAB MS: m/z 387 [M+H] +. 화합물 14 Amorphous powder; 1 H NMR (500 MHz, CD 3 OD) δ 5.87 (1H, s, H-4), 5.77 (1H, dd, J=15.5, 6.0 Hz, H-8), 5.63 (1H, dd, J=15.5, 9.0 Hz, H-7), 4.39 (1H, m, H-9), 4.34 (1H, d, J=7.5 Hz, H-1'), 3.83 (1H, dd, J=12.5, 2.0 Hz, H-6a'), 3.64 (1H, dd, J=12.5, 5.0 Hz, H- 6b'), 3.34 (1H, m, H-3'), 3.27 (2H, m, H-4', 5'), 3.13 (1H, t, J=8.5 Hz, H-2'), 2.67 (1H, d, J=16.5 Hz, H-2b), 2.43 (1H, d, J=16.5 Hz, H-2b), 2.03 (1H, d, J=16.5 Hz, H-2a), 1.93 (3H, s, H-13), 1.25 (3H, d, J=6.5 Hz, H-10), 1.02 (3H, s, H-11), 1.00 (3H, s, H-12); 13 C NMR (125 MHz, CD 3 OD) δ (C-3), (C-5), (C-8), (C-7), (C-4), (C-1'), 78.1 (C-3'), 78.0 (C-5'), 77.0 (C-9), 75.2 (C-2'), 71.4 (C-4'), 62.2 (C-6'), 56.8 (C- 6), 48.3 (C-2), 37.1 (C-1), 28.0 (C-12), 27.6 (C-11), 23.8 (C-13), 21.0 (C-10); FAB MS: m/z 371 [M+H] +. 화합물 15 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.96 (1H, d, J=16.5 Hz, H-8), 6.49 (1H, d, J=16.5 Hz, H-7), 4.36 (1H, d, J=7.5 Hz, H-1'), 4.25 (1H, m, H-3), 3.87 (1H, dd, J=12.5, 5.0 Hz, H-6b'), 3.80 (1H, dd, J=7.5,2.0 Hz, H-11b), 3.75 (1H, d, J=7.5 Hz, H-11a), 3.67 (1H, dd, J= 2.5, 2.0 Hz, H-6a'), 3.28 (1H, m, H-5'), 3.14 (1H, dd, J=9.0, 8.0 Hz, H-2'), 2.19 (1H, m, H-2b), 2.07 (3H, s, H-10), 1.98 (1H, m, H-2a, 4a), 1.80 (1H, m, H-4a), 1.17 (3H, s, H-13), 0.94 (3H, s, H-12); 13 C NMR (125 MHz, CD 3 OD) δ (C-9), (C-7), (C-8), (C-1'), 87.6 (C-5), 83.2 (C-6), 78.1 (C-3'), 78.0 (C-5'), 77.2 (C-11), 75.1 (C-2'), 73.9 (C-3), 71.7 (C- 4'), 62.8 (C-6'), 49.9 (C-1), 42.9 (C-4), 42.8 (C-2), 21.2 (C-10), 19.7 (C-13), 16.3 (C-12); FAB MS: m/z 445 [M+H] +. 화합물 16 Colorless gum; 1 H NMR (600 MHz, CD 3 OD) δ 7.59 (1H, d, J=8.5 Hz, H-6'), 7.53 (1H, s, H- 2'), 6.83 (1H, d, J=8.5 Hz, H-5'), 4.34 (1H, d, J=8.0 Hz, H-1''), 4.26 (1H, m, H-3), 3.97 (1H, m, H-3), 3.89 (3H, s, 3-OCH 3 ), 3.85 (1H, dd, J=13.0, 6.0 Hz, H-6''b), 3.68 (1H, dd, J=13.0, 4.5 Hz, H-6''a), 3.38 (1H, m, H-5''), 3.34 (2H, s, H-2), 3.17 (1H, dd, J=9.0, 8.0 Hz, H-2''); 13 C NMR (150 MHz, CD 3 OD) δ (C-1), (C-3), (C-4), (C-1), (C-6), (C-2), (C-5), (C-1'), 78.4 (C-3'), 78.4 (C-5'), 75.4 (C-2'), 71.9 (C- 4'), 66.8 (C-3), 63.9 (C-6'), 56.8 (3-OCH 3 ), 39.6 (C-2); FAB MS: m/z 359 [M+H] +. 화합물 17 White powder; 1 H NMR (500 MHz, CD 3 OD) δ 6.18 (1H, d, J=2.0 Hz, H-3), 5.94 (1H, d, J=2.0 Hz, H-5), 5.02 (1H, d, J=7.5 Hz, H-1'), 3.91 (1H, dd, J=12.0, 2.5 Hz, H-6''a), 3.72 (1H, dd, J=12.0, 5.0 Hz, H-6''b), 2.69 (2H, s, H-8); 13 C NMR (125 MHz, CD 3 OD) δ (C-7), (C-6), (C-4), (C-2), (C-1), (C-1'), 98.2 (C-5), 95.4 (C-3), 78.6 (C- 5'), 78.4 (C-3'), 74.8 (C-2'), 71.2 (C-4'), 62.5 (C-6'), 33.5 (C-8); FAB MS: m/z 331 [M+H] +. 화합물 18 Colorless gum; 1 H NMR (500 MHz, CD 3 OD) δ 7.62 (1H, d, J=16.5 Hz, H-7), 7.44 (1H, d, J=8.5 Hz, H-2, 6), 6.89 (2H, d, J=8.5 Hz, H-3, 5), 6.35 (1H, d, J=16.0 Hz, H-8); 13 C NMR (125 MHz, CD 3 OD) δ (C-9), (C-4), (C-7) (C-1), (C-2, 6), (C-3, 5), (C-8); FAB MS: m/z 165 [M+H] +. DPPH 라디칼소거능 각시료의전자공여능은 DPPH 의환원력을이용하여측정하였다. 즉각시료 100 μl에 100 μm DPPH 용액 100 μl를첨가하여상온에서 30분간반응시킨후 microplate reader를사용하여 517 nm에서흡광도를측정하였다. ABTS 저해활성도측정 ABTS 용액은 7.4 mm ABTS 와 2.45 mm K 2 S 2 O 8 를섞어 16시간동안냉암소에보관하여준비하였으며, 흡광도값이 사이에도달하게 50% 에탄올로희석하여사용하였다. 96 well plate에 ABTS용액과농도별시료를혼합하여실온에서 20분간반응시킨후, 734 nm에서흡광도를측정하였다. 그활성의크기는시료를넣지않은경우를대조군 (control) 으로하고시료를넣은것은실험군으로하여 ABTS의활성저해율을나타내었다. Fe 2+ Chelating 각화합물은일정한농도로준비하였으며, 이를 test tube에추출된시료 50 μl와 FeCl 2 (0.6 mm) 10 μl 증류수 90 μl를넣고혼합하였다. 이혼합물을상온에서 5분동안반응시킨후 ferrozine(5 mm) 20 μl을혼합물에첨가하였고, 이혼합물을다시상온에서 10분간반응시킨뒤혼합시료를 562 nm에서흡광도를측정하였다. FRAP 측정 반응액으로 acetate buffer(ph 3.6, 300 mm): 10 mm의 TPTZ: 20 mm의 FeCl 3 6H 2 O를 10:1:1의비율로

5 208 Kor. J. Pharmacogn. 혼합하여실험직전에만들었다. 반응액과화합물을각각의비율로혼합한후 10 분간상온에서보관후 595 nm 에서흡광도를측정하였다. 결과및고찰 배롱나무 (L. Indica) 가지의 80% MeOH 추출물을극성별용매분획을통해얻은분획물을각종 column chromatography 법을이용하여총 18종의화합물을분리하였고, 이들은기존논문에보고된 1 H, 13 C NMR, FAB MS 데이터를비교하여동정하였으며, 각각 tachioside(1), 7) isotachioside(2), 7) 2,4,6-trimethoxyphenyl β-d-glucopyranoside(3), 8) gallic acid 4-methyl ether(4), 9) protocatechuic acid(5), 10) gallic acid(6), 11) vanillic acid(7), 12) vanillin(8), 13) 2-methoxy-5-hydroxymethylphenyl-1-O-(6''-galloyl)-β-D-glucopyranoside(9), 14) 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside (10), 15) 4-hydroxy-3-methoxyphenyl-1-O-(6'-O-galloyl)-β-Dglucopyranoside(11), 16) vomifoliol(12), 17) vomifoliol 9-O-β-Dglucopyranoside(13), 17) 6R, 9R- 3- oxo- α- ionol- 9- O- β- D- glucopyranoside(14), 17) dihydrophaseic acid 4'-O-β-D-glucopyranoside (15), 18) β-hydroxypropiovanillone 3-O-β-D-glucopyranoside (16), 19) myrciaphenone A(17), 20) 그리고 coumaric acid(18) 21) 로확인하였다 (Fig. 1). 항산화와산화시스템의불균형은많은질병을유발하며, 라디칼소거능의활성은항산화시스템의중요한요소로많은질병예방에효과적이라고보고되어있다. 22) 본실험에서는배롱나무로부터분리한화합물 (1-18) 의항산화활성을탐색함으로써, 배롱나무의화합물의활용가치를증대시키고자하였다. 배롱나무로부터분리한물질의항산화능을측정하기위해먼저, DPPH 실험을실시하였다. DPPH 는안정화라디칼중하나로함황아미노산과, aromatic amine 등에의해서환원되어짙은자색이탈색되는점을이용하여, 수소공여능및유리기소거작용을측정하는데널리이용되고있는방법중하나이다. 22) 또한, ABTS 라디칼소거활성은수용성및지용성물질의항산화측정에모두적용할수있기때문에식품의항산화활성을측정하는데널리사용되는방법중하나로써, ABTS 라디칼이시료의항산화물질에의해제거되면특유의색인청록색이탈색되는점을이용하였다. 23) DPPH 와 ABTS 두실험에서화합물들의소거능이유사한결과를보였으며, 특히화합물 4, 10, 11 이 DPPH 와 ABTS 에서높은소거능을나타냈다 (Table I). Fig. 1. Chemical structures of compounds 1-18.

6 Vol. 47, No. 3, Table I. DPPH, ABTS radical scavenging capacity and Fe 2+ chelating and FRAP assay of isolated compounds (1-18)* Compound DPPH of IC 50 (µm) ABTS of IC 50 (µm) Fe 2+ chelating of IC 50 (µm) FRAP (mm/m) ± ± ± ± > ± ± ± >1000 >1000 > ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± > ± 1.24 > ± ± ± ± ± ± ± ± ± ± ± ± ± >1000 >1000 > ± >1000 >1000 > ± > ± 1.02 > ± >1000 >1000 > ± > ± 2.14 > ± > ± 9.87 > ± ± ± ± ± 2.57 Vit.C 6.73 ± ± ± ± *Data are expressed as the mean of triplicate ± S.D. Fe 2+ chelating 활성은생체내의철이온과잉으로발생할수있는 hydrogen peroxide 와의 fenton reaction 에의해단백질발현의손상, 산화적스트레스를통한 DNA 손상, 세포의노화등의원인으로알려진강력한활성산소종의억제를위한 Fe 2+ chelating 반응을이용한것이다. 24) 각추출물모두농도가높을수록 chelating 활성이높았으며, 특히화합물 4, 6, 11 이가장높은활성을나타내었다 (Table I). 환원력은일반적으로전자나수소, 산소등의전자의이동이나공여가가능한물질들과관련이높은것으로알려져있으므로, 산화반응촉매제로작용하는금속이온을환원시키는효력을측정하는방법중하나인 FRAP 법을이용하여각화합물들의환원력을측정하였다. 그중화합물 4, 6, 11 이가장높은환원력을나타냈으며, 이는 Fe 2+ chelating 과도유사한결과를나타내었다 (Table I). 결 배롱나무의가지 MeOH 추출물로부터컬럼크로마토그래피를실시하여 18 종의화합물을분리하였고, spectroscopy 법을이용하여구조동정하였다. 이들화합물중 DPPH, ABTS, Fe 2+ chelating, FRAP 모두유사한결과를나타내었 론 는데, 화합물 4, 6, 11 이환원력및라디칼소거능에서가장높은활성을나타내었으며, 이는이러한화합물들이질병치료에효과적일수있는물질이자미백활성이있을것으로사료되며, 기능성화장품소재및식품으로사용가능할것으로사료된다. 사 이논문은 2013 년도정부 ( 교육부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 (2013R1A1A2A ). 사 인용문헌 1. Lee, B. G., Kim, J. H., Ham, S. G. and Lee, C. E. (2014) Study on biological activities of extracts for cosmeceutical development from Lagerstroemia indica L. branch. Korean J. Plant Res. 27: 이창복 (2003) 원색대한식물도감, 789, 향문사, 서울. 3. Lee, I. S., Youn, U. J., Kim, H. J., Min, B. S., Kim, J. S. and Bae, K. H. (2011) Biphenyl and biphenyl ether quinolizidine N- oxide alkaloids from Lagerstroemia indica L. Planta Med.

7 210 Kor. J. Pharmacogn. 77: Woo, K. W., Suh, W. S., Subedi, L., Kim, S. Y., Choi, S. U., Kim, K. H. and Lee, K. R. (2015) Phenolic derivatives from the stems of Lagerstroemia indica and their biological activity. Heterocycles 91: Woo, K. W., Cha, J. M., Choi, S. U. and Lee, K. R. (2016) A new triterpene glycoside from the stems of Lagerstroemia indica. Arch. Pharm. Res. 39: Kim, H. J., Lee, I. S., Youn, U. J., Chen, Q. C., Ngoc, T. M., Ha, D. T., Liu, H., Min, B. S., Lee, J. Y., Seong, R. S. and Bae, K. H. (2009) Biphenylquinolizidine alkaloids from Lagerstroemia indica. J. Nat. Prod. 72: Cho, H. K., Suh, W. S., Kim, K. H., Kim, S. Y. and Lee, K. R. (2014) Phytochemical constituents of Salsola komarovii and their effects on NGF induction. Nat. Prod. Sci. 20: Chang, R., Wang, C., Zeng, Q., Guan, B., Zhang, W. and Jin, H. (2013) Chemical constituents of the stems of Celastrus rugosus. Arch. Pharm. Res. 36: Chaubal, R., Mujumdar, A. M., Misar, A. and Deshpande, N. R. (2005) Isolation of phenolic compounds from Acacia nilotica with topical antiinflammatory activity. Asian J. Chem. 17: Lee, S. Y., Kim, K. H., Lee, I. K., Lee, K. H., Choi, S. U. and Lee, K. R. (2012) A new flavonol glycoside from Hylomecon vernalis. Arch. Pharm. Res. 35: Zhang, H. M., Wang, C. F., Shen, S. M., Wang, G. L., Liu, P., Liu, Z. M., Wang, Y. Y., Du, S. S., Liu, Z. L. and Deng, Z. W. (2012) Antioxidant phenolic compounds from Pu- erh tea. Molecules 17: Sun, L. X., Fu, W., Ren, J., Xu, L., Bi, K. S. and Wang, M. W. (2006) Cytotoxic constituents from Solanum lyratum. Arch. Pharm. Res. 29: Kim, H., Yi, J. M., Kim, N. S., Lee, Y. J., Kim, J., Oh, D. S., Oh, S. M., Bang, O. S. and Lee, J. Cytotoxic compounds from the fruit of Vitex rotundifolia against human cancer cell lines. J. Korean Soc. Appl. Biol. Chem. 55: De Leo, M., Braca, A., De Tommasi, N., Norscia, I., Morelli, I., Battinelli, L. and Mazzanti, G. (2004) Phenolic compounds from Baseonema acuminatum leaves: isolation and antimicrobial activity. Planta Med. 70: Saijo, R., Nonaka, G. and Nishioka, I. (1989) Tannins and related compounds. Part 82. Phenol glucoside gallates from Mallotus japonicas. Phytochemistry 28: Ishimaru, K., Nonaka, G. and Nishioka, I. (1987) Tannins and related compounds. Part 54. Phenolic glucoside gallates from Quercus mongolica and Q. acutissima. Phytochemistry 26: Kuang, H., Yang, B., Xia, Y. and Feng, W. (2008) Chemical constituents from the flower of Datura metel L.. Arch. Pharm. Res. 31: Champavier, Y., Comte, G., Vercauteren, J., Allais, D. P. and Chulia, A. J. (1999) Norterpenoid and sesquiterpenoid glucosides from Juniperus phoenicea and Galega officinalis. Phytochemistry 80: Xiang, Y., Yang, S., Zhan, Z. and Yue, J. (2004) Terpenoids and phenols from Taiwania flousiana. Acta. Bot. Sin. 46: Lee, K. R., Hong, S. W., Kwak, J. H., Pyo, S. and Jee, O. P. (1996) Phenolic constituents from the aerial parts of Artemisia stolonifera. Arch. Pharm. Res. 19: Lee, S. Y., Choi, S. U., Lee, J. H., Lee, D. U. and Lee, K. R. (2010) A new phenylpropane glycoside from the rhizome of Sparganium stoloniferum. Arch. Pharm. Res. 33: Reis, F. S., Martins, A., Barros, L. and Ferreira, I.C. (2012) Antioxidant properties and phenolic profile of the most widely appreciated cultivated mushrooms: a comparative study between in vivo and in vitro samples. Food. Chem. Toxicol. 50: Kim, H. Y., Lee, Y. A. and Cho, E. J. (2012) Free radical scavenging effect and protective activity from oxidative stress of broccoli flowers and sprouts. CNU J. Agric. Sci. 39: Sakanaka, S., Tachibana, Y. and Okada, Y. (2005) Preparation and antioxidant properties of extracts of Japanese persimmon leaf tea (kakinoha-cha). J. Food Chem. 89: ( 접수 ; 심사 ; 게재확정 )

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< C0CCB0ADB3EB292E666D> 생약학회지 Kor. J. Pharmacogn. 47(1) : 1 6 (2016) 히어리나무의페놀성화합물및세포독성활성 권오길 1 김충섭 1 서원세 1 박경진 1 차준민 1 최상운 2 권학철 3 이강노 1 * 1 성균관대학교약학대학천연물약품화학연구실, 2 한국화학연구원, 3 한국과학기술연구원천연물연구소 Phenolic Compounds from the Twigs