全 文 : 2009 年 3 月 第 7 卷 第 2期 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 111
Chemical Constituents from the Leaves of Camellia
assamica var. kucha Chang et Wang
XU Jie-Kun1*, ZHANG Wei-Ku2, HIROSHI Kurihara3, YAO Xin-Sheng3,4 *
1School of Preclinical Medicine, Beijing University of Chinese Medicine, Beijing 100029;
2Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009;
3Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632;
4School of Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
【ABSTRACT】 AIM: To investigate the chemical constituents of the leaves of Camellia assamica var. kucha
Chang et Wang. METHODS: Chemical constituents were isolated by polyamide, Sephadex LH-20, ODS column and
HPLC chromatography. The structures were elucidated on the basis of physiochemical characteristics and spectral data.
RESULTS: Ten compounds were isolated and identified as theacrine (1), caffeine (2), theobromine (3), (+)-catechin (4),
(–)-epigallocatechin (5), (–)-gallocatechin (6), (–)-epigallocatechin 3-O-gallate (7), (–)-gallocatechin 3-O-gallate (8), 1,
2, 6-tri-O-galloyl-β-D-glucose (9), and gallic acid (10), respectively. CONCLUSION: Compounds 4-10 have been ob-
tained from the plant for the first time and NMR data of 1 are firstly reported.
【KEY WORDS】 Camellia assamica var. kucha Chang et Wang; Chemical constituents; Purine alkaloids; Tea poly-
phenols
【CLC Number】 R284.1 【Document code】 A 【Article ID】1672-3651(2009)02-0111-04
doi: 10.3724/SP. J. 1009.2009.00111
1 Introduction
Camellia assamica var. kucha Chang et Wang
(Theaceae) is an endemic tea plant grown in the wild wood
at 1 370 meters altitude in Yunnan province, People’s Re-
public of China and consumed as a daily beverage in this
region for a long time[1]. In order to understand the plant
further, we investigated systematically the chemical con-
stituents of C. assamica var. kucha. In the present paper,
three purine alkaloids and seven polyphenols were isolated
from the water extract of C. assamica var. kucha. On the
basis of spectroscopic data, their structures were elucidated
as theacrine (1), caffeine (2), theobromine (3), (+)-catechin
(4), (–)-epigallocatechin (5), (–)-gallocatechin (6), (–)- epi-
gallocatechin 3-O-gallate (7), (–)-gallocatechin 3-O- gallate
(8), 1, 2, 6-tri-O-galloyl-β-D-glucose (9), and gallic acid
(10), respectively.
2 Experimental
Optical rotations were determined on a Jasco P-1020
polarimeter (Jasco, Tokyo, Japan) at room temperature.
【Received on】 2008-10-28
【 *Corresponding author】 XU Jie-Kun: Dr., Tel: +86-10-
64286990, E-mail: jiekun_625@yahoo.com.cn; YAO Xin-Sheng:
Prof., Tel: +86-20-85225849, Fax: +86-20-85221559, E-mail:
yaoxinsheng@vip.tom.com
Infrared radiation (IR) spectra were recorded on a Jasco
FT/IR-480 Plus Fourier Transform infrared spectrometer
(Jasco, Tokyo, Japan) using KBr pellets. Electrospray ioni-
zation mass spectra (ESI-MS) were carried out on a Finni-
gan LCQ Deca mass spectrometer. Nuclear magnetic reso-
nance (NMR) spectra were measured on a Bruker AV-400
spectometer with tetramethyl-silane (TMS) as an internal
standard. ODS (Nomura Co. Ltd., Japan), C18 reverse-phase
(Merk, Germany) and Sephadex LH-20 (Pharmacia Biotec
AB, Sweden) were used for column chromatography. Sol-
vents used in column and HPLC chromatogram were of
analytical grade (Shanghai Chemical Plant, People’s Re-
public of China) and Fisher HPLC grade (Fisher Scientific,
USA).
C. assamica var. kucha was collected in March 2005
at Yaoshan village, Tongchang Town, Jinpin County, Yun-
nan Province, People’s Republic of China (Altitude 1 410 m,
N 22˚50.891, E 103˚05.745) and authenticated by Prof. YE
Chuang-Xin, Sun Yat-Sen University (Guangzhou, People’s
Republic of China). A voucher specimen was deposited in
Institute of Traditional Chinese Medicine & Natural Prod-
ucts, Jinan University (Guangzhou, People’s Republic of
China).
3 Extraction and Isolation
Fresh tea leaves were steamed for 3 min, and then put
into oven at 80°C to dry. 42 g of dry leaves were extracted
twice with 1 000 mL of boiling water for 30 min each time.
XU Jie-Kun, et al. /Chinese Journal of Natural Medicines 2009, 7(2): 111−114
112 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 2009年 3月 第 7卷 第 2期
After filtration, the filtrate was concentrated by evaporation
under vacuum to 450 ml, then partitioned with CHCl3,
EtOAc, and n-BtOH for 3 times to afford CHCl3 extract
(0.89 g), EtOAc extract (5.15 g), and n-BtOH extract (2.78
g), respectively.
The CHCl3 extract was subjected to polyamide col-
umn chromatography to afford the H2O extract and the
EtOH fraction. The H2O extract was further isolated by
ODS silica gel and then eluted with H2O, MeOH-H2O (1:9)
and MeOH, respectively. The fraction eluted with
MeOH-H2O (1:9) was further separated by preparative C18
reversed-phase HPLC to yield 1 (670.3 mg), 2 (12.5 mg),
and 3 (30.6 mg).
The EtOAc extract was applied to a Sephadex LH-20
column eluted with MeOH-H2O gradiently to give subfrac-
tions 1-5. After isolated by Diaion HP-20 column eluting
with MeOH-H2O (0:100, 20:80, 40:60, 60:40, 80:20, 100:0),
subfraction 2 (856 mg) was subjected by preparative C18
reversed-phase HPLC with MeOH-H2O (5:95) to afford 4
(3.7 mg), 5 (8.5 mg) and 6 (1.2 mg), respectively. Subfrac-
tion 4 (1.0 g) was isolated by preparative C18 re-
versed-phase HPLC with MeOH-H2O (5:95) to obtain 7
(199.0 mg), 8 (5.5 mg) and 9 (4.0 mg).
The n-BtOH extract was subjected by Sephadex LH-20
column eluting with MeOH-H2O (10:90, 30:70, 50:50,
80:20, 100:0) to obtain subfractions 1-5. Subfraction 1 (1.0
g) was recrystallized with MeOH-H2O to afford 10 (30.0
mg).
4 Structural Identification
Compound 1 Colorless needle (CH3OH-H2O), mp
228-229 °C. UV (CHCl3) λmax nm: 204 (log ε 3.36), 294
(log ε 3.02); IR νKBr max : 1 686, 1 544 cm−1; ESI-MS m/z: 225
[M + H]+, 247 [M + Na]+; 1H NMR (CD3OD, 400 MHz) δ:
3.37 (3H, s, 1-NCH3), 3.74 (3H, s, 3-NCH3), 3.58 (3H, s,
7-NCH3), 3.64 (3H, s, 9-NCH3); 13C NMR (CD3OD, 100
MHz) δ: 28.1 (1-NCH3), 150.7 (C-2), 31.5 (3-NCH3), 135.4
(C-4), 99.3 (C-5), 153.4 (C-6), 29.2 (7-NCH3), 151.9 (C-8),
30.5 (9-NCH3). These 1H, 13C NMR data about 1 were
firstly reported.
Compound 2 Colorless needle (CH3OH-H2O), mp
235-236 °C. UV (CHCl3) λmax nm: 209 (log ε 4.08), 274
(log ε 3.74); IR νKBr max : 1 600, 1 551 cm−1; ESI-MS m/z: 195
[M + H]+。1H NMR (CD3OD, 400 MHz) δ: 3.41 (3H, s,
1-NCH3), 3.99 (3H, s, 3-NCH3), 3.58 (3H, s, 7-NCH3), 7.52
(H, s, H-8). 13C NMR (CD3OD, 100 MHz) δ: 27.9
(1-NCH3), 148.7 (C-2), 33.6 (3-NCH3), 151.7 (C-4), 107.6
(C-5), 155.4 (C-6), 29.7 (7-NCH3), 141.4 (C-8) [4].
Compound 3 White powder. UV (CHCl3) λmax nm:
204 (log ε 2.78), 273 (log ε 2.24); IR νKBr max : 1 684, 1 547
cm−1; ESI-MS m/z: 181 [M + H]+; 1H NMR (DMSO-d6, 400
MHz) δ: 11.1 (1H, s, H-1), 3.85 (3H, s, 3-NCH3), 3.34 (3H,
s, 7-NCH3), 7.97 (H, s, H-8); 13C NMR (DMSO-d6, 100
MHz) δ: 150.9 (C-2), 33.0 (3-NCH3), 149.8 (C-4), 107.0
(C-5), 154.9 (C-6), 28.4 (7-NCH3), 142.7 (C-8) [5].
Compound 4 White powder, [α]23.8 D +1.67 (c 0.3,
CH3OH). UV (CH3OH) λmax nm: 211 (log ε 4.26), 281 (log
ε 3.22); IR νKBr max : 3 406, 1 607, 1 519, 1 468 cm−1; ESI-MS
m/z: 313 [M + Na]+, 289 [M – H]−, 579 [2M – H]−; 1H NMR
(400 MHz, DMSO-d6) δ: 4.47 (1H, d, J = 7.2 Hz, H-2), 3.81
(1H, m, H-3), 2.65 (1H, dd, J = 16.0, 5.2 Hz, H-4a), 2.46
(1H, dd, J = 16.0, 8.0 Hz, H-4b), 5.88 (1H, d, J = 2.3 Hz,
H-6), 5.68 (1H, d, J = 2.3 Hz, H-8), 6.72 (1H, d, J = 2.3 Hz,
H-2), 6.68 (1H, d, J = 8.0 Hz, H-5), 6.59 (1H, dd, J = 8.0,
2.3 Hz, H-6), 4.84 (1H, d, J = 5.2 Hz, 3-OH), 9.15 (1H, s,
5-OH), 8.91 (1H, s, 7-OH), 8.84 (1H, s, 4-OH), 8.79 (1H, s,
3-OH); 13C NMR (100 MHz, DMSO-d6) δ: 80.9 (C-2), 66.3
(C-3), 27.8 (C-4), 156.4 (C-5), 95.1 (C-6), 156.1 (C-7), 93.8
(C-8), 155.3 (C-9), 98.9 (C-10), 130.6 (C-1), 115.0 (C-2),
144.8 (C-3, 4), 114.5 (C-5), 118.3 (C-6) [4, 6, 7].
Compound 5 White powder, [α] 23.8 D –43 (c 0.1,
CH3OH,). UV (CH3OH) λmax nm: 209 (log ε 4.19), 270 (log
ε 4.05); IR νKBr max : 3 398, 1 632, 1 518, 1 450 cm−1; ESI-MS
m/z: 635 [2M + Na]+, 305 [M – H]−, 611 [2M – H]−; 1H
NMR (400 MHz, DMSO-d6) δ: 4.65 (1H, br s, H-2), 3.98
(1H, m, H-3), 2.66 (1H, dd, J = 16.4, 4.4 Hz, H-4a), 2.47
(1H, dd, J = 16.4, 3.6 Hz, H-4b), 5.88 (1H, d, J = 2.3 Hz,
H-6), 5.71 (1H, d, J = 2.3 Hz, H-8), 6.37 (2H, s, H-2, 6),
4.59 (1H, d, J = 4.5 Hz, 3-OH), 9.07 (1H, s, 5-OH), 8.87
(1H, s, 7-OH), 8.67 (2H, s, 3, 5-OH), 7.92 (1H, s, 4-OH);
13C NMR (100 MHz, DMSO-d6) δ: 78.1 (C-2), 64.9 (C-3),
28.1 (C-4), 156.4 (C-5), 94.9 (C-6), 156.2 (C-7), 94.0 (C-8),
155.7 (C-9), 98.5 (C-10), 129.6 (C-1), 106.0 (C-2, 6),
145.3 (C-3, 5), 132.1 (C-4) [8].
Compound 6 White powder, [α] 23.8 D –12 (c 0.2,
CH3OH). UV (CH3OH) λmax nm: 213 (log ε 4.44), 272 (log
ε 3.04). IR νKBr max : 3 359, 1 626, 1 521, 1 462 cm−1; ESI-MS
m/z: 307 [M + H]+, 305 [M − H]−; 1H NMR (400 MHz,
DMSO-d6) δ: 4.42 (1H, d, J = 7.2 Hz, H-2), 3.79 (1H, m,
H-3), 2.61 (1H, dd, J = 16.4, 4.6 Hz, H-4a), 2.34 (1H, dd, J
= 16.4, 8.0 Hz, H-4b), 5.68 (1H, d, J = 2.3 Hz, H-6), 5.88
(1H, d, J = 2.3 Hz, H-8), 6.25 (2H, s, H-2, 6), 4.82 (1H, d,
J = 5.0 Hz, 3-OH), 9.12 (1H, s, 5-OH), 8.90 (1H, s, 7-OH),
8.73 (2H, s, 3, 5-OH), 7.98 (1H, s, 4-OH); 13C NMR (100
MHz, DMSO-d6) δ: 81.0 (C-2), 66.3 (C-3), 27.3 (C-4),
156.4 (C-5), 95.0 (C-6), 156.1 (C-7), 93.8 (C-8), 155.2
(C-9), 98.9 (C-10), 129.8 (C-1), 105.9 (C-2, 6), 145.6
(C-3, 5), 132.4 (C-4) [4, 9].
Compound 7 White powder, [α] 23.8 D –144 (c 0.2,
CH3OH,). UV (CH3OH) λmax nm: 212 (log ε 5.08), 276 (log
ε 4.28); IR νKBr max : 3 361, 1 692, 1 617, 1 541, 1 447 cm−1;
ESI-MS m/z: 481 [M + Na]+, 457 [M − H]−; 1H NMR (400
MHz, DMSO-d6) δ: 4.95 (1H, s, H-2), 5.36 (1H, br s, H-3),
2.94 (1H, dd, J = 16.0, 4.4 Hz, H-4a), 2.65 (1H, dd, J =
16.0, 3.2 Hz, H-4b), 5.93 (1H, d, J = 2.3 Hz, H-6), 5.82 (1H,
d, J = 2.3 Hz, H-8), 6.40 (2H, s, H-2, 6), 6.81 (2H, s, H-2,
XU Jie-Kun, et al. /Chinese Journal of Natural Medicines 2009, 7(2): 111−114
2009 年 3 月 第 7 卷 第 2期 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 113
6), 9.27 (1H, s, 5-OH), 9.03 (1H, s, 7-OH), 8.69 (2H, s, 3,
5-OH), 8.01 (1H, s, 4-OH), 9.18 (2H, s, 3, 5-OH), 8.89
(1H, s, 4-OH); 13C NMR (100 MHz, DMSO-d6) δ: 76.4
(C-2), 67.9 (C-3), 25.8 (C-4), 156.5 (C-5), 95.5 (C-6), 156.4
(C-7), 94.3 (C-8), 155.6 (C-9), 97.3 (C-10), 128.6 (C-1),
105.5 (C-2, 6), 145.6 (C-3, 5), 132.3 (C-4), 119.3 (C-1),
108.6 (C-2, 6), 145.3 (C-3, 5), 138.5 (C-4), 165.1
(C-7)[8].
Compound 8 White powder, [α] 23.8 D –24 (c 0.1,
CH3OH). UV (CH3OH) λmax nm: 212 (log ε 4.52), 276 (log
ε 3.71); IR νKBr max : 3 426, 1 704, 1 631, 1 531, 1 450 cm−1;
ESI-MS m/z: 481 [M+Na]+, 457 [M−H]−; 1H NMR (400
MHz, DMSO-d6) δ: 5.05 (1H, d, J = 4.8 Hz, H-2), 5.27 (1H,
d, J = 5.2 Hz, H-3), 2.59 (2H, br s, H-4), 5.92 (1H, d, J =
2.3 Hz, H-6), 5.82 (1H, d, J = 2.3 Hz, H-8), 6.26 (2H, s,
H-2, 6), 6.86 (2H, s, H-2, 6), 9.29 (1H, s, 5-OH), 9.05
(1H, s, 7-OH), 8.83 (2H, s, 3, 5-OH), 8.07 (1H, s, 4-OH),
9.24 (2H, s, 3, 5-OH), 8.93 (1H, s, 4-OH); 13C NMR
(100 MHz, DMSO-d6) δ: 76.9 (C-2), 68.8 (C-3), 22.3 (C-4),
156.8 (C-5), 95.4 (C-6), 156.1 (C-7), 94.0 (C-8), 154.5
(C-9), 97.2 (C-10), 128.6 (C-1), 104.9 (C-2, 6), 145.9
(C-3, 5), 132.9 (C-4), 119.2 (C-1), 108.6 (C-2, 6), 145.4
(C-3, 5), 138.6 (C-4), 165.2 (C-7) [10].
Compound 9 White powder, [α] 29 D −70.8 (c1.0,
CH3OH). UV (CH3OH) λmax nm: 219 (log ε 4.26), 280 (log
ε 3.88); IR νKBr max : 3 423, 1 696, 1 619, 1 538, 1 446 cm−1.
ESI-MS m/z: 659 [M+Na]+, 635 [M–H]−; 1H NMR (400
MHz, DMSO-d6) δ: 5.72 (1H, d, J = 8.0 Hz, H-1), 5.01 (1H,
t, J = 9.0 Hz, H-2), 3.68 (1H, m, H-3), 4.02 (1H, m, H-4),
3.45 (1H, m, H-5), 4.22 (1H, d, J = 13.0 Hz, H-6a), 4.10
(1H, dd, J = 13.0, 4.0 Hz, H-6b), 7.04 (2H, s, H-2, 6), 6.97
(2H, s, H-2, 6), 6.93 (2H, s, H-2, 6), 9.29 (2H, s, 3,
5-OH), 9.23 (1H, s, 4-OH), 9.03 (2H, s, 3, 5-OH), 9.23
(1H, s, 4-OH), 8.94 (2H, s, 3, 5-OH), 9.23 (1H, s,
4-OH); 13C NMR (100 MHz, DMSO-d6) δ: 94.2 (C-1),
70.5 (C-2), 74.6 (C-3), 72.3 (C-4), 72.8 (C-5), 62.1 (C-6),
165.5 (C-7), 164.8 (C-8), 164.4 (C-9), 119.0 (C-1), 109.0
(C-2, 6), 145.5 (C-3, 5), 139.0 (C-4), 118.0 (C-1), 108.9
(C-2, 6), 145.4 (C-3, 5), 138.6 (C-4), 117.8 (C-1),
108.7 (C-2, 6), 145.3 (C-3, 5), 138.5 (C-4) [11].
Compound 10 Colorless needle (CH3OH), mp
252-253 °C. UV (CH3OH) λmax nm: 217 (log ε 4.14), 272
(log ε 3.92); IR νKBr max : 3 289, 1 702, 1 618, 1 541, 1 469 cm−1;
ESI-MS m/z: 169 [M − H]−; 1H NMR (400 MHz, DMSO-d6)
δ: 6.92 (2H, s, H-2, 6), 9.15 (1H, s, 3-OH), 8.79 (1H, br s,
4-OH), 9.15 (1H, s, 5-OH), 12.20 (1H, br s, 7-OH); 13C
NMR (100 MHz, DMSO-d6) δ: 121.0 (C-1), 109.2 (C-2),
145.9 (C-3, 5), 138.5 (C-4), 109.2 (C-6), 167.9 (COOH) [12].
Acknowledgements
The authors thank Prof. YE Chuang-Xin (Sun Yat-Sen
University, Guangzhou, China) for identifying plant mate-
rial. We are also grateful to Dr. LI Qian (Department of
Phytochemistry, China Pharmaceutical University, Nanjing,
China) and Dr. DAI Yi (College of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University,
Shenyang, China) for providing NMR and MS data.
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114 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 2009年 3月 第 7卷 第 2期
苦茶的化学成分
续洁琨 1*, 张维库 2, 栗原博 3, 姚新生 3,4*
1北京中医药大学基础医学院, 北京 100029;
2中国药科大学天然药物化学教研室, 南京 210009;
3暨南大学中药及天然药物研究所, 广州 510632;
4沈阳药科大学中药学院, 沈阳 110016
【摘 要】 目的:对苦茶(Camellia assamica var. kucha Chang et Wang)叶的化学成分进行研究。方法:采用水提取, 聚酰
胺、Sephadex LH-20、ODS柱和 HPLC等手段分离纯化, 通过理化常数和谱学数据鉴定化合物的结构。结果:分离鉴定了
10个化合物, 其中 3个为嘌呤生物碱类化合物, 分别为 theacrine (1), caffeine (2), theobromine (3); 7个为茶多酚类, 分别为
(+)-catechin (4), (–)-epigallocatechin (5), (–)-gallocatechin (6), (–)-epigallocatechin 3-O-gallate (7), (–)-gallocatechin 3-O-gallate
(8), 1, 2, 6-tri-O-galloyl-β-D-glucose (9), gallic acid (10)。结论:化合物 4~10为首次从该植物中分离得到, 并首次对化合物 1
的核磁数据进行了全归属。
【关键词】 苦茶; 化学成分; 嘌呤生物碱类; 茶多酚类
·会 讯·
FIP2009年世界药学大会暨第 69届年会
由国际药学联合会(FIP)主办的 2009 年世界药学大会暨 FIP 第 69 届年会将于 2009年 9 月 3日~9
月 8日在土耳其伊斯坦布尔召开,会期 6天。会议主题:为患者的利益负责-你准备好了吗?
国际药学联合会成立于 1912年, 英文名称是“International Pharmaceutical Federation”, 简称 FIP, 总
部设在荷兰海牙, 目前有来自 80多个国家的 130多个团体会员, 是世界上最大的国际性药学组织。中国
药学会是 FIP的团体会员单位。FIP每年召开一次世界药学大会, 参加会议人数约 3000人, 是世界药学
领域的传统盛会。会议内容广泛, 涉及药学科研、教育、实践等各个领域, 至今已经召开了 68届。
自 1992年以来, 中国药学会每年组团参加 FIP世界药学大会, 今年中国药学会将继续组织国内药学
人员参加大会。出访团组拟于 2009年 9月 2日北京出发, 9月 10日返回。
联系方式:中国药学会国际交流部
地 址:北京朝阳区建外大街四号建外 SOHO九号楼 1802室 邮 编:100022
电 话:010-58699271 传 真:010-58699272
E-mail: gjh6565@163.com; sinopharmacy@163.net
联系人:葛军华 刘春光