Medicinal flowers. XXXVI.1) Acylated oleanane-type triterpene saponins with inhibitory effects on melanogenesis from the flower buds of Chinese Camellia japonica

Article abstract of DOI:10.1248/cpb.60.752

Four acylated oleanane-type triterpene oligoglycosides, sanchakasaponins E-H, were isolated from the flower buds of Camellia japonica cultivated in Yunnan province, China, together with four known triterpene oligoglycosides. The chemical structures of the new triterpene oligoglycosides were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the triterpene oligoglycoside constituents on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were investigated.

Full text of DOI:10.1248/cpb.60.752

752
Regular Article
Chem. Pharm. Bull. 60(6) 752–758 (2012)
Vol. 60, No. 6
Medicinal Flowers. XXXVI.1) Acylated Oleanane-Type Triterpene
Saponins with Inhibitory Effects on Melanogenesis from the Flower Buds
of Chinese Camellia japonica
Seikou Nakamura, Katsuyoshi Fujimoto, Souichi Nakashima, Takahiro Matsumoto,
Tomoko Miura, Kaoru Uno, Hisashi Matsuda, and Masayuki Yoshikawa*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received February 10, 2012; accepted March 18, 2012; published online March 28, 2012
Four acylated oleanane-type triterpene oligoglycosides, sanchakasaponins E–H, were isolated from the
flower buds of Camellia japonica cultivated in Yunnan province, China, together with four known triterpene
oligoglycosides. The chemical structures of the new triterpene oligoglycosides were elucidated on the basis of
chemical and physicochemical evidence. The inhibitory effects of the triterpene oligoglycoside constituents on
melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were investigated.
Key words Camellia japonica; sanchakasaponin; triterpene oligoglycoside; melanogenesis inhibitor; medici-
nal flower
The Theaceae plant, Camellia (C.) japonica L., has been with negative optical rotation ([α]D25 −38.8 in MeOH). Its IR
°
widely cultivated as an ornamental in Japan, China, and Tai- spectrum showed absorption bands at 3350, 1730, 1713, 1695,
wan. The flower buds of C. japonica have been used for the and 1050cm−1 due to hydroxyl, ester, α,β-unsaturated ester,
treatment of blood stagnation, vomiting of blood and bleeding carboxyl and ether components. FAB-MS in the positive-ion
due to internal and external injury, and also as an anti-inflam- mode revealed a quasimolecular ion peak [M+Na]+ at m/z
matory, tonic, and stomatic in Japanese folk medicine and 1283 from which the molecular formula C61H96O27 was de-
Chinese traditional medicine. In the course of characterizing termined by high-resolution (HR)-MS. Treatment of 1 with
the flower buds of Camellia spiecies,1–9) we have reported the a 10% aqueous KOH–1,4-dioxane (1:1, v/v) mixture yielded
isolation and structural elucidation of camelliosides A–F and a desacyl-derivative [desacyl-jegosaponin],13) together with
sanchakasaponins A–D from the flower buds of Japanese, Chi- angelic acid. The angelic acid was identified from the reten-
nese, and Korean C. japonica as well as their gastroprotective, tion time in a HPLC analysis of its p-nitrobenzyl ester.9)
platelet-aggregating, and inhibitory effects on melanogen- Acid hydrolysis of 1 with 5% aqueous H2SO4-1,4-dioxane
esis.10,11) Our continuing search led us to the additional isola- yielded barringtogenol C14) and monosaccharides (D-glucose,
tion of four new acylated oleanane-type triterpene oligogly- D-galactose, L-rhamnose, and D-glucuronic acid), which were
cosides, sanchakasaponins E (1), F (2), G (3), and H (4), from identified by HPLC of their tolylthiocarbamoyl thiazolidine
the flower buds of C. japonica cultivated in Yunnan province, derivatives.15) The 1H-NMR (pyridine-d5) and 13C-NMR
China, together with four known triterpene oligoglycosides. In (Table 1) spectra of 1, which were assigned by various NMR
addition, the inhibitory effects of the saponin constituents on experiments,16) showed signals assignable to a desacyl-jegosa-
melanogenesis in theophylline-stimulated B16 melanoma 4A5 ponin component: seven methyls [δ 0.76, 0.82, 1.05, 1.08, 1.16,
cells were investigated. In this paper, we describe the isola- 1.30, 1.90 (all s, H3-25, 26, 24, 29, 23, 30, 27)], a methylene
tion, structural elucidation, and inhibitory effects on melano- [δ 3.37, 3.62 (both d, J=10.4Hz, H2-28)] and four methines
genesis in theophylline-stimulated B16 melanoma 4A5 cells of with an oxygen function [δ 3.14 (dd, J=3.8, 11.8Hz, H-3),
the triterpene oligoglycoside constituents.
4.44 (m, H-16), 6.21 (d, J=10.0Hz, H-22), 6.62 (d, J=10.0Hz,
The methanolic extract (10.0% from the dried flower buds H-21)], an olefin [δ 5.36 (brs, H-12)], and four glycopyranosyl
of C. japonica cultivated in Yunnan province, China) was moieties {β-D-glucuronopyranosyl [δ 4.87 (d like, J=7.0Hz,
partitioned into an EtOAc–H2O (1:1, v/v) mixture to fur- H-1′)], β-D-glucopyranosyl [δ 5.92 (d, J=7.5Hz, H-1″)], β-D-
nish an EtOAc-soluble fraction (2.14%) and aqueous layer. galactopyranosyl [δ 6.22 (d like, J=7.0Hz, H-1‴)], and α-L-
The aqueous layer was further extracted with 1-butanol to rhamnopyranosyl [δ 6.26 (brs, H-1″″)]} together with an ange-
give 1-butanol- (5.69%) and H2O- (2.12%) soluble fractions loyl group [δ 2.01 (s, H3-5″‴), 2.10 (d, J=7.2Hz, H3-4‴‴), 5.96
as described previously.1) The 1-butanol-soluble fraction was (q, J=7.0Hz, H-3″‴)] and an acetyl group [δ 2.15 (s, H-2‴‴)].
subjected to normal- and reversed-phase silica-gel column The positions of the acyl groups and the structure of the oli-
chromatographies and repeated HPLC to give four new acyl- goglycoside moiety were confirmed based on double quantum
ated oleanane-type triterpene oligoglycosides, sanchakasapo- filter correlation spectroscopy (DQF COSY) and heteronuclear
nin E (1, 0.0021%), F (2, 0.0057%), G (3, 0.0036%) and H (4, multiple bond connectivity (HMBC) spectroscopy. Long-range
0.0034%) together with four known compounds, yuchasaponin correlations were observed between the following proton and
A (5, 0.0034%),8) sasanquasaponins I (6, 0.0080%)9) and III (7, carbon pairs: H-1′ and C-3; H-1″ and C-2′; H-1‴ and C-3′;
0.017%),9) and ternstoemiaside C (8, 0.0050%).12)
H-1″″ and C-2‴; H-21 and C-1″‴; H-22 and C-1‴‴. From all this
Structure of Sanchakasaponins E–H (1–4) Sanchakasa- evidence, the chemical structure of sanchakasaponin E was
ponin E (1) was isolated as a colorless amorphous powder determined to be 21-O-angeloyl-22-O-acetyl-barringtogenol C
3-O-[β-D-glucopyranosyl(1
→2)][α-L-rhamnopyranosyl(1→2)-β-
D-galactopyranosyl(1 3)]-β-D-glucuronopyranoside (1).
→
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2012 The Pharmaceutical Society of Japan

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753
Table 1. 13C-NMR (125MHz) Spectroscopic Data for Compounds 1–4 in C5D5N
Position
1
2
3
4
Position
1
2
3
4
1
2
38.7
26.4
89.7
39.7
55.7
18.5
33.1
40.1
46.9
36.7
23.8
123.1
142.8
41.7
34.7
68.0
44.6
40.0
47.2
36.3
78.9
74.4
16.7
28.0
15.6
16.9
27.5
63.8
20.2
29.5
167.8
129.0
137.1
15.9
21.0
170.0
22.1
—
39.0
26.5
89.8
39.7
55.6
18.9
36.8
41.5
47.1
37.0
24.0
125.5
143.7
47.8
67.6
73.4
48.4
41.0
46.9
36.4
78.7
73.7
16.8
27.9
15.8
17.6
21.3
63.2
20.3
29.6
168.2
129.2
136.6
16.0
21.1
167.8
129.0
137.5
15.8
20.7
38.7
26.4
89.6
39.7
55.7
18.5
33.1
41.7
46.9
36.8
23.8
123.1
143.8
40.1
35.2
70.0
45.1
40.9
47.5
32.1
41.8
73.1
16.8
28.0
15.7
16.9
27.6
63.6
25.2
33.5
167.9
130.0
136.3
14.1
12.3
—
38.8
26.5
89.8
39.7
55.8
18.5
33.1
41.7
46.9
36.8
23.8
123.1
143.7
40.1
35.2
70.2
44.9
40.9
47.5
32.1
41.8
73.0
16.9
28.2
15.7
16.9
27.6
63.7
25.2
33.5
168.0
129.6
136.5
15.9
21.0
—
3-O-β-D-Glucuronopyranosyl
1′
2′
3′
4′
5′
6′
105.4
79.4
82.7
71.3
77.0
172.4
105.5
79.4
82.5
71.3
77.1
172.5
105.4
79.4
82.7
71.2
77.0
172.2
105.4
79.7
83.1
71.1
77.1
172.3
3
4
5
6
7
8
2′-O-β-D-Glucopyranosyl
9
1″
2″
3″
4″
5″
6″
102.7
76.3
78.5
72.6
78.2
63.6
102.7
76.3
78.5
72.6
78.3
63.7
102.7
76.3
78.5
72.6
78.2
63.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1″‴
2″‴
3″‴
4″‴
5″‴
1‴‴
2‴‴
31‴‴
41‴‴
51‴‴
2′-O-β-D-Galactopyranosyl
1″
2″
3″
4″
5″
6″
103.7
73.5
75.3
70.3
76.9
62.9
3′-O-β-D-Galactopyranosyl
1‴
2‴
3‴
4‴
5‴
6‴
101.4
76.5
75.7
71.2
77.5
61.9
101.3
76.5
76.1
71.4
77.5
62.1
101.4
76.5
76.1
71.3
77.5
61.9
101.5
77.0
76.1
70.9
77.3
61.9
3′-O-β-D-Galactopyranosyl
1″″
2″″
3″″
4″″
5″″
6″″
102.4
72.6
72.6
73.9
69.8
18.3
102.4
72.6
72.7
73.9
69.9
18.3
102.4
72.6
72.7
73.9
69.8
18.3
102.7
72.6
72.8
74.0
69.9
18.4
—
—
—
—
—
—
—
—
—
—
Sanchakasaponin F (2), obtained as a colorless amor- (all s, H3-25, 26, 24, 29, 23, 30, 27)], a methylene [δ 3.48,
phous powder with a negative optical rotation ([α]D25 −12.3
3.74 (both d, J=10.2Hz, H2-28)] and five methines with an
°
in MeOH), showed absorption bands in the IR spectrum oxygen function [δ 3.18 (dd-like, H-3), 4.21 (m, H-15), 4.45 (m,
due to hydroxyl, α,β-unsaturated ester, carboxyl and ether H-16), 6.31 (d, J=10.4Hz, H-22), 6.69 (d, J=10.4Hz, H-21)],
functions. FAB-MS in the positive-ion mode revealed a qua- and four glycopyranosyl moieties {β-D-glucuronopyranosyl
simolecular ion peak [M+Na]+ at m/z 1339 from which the [δ 4.82 (d-like, J=7.0Hz, H-1′)], β-D-glucopyranosyl [δ 5.91
molecular formula C64H100O28 was determined by HR-MS. Al- (d-like, J=7.0Hz, H-1″)], β-D-galactopyranosyl [δ 6.20 (d-
kaline hydrolysis of 2 provided a desacyl-derivative [desacyl- like, J=7.0Hz, H-1‴)], and α-L-rhamnopyranosyl [δ 6.23
yuchasaponin A],8) together with angelic acid. The angelic (brs, H-1″″)]} together with two angeloyl groups [δ 1.73 (s,
acid was identified from retention time in a HPLC analysis H3-5″‴), 1.95 (d, J=7.1Hz, H3-4″‴), 2.00 (s, H3-5‴‴), 2.08
of its p-nitrobenzyl ester.9) Acid hydrolysis of 2 yielded R1- (d, J=7.1Hz, H3-4‴‴), 5.77 (d, J=7.1Hz, H-3″‴), 5.94 (d,
barrigenol17) and monosaccharides (D-glucose, D-galactose, J=7.1Hz, H-3‴‴)]. The positions of the acyl groups and the
L-rhamnose, and D-glucuronic acid), which were identified by structure of the oligoglycoside moiety were confirmed based
HPLC of their tolylthiocarbamoyl thiazolidine derivatives.15) on DQF COSY and HMBC experiments. Long-range correla-
1
The H-NMR (pyridine-d5) and 13C-NMR (Table 1) spectra tions were observed between the following proton and carbon
of 2, which were assigned by various NMR experiments,16) pairs: H-1′ and C-3; H-1″ and C-2′; H-1‴ and C-3′; H-1″″ and
showed signals assignable to a desacyl-yuchasaponin A com- C-2‴; H-21 and C-1″‴; H-22 and C-1‴‴. On the basis of all
ponent: seven methyls [δ 0.78, 0.98, 1.05, 1.08, 1.11, 1.31, 1.84 this evidence, the chemical structure of sanchakasaponin F

754
Vol. 60, No. 6
Fig. 1. Structure of Saponin Constituents Isolated from Flower Buds of Chinese C. japonica
was determined as 21,22-O-di-angeloyl-R1-barrigenol 3-O- seven methyls [δ 0.78, 0.86, 1.06, 1.07, 1.17, 1.29, 1.87 (all s,
[β-D-glucopyranosyl(1
→
2)][α-L-rhamnopyranosyl(1
→
2)-β-D- H3-25, 26, 24, 29, 23, 30, 27)], a methylene [δ 3.54, 3.62 (both
d, J=10.4Hz, H2-28)] and three methines with an oxygen
galactopyranosyl(1 3)]-β-D-glucuronopyranoside (2).
→
Sanchakasaponins G (3) and H (4), each obtained as a col- function [δ 3.19 (dd-like, H-3), 4.67 (m, H-16), 6.19 (m, H-22)],
orless amorphous powder with a negative optical rotation (3: and four glycopyranosyl moieties {β-D-glucuronopyranosyl
25
[α]D25 −27.7 , 4: [α]D −12.8 in MeOH), showed absorption [δ 4.88 (d-like, J=7.0Hz, H-1′)], β-D-glucopyranosyl [δ 5.92
bands in their IR spectra due to hydroxy1, α,β-unsaturated es- (d-like, J=7.3Hz, H-1″)], β-D-galactopyranosyl [δ 6.22 (d-
ter, carboxy1, and ether functions. The same molecular formu- like, J=7.0Hz, H-1‴)], and α-L-rhamnopyranosyl [δ 6.27 (brs,
la C59H94O25 was determined for both compounds from a qua- H-1″″)]} together with tigloyl groups [δ 1.54 (d, J=7.1Hz,
simolecular ion peak (m/z 1225 [M+Na]+) in positive-ion FAB- H3-4″‴), 1.87 (s, H3-5″‴), 6.99 (q, J=7.1Hz, H-3″‴)]. On the
°
°
1
MS and by HR-MS measurements. Alkaline treatment yielded other hand, the proton and carbon signals in the H- and 13C-
a desacyl-derivative [ternstroemiaside A12) from 3 and 4a from NMR (Table 1) spectra16) of 4 were superimposable on those
4] together with organic acids (tiglic acid from 3; angelic acid of 3, except for the signals due to the 22-acyl group and the
1
from 4), which were identified by HPLC of their p-nitrobenzyl glycopyranosyl moieties of 3. The H-NMR (pyridine-d5) and
derivatives.9) Acid hydrolysis yielded camelliagenin A18–20) 13C-NMR spectra of 4 showed signals assignable to an ange-
and monosaccharides (D-glucose, D-galactose, L-rhamnose, loyl group [δ 1.93 (s, H3-5″‴), 2.07 (d, J=7.0Hz, H3-4″‴), 5.90
and D-glucuronic acid from 3; D-galactose, L-rhamnose, and (q, J=7.0Hz, H-3″‴)] and four glycopyranosyl moieties {β-D-
D-glucuronic acid from 4), which were identified by HPLC glucuronopyranosyl [δ 4.90 (d-like, J=7.0Hz, H-1′)], two β-D-
of their tolylthiocarbamoyl thiazolidine derivatives.15) The galactopyranosyl [δ 5.77 (d, J=7.3Hz, H-1″), 6.14 (d, J=7.6Hz,
1H-NMR (pyridine-d5) and 13C-NMR (Table 1) spectra of 316) H-1‴)], and α-L-rhamnopyranosyl [δ 6.24 (brs, H-1″″)]} togeth-
showed signals assignable to a ternstroemiaside A component: er with an camelliagenin A component. The positions of the

June 2012
755
Fig. 2. Important 2D-NMR Correlations of 1–4
Table 2. Inhibitory Effects of Constituents on Melanogenesis in B16 Melanoma 4A5 Cells
Inhibition (%) of melanogenesis
Sample
IC50 (µM)
Conc. (µM)
0µM
1µM
3µM
10µM
Sanchakasaponin E (1)a)
Sanchakasaponin F (2)a)
Sanchakasaponin H (4)a)
Yuchasaponin A (5)a)
Sasanquasaponin I (6)b)
Sasanquasaponin III (7)b)
Ternstoemiaside C (8)b)
0.0 0.7
0.0 1.5
0.0 9.0
0.0 4.1
0.0 4.4
0.0 1.8
0.0 3.0
13.3 2.5**
16.7 2.4**
69.2 2.6**
51.4 2.9**
15.9 6.4*
56.4 6.1**
51.1 3.2**
59.8 9.9**
13.6 1.4*
94.8 0.5**
91.7 0.4**
87.2 1.6**
98.8 0.3**
96.3 0.3**
98.7 0.3**
14.7 4.6*
2.1
2.9
0.4 7.7
4.7
22.1 5.3**
2.5
–5.7 4.3
3.0
12.5 1.1*
2.5
9.8 5.4
>10
Inhibition (%) of melanogenesis
30µM
—
Conc. (µM)
0µM
100µM
300µM
Arbutinb)
0.0 1.4
20.4 0.5**
38.1 0.9**
61.5 0.6**
174
Each value represents the mean S.E.M. (N=4). Significantly different from the control, *p<0.05, **p<0.01. a) The cell viability for 1, 2, 4, and 5 at 10µM is less than
27% (cytotoxic effects were observed). b) The cell viability for 6, 7, 8 and arbutin at 10µM is more than 70%.
acyl groups and the structure of the oligoglycoside moieties sterol glycosides in theophylline-stimulated B16 melanoma
of 3 and 4 were confirmed on the basis of DQF COSY and 4A5 cells.21–23) As a continuation of these studies, the in-
HMBC experiments (Fig. 2). From this evidence, the chemi- hibitory effects of constituents from the flowers buds of C. ja-
cal structure of sanchakasaponin G and H was determined as ponica on melanogenesis were examined. Among the isolates,
22-O-tigloyl-camelliagenin A 3-O-[β-D-glucopyranosyl(1
[α-L-rhamnopyranosyl(1 2)-β-D-galactopyranosyl(1 3)]-β-D- with an acyl group at the 22-position substantially inhibited
glucuronopyranoside (3) and 22-O-angeloyl-camelliagenin melanogenesis without cytotoxic effects. The effects of 6 and
→2)] sasanquasaponins I (6, IC50=3.0µM) and II (7, IC50=2.5µM)
→
→
A
3-O-[β-D-galactopyranosyl(1
2)-β-D-galactopyranosyl(1 3)]-β-D-glucuronopyranoside
respectively.
→
2)][α-L-rhamnopyranosyl(1
→
7 were stronger than that of a reference compound, arbutin
→
(4), (IC50=174µM).22,23) Sanchakasaponins E (1) and F (2) and yu-
chasaponin A (5) with two acyl groups at the 21- and 22-posi-
Melanogenesis Inhibitory Effects of Triterpene Oligogly- tions also inhibited melanogenesis, but had cytotoxic effects
coside Constituents Melanin production is principally re- at 3µM.
sponsible for skin color, and melanin pigmentation is a major
In conclusion, four new acylated oleanane-type triterpene
defense mechanism against UV rays from the sun. However, saponins, sasanquasaponins E–H (1–4), were isolated from
excess production of melanin after long periods of exposure to the flower buds of C. japonica cultivated in Yunnan province,
the sun can cause dermatological disorders such as melasma, China, and their chemical structures were elucidated on the
freckles, postinflammatory melanoderma, and solar lentigines. basis of chemical and physicochemical evidence. In addition,
To develop inhibitors of melanogenesis, we have examined the the isolated constituents, sasanquasaponins I (6) and II (7)
inhibitory effects of several diaryheptanoids, flavonoids, and with an acyl group at the 22-position, substantially inhibited

756
Vol. 60, No. 6
melanogenesis without cytotoxic effects.
MeOH] to give three fractions [Fr.5-7-1, Fr.5-7-2 (2.62g), Fr.5-
7-3 (7.00g)]. A part of fraction 5-7-2 (1.62g) was purified
by HPLC [H2O–MeCN–AcOH (650:350:3, v/v/v), COS-
Experimental
General Experimental Procedures The following instru- MOSIL 5C18-MS-II] to give 7 (194mg). A part of fraction
ments were used to obtain physical data: specific rotations, 5-7-3 (2.40g) was further separated by HPLC [H2O–MeOH–
a Horiba SEPA-300 digital polarimeter (l=5cm); IR spectra, MeCN–AcOH (300:575:175:3, v/v/v/v), COSMOSIL 5C18-
a Thermo Electron Nexus 470; FAB-MS and HR-FAB-MS, MS-II] to give 10 fractions [Fr.5-7-3-1, Fr.5-7-3-2, Fr.5-7-3-3,
a JEOL JMS-SX 102A mass spectrometer; 1H-NMR spec- Fr.5-7-3-4 (145mg), Fr.5-7-3-5 (48.2mg), Fr.5-7-3-6, Fr.5-7-3-7
tra, JEOL JNM-LA 500 (500MHz) spectrometer; 13C-NMR (113mg), Fr.5-7-3-8 (127mg), Fr.5-7-3-9, Fr.5-7-3-10 (68.4mg)].
spectra, JEOL JNM-LA 500 (125MHz) spectrometer; HPLC, Fraction 5-7-3-4 (145mg) was purified by HPLC [H2O–
a Shimadzu SPD-10AVP UV-VIS detector. COSMOSIL 5C18- MeCN–AcOH (550:450:3, v/v/v), COSMOSIL 5C18-MS-II]
MS-II (250×4.6mm i.d. and 250×20mm i.d.) and 5C18-AR-II to give 1 (13.2mg) and 3 (19.2mg). Fraction 5-7-3-5 (48.2mg)
(250×4.6mm i.d.) columns were used for analytical and pre- was purified by HPLC [H2O–MeCN–AcOH (550:450:3,
parative purposes.
v/v/v), COSMOSIL 5C18-MS-II] to give 4 (18.0mg). Frac-
The following materials were used for chromatography: tion 5-7-3-7 (113mg) was purified by HPLC [H2O–MeCN–
normal-phase silica gel column chromatography, Silica gel AcOH (550:450:3, v/v/v), COSMOSIL 5C18-MS-II] to give
BW-200 (Fuji Silysia Chemical, Ltd., 150–350 mesh); re- 6 (50.4mg). Fraction 5-7-3-8 (127mg) was purified by HPLC
versed-phase silica gel column chromatography, Chromatorex [H2O–MeCN–AcOH (550:450:3, v/v/v), COSMOSIL 5C18-
ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); MS-II] to give 5 (21.4mg). Fraction 5-7-3-10 (68.8mg) was pu-
TLC, precoated TLC plates with Silica gel 60F254 (Merck, rified by HPLC [H2O–MeOH–MeCN–AcOH (250:562:188:3,
0.25mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, v/v/v/v), COSMOSIL 5C18-MS-II] to give 2 (36.0mg). The
0.25mm) (reversed phase); reversed-phase HPTLC, precoated known saponins were identified by comparison of their physi-
TLC plates with Silica gel RP-18 WF254S (Merck, 0.25mm). cal data ([α]D, 1H-NMR, 13C-NMR, and MS) with reported
Detection was achieved by spraying with 1% Ce(SO4)2–10% values.
aqueous H2SO4 followed by heating.
Sanchakasaponin E (1): A colorless amorphous powder;
Plant Material The dried flower buds of C. japonica [α]D25 −38.8 (c=0.26, MeOH); IR attenuated total reflectance
°
cultivated in Yunnan province, China, were purchased from (ATR): νmax 3350, 2910, 1730, 1713, 1695, and 1050cm−1; H-
1
Tochimoto Tenkaido Co., Ltd. (Osaka, Japan) in May 2010, NMR (pyridine-d5, 500MHz) δ: 0.76, 0.82, 1.05, 1.08, 1.16,
and identified by one of authors (M.Y.). A voucher specimen is 1.30, 1.90 (3H each, all s, H3-25, 26, 24, 29, 23, 30, 27), 1.42
on file in our laboratory.
(3H, d, J=6.0Hz, H-6″″), 2.01 (3H, s, H-5″‴), 2.10 (3H, d,
Extraction and Isolation The dried flower buds (4.0kg) J=7.2Hz, H-4″‴), 2.15 (3H, s, H-2‴‴), 3.14 (3H, dd, J=3.8,
were extracted three times with methanol under reflux for 3h. 11.8Hz, H-3), 3.37 (1H, d, J=10.4Hz, H-28a), 3.62 (1H, d,
Evaporation of the solvent under reduced pressure provided J=10.4Hz, H-28b), 4.44 (1H, m, H-16), 4.87 (1H, d like,
a MeOH extract (398g, 10.0%). A part of the MeOH extract J=7.0Hz, H-1′), 5.36 (1H, brs, H-12), 5.92 (1H, d, J=7.5Hz,
(236g) was partitioned into an EtOAc–H2O (1:1, v/v) mixture H-1″), 5.96 (1H, q, J=7.2Hz, H-3″‴), 6.21 (1H, d, J=10.0Hz,
to furnish an EtOAc-soluble fraction (50.8g, 2.14%) and an H-22), 6.22 (d like, J=7.0Hz, H-1‴), 6.26 (1H, brs, H-1″″),
aqueous phase. The aqueous phase was further extracted with 6.62 (1H, d, J=10.0Hz, H-21); 13C-NMR: given in Table 1;
1-butanol to give a 1-butanol-soluble fraction (135g, 5.69%) positive-ion FAB-MS: m/z 1283 [M+Na]+; high-resolution
and an H2O-soluble fraction (50.3g, 2.12%). A part of the 1-bu- positive-ion FAB-MS: m/z 1283.6037 (Calcd for C61H96O27Na
tanol-soluble fraction (104g) was subjected to normal phase [M+Na]+: m/z 1283.6037).
silica gel column chromatography [3.90kg, CHCl3–EtOAc–
isopropanol–MeOH (3:3:1:1 2:2:1:1 1:1:1:1, v/v/v/v)
CHCl3–EtOAc–isopropanol–MeOH–H2O (10:10:10:10:1
5:5:5:5:1 5:5:10:10:4, v/v/v/v/v)
Sanchakasaponin F (2): A colorless amorphous powder;
→
→
→
→
[α]D25 −12.3 (c=0.92, MeOH); IR(ATR): νmax 3350, 2914, 1720,
°
1
1695, and 1050cm−1; H-NMR (pyridine-d5, 500MHz) δ: 0.78,
→
→
MeOH] to give six 0.98, 1.05, 1.08, 1.11, 1.31, 1.84 (3H each, all s, H3-25, 26, 24,
fractions [Fr.1 (3.67g), Fr.2 (13.7g), Fr.3 (10.7g), Fr.4 (5.70g), 29, 23, 30, 27), 1.41 (3H, d like, H-6″″), 1.73 (3H, s, H-5″‴),
Fr.5 (24.6g), Fr.6 (0.56g)]. Fraction 5 (24.6g) was further 1.95 (3H, d, J=7.1Hz, H-4″‴), 2.00 (3H, s, H-5‴‴), 2.08 (3H,
separated by reversed phase silica gel column chromatography d, J=7.1Hz,, H-4‴‴), 3.18 (3H, dd-like, H-3), 3.48 (1H, d,
[750g, MeCN–H2O (10:90 15:85
→
→
20:80
→
25:75
→
30:70
→
J=10.2Hz, H-28a), 3.74 (1H, d, J=10.2Hz, H-28b), 4.21 (1H,
40:60 50:50 60:40, v/v)→MeOH] to give eight fractions m, H-15), 4.45 (1H, m, H-16), 4.82 (1H, d like, J=7.0Hz,
→
→
[Fr.5-1, Fr.5-2, Fr.5-3, Fr.5-4, Fr.5-5, Fr.5-6 (2.10g), Fr.5-7 H-1′), 5.49 (brs, H-12), 5.77 (1H, d, J=7.1Hz, H-3″‴), 5.91 (1H,
(17.9g), Fr.5-8]. Fraction 5-6 (2.10g) was further separated d-like, J=7.0Hz, H-1″), 5.94 (1H, d, J=7.1Hz, H-3‴‴), 6.20
by reversed phase silica gel column chromatography [60g, (1H, d-like, J=7.0Hz, H-1‴), 6.23 (1H, brs, H-1″″), 6.31 (1H,
MeOH–H2O (50:50, v/v)→
MeOH] to give four fractions [Fr.5- d, J=10.4Hz, H-22), 6.69 (1H, d, J=10.4Hz, H-21); 13C-NMR:
6-1, Fr.5-6-2, Fr.5-6-3 (630mg), Fr.5-6-4 (364mg)]. Fraction given in Table 1; positive-ion FAB-MS: m/z 1339 [M+Na]+;
5-6-3 (630mg) was purified by HPLC [H2O–MeOH–MeCN– high-resolution positive-ion FAB-MS: m/z 1339.6296 (Calcd
AcOH (300:525:175:3, v/v/v/v), COSMOSIL 5C18-MS-II] for C64H100O28Na [M+Na]+: m/z 1339.6293).
to give 8 (58.9mg). Fr.5-6-4 (364mg) was purified by HPLC
Sanchakasaponin G (3): A colorless amorphous powder;
[H2O–MeOH–MeCN–AcOH (450:275:275:3, v/v/v/v), COS- [α]D25 −27.7 (c=0.30, MeOH); IR(ATR): νmax 3350, 2906,
°
MOSIL 5C18-MS-II] to give 8 (33.7mg). Fraction 5-7 (17.9g) 1720, 1698, and 1050cm−1; H-NMR (pyridine-d5, 500MHz)
1
was further separated by reversed phase silica gel column δ: 0.76, 0.86, 1.06, 1.07, 1.17, 1.29, 1.87 (all s, H3-25, 26, 24,
chromatography [600g, MeOH–H2O (55:45→70:30, v/v)
→
29, 23, 30, 27), 1.44 (3H, d, J=5.8Hz, H-6″″), 1.54 (3H, d,

June 2012
757
J=7.1Hz, H-4″‴), 1.87 (3H, s, H-5″‴), 3.19 (3H, dd-like, H-3), dissolved in 5% aqueous H2SO4–1,4-dioxane (1:1, v/v,
°
3.54 (1H, d, J=10.4Hz, H-28a), 3.62 (1H, d, J=10.4Hz, H- 2.0mL), and each solution was heated at 90 C for 3h. After
28b), 4.67 (1H, m, H-16), 4.88 (d-like, J=7.0Hz, H-1′), 5.37 extraction three times with EtOAc, the EtOAc fraction was
(1H, brs, H-12), 5.92 (1H, d-like, J=7.3Hz, H-1″), 6.19 (1H, m, purified by normal-phase silica gel column chromatography
H-22), 6.22 (1H, d-like, J=7.0Hz, H-1‴), 6.27 (1H, brs, H-1″″), [CHCl3 :MeOH:H2O [10:3:1, lower phase] to give barringto-
6.99 (1H, q, J=7.1Hz, H-3″‴); 13C-NMR: given in Table 1; genol C (from 1), R1-barrigenol (from 2), and camelliagenin A
positive-ion FAB-MS: m/z 1225 [M+Na]+; high-resolution (from 3 and 4), which were identified by comparison of their
positive-ion FAB-MS: m/z 1225.5969 (Calcd for C59H94O25Na physical data with authentic samples and reported values.
[M+Na]+: m/z 1225.5976).
The aqueous layer was neutralized with Amberlite IRA-400
Sanchakasaponin H (4): A colorless amorphous powder; (OH− form). After drying in vacuo, the residue was dissolved
[α]D25 −12.8 (c=0.40, MeOH); IR(ATR): νmax 3350, 2918, in pyridine (0.1mL) containing L-cysteine methyl ester hydro-
°
1720, 1700, and 1050cm−1; H-NMR (pyridine-d5, 500MHz) chloride (0.5mg) and heated at 60 C for 1h. o-Torylisothiocy-
1
°
δ: 0.76, 0.83, 1.03, 1.16, 1.25, 1.27, 1.85 (3H each, all s, H3- anate (0.5mg) in pyridine (0.1mL) was added to the mixture
°
25, 26, 24, 29, 23, 30, 27), 1.43 (3H, d, J=5.8Hz, H-6″″), 1.93 and heated at 60 C for 1h. The reaction mixture was analyzed
(3H, s, H-5″‴), 2.07 (3H, d, J=7.0Hz, H-4″‴), 3.20 (3H, dd, by reversed-phase HPLC [column: COSMOSIL 5C18-AR-II,
J=3.1, 11.6Hz, H-3), 3.54 (1H, d, J=10.7Hz, H-28a), 3.68 (1H, 250×4.6mm i.d.; mobile phase: MeCN–0.05M H3PO4 (77:23,
d, J=10.7Hz, H-28b), 4.62 (1H, m, H-16), 4.90 (1H, d-like, v/v); detection: UV (250nm); flow rate: 0.8mL/min; column
°
J=7.0Hz, H-1′), 5.34 (1H, brs, H-12), 5.77 (1H, d, J=7.3Hz, temperature: 35 C] to identify the derivatives of constituent
H-1″), 5.90 (1H, q, J=7.0Hz, H-3″‴), 6.14 (1H, d, J=7.6Hz, monosaccharides in sanchakasaponins E–H (1−4) by compari-
H-1‴), 6.21 (1H, dd, J=5.7, 11.9Hz, H-22), 6.24 (1H, brs, son of their retention times with those of authentic samples
H-1″″); 13C-NMR: given in Table 1; positive-ion FAB-MS: (tR: D-glucose; 19.7min, D-galactose; 17.0min, L-rhamnose;
m/z 1225 [M+Na]+; high-resolution positive-ion FAB-MS: m/z 33.7min and D-glucuronic acid; 20.4min).
1225.5983 (Calcd for C59H94O25Na [M+Na]+: m/z 1225.5976).
’ ’
Reagents for Bioassays Dulbecco s modified Eagle s me-
Alkaline Hydrolysis of Sanchakasaponins Solutions of dium (DMEM, 4.5g/L glucose) was purchased from Sigma-
sanchakasaponins E–H (1–4, each 4.0mg) were treated with Aldrich (St. Louis, MO, U.S.A.); fetal bovine serum (FBS),
10% aqueous KOH–1,4-dioxane (1:1, v/v, 1.0mL) and stirred penicillin, and streptomycin were purchased from Gibco
at 37 C for 6h. Each reaction mixture was neutralized with (Invitrogen, Carlsbad, CA, U.S.A.); the Cell Counting Kit-8TM
°
Dowex HCR W2 (H+ form) and the resin was removed by was from Dojindo Lab. (Kumamoto, Japan); and the other
filtration. Evaporation of the solvent from the filtrate under chemicals were purchased from Wako Pure Chemical Co.,
reduced pressure yielded a crude product, most of which was Ltd. (Osaka, Japan).
subjected to reversed-phase silica gel solid-phase extraction
(Sep-Pak-C18, Waters Co., Ltd.) [H2O MeOH] to give desac- were obtained from Riken Cell Bank (Tsukuba, Japan), and
yl-saponins (desacyl-jegosaponin from 1, desacyl-yuchasapo- grown in DMEM supplemented with 10% FBS, penicillin
Cell Culture Murine B16 melanoma 4A5 cells (RCB0557)
→
°
nin from 2, ternstroemiaside A from 3, and 4a from 4). Their (100units/mL), and streptomycin (100µg/mL) at 37 C in 5%
1
desacyl-saponins were identified by comparison of their H- CO2/air. The cells were harvested by incubation in phosphate-
and 13C-NMR data with authentic samples or reported values.
buffered saline (PBS) containing 1mM ethylenediaminetetra-
°
4a: Colorless amorphous powder; 1H-NMR (pyridine-d5, acetic acid (EDTA) and 0.25% trypsin for ca. 5min at 37 C
500MHz) δΗ: 0.77, 0.88, 1.05, 1.17, 1.26, 1.27, 1.85 (all s, and used for the subsequent bioassays.
H3-25, 26, 24, 29, 23, 30, 27), 1.44 (3H, d, J=6.1Hz, H-6″″),
Melanogenesis The melanoma cells (2.0×104 cells/400µL/
3.24 (3H, dd, J=4.0, 11.6Hz, H-3), 3.67 (2H, m, H2-28), 4.05 well) were seeded into 24-well multiplates. After 24h of cul-
(1H, m, H-22), 4.59 (1H, m, H-16), 4.89 (1H, d-like, H-1′), ture, a test compound and theophylline 1mM were added and
5.33 (brs, H-12), 5.79 (1H, d, J =7.7Hz, H-1″), 6.18 (1H, d, the cells incubated for 72h. The cells were harvested by incu-
J=7.9Hz, H-1‴), 6.26 (1H, brs, H-1″″); 13C-NMR (pyridine- bating with PBS containing 1mM EDTA and 0.25% trypsin,
d5, 500MHz) δC: 15.7, 16.9×2, 18.3×2, 23.8, 25.5, 26.5, 27.5, and then washed with PBS. The cells were treated with NaOH
°
28.1, 31.8, 33.2, 33.8, 34.7, 36.8, 38.9, 39.8, 40.1, 41.7, 42.2, 1M (120µL/tube, 80 C, 30min) to yield a lysate. An aliquot
44.3, 44.9, 47.0, 47.9, 55.8, 62.0, 62.9, 68.4, 69.9, 70.3, 71.0, (100µL) of the lysate was transferred to a 96-well microplate,
71.2×2, 72.6×2, 72.8, 73.5, 74.0, 75.3, 76.1, 77.0, 77.1×2, 77.4, and the optical density of each well was measured with a mi-
79.8, 83.1, 89.9, 101.5, 102.7, 103.6, 105.4, 123.1, 144.3, 172.2; croplate reader (Model 550, Bio-Rad Laboratories) at 405nm
positive-ion FAB-MS: m/z 1143 [M+Na]+.
(reference: 655nm). The test compound was dissolved in di-
Next, a part of the crude product (each 1.0mg) obtained methyl sulfoxide (DMSO). The final concentration of DMSO
from 1–4 was dissolved in (CH2)2Cl2 (0.5mL), respec- in the medium was 0.1%. The production of melanin was cor-
tively. The solution was treated with p-nitrobenzyl-N,N′- rected based on cell viability. Inhibition (%) was calculated
°
diisopropylisourea (4mg), then stirred at 80 C for 1h. The using the following formula, and IC50 values were determined
reaction solution was subjected to HPLC [column: COSMO- graphically.
SIL 5C18-MS-II, 250×4.6mm i.d.; mobile phase: MeCN–
H2O (50:50, v/v); detection: UV (254nm); flow rate: 1.0mL/
inhibition (%)=[(A B)] / (C /100)×100
min; column temperature: room temperature] to identify the where A and B indicate the optical density of vehicle- and test
p-nitrobenzyl esters of tiglic acid (tR: 20.9min, from 3) and compound-treated groups, respectively, and C indicates cell
angelic acid (tR: 23.0min, from 1, 2 and 4).
Acid Hydrolysis and Monosaccharide Composition of
Sanchakasaponins Compounds 1–4 (1.0–2.0mg) were well) were seeded into 96-well microplates and incubated for
viability (%).
Cell Viability The melanoma cells (5.0×103 cells/100µL/

758
Vol. 60, No. 6
8) Sugimoto S., Chi G., Kato Y., Nakamura S., Matsuda H., Yoshi-
kawa M., Chem. Pharm. Bull., 57, 269–275 (2009).
9) Matsuda H., Nakamura S., Fujimoto K., Moriuchi R., Kimura Y.,
Ikoma N., Hata Y., Muraoka O., Yoshikawa M., Chem. Pharm.
Bull., 58, 1617–1621 (2010).
10) Yoshikawa M., Morikawa T., Fujiwara E., Ohgushi T., Asao Y.,
Matsuda H., Heterocycles, 55, 1653–1657 (2001).
11) Yoshikawa M., Morikawa T., Asao Y., Fujiwara E., Nakamura S.,
Matsuda H., Chem. Pharm. Bull., 55, 606–612 (2007).
12) Shin M. H., Wang W., Nam K. I., Jo Y., Jung J. H., Im K. S., J. Nat.
Prod., 66, 1351–1355 (2003).
24h. After 70h of incubation with theophylline 1mM and a
test compound, 10µL of WST-8 solution (Cell Counting Kit-
8
the optical density of the water-soluble formazan produced by
the cells was measured with a microplate reader (Model 550,
Bio-Rad Laboratories, Hercules, CA, U.S.A.) at 450nm (refer-
ence: 655nm). The test compound was dissolved in DMSO.
The final concentration of DMSO in the medium was 0.1%.
Cell viability (%) was calculated using the following formula.
TM) was added to each well. After a further 2h in culture,
cell viability (%)= A / B×100
13) Kitagawa I., Yoshikawa M., Kobayashi K., Imamura Y., Im K. S.,
Ikenishi Y., Chem. Pharm. Bull., 28, 296–300 (1980).
14) Yosioka I., Nishimura T., Matsuda A., Kitagawa I., Chem. Pharm.
Bull., 18, 1610–1620 (1970).
where A and B indicate the optical density of vehicle- and test
compound-treated groups, respectively.
Statistical Analyses Values are expressed as the mean
15) Tanaka T., Nakashima T., Ueda T., Tomii K., Kouno I., Chem.
Pharm. Bull., 55, 899–901 (2007).
’
S.E.M. A one-way analysis of variance followed by Dunnett s
test was used for statistical analyses.
1
16) The H- and 13C-NMR spectra of 1–4 were assigned with the aid of
distortionless enhancement by polarization transfer (DEPT), DQF
COSY, heteronuclear multiple quantum coherence spectroscopy
(HMQC), and HMBC experiments.
References and Notes
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Bioorg. Med. Chem., 17, 6048–6053 (2009).
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cycles, 78, 1023–1029 (2009).
22) Nakashima S., Matsuda H., Oda Y., Nakamura S., Xu F., Yoshikawa
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