414
Vol. 58, No. 3
further separated by HPLC (MeCN–0.05% TFA in H2O, 35 : 65, UV detec-
tion at 210 nm), to yield 1 (20 mg) and 3 (25 mg) respectively. Fraction 2
was subjected to RP-C18 column with MeOH–H2O (4 : 6→8 : 2) to give 3
fractions and compound 2 (9 mg) was obtained by further HPLC purification
(MeCN–0.05% TFA in H2O, 30 : 60, UV detection at 210 nm) from fraction
2.2.
Compound 1: White amorphous powder; [a]D25 ꢀ3.5 (cꢁ0.13, MeOH);
IR (KBr) cmꢂ1: 3419, 2948, 1724, 1678, 1447, 1387, 1080, 1042; ESI-MS
m/z: 793 [MꢂH]ꢂ; HR-ESI-MS m/z 793.4038 [MꢂH]ꢂ (Calcd for
1
C41H61O15: 793.4015); H-NMR (C5D5N, 600 MHz) and 13C-NMR (C5D5N,
150 MHz) are given in Tables 1 and 2.
Compound 2: White amorphous powder; [a]D25 ꢂ13.0 (cꢁ0.02, MeOH);
IR (KBr) cmꢂꢀ: 3443, 2925, 1680, 1642, 1139; ESI-MS m/z: 1584
[MꢂH]ꢂ; HR-ESI-MS m/z 1583.6809 [MꢂH]ꢂ (Calcd for C72H111O38:
1583.6758); H-NMR (C5D5N, 600 MHz) and 13C-NMR (C5D5N, 150 MHz)
1
Fig. 2. Selected NOESY and HMBC Correlations for Compound 2
are given in Tables 1 and 2.
Compound 3: White amorphous powder; [a]D25 0 (cꢁ0.15, MeOH); IR
(KBr) cmꢂ1: 3424, 2948, 1724, 1681, 1142, 1080; ESI-MS m/z: 837
[MꢂH]ꢂ; 1H-NMR (C5D5N, 600 MHz) and 13C-NMR (C5D5N, 150 MHz)
are given in Tables 1 and 2.
of 2, an extra 42 mass unit revealed the presence of an acetyl
group combining with the information of the dC 20.8, 171.8
and dH 2.14. The downfield signals of Glc-6 at dH 4.88 (br d,
Jꢁ12.0 Hz), 4.63 (br d, Jꢁ12.0 Hz) indicated that the loca-
tion of the acetyl group was at this position, which was fur-
ther confirmed by the correlations between these two protons
and the carbonyl carbon (dC 171.8) of acetyl group in the
HMBC spectrum, respectively. From the above evidences,
the structure of 2 was elucidated as 3-O-b-D-galactopyra-
Acid Hydrolysis of Compounds 1 and 2 and Determination of Ab-
solute Configuration of Monosaccharides Each compound (3 mg) was
heated in 2 M HCl (5 ml) at 90 °C for 4 h. The reaction mixture was extracted
with CHCl3 (5 mlꢄ3). The CHCl3 extract was purified by chromatography
on Sephadex LH-20 (2.0ꢄ100 cm). Comparing TLC with authentic samples,
the aglycone was determined to be quillaic acid (1a, Rf: 0.25, CHCl3–
MeOH, 20 : 1). Each remaining aqueous layer was concentrated to dryness
to give a residue and dissolved in pyridine (2 ml), and then L-cysteine methyl
nosyl-(1→2)-[b-D-xylopyranosyl-(1→3)]-b-D-glucuronopy- ester hydrochloride (2 mg) was added to the solution. The mixture was
heated at 60 °C for 1 h, and trimethylchlorosilane (0.5 ml) was added, fol-
ranosyl quillaic acid 28-O-(6-O-acetyl)-b-D-glucopyranosyl-
lowed by heating at 60 °C for 30 min. Then, the solution was concentrated to
dryness and dissolved in water (1 mlꢄ3), followed by extraction with n-
hexane (1 mlꢄ3). The hexane extract was subjected to GC/MS analysis. The
absolute configurations of the monosaccharides were confirmed to be D-fu-
cose, L-rhamnose, D-xylose, D-glucuronic acid, D-galactose, and D-glucose by
comparison of the retention times of monosaccharide derivatives with those
of standard samples: D-fucose (12.85 min), L-rhamnose (12.67 min), D-xy-
(1→3)-[b-D-xylopyranosyl-(1→4)]-a-L-rhamnopyranosyl-
(1→2)-b-D-fucopyranoside.
Compound 3 was determined as 3-O-b-D-galactopyra-
nosyl-(1→2)-6-O-methyl-b-D-glucuronopyranosyl quillaic
acid first obtained as the hydrolyzate of lucyoside N.10)
lose (11.89 min), D-glucuronic acid (14.13 min), D-galactose (14.32 min),
and D-glucose (14.01 min), respectively.
Experimental
General Experimental Procedures Optical rotations were measured
with a JASCO P-1020 polarimeter (cell length: 1.0 dm). IR (KBr-disks)
spectra were recorded by Brucker Tensor 27 spectrometer. Mass spectra
were obtained on a MS Agilent 1100 Series LC/MSD Trap mass spectrome-
ter (ESI-MS) and a G1969A TOF MS (HR-ESI-MS), respectively. 1D and
2D NMR spectra were recorded in C5D5N at 300 K on Bruker ACF-600
NMR (1H: 600 MHz, 13C: 150 MHz) spectrometers, in which coupling con-
stants were given in Hz. Gas chromatography was done on Varian CP-3800
Gas Chromatograph equipped with a Saturn 2200 Mass detector (detection
temperature 220 °C). Column: CP-sil 5 CB capillary column (30 m, 0.25 mm
i.d., 0.25 mm). Column temperature: 150—260 °C with the rate of 8 °C/min,
and the carrier gas was He (0.8 ml/min), split ratio 1/10, injection tempera-
ture: 250 °C. Injection volume: 0.5 ml. All solvents used were of analytical
or chromatographic grade (Jiangsu Hanbang Sci. & Tech. Co., Ltd). TLC
was performed on precoated silica gel 60 F254 plates (Qingdao Haiyang
Chemical Co., Ltd.), and detection was achieved by 10% H2SO4–EtOH for
saponins. Sephadex LH-20 (20ꢄ100 mm, Pharmacia), macroporous resin
D101 (pore size B 13—14 nm, 26—60 mesh), and ODS-C18 (40—63 mm,
Fuji) were used for column chromatography. Preparative HPLC was carried
out using Agilent 1100 Series with Shim-park RP-C18 column (200ꢄ20 mm
i.d.) and 1100 Series Multiple Wavelength detector.
1
Quillaic Acid (1a): White needles; ESI-MS m/z: 487 [MꢀH]ꢀ; H-NMR
(DMSO-d6, 500 MHz) d: 0.77 (3H, s, Me), 0.92 (3H, s, Me), 0.95 (3H, s,
Me), 0.97 (3H, s, Me), 0.99 (3H, s, Me), 1.43 (3H, s, Me), 3.75 (1H, m, 3-
H), 4.40 (1H, br s, –OH), 4.80 (1H, br s, 16-H), 5.30 (1H, t, Jꢁ3.2 Hz, 12-
H), 9.40 (1H, s, –CHO), 12.09 (1H, br s, –COOH). 13C-NMR (DMSO-d6,
125 MHz) d: 37.9 (C-1), 26.2 (C-2), 70.6 (C-3), 55.3 (C-4), 46.6 (C-5), 20.4
(C-6), 31.6 (C-7), 39.4 (C-8), 47.5 (C-9), 35.6 (C-10), 24.3 (C-11), 121.2
(C-12), 144.2 (C-13), 40.0 (C-14), 35.3 (C-15), 73.1 (C-16), 46.2 (C-17),
41.3 (C-18), 46.6 (C-19), 30.4 (C-20), 34.8 (C-21), 32.0 (C-22), 207.3 (C-
23), 9.1 (C-24), 15.5 (C-25), 17.0 (C-26), 26.7 (C-27), 178.4 (C-28), 33.1
(C-29), 23.0 (C-30).
Acknowledgment This research work was supported by the National
Natural Science Foundation of China for Outstanding Young Scientists (No.
30525032).
References
1) Lu D. Q., Bull. Bot. Res., 4, 329—337 (1994).
2) “The Chinese Medicine Dictionary,” Shanghai People’s Publishing
House, Shanghai, 1977, p. 2170.
3) Luo J. G., Kong L. Y., Takaya Y., Niwa M., Chem. Pharm. Bull., 54,
1200—1202 (2006).
4) Luo J. G., Kong L. Y., Helv. Chim. Acta, 89, 947—953 (2006).
5) Luo J. G., Ma L., Kong L. Y., Bioorg. Med. Chem., 16, 2912—2920
(2008).
6) Frechet D., Christ B., du Sorbier B. M., Fischer H., Vuilhorgne M.,
Phytochemistry, 30, 927—931 (1991).
7) Mahato S. B., Kundu A. P., Phytochemistry, 37, 1517—1575 (1994).
8) Glensk M., Wray V., Nimtz M., Schöpke T., J. Nat. Prod., 62, 717—
721 (1999).
Plant Material G. altissima was collected from Zhaosu County, Xin-
jiang Province, People’s Republic of China, in August 2007, and the botani-
cal origin of material was identified by Prof. Rena Kasimu, College of Phar-
maceutical Sciences, Xinjiang Medical University, China. The voucher spec-
imens (No. 070806) were deposited at the Department of Natural Medicinal
Chemistry, China Pharmaceutical University, Nanjing, China.
Extraction and Isolation The air-dried plants (5 kg) were extracted
with 70% aqueous ethanol (v/v) three times (10 l, 2 h each) under reflux.
After evaporation, the residue was suspended in H2O and partitioned be-
tween EtOAc and H2O. Then the water layer was chromatographed over a
macroporous resin D101 column eluted with 30%, 70% and 100% EtOH, re-
spectively. The 70% EtOH portion was fractionated by MCI (MeOH/H2O,
3 : 7, 5 : 5, 6 : 4, 7 : 3 and 10 : 0) to give five fractions (Fractions 1—5). Frac-
tion 4 was further subjected to repeated RP-C18 column with MeOH–H2O
(4 : 6→9 : 1) and then the eluents of fraction 4.2 (MeOH–H2O, 7 : 3) were
9) Sang S. M., Lao A. N., Leng Y., Gu Z. P., Chen Z. L., Uzawa J., Fuji-
moto Y., Tetrahedron Lett., 41, 9205—9207 (2000).
10) Yoshikawa K., Arihara S., Wang J. D., Narui T., Okuyama T., Chem.
Pharm. Bull., 39, 1185—1188 (1991).