522
Can. J. Chem. Vol. 88, 2010
25
thors (K.R.L.). A voucher specimen (SKKU-2008–6) of the
plant was deposited in the herbarium of the School of Phar-
macy, Sungkyunkwan University, Suwon, Korea.
rotation value, ½aꢁD + 26.6 (c 0.15, MeOH) with that of an
authentic sample14 and by co-TLC comparison with an au-
thentic sample (CHCl3/MeOH/H2O, 9:7:1.5; Rf = 0.31). The
sucrose was detected by co-injection of an authentic sample
on preparative normal-phase HPLC using a solvent system
of CHCl3/MeOH/H2O (9:7:1.5) at a flow rate of 2.0 mL/min
in the same manner above, giving a single peak at 11.03 min.
Extraction and isolation
The dried aerial parts of B. manshuriensis (2.9 kg) were
extracted three times at room temperature with 80% MeOH
over a period of 3 days and evaporated under reduced pres-
sure to give a crude extract (219 g). The crude extract was
dissolved in distilled water (800 mL) and successively ex-
tracted with n-hexane, CH2Cl2, EtOAc, and n-BuOH to pro-
vide n-hexane (10 g), CH2Cl2 (2 g), EtOAc (9 g), and
n-BuOH extracts (32 g). The EtOAc-soluble extract (9 g)
was subjected to normal-phase column chromatography
(CC) over silica gel using CHCl3/MeOH of increasing polar-
ity (7:1 to 1:1) to give six fractions (E1–E6). Fraction E2
(1.1 g) was subjected to reversed-phase column chromatog-
raphy over an RP-C18 silica gel using MeOH/H2O (3:2) to
provide five subfractions (E21–E25). Subfraction E21
(220 mg) was chromatographed on LiChroprep Lobar1-A
RP-18 using MeOH/H2O (1:1) to give four subfractions
(E211–E214). Subfraction E213 (45 mg) was separated on
Sephadex LH-20 column using 100% MeOH to yield bis-
toroside A (1, 8 mg) and helonioside A (3, 5 mg). Subfrac-
tion E22 (130 mg) was subjected to LiChroprep Lobar1-A
RP-18 column using MeOH/H2O (7:3) to afford three sub-
fractions (E221–E223). Subfraction E223 (52 mg) was sepa-
rated by preparative normal-phase HPLC using a solvent
system of CHCl3/MeOH (6:1) over 30 min at a flow rate of
2.0 mL/min to obtain bistoroside B (2, 26 mg) and helonio-
side B (4, 5 mg). Subfraction E23 (150 mg) was separated
over LiChroprep Lobar1-A RP-18 column using MeOH/H2O
(1:1) to afford smilaside L (5, 8 mg).
Acetylation of 1 and 2
To each compound (1: 3.0 mg; 2: 3.0 mg) mixed with
pyridine (1 mL), acetic anhydride (1 mL) was added. Each
solution was stirred at room temperature for 24 h. Each re-
action solution was extracted with CHCl3 three times to give
each octaacetate, which was purified by a silica gel Waters
Sep-Pak1 Vac 6cc (hexane/EtOAc, 5:1). Octaacetate of 1
(1a): colorless gum. FAB-MS m/z: 1031 [M + H]+. 1H
NMR (CDCl3, 500 MHz) d: 7.93, 7.91 (1H each, d, J =
2.0 Hz, H-2@, H-2@’), 7.34–7.10 (4H, m, H-5@, H-5@’, H-6@,
H-6@’), 7.02, 6.97 (1H each, d, J = 13.0 Hz, H-7@, H-7@’),
5.95, 5.88 (1H each, d, J = 13.0 Hz, H-8@, H-8@’), 5.71 (1H,
d, J = 3.5 Hz, H-1’), 5.63 (1H, d, J = 6.0 Hz, H-3), 5.55
(1H, t, J = 6.0 Hz, H-4), 5.45 (1H, t, J = 9.5 Hz, H-3’),
5.01 (1H, t, J = 9.5 Hz, H-4’), 4.90 (1H, dd, J = 9.5,
3.5 Hz, H-2’), 4.40, 4.17 (1H each, d, J = 12.0 Hz, H2-1),
4.55–4.15 (6H, m, H-5, H2-6, H-5’, H2-6’), 3.91, 3.88 (3H
each, s, OCH3), 2.30, 2.28 (3H each, s, OAc ꢀ 2), 2.13, 2.12,
2.10, 2.05, 1.97, 1.81 (3H each, s, OAc ꢀ 6). Octaacetate of 2
1
(2a) was identical to 1a in terms of H NMR and TLC.
Acknowledgements
The authors would like to thank Mr. Do Kyun Kim, Dr.
Eun Jung Bang, and Dr. Jung Ju Seo at the Korea Basic Sci-
ence Institute for the NMR and MS spectra measurements.
Bistoroside A (1)
25
References
Yellowish gum. ½aꢁ – 6.5 (c 0.25, MeOH). UV lmax
(MeOH) nm: 326, 299D(sh), 235, 217. IR (KBr) nmax: 3354,
2945, 2833, 1699, 1453, 1597, 1278, 1030 cm–1. 1H
(500 MHz) and 13C (125 MHz) NMR data: see Table 1.
HR-FAB-MS m/z: 695.2180 [M + H]+ (calcd. for C32H39O17:
695.2187).
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Republic of Korea; World Health Organization: Seoul, 1998;
p. 53.
(2) Editorial Committee of the Pharmacopoeia of People’s Re-
public of China. The Pharmacopoeia of People’s Republic
of China, Part 1; Chemical Industry Press: Beijing, 2000; p.
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(3) Duwiejua, M.; Zeitlin, I. J.; Waterman, P. G.; Gray, A. I. J.
Pharm. Pharmacol. 1994, 46 (4), 286. PMID:8051612.
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Planta Med. 1999, 65 (4), 371. doi:10.1055/s-2006-960791.
PMID:10364846.
Bistoroside B (2)
25
Yellowish gum. ½aꢁ – 4.7 (c 0.65, MeOH). UV lmax
(MeOH) nm: 327, 299D(sh), 235, 218. IR (KBr) nmax: 3357,
2945, 2833, 1701, 1598, 1518, 1453, 1278, 1121, 1029 cm–1.
1H (500 MHz) and 13C (125 MHz) NMR data: see Table 1.
HR-FAB-MS m/z: 737.2298 [M + H]+ (calcd. for C34H41O18:
737.2293).
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Alkaline hydrolysis of 1 and 2
(7) Manoharan, K. P.; Benny, T. K. H.; Yang, D. Phytochemis-
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Compounds (1: 2.5 mg and 2: 6.0 mg) were hydrolyzed
with 3% KOH/MeOH (3 mL) at room temperature for
15 min. The reaction mixture was neutralized with 2N HCl
(monitored with indicator paper) and filtered. The filtrate was
concentrated under reduced pressure to give a residue that
was chromatographed over Sephadex LH-20 (100% MeOH)
and preparative normal-phase HPLC (CHCl3/MeOH/H2O,
9:7:1.5) to afford methyl (Z)-ferulate13 (0.3 mg from 1;
1.2 mg from 2) and sucrose (0.8 mg from 1; 2.7 mg from
2). The sucrose was identified by comparison of its optical
(8) Liu, X.; Chen, F.; Wu, L.; Wang, S.; Li, W. Shenyang Yaoke
Daxue Xuebao 2004, 21, 187.
(9) Xiao, K.; Xuan, L.; Xu, Y.; Bai, D. Zhongcaoyao 2003, 34,
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cogn. 2000, 31, 426.
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Published by NRC Research Press