Triterpene glycosides from the roots of Sanguisorba officinalis

Article abstract of DOI:10.1016/S0031-9422(01)00083-8

Five triterpene glycosides were isolated from the MeOH extract of Sanguisorba officinalis (Rosaceae) roots, as confirmed by detailed analysis of their ¹H, ¹³C, and two-dimensional NMR data, and by the results of hydrolytic cleavage.

Full text of DOI:10.1016/S0031-9422(01)00083-8

Phytochemistry 57 (2001) 773–779
www.elsevier.com/locate/phytochem
Triterpene glycosides from the roots of Sanguisorba officinalis
Yoshihiro Mimakia,*, Masato Fukushimaa, Akihito Yokosukaa, Yutaka Sashidaa,
Shigenori Furuyab, Hiroshi Sakagamib
aLaboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science,
1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
bDepartment of Dental Pharmacology, Meikai University School of Dentistry, 1-1, Keyaki-dai, Sakado, Saitama 350-0283, Japan
Received 2 November 2000; received in revised form 15 February 2001
Abstract
Five triterpene glycosides were isolated from the MeOH extract of Sanguisorba officinalis (Rosaceae) roots, as confirmed by
1
detailed analysis of their H, 13C, and two-dimensional NMR data, and by the results of hydrolytic cleavage. Three known tri-
terpenes and six known triterpene glycosides were also isolated and identified. The isolated compounds were evaluated for their
cytotoxic activities against HSC-2cells and HGF. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Sanguisorba officinalis; Rosaceae; Triterpene glycosides; Cytotoxic activity; HSC-2cells
1. Introduction
completely known and thus deserved further study. We
systematically examined the triterpene glycosides com-
ponents of S. officinalis and isolated five new triterpene
glycosides, along with three known triterpenes and six
known triterpene glycosides. This paper deals with the
structural elucidation of the new triterpene glycosides on
the basis of spectroscopic analysis, including two-
dimensional (2D) NMR spectroscopic techniques, and of
the results of hydrolytic cleavage. The cytotoxic activities
of the isolated triterpenes against human oral squamous
cell carcinoma (HSC-2) cells and normal human gingival
fibroblasts (HGF) are also reported.
Sanguisorba officinalis L. (Rosaceae) is a perennial
plant that occurs in the northern districts in China. Its
roots have hemostatic, analgesic, and astringent prop-
erties, and have been used in traditional Chinese medi-
cine for the treatment of burns, scalds, and internal
hemorrhage (Cheng and Cao, 1992). A series of hydro-
lysable tannins were reported as characteristic con-
stituents of S. officinalis roots and are considered to be
partially responsible for the therapeutic effects of this
crude drug (Tanaka et al., 1985). Apart from medicinal
folklore background of S. officinalis as mentioned above,
the discovery of a cytotoxic activity associated with
inhibition of DNA topoisomerases of sanguiin H-6, a
dimeric ellagitannin from S. officinalis, is noteworthy
(Bastow et al., 1993). A communication dealing with the
nitric oxide production-suppressing activity of sanguiin
H-6 and H-11 has recently been published (Yokozawa
et al., 2000). Furthermore, S. officinalis is known to
contain a triterpene glycoside, ziyu-glycoside I, and its
derivatives, including their corresponding aglycones
(Cheng and Cao, 1992; Yosioka et al., 1971). However,
triterpene constituents of S. officinalis seemed not to be
2. Results and discussion
A concentrated MeOH extract of S. officinalis roots was
passed through a polystyrene resin (Diaion HP-20) col-
umn, and the MeOH eluate fraction, in which triterpenes
and triterpene glycosides were enriched, was subjected to
column chromatography using silica gel and octadecylsi-
lanized (ODS) silica gel, as well as preparative HPLC, to
yield a total of 14 triterpenes and triterpene glycosides
(compounds 1–14). Compounds 6–14 are known con-
stituents and identified as 3b,19a-dihydroxyurs-12-
en-28-oic acid (6) (Cheng and Cao, 1992), 2a,3a,19a-
trihydroxyurs-12-en-28-oic acid (7) (Cheng and Cao,
1992), 1b,2a,3a,19a-tetrahydroxyurs-12-en-28-oic acid
* Corresponding author. Tel.: +81-426-76-4577; fax: +81-426-76-
4579.
E-mail address: mimakiy@ps.tokaku.ac.jp (Y. Mimaki).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00083-8

774
Y. Mimaki et al. / Phytochemistry 57 (2001) 773–779
(8) (Guang-Yi et al., 1989), 3b,19a-dihydroxyurs-12-en-
28-oic acid 28-b-d-glucopyranosyl ester (9) (Cheng and
Cao, 1992), 2a,3b,19a-trihydroxyurs-12-en-28-oic acid
28-b-d-glucopyranosyl ester (10) (Jia et al., 1993),
2a,3a,19a-trihydroxyurs-12-en-28-oic acid 28-b-d-glu-
copyranosyl ester (11) (Jia et al., 1993), 3b-[(a-l-arabi-
nopyranosyl)oxy]-19a-hydroxyurs-12-en-28-oic acid (12)
(Cheng and Cao, 1992), 3b-[(a-l-arabinopyranosyl)oxy]-
19a-hydroxyurs-12-en-28-oic acid 28-b-d-glucopyranosyl
ester (13) (Cheng and Cao, 1992), 3b-[(a-l-arabinopyr-
anosyl)oxy]-23-hydroxyolean-12-en-28-oic acid 28-b-d-
glucopyranosyl ester (14) (Kizu et al., 1985), respec-
tively. The physical and spectral data were consistent
with the literature values.
proton at ꢀ 5.52( t-like, J=3.2Hz), which were char-
acteristic of the oleanolic acid skeleton. Furthermore,
two anomeric proton signals were identified at ꢀ 6.39 (d,
J=8.1 Hz) and 4.78 (d, J=7.0 Hz). Acid hydrolysis of 1
with 1 M HCl in dioxane–H2O (1:1) gave a triterpene
(C30H48O4) (1a), and d-glucose and l-arabinose as the
carbohydrate components. The monosaccharides, includ-
ing their absolute configurations, were identified by direct
HPLC analysis of the hydrolysate, with detection being
carried out by using an optical rotation (OR) detector.
Comparison of the 1H and 13C NMR spectroscopic
assignments of 1a with those of oleanolic acid (Kizu et
al., 1995), taking into account the molecular formula of
1a, suggested that 1a was an oleanolic acid derivative
with a hydroxyl group at the ring E portion. The proton
signal at ꢀ 3.66 showed long-range correlations with C-13
(ꢀ 144.9), C-17 (ꢀ 46.1), and C-28 (ꢀ 180.9) in the HMBC
spectrum, and was assigned to H-18. This proton had a
proton spin-coupling correlation with the signal at ꢀ 3.64,
which was associated with the carbon signal at ꢀ 81.3 (CH)
in the HMQC spectrum. Thus, the presence of a hydroxyl
3
group at C-19 was evident. The JH,H value of 2.8 Hz
between H-18 and H-19, and NOE correlations from H-
19 to Me-29 (ꢀ 1.21) and Me-30 (ꢀ 1.13) gave evidence
1
for the C-19a hydroxy configuration. The H and 13C
NMR signals of the glycoside moieties were readily
assigned by interpretation of the 1H–1H COSY and
HMQC spectra. Proton multiplet patterns and coupling
constants, as well as the proton and carbon chemical
shifts revealed that 1 contained that an a-l-arabinopyr-
anosyl unit and a b-d-glucopyranosyl unit, which were
directly linked to the aglycone. In the HMBC spectrum
of 1, correlation peaks were observed from ꢀ 4.78
(anomer of arabinosyl) to ꢀ 88.7 (C-3 of aglycone), and ꢀ
6.39 (anomer of glucosyl) to ꢀ 177.2(C-28 of aglycone).
Accordingly, the structure of 1 was characterized as 3b-
[(a-l-arabinopyranosyl)oxy]-19a-hydroxyolean-12-en-28-
oic acid 28-b-d-glucopyranosyl ester.
Compound 2 was obtained as an amorphous solid,
and its molecular formula, C41H64O12, was deduced
from the positive-ion FABMS (m/z 771 [M+Na]+) and
the result of elemental analysis. The 1H NMR spectrum
of 2 (C5D5N) contained signals for five tertiary methyl
groups at ꢀ 1.29ꢁ2, 1.14, 0.97, and 0.89 (each s), a sec-
ondary methyl group at ꢀ 1.07 (d, J=6.4 Hz), an exo-
methylene group at ꢀ 5.21 and 5.06 (each br s), an olefinic
proton at ꢀ 5.55 (t-like, J=3.3 Hz), and two anomeric
protons at ꢀ 6.34 (d, J=8.1 Hz) and 4.79 (d, J=7.1 Hz).
These data and the 13C NMR spectral feature suggested
that 2 was a triterpene bisdesmoside based upon ursolic
acid. In the HMBC spectrum, the exomethylene protons
showed long-range correlations with the carbons due to
C-18 (ꢀ 52.2) and C-20 (ꢀ 37.5), indicating the location
of the exomethylene group at C-19(29). Based on an
NOE correlation between the protons of Me-30 and H-
21a, Me-30 was assigned as a-configuration. Acid
Compound 1 was isolated as an amorphous solid with
a molecular formula, C41H66O13, as determined by ele-
mental analysis combined with data of the positive-ion
FABMS exhibiting an [M+Na]+ ion at m/z 789. The
IR spectrum showed a characteristic absorption attri-
butable to an ester carbonyl group at 1731 cmꢀ1, as well
as a broad absorption due to hydroxyl groups near 3418
1
cmꢀ1. The H NMR spectrum of 1 (C5D5N) exhibited
signals for seven tertiary methyl groups at ꢀ 1.65, 1.27,
1.16ꢁ2, 1.00, 0.97, and 0.91 (each s), and an olefinic

Y. Mimaki et al. / Phytochemistry 57 (2001) 773–779
775
hydrolysis of 2 with 1 M HCl resulted in the production
of two artifactual triterpenes (2a, 2b), together with d-
glucose and l-arabinose. The H NMR spectrum of 2a
Compound 5 was obtained as a pale-yellow solid. Its
molecular formula was determined as C48H70O17 on the
basis of the positive-ion FABMS (m/z 941 [M+Na]+)
and elemental analysis. The 1H NMR spectrum included
signals for seven triterpene methyls at ꢀ 1.68 (s), 1.33 (s),
1.25 (s), 1.19 (s), 1.02( d, J=6.7 Hz), 0.99 (s), and 0.93
(s), an olefinic proton at ꢀ 5.53 (t-like, J=3.3 Hz), and
two anomeric protons at ꢀ 6.30 (d, J=8.0 Hz) and 4.76
(d, J=7.0 Hz). In addition, the presence of a galloyl ester
group was suggested by the UV [lmax 278 nm (log " 3.75)],
1H NMR [ꢀ 7.87 (2H, s)], and 13C NMR [ꢀ 167.4 (C¼O),
147.4 (Cꢁ2), 140.8 (C), 121.1 (C), 110.3 (CHꢁ2)]. Alka-
line treatment of 5 with 10% NH3aq yielded 13 and gallic
acid. When the 1H and 13C NMR spectra of 5 were com-
pared with those of 13, downfield shifts by O-acylation
were detected at the H2-6 signal at ꢀ 5.01 (2H, d, J=9.2
Hz) and the C-6 signal at ꢀ 64.6 of the glucosyl residue.
Furthermore, an HMBC correlation was observed from
ꢀ 5.01 to the carbonyl carbon resonance of the galloyl
group at ꢀ 167.4. All of these data for 5 were consistent
with the structure 3b-[(a-l-arabinopyranosyl)oxy]-19a-
hydroxyurs-12-en-28-oic acid 28-(6-O-galloyl-b-d-glu-
copyranosyl) ester.
1
showed seven three-proton singlet signals, and two of
them were assigned as being located on a double bond.
The UV spectrum of 2b showed a lmax at 226 nm (log "
3.65), which implied the presence of a conjugated diene
1
group, and the H NMR spectrum included signals for
five tertiary methyl groups, a secondary methyl group,
and a methyl group on a double bond. These spectral
properties combined with information from the MS, 13
C
NMR, and various 2D NMR spectra allowed the iden-
tification of 2a and 2b as 3b-hydroxyurs-12,19(20)-dien-
28-oic acid and 3b-hydroxyurs-12,18-dien-28-oic acid,
respectively (Ahmad and Basha, 2000). These com-
pounds were considered to be produced through migra-
tion of the C-19(29) double bond under acidic
1
conditions. The H, 13C, and 2D NMR spectral data of
2 indicated that it had an a-l-arabinopyranosyl unit at
C-3 and a b-d-glucopyranosyl unit at C-28 in the mole-
cule, as in 1. The structure of 2 was characterized as 3b-
[(a-l-arabinopyranosyl)oxy]urs-12,19(29)-dien-28-oic
acid 28-b-d-glucopyranosyl ester.
Compound 3 was shown to have the molecular for-
mula, C41H64O13, by the positive-ion FABMS (m/z 787
[M+Na]+) and elemental analysis. The spectral prop-
erties of 3 were very similar to those of 2. However, the
molecular formula of 3 differed from 2 by containing an
Compounds 1–5 are new triterpene glycosides, and
this is the first report on the isolation of 8, 10, 11, and
14 from S. officinalis.
The isolated compounds were evaluated for their
cytotoxic activities against HSC-2cells and HGF
(Table 2). Compounds 3 and 14, which are bisdesmosidic
triterpenes with the oleanane or ursane skeleton bearing
a hydroxyl group at C-23, showed cytotoxic activity
against HSC-2cells with the respective IC 50 values of 15
and 18 mg/ml, although they were not cytotoxic against
HGF at all (IC50 >200 mg/ml).
1
additional oxygen atom. When the H NMR spectrum
of 3 was compared with that of 2, the signal for Me-23,
which was observed at ꢀ 1.29 in 2, disappeared in 3, and
instead signals for an hydroxymethyl group were detec-
ted at ꢀ 4.30 and 3.70 (ABq, J=10.6 Hz). The 13C NMR
spectrum of 3, in which a signal at ꢀ 64.5 was observed
for an hydroxymethyl group rather than a tertiary
methyl group, and an NOE correlation between the
hydroxymethyl protons and H-6a equatorial proton (ꢀ
1.67), also supported this structural modification. The
structure of 3 was shown to be 3b-[(a-l-arabinopyr-
anosyl)oxy]-23-hydroxyurs-12,19(29)-dien-28-oic acid 28-
b-d-glucopyranosyl ester.
3. Experimental
3.1. General
FABMS: Finnigan MAT TSQ-700 (San Jose, CA,
USA) (matrix: Magic Bullet, a mixture of dithiothreitol
and dithioerythritol, 3:1; Tokyo-Kasei, Tokyo, Japan).
Compound 4 had the molecular formula C35H54O7 on
the basis of the positive-ion FABMS (m/z 609 [M+Na]+)
1
1
and elemental analysis. The H NMR spectrum showed
NMR (ppm, J Hz): Bruker DPX-400 (400 MHz for H
NMR, Karlsruhe, Germany) or Bruker AM-500 (500
signals for six tertiary methyl groups at ꢀ 1.89, 1.31, 1.18,
1.04, 0.97, and 0.89 (each s ), a secondary methyl group at
ꢀ 1.13 (d, J=6.9 Hz), an olefinic proton at ꢀ 5.73 (t-like,
J=2.8 Hz), and an anomeric proton at ꢀ 4.80 (d, J=7.1
Hz). The UV absorption at lmax 2 2 6 (log" 3.79) were
attributable to a conjugated diene system. Acid hydrolysis
of 4 gave 2b and l-arabinose. The 13C NMR spectrum of
4 showed data consistent with an arabinose moiety, and it
was found to be attached at C-3 of the aglycone by an
HMBC correlation from ꢀ 4.80 to ꢀ 88.7 (C-3 of aglycone).
The structure of 4 was determined to be 3b-[(a-l-arabi-
nopyranosyl)oxy]urs-12,18-dien-28-oic acid.
1
MHz for H NMR). CC: Diaion HP-20 (Mitsubishi-
Kasei, Tokyo, Japan); silica gel (Fuji-Silysia Chemical,
Aichi, Japan); ODS silica gel (Nacalai Tesque, Kyoto,
Japan); Sephadex LH-20 (Pharmacia, Freiburg, Ger-
many). TLC: precoated Kieselgel 60 F254 (0.25 mm
thick, Merck, Darmstadt, Germany) and RP-18 F254S
(0.25 mm thick, Merck). Preparative HPLC: a Tosoh
HPLC system (pump, CCPM; controller, CCP con-
troller PX-8010) equipped with an RI detector (Tosoh
RI-8010, Tokyo, Japan) and a Capcell Pak C18 UG80
column (10 mm i.d.ꢁ250 mm, ODS, 5 mm, Shiseido,

776
Y. Mimaki et al. / Phytochemistry 57 (2001) 773–779
Tokyo, Japan). Cell culture and assay for cytotoxic activ-
ity: Dulbecco’s modified Eagle medium (DMEM) (Gibco,
Grand Island, NY, USA); fetal bovine serum (FBS) (JRH
Biosciences, Lenexa, KS, USA); penicillin, streptomycin,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT), and a-minimum essential medium (a-
MEM) (Sigma, St. Louis, MO, USA). All other chemicals
used were of biochemical reagent grade.
to give 3 (21.4 mg) and 5 (23.8 mg), and 14 with a few
impurities. Compound 14 was purified by preparative
HPLC using using MeOH–H2O (2:1) to afford 14 (9.4
mg) as a pure compound.
3.4. Compound 1
An amorphous solid. [a]2D5 +14.0ꢂ (MeOH: c 0.10).
FABMS (positive mode) m/z 789 [M+Na]+. (Found:
.
3.2. Plant material
C, 60.68; H, 8.64. Calc. for C41H66O13 5/2H2O: C,
60.66; H, 8.81%). IR ꢁmax (film) cmꢀ1: 3418 (OH), 2927
(CH), 1731 (C¼O), 1455, 1388, 1260, 1190, 1071, 1028,
Sanguisorba officinalis was purchased from a whole-
sale firm in Uchida-Wakanyaku, Tokyo Japan in 1997.
One of the authors, Y. Sashida., identified it by its
morphological characters. A voucher specimen has been
deposited in the laboratory of Y. Sashida (voucher No.
SO-97-7, Laboratory of Medicinal Plant Science).
1
866. H NMR (pyridine-d5): ꢀ; 6.39 (1H, d, J=8.1 Hz,
H-100), 5.52(1H, t-like, J=3.2Hz, H-12), 4.78 (1H, d,
J=7.0 Hz, H-10), 3.59 (1H, d, J=2.8 Hz, H-19), 3.34
(1H, dd, J=11.7, 4.3 Hz, H-3), 1.65 (3H, s, Me-27), 1.27
(3H, s, Me-23), 1.16 (3Hꢁ2, s, Me-26 and Me-29), 1.00
(3H, s, Me-30), 0.97 (3H, s, Me-24), 0.91 (3H, s, Me-25).
13C NMR: Table 1.
3.3. Extraction and isolation
The dessicated roots of the plant material (3 kg) were
extracted with hot MeOH. The MeOH extract was
concentrated under reduced pressure, and the viscous
concentrate (320 g) was passed through a Diaion HP-20
column eluting with increased amounts of MeOH in
H2O (30% MeOH, 50% MeOH, and 80% MeOH),
MeOH, EtOH, and EtOAc. Preliminary TLC analysis
of these fractions suggested that the MeOH eluate por-
tion contained a variety of triterpene and triterpene
glycosides, on which further fractionation was carried
out. CC of the MeOH eluate portion on silica gel and
elution with a stepwise gradient mixture of CHCl3–
MeOH (9:1; 4:1; 2:1), and finally with MeOH alone,
gave five fractions (I–V). Fraction I was applied to a
silica gel column this being eluted with hexane–Me2CO
(1:1) and CHCl3–MeOH (19:1) and ODS silica gel with
MeOH–H2O (4:1) to give 4 (9.0 mg), 6 (43.2mg), 7 (7.5
mg), and 8 (5.5 mg). Fraction II was subjected to CC on
silica gel eluting with CHCl3–MeOH–H2O (80:10:1;
50:10:1) and ODS silica gel with MeOH–H2O (3:1) and
MeCN–H2O (2:3; 1:2) to yield 9 (7.0 mg), 11 (115 mg),
and 12 (104 mg). Fraction III was subjected to a silica
gel column eluting with CHCl3–MeOH–H2O (80:10:1;
50:10:1) and an ODS silica gel column with MeOH–
H2O (2:1) and MeCN–H2O (1:2) to give 10 (3.2mg).
Fraction IV was applied to a silica gel column which
was eluted with CHCl3–MeOH–H2O (50:10:1) and ODS
silica gel with MeOH–H2O (2:1) to give a mixture of 1
and 13, and 2 with a few impurities. The mixture was
separated by preparative HPLC using MeOH–H2O
(3:2) into 1 (40.2mg) and 13 (180 mg). Compound 2
was completely refined by preparative HPLC using
MeOH–H2O (4:1) to furnish 2 (140 mg) as a pure com-
pound. Fraction V was applied to a silica gel column
eluted with CHCl3–MeOH–H2O (40:10:1) and ODS
silica gel with MeOH–H2O (2:1) and MeCN–H2O (1:2)
3.5. Acid hydrolysis of 1
A solution of 1 (25 mg) in 1 M HCl (dioxane–H2O, 1:1,
4 ml) was heated at 95ꢂ for 2h under an Ar atmosphere.
After cooling, the reaction mixture was neutralized by
passage through an Amberlite IRA-93ZU (Organo,
Tokyo, Japan) column and subjected to silica gel chro-
matography using a gradient mixture of CHCl3–MeOH
(19:1; 9:1; 1:1) to give an aglycone fraction and a sugar
fraction (5.9 mg). The aglycone fraction was purified by
silica gel CC eluting with hexane–Me2CO (4:1) to give an
aglycone (1a) (3.2mg). HPLC analysis of the sugar frac-
tion under the following conditions showed the presence
of d-glucose and l-arabinose. Column: Capcell Pak NH2
UG80 (4.6 mm i.d.ꢁ250 mm, 5 mm, Shiseido); detector:
Shodex OR-2(Showa-Denko, Tokyo, Japan); solvent:
MeCN–H2O (17:3); flow rate: 1.0 ml/min. Rt (min): 8.27
(l-arabinose, positive optical rotation); 13.39 (d-glucose,
positive optical rotation).
3.6. Compound 1a
An amorphous solid. [a]2D5 +20.0ꢂ (MeOH: c 0.10).
EIMS m/z 472[M] +. IR ꢁmax (film) cmꢀ1: 3449 (OH),
2927 (CH), 1688 (C¼O), 1455, 1388, 1260, 1071, 1028,
1
866. H NMR (pyridine-d5): ꢀ 5.60 (1H, t-like, J=3.3
Hz, H-12), 3.66 (1H, d, J=2.8 Hz, H-18), 3.64 (1H, d,
J=2.8 Hz, H-19), 3.46 (1H, dd, J=11.7, 4.3 Hz, H-3),
1.67 (3H, s, Me-27), 1.26 (3H, s, Me-23), 1.21 (3H, s,
Me-29), 1.13 (3H, s, Me-30), 1.10 (3H, s, Me-26), 1.05
(3H, s, Me-24), 0.95 (3H, s, Me-25). 13C NMR: Table 2.
3.7. Compound 2
An amorphous solid. [a]2D5 +12.0ꢂ (MeOH: c 0.10).
FABMS (positive mode) m/z 771 [M+Na]+. (Found:

Y. Mimaki et al. / Phytochemistry 57 (2001) 773–779
777
Table 1
13C NMR spectral data for compounds 1, 1a, 2, 2a, 2b and 3–5 in
pyridine-d5
Table 2
Cytotoxic activites of compounds 1–14 against HSC-2cells and HGF
IC50 (mg/ml)
C
1
1a
2
2a
39.3
2b
39.4
3
4
5
Compounds
HSC-2HGF
1
38.7
26.6
38.8
28.4
38.9
26.7
38.9
26.1
39.3
27.1
38.9
26.6
1
>200
50
15
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
2
28.1
28.2
2
3
88.7
39.6
78.288.7
39.4
56.0
78.1
78.1
81.9
88.7
88.8
3
4
39.6
56.0
18.5
39.6
55.9
18.7
39.5
56.0
18.7
43.5
47.7
39.6
56.1
39.6
55.9
4
>200
79
5
56.0
18.7
5
6
19.0
18.218.5
35.6
39.4
18.6
33.5
40.5
6
>200
>200
100
7
33.233.7
40.240.0
48.3
33.4
39.8
34.3
39.3
35.6
39.3
33.1
39.9
48.1
36.9
7
8
8
9
48.4
48.0
48.248.3
48.247.7
37.0
9
50
10
11
37.237.6
24.1
37.1
37.237.237.0
23.6
10
11
12
13
14
>200
>200
>200
153
24.2
23.8
23.4
23.9
23.4
24.0
12 123.5 123.5 128.5 127.3 125.9 128.6 125.9 128.5
13 144.3 144.9 137.5 138.5 139.4 137.5 139.5 139.2
14
15
16
17
18
19
20
21
22
23
24
25
26
27
42.1
29.0
28.0
46.5
44.6
81.0
35.5
28.9
33.0
28.2
16.8
15.5
17.6
24.9
42.2
30.0
28.1
46.1
44.9
42.8
29.1
25.8
49.8
52.2
43.8
28.6
23.9
46.9
45.0
29.1
27.0
49.7
42.8
29.1
25.8
49.8
45.0
29.2
26.8
49.8
42.0
29.1
26.0
48.7
54.3
72.6
42.0
26.6
37.8
28.2
17.4
15.6
16.9
24.6
18
50.7 123.7
52.2 123.9
3.8. Acid hydrolysis of 2
81.3 153.4 129.0 134.6 153.4 134.7
35.7
29.2
33.4
29.2
16.5
15.5
17.5
24.9
37.5 123.9
34.8
31.8
35.1
28.8
16.6
16.3
18.3
22.1
37.5
30.6
37.1
64.5
13.6
16.3
17.4
26.2
34.8
31.9
35.1
28.3
17.0
16.3
18.3
22.1
Compound 2 (20 mg) was subjected to acid hydrolysis
as described for 1 to give 2a (4.8 mg) and 2b (3.5 mg),
and a sugar fraction (4.1 mg). HPLC analysis of the
sugar fraction under the same conditions as in the case
of that of 1 showed the presence of d-glucose and l-
arabinose.
30.6
37.1
28.2
17.0
15.7
17.3
26.2
28.7
33.5
28.8
16.6
16.1
18.0
22.1
28 177.2 180.9 176.0 180.0 178.5 176.1 178.7 177.0
29
30
10
28.7
24.7
107.6
72.9
74.6
69.5
66.8
95.9
74.1
78.9
71.1
79.3
62.2
28.8 110.5
17.3
20.5
19.6 110.4
18.9 19.4
19.6
18.9
26.9
16.6
3.9. Compound 2a
24.9
19.4
107.6
72.9
74.7
69.6
66.8
95.6
74.1
78.9
71.1
79.4
62.2
106.6 107.5 107.4
An amorphous solid. [a]2D5 +4.0ꢂ (MeOH: c 0.10).
EIMS m/z 454 [M]+. IR ꢁmax (film) cmꢀ1: 3383 (OH),
2927 (CH), 1689 (C¼O), 1449, 1378, 1256, 1091, 1025,
20
73.1
74.7
69.6
67.0
95.9
74.1
78.9
71.1
79.3
62.3
72.9
74.6
69.5
66.7
72.9
74.6
30
400
69.5
66.6
1
500
100
200
300
400
500
6000
1
866. H NMR (pyridine-d5): ꢀ 5.71 (1H, t-like, J=3.5
95.7
74.0
Hz, H-12), 3.64 (1H, br s, H-18), 3.46 (1H, dd, J=11.3,
4.3 Hz, H-3), 1.71 (3H, s, Me-29), 1.64 (3H, s, Me-30),
1.23 (3H, s, Me-23), 1.15 (3H, s, Me-27), 1.04 (3H, s,
Me-26), 1.03 (3H, s, Me-25), 0.92 (3H, s, Me-24). 13C
NMR: Table 2.
78.8
71.3
76.1
64.6
121.1
110.3
147.4
140.8
147.4
110.3
167.4
2000
3000
4000
5000
6000
7000
3.10. Compound 2b
An amorphous solid. [a]2D5 +166ꢂ (MeOH: c 0.10).
EIMS m/z 454 [M]+. UV lmax (MeOH) nm: 226 (log "
3.65). IR ꢁmax (film) cmꢀ1: 3412 (OH), 2927 (CH), 1689
1
(C¼O), 1460, 1376, 1258, 1096, 1034. H NMR (pyri-
dine-d5): ꢀ 5.74 (1H, t-like, J=2.8 Hz, H-12), 3.48 (1H,
dd, J=11.3, 4.3 Hz, H-3), 1.88 (3H, s, Me-29), 1.25 (3H,
s, Me-23), 1.15 (3H, s, Me-27), 1.12 (3H, d, J=6.9 Hz,
Me-30), 1.06 (3H, s, Me-26), 1.04 (3H, s, Me-24), 0.93
(3H, s, Me-25). 13C NMR: Table 2.
.
C, 59.97; H, 8.66. Calc. for C41H64O12 4H2O: C, 59.98; H,
8.84%). IR ꢁmax (film) cmꢀ1: 3375 (OH), 2927 (CH), 1731
(C¼O), 1455, 1388, 1260, 1200, 1136, 1071, 986. 1H
NMR (pyridine-d5): ꢀ 6.34 (1H, d, J=8.1 Hz, H-100), 5.55
(1H, t-like, J=3.3 Hz, H-12), 5.21 and 5.06 (each 1H, br s,
CH2-29), 4.79 (1H, d, J=7.1 Hz, H-10), 3.39 (1H, dd,
J=11.8, 4.2Hz, H-3), 1.29 (3H ꢁ2, s, Me-23 and Me-27),
1.14 (3H, s, Me-2 6), 1.07 (3H,d, J=6.4 Hz, Me-30), 0.97
(3H, s, Me-24), 0.89 (3H, s, Me-25). 13C NMR: Table 2.
3.11. Compound 3
An amorphous solid. [a]2D5 +24.0ꢂ (MeOH: c 0.10).
FABMS (positive mode) m/z 787 [M+Na]+. (Found:

778
Y. Mimaki et al. / Phytochemistry 57 (2001) 773–779
.
C, 63.28; H, 8.75. Calc. for C41H64O13 1/2H2O: C,
63.63; H, 8.47%). IR ꢁmax (film) cmꢀ1: 3376 (OH), 2935
(CH), 1733 (C¼O), 1455, 1388, 1260, 1198, 1136, 1073.
of solvent, the reaction mixture was chromatographed
on Sephadex LH-20 eluting with MeOH to furnish 13
(6.3 mg) and gallic acid (1.2mg).
00
1H NMR (pyridine-d5): ꢀ 6.32(1H, d, J=8.2Hz, H-1 ),
5.54 (1H, t-like, J=3.1 Hz, H-12), 5.18 and 5.03 (each
1H, ABq, J=1.4 Hz, CH2-29), 4.79 (1H, d, J=7.2Hz,
H-10), 4.31 (1H, overlapping, H-3), 4.30 and 3.70 (each
1H, ABq, J=10.6 Hz, CH2-23), 1.19 (3H, s, Me-27),
1.16 (3H, s, Me-25), 1.06 (3H, d, J=6.4 Hz, Me-30),
0.99 (3H, s, Me-26), 0.93 (3H, s, Me-24). 13C NMR:
Table 2.
3.16. Cell culture
HSC-2cells were maintained as monolayer cultures at
37ꢂ in DMEM medium supplemented with 10% heat-
inactivated FBS in a humidified 5% CO2 atmosphere.
HGF were isolated, as described previously (Ito et al.,
2000). Briefly, gingival tissues were obtained from heal-
thy gingival biopsies from a 10-year-old girl, undergoing
periodontal surgery. The tissue were cut into 1 to 2mm 3
pieces, washed twice with phosphate-buffered saline (PBS,
0.01 M phosphate buffer, 0.15 M NaCl, pH 7.4) supple-
mented with 100 U/ml penicillin and 100 mg/ml strepto-
mycin, and placed into 25 cm2 tissue culture flasks. The
explants were incubated in a-MEM supplemented with
30% FBS and antibiotics. When outgrowth of the cells
was observed, the medium was replaced twice until the
cells reached confluence. The cells were detached from
the monolayer by trypsinization and recultured in 100
cm2 tissue culture flasks until confluent monolayers
were again obtained. Cells between the fifth and seventh
passages were used.
3.12. Compound 4
An amorphous solid. [a]2D5 +112ꢂ (MeOH: c 0.10).
FABMS (positive mode) m/z 609 [M+Na]+. (Found:
.
C, 63.86; H, 9.20. Calc. for C35H54O7 4H2O: C, 63.80;
H, 9.48%). UV lmax (MeOH) nm: 226 (log " 3.79). IR
ꢁmax (film) cmꢀ1: 3367 (OH), 2929 (CH), 1688 (C¼O),
1449, 1388, 1367, 1256, 1136, 1083, 998. 1H NMR
(pyridine-d5): ꢀ 5.73 (1H, t-like, J=2.8 Hz, H-12), 4.80
(1H, d, J=7.1 Hz, H-10), 3.40 (1H, dd, J=12.0, 4.1 Hz,
H-3), 1.89 (3H, s, Me-29), 1.31 (3H, s, Me-23), 1.18 (3H,
s, Me-27), 1.13 (3H, d, J=6.9 Hz, Me-30), 1.04 (3H, s,
Me-26), 0.97 (3H, s, Me-24), 0.89 (3H, s, Me-25). 13C
NMR: Table 2.
3.17. Assay for cytotoxic activity
3.13. Acid hydrolysis of 4
Cells were trypsinized and inoculated at 6ꢁ103 per
each 96-microwell plate (Falcon, flat bottom, treated
polystyrene, Becton Dickinson, San Jose, CA, USA),
and incubated for 24 h. After washing once with PBS,
they were treated with 24 h without or with test com-
pounds. They were washed once with PBS and incu-
bated for 4 h with 0.2mg/ml MTT in DMEM medium
supplemented with 10% FBS. After the medium was
removed, the cells were lysed with 0.1 ml DMSO and
the relative viable cell number was determined by mea-
suring the absorbance at 540 nm of the cell lysate, using
Labsystems MultiskanR (Biochromatic, Helsinki, Fin-
land) connected to a Star/DOT Matrix printer JL-10.
The LD50 value, which reduces the viable cell number
by 50%, was determined from the dose-response curve.
Compound 4 (5 mg) was subjected to acid hydrolysis
as described for 1 to give 2b (2.7 mg) and a sugar frac-
tion (0.7 mg). HPLC analysis of the sugar fraction
under the same conditions as in the case of that of 1
showed the presence of l-arabinose.
3.14. Compound 5
A pale-yellow amorphous solid. [a]2D5 +10.0ꢂ (MeOH:
c 0.10). FABMS (positive mode) m/z 941 [M+Na]+.
.
(Found: C, 55.65; H, 7.74. Calc. for C48H70O17 13/
2H2O: C, 55.63; H, 8.07%). UV lmax (MeOH) nm: 278
(log " 3.75). IR ꢁmax (film) cmꢀ1: 3418 (OH), 2931 and
2878 (CH), 1723 and 1714 (C¼O), 1614 and 1538 (aro-
1
matic ring), 1455, 1350, 1228, 1137, 1078, 938, 870. H
NMR (pyridine-d5): ꢀ 7.87 (2H, s, H-2000 and H-6000), 6.30
(1H, d, J=8.0 Hz, H-100), 5.53 (1H, t-like, J=3.3 Hz, H-
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