4946 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Ling et al.
MATERIALS AND METHODS
General. The melting point was uncorrected. Optical rotations
were measured on a P-1020 Polarimeter (Jasco, Tokyo, Japan). IR spectra
were measured on a Nicolet 8700 FT-IR spectrophotometer (Thermo,
1
13
United States). The H NMR (400 MHz), C NMR (100 MHz),
distortionless enhancement by polarization transfer (DEPT), and 2D
1
1
NMR spectra such as H- H correlation spectroscopy (COSY), HXCO,
heteronuclear single quantum coherence (HSQC), heteronuclear multiple
bond correlation (HMBC), and nuclear Overhauser effect spectroscopy
(NOESY) were recorded on an Avance-400 instrument (Bruker, United
States). Chemical shifts were expressed in ppm (δ). The high-resolution
time-of-flight-mass spectrometry (HRTOF-MS) were recorded on a
GCT-MS instrument (Micromass Ltd., United Kingdom) by direct inlet.
The electrospray ionization-mass spectrometry (ESI-MS) data were
obtained with a Finnigan LTQ LC/MS system (Thermo) by direct inlet
(ESI-MS conditions: sheath gas flow rate, 14 arb; aux/sweep gas flow rate,
0
arb; spray voltage, 4.20 kV; capillary temperature, 300 °C; capillary
voltage, 4.30 V; and tube lens offset, 30.0 V). The gas chromatography-
mass spectrometry (GC-MS) analyses were performed with a Shimadzu
GCMS-QP2010S instrument [GC conditions: DB-5 capillary column
(
5
30 m ꢀ 0.25 mm); column temperature, 50 °C (hold time, 2 min),
Figure 2. Flowchart of extraction and isolation of 1-14 from Fuzhuan
brick tea.
0f250 °C (rate of temperature increase, 8 °C/min), 250f300 °C (rate
of temperature increase, 3 °C/min), 300 °C (hold time, 18 min); injector
temperature, 250 °C; and He at 1.0 mL/min]. Silica gel 60 (200-300 mesh,
Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of
China) and YMC GEL ODS-A (50 μm, YMC Co. Ltd., Japan; column
dimension: 500 mm ꢀ 25 mm i.d.; flow rate, 7 mL/min) were used for
column chromatography (CC). The column eluant was monitored by thin-
layer chromatography (TLC), which was performed on precoated silica gel
plates (GF254, Liangchen Chemical Co. Ltd., Huoshan, Anhui province,
People’s Republic of China) or on precoated RP-18 silica gel plates (F254,
Merck, Germany), with detection by spraying in sequence with 2%
CHCl -MeOH (98: 2) resulting in three subfractions (5A-5C). Subfrac-
3
tions 5A and 5B were purified by ODS CC eluting with methanol to afford
2 (5 mg) and 3 (11 mg), respectively. Subfraction 5C was sequentially
chromatographed on a silica gel column eluted with petroleum ether-
acetone (19: 1) mixture and an ODS CC using methanol-H
2
O (90: 10) as
the eluant to afford 1 (20 mg).
The CHCl3 extract (fraction B) was concentrated to give the crude
compound 14 (40 g). The remaining aqueous solution was named as
fraction C.
ethanolic FeCl
followed by heating.
3 2 4
and 1% methanolic p-anisaldehyde H SO reagent
25
3β,6r,13β-Trihydroxyolean-7-one (1). Colorless gum, [R]D þ 15
-1
Materials. Fuzhuan brick tea (produced in December 2006) was
(c 0.002, MeOH). IR (KBr) νmax (cm ): 3435, 2919, 2850, 1707, 1464,
-
purchased from Yiyang Fu Cha Industry Development Co. Ltd.
1395, 1364, 1294, 1101, 1031, 989. ESI-MS: m/z 473 [M - H] in negative
þ
(Yiyang, Hunan province, People’s Republic of China). Butyl p-hydro-
mode, 497 [M þ Na] in positive mode. HRTOF-MS: m/z 474.3712 (calcd
xybenzoate was purchased from Beijing Chemical Co. Ltd. (Beijing,
People’s Republic of China). Noroxin was purchased from Shanghai
Yan-an Pharmaceutical Co. Ltd. (Shanghai, People’s Republic of China).
Berberine hydrochloride was purchased from Yabaoguangtai Pharma-
ceutical Co. Ltd. (Pengzhou, Sichuan province, People’s Republic of
China). Beef extract and peptone were purchased from Beijing Aobo-xing
Biotechnology Co. Ltd. (Beijing, People’s Republic of China). Agar
powder was purchased form Hangzhou Microbial Reagent Co. Ltd.
for C H O , 474.3709). GC-MS: GC t 48.71 min. EI-MS (70 eV): m/z
30
50
4
R
456 (45), 441 (79), 405 (17), 355 (9), 303 (21), 281 (14), 263 (16), 219 (100),
175 (27), 151 (28), 135 (36), 121 (52), 109 (39), 95 (64), 83 (67), 69 (68), 55
1
13
(71), 41 (74). H and C NMR data, see Table 1.
3β-Acetoxy-6r,13β-dihydroxyolean-7-one (2). Colorless needles,
2
5
-1
mp 285-289 °C, [R]
D
þ 14 (c 0.001, MeOH). IR (KBr) νmax (cm ):
3485, 3434, 3003, 2923, 2854, 1716, 1463, 1398, 1377, 1269, 1098, 1026.
ESI-MS: m/z 515 [M - H]- in negative mode, 539 [M þ Na] in positive
mode. HRTOF-MS: m/z 516.3812 (calcd for C H O , 516.3815). GC-
þ
(Hangzhou, Zhejiang province, People’s Republic of China). Sodium
32
52 5
chloride and sodium hydroxide were purchased from Xuzhou Chemical
Co. Ltd. (Xuzhou, Jiangsu province, People’s Republic of China).
Caffeine was purchased from Sigma Chemical Co. Ltd. (United States).
Extraction and Isolation. Fuzhuan brick tea (3.6 kg) was ground
and extracted with 70% aqueous acetone at room temperature three times
R
MS: GC t 51.02 min. EI-MS (70 eV): m/z 498 (14), 483 (38), 355 (3), 302
(7), 219 (61), 175 (17), 151 (15), 136 (15), 121 (23), 107 (20), 95 (32), 83 (22),
1
69 (35), 55 (24), 43 (100, CH CO). H NMR (CDCl ) δ: 1.11 (1H,
3
3
overlapped, H-1R), 2.589 (1H, ddd, J = 14.0, 3.6, 3.6 Hz, H-1β), 1.61
(2H, overlapped, H-2), 4.491 (1H, dd, J = 10.4, 6.0 Hz, H-3R), 1.53 (1H,
overlapped, H-5), 4.907 (1H, d, J = 12.0 Hz, H-6β), 0.811 (1H, m, H-9),
1.58 (2H, overlapped, H-11), 1.19 (2H, overlapped, H-12), 1.08 (1H,
overlapped, H-15R), 2.097 (1H, ddd, J = 13.6, 13.6, 4 Hz, H-15β), 1.823
(1H, ddd, J = 13.6, 13.6, 4.0 Hz, H-16R), 1.18 (1H, overlapped, H-16β),
1.51 (1H, overlapped, H-18), 1.989 (1H, dd, J = 12.8, 2.0 Hz, H-19R), 1.26
(1H, overlapped, H-19β), 1.47 (1H, overlapped, H-21a), 1.32 (1H, over-
lapped, H-21b), 1.38 (1H, overlapped, H-22a), 1.11 (1H, overlapped, H-
(10, 6, and 6 L). After acetone was removed under reduced pressure, the
aqueous solution was extracted with petroleum ether and chloroform,
respectively. The petroleum ether solution (fraction A) was concentrated
under reduced pressure to afford a residue (50 g). This residue was applied
to a silica gel CC, eluting with a petroleum ether-acetone mixture of
increasing polarity (20:1 to 5:1), yielding five fractions (Figure 2).
Fraction 1 (4 g) was fractionated by silica gel CC using petroleum
ether-acetone (99: 1) as the eluant, resulting in three subfractions
3 3 3
22b), 0.872 (3H, s, CH -23), 0.895 (3H, s, CH -24), 1.146 (3H, s, CH -25),
(
1A-1C). Compound 4 (65 mg) was obtained from subfraction 1A by
recrystallization. Subfraction 1B was subjected to a silica gel CC using
petroleum ether-CHCl (5: 1) as the eluant, yielding 5 (70 mg). Subfrac-
tion 1C was subjected to repeated ODS CC eluted with methanol to yield 7
30 mg) and 9 (45 mg). Fraction 2 (5 g) was subjected to a silica gel CC,
1.373 (3H, s, CH -26), 0.923 (3H, s, CH -27), 1.233 (3H, s, CH -28), 0.955
3
3
3
13
(3H, s, CH -29), 0.903 (3H, s, CH -30), 2.041 (3H, s, CH CO). C NMR
3
3
3
3
3
(CDCl ) δ: 39.95 (C-1), 23.83 (C-2), 80.52 (C-3), 39.42 (C-4), 56.87 (C-5),
72.35 (C-6), 211.17 (C-7), 43.39 (C-8), 55.46 (C-9), 39.04 (C-10),
17.57 (C-11), 34.04 (C-12), 82.39 (C-13), 45.19 (C-14), 22.58 (C-15),
30.57 (C-16), 33.59 (C-17), 49.19 (C-18), 38.33 (C-19), 31.39 (C-20),
33.99 (C-21), 38.53 (C-22), 28.20 (C-23), 16.47 (C-24), 16.05 (C-25),
20.51 (C-26), 18.44 (C-27), 31.27 (C-28), 31.86 (C-29), 25.04 (C-30),
170.97 (CH CO), 21.32 (CH CO).
(
eluted with petroleum ether-acetone (99: 1) and an ODS CC eluted with
methanol to yield 12 (50 mg). Fraction 3 (2 g) was subjected to an ODS CC
using methanol as the eluant to yield 6 (62 mg), 11 (106 mg), and 13 (11
mg). Fraction 4 (5 g) was sequentially chromatographed on a silica gel
3
3
column eluted with CHCl
using methanol as the eluant, yielding 8 (7 mg) and 10 (9 mg). Fraction 5
6 g) was fractionated by silica gel CC employing a gradient of CHCl to
3
-MeOH (99: 1) mixture and repeated ODS CC
3β-O-(8-Hydroxyoctanoyl)-12-oleanene (3). White amorphous
25
powder, [R]
D
þ 74.5 (c 0.001, MeOH). HRTOF-MS: m/z 568.4861
1
(
3
(calcd.forC H O , 568.4855). HNMR(CDCl ) δ:5.140(1H, t, J=3.6Hz,
38
64
3
3