Anti-complement activity of norlignans and terpenes from the stem bark of Styrax japonica

Article abstract of DOI:10.1055/s-2004-835853

A new norlignan, styraxlignolide A (1), and two new terpenes, styraxosides A (2) and B (3), were isolated from the MeOH-soluble fraction of Styrax japonica Sieb. et Zucc. (Styracaceae) stem bark, together with two known compounds, egonol (4) and masutakeside I (5). The new compounds were determined as 5-(3″-hydroxypropyl)-7-methoxy-2-(3′,4′-dimethoxyphenyl) -benzofuran 3″-O-[β-D-xylopyranoside-(1→6)-β-D- glucopyranoside] (1), 3β,7β-dihydroxy-4α,4β,8β, 10β,14α-pentamethyl-5α-gon-16-en-2-one 3-O-[β-D- glucopyranoside-(1→2)-β-D-glucopyranoside] (2), and 3β,17β-dihydroxy-28-norolean-12-en-16-one 3-O-[α-L- rhamopyranoside-(1→2)-β-D-glucuronopyranoside] (3) by spectroscopic means including 2D-NMR. The five compounds were tested in vitro for anti-complement activity against the complement system. Compounds 1, 3, 4, and 5 displayed inhibitory activity in the anti-complement assay, with IC ₅₀ values of 123, 65, 33, and 166 μM, respectively. Compound 1a and camellenodiol (3a), obtained from acid hydrolysis of 1 and 3, respectively, did not affect the hemolytic activity of human serum against sensitized erythrocytes. This shows that a sugar seems to play a role of enhancing significantly anti-complement activity.

Full text of DOI:10.1055/s-2004-835853

Byung-Sun Min1
Sei-Ryang Oh1
Kyung-Seop Ahn1
Jung-Hee Kim1
Joongku Lee1
Doo-Young Kim1
Eun-Hee Kim2
Hyeong-Kyu Lee1
Anti-Complement Activity of Norlignans and Terpenes
from the Stem Bark of Styrax japonica
Abstract
troscopic means including 2D-NMR. The five compounds were
tested in vitro for anti-complement activity against the comple-
A new norlignan, styraxlignolide A (1), and two new terpenes, ment system. Compounds 1, 3, 4, and 5 displayed inhibitory ac-
styraxosides A (2) and B (3), were isolated from the MeOH-solu- tivity in the anti-complement assay, with IC50 values of 123, 65,
ble fraction of Styrax japonica Sieb. et Zucc. (Styracaceae) stem 33, and 166 mM, respectively. Compound 1a and camellenodiol
bark, together with two known compounds, egonol (4) and ma- (3a), obtained from acid hydrolysis of 1 and 3, respectively, did
sutakeside I (5). The new compounds were determined as 5-(3¢¢- not affect the hemolytic activity of human serum against sensi-
hydroxypropyl)-7-methoxy-2-(3¢,4¢-dimethoxyphenyl)-benzo- tized erythrocytes. This shows that a sugar seems to play a role
furan 3¢¢-O-[b-D-xylopyranoside-(1®6)-b-D-glucopyranoside] (1), of enhancing significantly anti-complement activity.
3b,7b-dihydroxy-4a,4b,8b,10b,14a-pentamethyl-5a-gon-16-en-
2-one 3-O-[b-D-glucopyranoside-(1®2)-b-D-glucopyranoside] Key words
(2), and 3b,17b-dihydroxy-28-norolean-12-en-16-one 3-O-[a-L- Styrax japonica ´ Styracaceae ´ anti-complement activity ´ nor-
rhamopyranoside-(1®2)-b-D-glucuronopyranoside] (3) by spec- lignan ´ octanordammarane noroleanane ´ egonol
1210
Introduction
and immunomodulation. However, the smaller molecules, C3a,
C4a, and C5a, designated anaphylatoxins, induce the release of
The complement system is activated either by a cascade mecha- mediators from mast cells and lymphocytes, which cause a vari-
nism of the classical pathway (CP), an alternative pathway (AP), ety of inflammatory diseases, and may be fatal if they occur after
or the mannan-binding lectin (MBL)-associated serine protease organ transplantation [2], [3]. Therefore, modulation of comple-
(MBL/MASP) pathway [1]. The thirty odd complement fragments ment activity should be beneficial in the therapy of inflamma-
that make up the system include proteolytic pro-enzymes, non- tory diseases.
enzymatic components that form functional complexes, co-fac-
tors, regulators, and receptors [2]. The proteolytic cascade allows Styrax japonica Sieb. et Zucc. (Styracaceae) is a deciduous tree
for significant amplification since each proteinase molecule grown in the Southern areas of Korea and Japan. The pericarps
activated at one step can generate multiple copies of activated are used as washing soap, cough medicine, and as a piscicidal
enzyme later in the cascade which, in turn, cleave non-enzymat- agent [4]. Previous phytochemical studies performed on this
ic components, such as C3, C4, and C5. The larger fragments de- plant included the isolation of jegosaponins and benzofurans
rived from C3, C4, and C5 (i.e., C3b, C4b, and C5b) are involved in [5], [6], [7], [8]. Jegosaponins from fresh fruits display an anti-
biological effector functions, such as opsonization, phagocytosis, sweet activity [4], and egonol from the seeds has attracted the
Affiliation
1 Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
2 Korea Basic Science Institute, Daejeon, Korea
Correspondence
Hyeong-Kyu Lee ´ Korea Research Institute of Bioscience and Biotechnology ´ Yusung P. O. Box 115 ´ Daejeon ´
Korea ´ Phone: +82-42-860-4413 ´ Fax: +82-42-860-4309 ´ E-mail: hykylee@kribb.re.kr
Received March 26, 2004 ´ Accepted July 10, 2004
Bibliography
Planta Med 2004; 70: 1210±1215 ´ ꢀ Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2004-835853
ISSN 0032-0943

attention of synthetic organic chemists, due to its activity against Enzymatic hydrolysis of 1: Naringinase (200 mg, from Penicillium
human leukemic HL-60 cells [9], [10]. In this study, we report the decumbens) was added to a suspension of 1 (20 mg) in 50 mM
isolation, structural elucidation, and anti-complement activity of acetate buffer (pH 5.5) and the mixture was stirred at 378C for
lignans and terpenes isolated from the stem bark of S. japonica.
5 h. The reaction mixture was extracted with EtOAc (10 mL”3)
and the organic layer was evaporated to dryness. The residue
was separated by preparative TLC with CHCl3/MeOH (9:1;
Rf = 0.55) to give 5-(3¢¢-hydroxypropyl)-7-methoxy-2-(3¢,4¢-di-
methoxyphenyl)-benzofuran (1a, 10 mg) as white amorphous
powder. 1H- and 13C-NMR data, see Table 1.
Materials and Methods
General experimental procedures
Optical rotations were measured with a JASCO DIP-370 digital
polarimeter in MeOH. UV spectra were recorded on a Shimadzu Styraxoside A (2): white amorphous powder. UV (MeOH): lmax
UV-2450 spectrometer. 1H- and 13C-NMR spectra were recorded (log e) = 202 (3.82) nm; [a]D26: ±11.08 (c 0.1, MeOH); FAB-MS:
on a Bruker AMX 600 spectrometer. FAB-MS were measured on m/z = 693 [M + Na]+; HR-FAB-MS: m/z = 693.3463 [M + Na]+
1
a JMS-HX 110/110A spectrometer (JEOL).
(calcd. for C34H54O13Na: 693.3462). H- and 13C-NMR data, see
Table 2.
Plant material
The stem bark of Styrax japonica Sieb. et Zucc. (Styracaceae) was Styraxoside B (3): white amorphous powder. UV (MeOH): lmax
collected at Jeju (Korea) in July 2002. A voucher specimen veri- (log e) = 201 (3.77) nm; [a]D26: ±43.78 (c 0.9, MeOH); FAB-MS:
fied by Dr. Tae-Jin Kim of Korea Research Institute of Bioscience m/z = 787 [M + Na]+; HR-FAB-MS: m/z = 787.4240 [M + Na]+
and Biotechnology (KRIBB), Korea, is deposited at the Plant Ex- (calcd. for C41H64O13Na: 787.4245). 1H- and 13C-NMR data, see
tract Bank (00250), KRIBB, Korea.
Table 2.
Extraction and isolation
Enzymatic hydrolysis of 3: Naringinase (200 mg) was added to a
The stem bark of S. japonica (12.0 kg) was extracted with MeOH suspension of 3 (20 mg) in 50 mM acetate buffer (pH 5.5) and the
at room temperature (4”5 L) to obtain 1.4 kg of a solid extract. mixture was stirred at 378C for 5 h. Work-up as mentioned above
The MeOH extract was suspended in H2O and extracted with (preparative TLC, hexane/EtOAc, 3:1; Rf = 0.6) gave 3b,17b-dihy-
hexane (3”3 L) and EtOAc (3”3 L), successively, to give the hex- droxy-28-norolean-12-en-16-one (3a, 8 mg) as white amor-
ane- (118 g) and EtOAc-soluble fractions (78 g), respectively. The phous powder. 13C-NMR (CDCl3; 150 MHz): d = 15.3 (C-25), 15.6
resulting H2O solution was passed through a Diaion HP-20 col- (C-24), 17.3 (C-26), 18.2 (C-6), 23.6 (C-11), 23.7 (C-30), 27.0 (C-
umn (10.0”85.0 cm) eluting with H2O (8 L), 50% aqueous MeOH 27), 27.2 (C-2), 28.1 (C-23), 30.3 (C-22), 30.8 (C-20), 32.4 (C-29),
(8 L), and MeOH (8 L), respectively. The MeOH-soluble fraction 32.7 (C-7), 36.4 (C-10), 37.0 (C-21), 38.4 (C-1), 38.8 (C-4), 39.8 (C-
(145 g) was chromatographed on an RP C-18 column (6.0”90.0 8), 42.9 (C-15), 46.7 (C-9), 47.1 (C-19), 47.4 (C-14), 52.5 (C-18),
cm) eluted with 50% aqueous MeOH (4 L), 25% aqueous MeOH 55.2 (C-5), 76.5 (C-17), 78.9 (C-3), 125.6 (C-12), 140.4 (C-13),
(4 L), and MeOH (4 L) to yield five fractions (Fr. A±E: 95 g, 6 L; 213.6 (C-16).
1211
35 g, 2.5 L; 8.2 g, 1.5 L; 4.6 g, 1 L; 1.2 g, 1 L). Fr. B was chromato-
graphed on RP C-18 eluted with 50% aqueous MeOH Egonol (4): white amorphous powder. UV (MeOH): lmax (log
(6.0”60 cm, 6 L) to yield five sub-fractions (Fr. B1±B5:2.0 g, 0.8 e) = 219 (4.26), 316 (4.25) nm.
L; 1.3 g, 0.8 L; 1.2 g, 1.0 L; 28.5 g, 2.6 L; 1.6 g, 0.8 L). Fr. B4 was crys-
tallized from MeOH to yield 1 (12.92 g). The mother liquor of Fr. Masutakeside I (5): white amorphous powder. UV (MeOH): lmax
B4 (14.0 g) was subjected to an RP C-18 column (5.0”50 cm, 65% (log e) = 217 (4.48), 312 (4.49) nm; [a]D22: ±41.38 (c 0.1, MeOH).
aqueous CH3CN) to obtain three sub-fractions (Fr. B41±
B43:3.12 g, 0.5 L; 8.29 g, 3.5 L; 2.18 g, 1.0 L). Fr. B41 was further Determination of sugars in 1±3
chromatographed on silica gel (3.0”50 cm, CHCl3/MeOH, Each sample (2 mg) was refluxed with 2 N HCl/dioxane (1:1, 2
8.5:1.5, 3 L) to give 4 (1.4±1.9 L, 2.02 g). Fr. C was chromato- mL) for 2 h. The mixture was extracted with EtOAc (5 mL”3).
graphed on a RP C-18 column (4.5”60 cm, 70% aqueous CH3CN, The residual water layer was neutralized with Amberlite MB-3
6.0 L) to yield five sub-fractions (Fr. C1±C5:0.2 g, 0.5 L; 0.6 g, 0.5 and dried to obtain a residue. The residue was trimethylsilylated
L; 1.2 g, 1.5 L; 5.2 g, 2.5 L; 0.8 g, 1.0 L). Fr. C4 was crystallized from by the method of Hara [10], and analyzed by GC (column, DB-1,
MeOH to yield 5 (2.64 g). Fr. C3 was chromatographed on an 0.25 mm i.d.”30 m; column temperature, 50±2308C, 158C/min,
MPLC/RP C-18 (Merck size B, 70% aqueous CH3CN) to give 2 2308C for 18 min; carrier gas, He). The sugar derivatives obtained
(110±140 mL, 172 mg). Fr. D was subjected to an RP C-18 column exhibited a retention time of 21.30 min, identical with that of au-
(3.0”50 cm, 35% aqueous MeOH, 2 L) to obtain three sub-frac- thentic D-glucose (L-glucose; 22.00 min).
tions (Fr. D1±D3:1.4 g, 0.4 L; 1.0 g, 0.5 L; 2.1 g, 1.1 L). Fr. D2 was
further subjected to MPLC/RP C-18 (B size, 60% aqueous CH3CN) Anti-complement assay
to give 3 (55±80 mL, 60 mg).
A diluted solution of normal human serum (complement serum,
80 mL) was mixed with a gelatin veronal buffer (GVB2+, 80 mL)
Styraxlignolide A (1): white amorphous powder. UV (MeOH): lmax with sample dissolved in DMSO. The mixture was pre-incubated
(log e) = 217 (4.39), 312 (4.38) nm; [a]D22: ±53.78 (c 0.8, MeOH); at 378C for 30 min, and sensitized erythrocytes (sheep red blood
FAB-MS: m/z = 659 [M + Na]+; HR-FAB-MS: m/z = 659.2311 [M cells, 40 mL) were added. After incubation under the same condi-
+ Na]+ (calcd. for C31H40O14Na: 659.2316); 1H- and 13C-NMR tions, the mixture was centrifuged (48C, 1500 rpm) and the opti-
data: see Table 1.
cal density of the supernatant (100 mL) was measured at 405 nm
Min B-S et al. Anti-Complement Activity of¼ Planta Med 2004; 70: 1210±1215

Table 1 1H- (600 MHz) and 13C- (150 MHz) NMR data of compounds 1 (pyridine-d5) and 1a (CDCl3)a
Position
1
1a
1H, multi (J in Hz)
13C
1H, multi (J in Hz)
13C
2
157.6
101.6
113.4
138.9
108.6
145.9
143.5
132.2
124.5
109.6
151.0
151.0
113.0
118.8
33.3
156.8
100.7
112.7
137.9
107.8
145.2
143.0
131.6
124.0
108.7
150.0
149.6
111.8
118.5
32.8
3
7.21 s
7.28 s
4
7.15 br. s
7.00 (d, 1.2)
5
6
6.85 br. s
6.66 (d, 1.2)
7
8
9
1¢
2¢
7.63 (d, 2.0)
7.39 (d, 1.8)
3¢
4¢
5¢
7.04 (d, 8.0)
7.68 (dd, 8.0, 2.0)
2.89 m
6.95 (d, 8.3)
7.47 (dd, 8.3, 1.8)
2.81 (t, 7.2)
1.97 (dt, 15.0, 6.3)
3.74 (t, 6.3)
4.06 s
6¢
1¢¢
2¢¢
2.06 m
33.0
35.1
3¢¢
3.73 m, 4.26 m
3.95 s
69.4
62.7
OCH3±7
OCH3±3¢
OCH3±4¢
Glc 1¢¢¢
2¢¢¢
56.4
56.5
3.76 s
56.3b
56.5b
105.2
75.4
3.94 sb
4.00 sb
56.5b
56.4b
3.76 s
4.83 (d, 8.0)
4.04 m
3¢¢¢
4.23 m
78.8
4¢¢¢
4.25 m
71.6
5¢¢¢
4.11 m
77.7
6¢¢¢
4.36 (dd, 11.4, 5.6)
4.88 (dd, 11.4, 1.8)
70.5
Xyl 1¢¢¢¢
2¢¢¢¢
5.03 (d, 7.0)
4.07 m
106.5
76.0
79.1
72.1
67.7
1212
3¢¢¢¢
4.22 m
4¢¢¢¢
3.69 m
5¢¢¢¢
4.38 (dd, 11.0, 2.8)
a Assignments made on the basis of COSY, HMQC, and HMBC.
b Chemical shift value assignments may be interchangeable.
[11]. Rosmarinic acid was employed as positive control [11], [12]. tablished by ABX type signals at d = 7.68 (dd; 8.0, 2.0), 7.63 (d;
The purity of the compounds used for the assay was above 95% 2.0) and 7.04 (d; 8.0) in the 1H-NMR spectrum. This observation
(determined by HPLC).
was confirmed by signals for three sp2 quaternary carbons and
three sp2 methines in the 13C-NMR spectrum. The 1H-NMR spec-
trum additionally displayed three methoxy groups at d = 3.95
and 3.76 (”2), which correlated with quaternary carbons at
d = 145.9 (C-7), 151.0 (C-3¢ and C-4¢), respectively, in HMBC, and
Results and Discussion
The molecular formula of styraxlignolide A (1) was deduced as indicated the positions of the three methoxy groups. Long-range
C
31H40O14, based on the molecular ion at m/z = 659.2311 [M + correlations between at dH = 7.21 (H-3) and dC = 124.5 (C-1¢),
Na]+ on HR-FAB-MS. The spectrum of this compound contained and dH = 7.63 (H-2¢)/dH = 7.68 (H-6¢) and dC = 157.6 (C-2) led to
signals for a hydroxypropanol group at d = 4.26 (1H), 3.73 (1H), the assignment of an aglycone moiety in 1, namely 5-(3¢¢-hydro-
2.89 (2H) and 2.06 (2H), an independent olefinic proton at xypropyl)-7-methoxy-2-(3¢,4¢-dimethoxyphenyl)-benzofuran.
1
d = 7.21, and two olefinic protons at d = 7.15 and 6.85, which On the other hand, the H-NMR spectrum contained signals for
were assigned to a hydroxypropylbenzofuran moiety in the H- two anomeric protons at d = 4.83 and 5.03, and ten oxygenated
1
NMR spectrum (Table 1), as compared with that of benzofurans protons due to a pentose unit and a hexose unit in combination
isolated from Styrax species [13]. The HMBC correlation between with the 13C-NMR data. On enzymic hydrolysis, 1 yielded mono-
at dH = 2.89 (H-1¢¢) and dC = 138.9 (C-5)/dC = 113.4 (C-4)/ saccharide units, which were identified by co-TLC (EtOAc:-
dC = 108.6 (C-6) implies that the hydroxypropyl moiety (C1¢¢H2- MeOH:H2O:AcOH, 65:20:15:15) with authentic samples, as
C2¢¢H2-C3¢¢H-OH) is linked to benzofuran at C-5 (Fig.1). Further- glucose and xylose. Their absolute configurations were deter-
more, the presence of a three-substituted phenyl group was es- mined as D-glucose and D-xylose, by using gas chromatography.
Min B-S et al. Anti-Complement Activity of¼ Planta Med 2004; 70: 1210±1215

Table 2 1H- (600 MHz) and 13C- (150 MHz) NMR data of compounds 2 and 3 in pyridine-d5
a
Position
2
3
1H, multi (J in Hz)1)
13C
1H, multi (J in Hz)1)
13C
1
2
2.21 (d, 11.4), 2.54 (d, 11.4)
4.51 s
51.6
0.83 m, 1.30 m
38.7
207.8
83.2
45.0
44.4
30.7
69.2
45.1
47.5
41.7
24.3
23.9
48.3
53.6
42.9
133.7
130.8
15.1
25.2
29.1
17.1
17.7
1.85 m, 2.29 (dd, 10.0, 3.1)
3.36 (dd, 13.7, 4.0)
26.7
88.9
39.4
55.9
18.3
33.2
40.1
46.9
36.9
23.8
123.3
142.6
48.3
43.4
215.3
76.4
52.8
48.1
31.0
37.2
31.6
28.0
17.1
15.4
17.7
27.2
3
4
5
2.69 m
0.74 (d, 11.2)
6
1.88 m, 1.98 m
4.31 m
1.32 m, 1.47 (d, 11.2)
1.21 m, 1.36 m
7
8
9
1.58 m
1.53 (dd, 10.6, 6.7)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1¢
1.14 m, 1.43 m
1.33 m, 1.56 m
2.71 m
1.89 m
5.98 s
2.20 (d, 15.3), 2.78 (d, 15.3)
1.92 (d, 13.2), 3.69 (d, 13.2)
5.74 br. s
5.62 br. s
1.36 s
3.09 (dd, 14.7, 3.4)
0.93 s
1.33 m, 1.67 (t, 13.9)
1.47 s
1.18 s
1.78 m, 1.92 m
1.11 s
1.78 (dd, 12.9, 4.0), 2.50 (d, 12.9)
1.25 s
1.20 s
0.85 s
1.23 s
1.33 s
1213
0.88 s
32.7
23.6
105.6
77.4
79.3
73.7
77.6
172.6
101.8
72.4
72.5
73.7
69.7
18.7
0.96 s
4.94 (d, 7.5)
4.37 (t, 8.1)
4.25 m
104.8
83.2
78.1
71.4
78.2
62.6
105.9
77.1
77.9
71.6
78.3
62.6
5.05 (d, 7.4)
4.40 (t, 7.4)
4.38 m
2¢
3¢
4¢
4.11 m
4.54 (t, 9.3)
4.65 (t, 9.3)
5¢
3.78 m
6¢
4.27 m, 4.46 m
5.37 (d, 7.6)
4.08 m
1¢¢
2¢¢
3¢¢
4¢¢
5¢¢
6¢¢
6.60 s
4.86 (d, 1.7)
4.69 (9.3, 1.7)
4.35 m
4.21 (d, 9.0)
4.29 m
3.90 m
4.80 m
4.40 m, 4.42 m
1.73 (d, 6.2)
a Assignments made on the basis of COSY, HMQC, and HMBC.
The configurations of the glycosidic linkages for the glucopyra- The 1H-NMR spectrum contained signals for isolated methylene
noside and xylopyranoside units were determined as b on the ba- protons at d = 2.54 (d, 11.5) and 2.21 (d, 11.5), and methine at
sis of the J1,2 values of the anomeric protons of 8.0 Hz (d = 4.83) d = 4.51 with geminal and angular methyls (d = 1.47, 1.18, 0.93)
and 7.0 Hz (d = 5.03). Sugar connectivity was determined on the on the other cyclohexane ring (Table 2), supporting the presence
basis of correlations between at dH = 4.83 (H-1¢¢¢) and dC = 69.4 of a C1H2-C2O-C3H-OH moiety, compared with that of yucalexin
(C-3¢¢), and dH = 5.03 (H-1¢¢¢¢) and dC = 70.5 (C-6¢¢¢), supporting B-9 isolated from cassava root [15]. The existence of this moiety
the presence of a primeverose moiety (-Glc6-Xyl) [14].
was further supported by diaxial coupling between H-1ax
(d = 2.54) and H-3ax (d = 4.51), and W-type coupling between
Styraxoside A (2) was obtained as a white amorphous powder, H-3ax and H-21-methyl in the COSY spectrum, as well as long-
[a]D: ±11.08. A molecular formula of C34H54O13 was established range correlations between at dH = 2.54/4.51 and dC = 207.8 (C-
using HR-FAB-MS, with a mass of m/z = 693.3463 [M + Na]+. 2) in the HMBC spectrum (Fig. 2). The compound exhibited sig-
Min B-S et al. Anti-Complement Activity of¼ Planta Med 2004; 70: 1210±1215

Fig. 1 Chemical structures of compounds from S.
japonica.
nals for a methine (d = 2.69), a methylene (d = 1.98, 1.88) and a displayed W-type coupling to the H-18 methyl group. The partial
hydroxymethine (d = 4.31), indicating the presence of a C5H- structures were linked using an HMBC spectrum. NMR studies
C6H2-C7H-OH spin system. Diaxial coupling between H-5 and H- led to the assignment of the aglycone moiety in 2 as 3b,7b-dihy-
7, and correlations between H-6 (d = 1.98, 1.88) and H-5/H-7 droxy-4a,4b,8b,10b,14a-pentamethyl-5a-gon-16-en-2-one, de-
were observed in the COSY and ROESY spectra, respectively. Fur- rived by the loss of the C-17 side-chain from a dammarane triter-
thermore, the 1H-NMR and COSY spectral data disclosed two ole- pene [16]. Acid hydrolysis of 2 with 2 N HCl-dioxane produced a
finic protons, a methine, and a methylene, suggesting the pres- sugar component, which was identified as D-glucose. The config-
1214
ence of a spin system C13H (d = 2.71)-C17H (d = 5.62) = C16
H
urations of the glycosidic linkage for glucopyranoside units were
(d = 5.74)-C15H2 (d = 2.78, 2.20) with a d = 2.78 signal exhibit- determined as b on the basis of the J1,2 values of the anomeric
ing additional W-type coupling to the C-14 methyl substituent. protons of 7.5 Hz (d = 4.94) and 7.6 Hz (d = 5.37). The sugar con-
The presence of a double bond at C-16(17) was further supported nectivity was established from correlations between dH = 4.94
by the low value of the couplings between the H-16 and Hax-15, (H-1¢) and dC = 83.2 (C-3), and dH = 5.37 (H-1¢¢) and dC = 83.2
and H-17 and Hax-13 protons, with dihedral angles of approxi- (C-2¢), consistent with the presence of a -Glc2-Glc moiety
mately 1108 in each case. In addition, a methine (d = 1.58) cou- (Fig.1). This is the first isolation of a trioxygenated octanordam-
pled to a methylene (d = 1.43, 1.14) that was further linked to a marane-type saponin from a natural source.
second methylene (d = 1.56, 1.33), was suggestive of a molecular
sequence of C9H-C11H2-C12H2, in which the d = 1.58 (H-9) signal Styraxoside B (3) was obtained as a white amorphous powder
with an [a]D22 of ±43.78. HR-FAB-MS of 3 displayed a quasi-mole-
cular ion at m/z = 787.4240 [M + Na]+ giving the molecular for-
mula C41H64O13. The 1H-NMR spectrum of 3 contained seven ter-
tiary methyls at d = 0.85±1.33, as well as a hydroxy methine at
d = 3.36 and a trisubstituted double bond at d = 5.98 (Table 2).
The 13C-NMR spectrum of 3 contained signals for secondary
(d = 88.9) and tertiary (d = 76.4) carbinol carbons, and a double
bond at d = 142.6 and 123.3. Detailed examination of the NMR
spectral data, compared with that of camellioside A isolated
from Camellia japonica (Fig.1), disclosed that 3 was a 28-nor-
olean-12-en-16-one triterpene [17]. The b-orientation of the hy-
droxy groups at C-3 and C-17 was deduced from the multiplicity
of H-3 (dd, J = 10.0, 3.1 Hz) and the chemical shift of C-17
(d = 76.4), compared with that of camellenodiol isolated from
Fig. 2 Significant COSY (<Ð>) and HMBC (®) correlations for com-
pound 2.
Min B-S et al. Anti-Complement Activity of¼ Planta Med 2004; 70: 1210±1215

search Program funded by the Ministry of Science and Technolo-
gy of the Korean government.
Table 3 Inhibitory effects of the compounds 1±5 isolated form S. ja-
ponica and analogues (1a and 3a) on complement system of
classical pathway
References
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Acknowledgements
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This research was supported by a grant (PF0300401-00) from the
Plant Diversity Research Center of the 21st Century Frontier Re-
Min B-S et al. Anti-Complement Activity of¼ Planta Med 2004; 70: 1210±1215