Oleanene glycosides of the aerial parts and seeds of Bupleurum falcatum and the aerial parts of Bupleurum rotundifolium, and their evaluation as anti-hepatitis agents

Article abstract of DOI:10.1248/cpb.59.1329

To facilitate effective resource utilization, we have investigated triterpene saponins such as saikosaponin from the aerial parts of Bupleurum (B.) falcatum L., which are commonly discarded. Seven oleanene saponins were isolated from this plant; they were classified as the 13,28-epoxy type, 12-ene type, 9(11),12-diene type, and 28-acid type on the basis of their structural characteristics. For comparison, we also examined the oleanene saponins of the seeds of B. falcatum and the aerial parts of B. rotundifolium L. to obtain seven saponins and one sapogenol from the former and thirteen oleanene saponins from the latter. Several compounds obtained from them were investigated for their hepatoprotective activity and hepatotoxicity. The 13,28-epoxy type saponins had hepatoprotectivity. Ursane type showed hepatotoxicity from middle concentration. The 11,13(18)-diene type saponins did not express hepatoprotective activity. The 28-acid type saponin which has a glucosyl carboxy group showed hepatoprotective action.

Full text of DOI:10.1248/cpb.59.1329

November 2011
Regular Article
Chem. Pharm. Bull. 59(11) 1329—1339 (2011)
1329
Oleanene Glycosides of the Aerial Parts and Seeds of Bupleurum falcatum
and the Aerial Parts of Bupleurum rotundifolium, and Their Evaluation as
Anti-hepatitis Agents
a
b
b
,c
*
Yuko NAKAHARA, Masafumi OKAWA, Junei KINJO, and Toshihiro NOHARA
a Graduate School of Pharmaceutical Sciences, Faculty of Life Sciences, Kumamoto University; 5–1 Oe-honmachi,
b
Kumamoto 862–0973, Japan: Faculty of Pharmaceutical Sciences, Fukuoka University; 8–19–1 Nanakuma, Higashi-ku,
Fukuoka 814–0180, Japan: and c Faculty of Pharmaceutical Sciences, Sojo University; 4–22–1 Ikeda, Kumamoto
860–0082, Japan. Received May 25, 2011; accepted August 18, 2011; published online August 23, 2011
To facilitate effective resource utilization, we have investigated triterpene saponins such as saikosaponin
from the aerial parts of Bupleurum (B.) falcatum L., which are commonly discarded. Seven oleanene saponins
were isolated from this plant; they were classified as the 13,28-epoxy type, 12-ene type, 9(11),12-diene type, and
28-acid type on the basis of their structural characteristics. For comparison, we also examined the oleanene
saponins of the seeds of B. falcatum and the aerial parts of B. rotundifolium L. to obtain seven saponins and one
sapogenol from the former and thirteen oleanene saponins from the latter. Several compounds obtained from
them were investigated for their hepatoprotective activity and hepatotoxicity. The 13,28-epoxy type saponins had
hepatoprotectivity. Ursane type showed hepatotoxicity from middle concentration. The 11,13(18)-diene type
saponins did not express hepatoprotective activity. The 28-acid type saponin which has a glucosyl carboxy group
showed hepatoprotective action.
Key words Bupleurum falcatum; saikosaponin; hepatoprotective activity; Bupleurum rotundifolium
Bupleuri Radix appeared in ‘Shen hong ben cao jing’ as BFA-6 (264 mg), BFA-8 (45 mg), BFA-11 (21 mg), BFA-13
a drug harmless to humans and one of the most important (110 mg), BFA-17 (21 mg), BFA-20 (20 mg), BFA-22
Chinese crude drugs used as an antipyretic and anti-inflam- (72 mg), BFA-25 (17 mg), BFA-27 (110 mg), BFA-28
matory. The occurrence of saikosaponins (saikosaponin (680 mg), and BFA-29 (20 mg).
a : c : dꢀ2 : 1 : 3) in this drug and their functions in peptide
BFA-8 (1), BFA-22 (2), BFA-5 (3), BFA-6 (4), and BFA-
synthesis and detoxification in the liver are well known. 25 (5) were identified as saikosaponin a,3) saikosaponin c,3)
Many earlier works were reported, as follows. Shibata et al. saikosaponin f,3) saikosaponin b2,2) and 4ꢁ-O-acetylsaiko-
reported isolation of various saponins from Bupleuri Radix saponin b2,13) respectively, by fast atom bombardment-mass
originating from Bupleurum falcatum cultivated in Japan and spectrometry (FAB-MS), 1H- and 13C-NMR spectra, and
the liver-protecting activity of a mixture of the major chemical reactions.
saponin, saikosaponin a, with other saponins.1) Shimaoka et
BFA-20 (6) was obtained as an amorphous powder having
al. reinvestigated the structure of saikosaponin,2) and Tori et [a]D ꢂ11.8° (MeOH). Positive high resolution (HR)-FAB-
al. conducted a 13C-NMR investigation of saikosapogenins.3) MS revealed a molecular formula of C48H78O18Na at m/z
Ogihara and colleagues synthesized artificial derivatives of 965.5088 [MꢃNa]ꢃ. The H-NMR spectrum displayed sig-
1
saikosaponins by using an acid or enzyme4,5) and examined nals due to six tertiary methyl groups at d 0.90, 0.96, 1.00,
their function of accelerating corticosteroid secretion.6) 1.00, 1.06, and 1.66 (each 3H, s); one methyl pentosyl
Mitsuhashi and colleagues isolated a new saikosaponin mal- methyl group (3H, d, Jꢀ6.1 Hz, H3-6) at d 1.66; two hexosyl
onate from B. scorzonerifolium,7) and Yoshikawa et al. iso- anomeric protons at d 4.94 and 5.01 (each 1H, anomeric pro-
lated a new saponin from B. scorzonerifolium and studied its ton, d, Jꢀ7.9 Hz); one methyl pentosyl anomeric proton at d
liver-protecting components.8,9) Moreover, agricultural stud- 5.87 (1H, s); and two olefinic protons at d 5.67 and 6.68 (1H,
ies of the constituents of the seeds have been begun by Ono d, Jꢀ11.0 Hz and 1H, br d, Jꢀ8.5 Hz, respectively). The 13C-
et al.,10) and studies of anti-proliferation of tumor cells have NMR spectrum (Table 1) displayed a total of 48 carbon sig-
been initiated by Mihashi and colleagues.11,12)
nals, of which 30 were attributable to saikogenin D com-
In some areas in Kumamoto Prefecture, this plant is culti- prised of BFA-6 (4). The remaining carbon signals arose
vated, and its aerial parts are discarded. We investigated the from sugar moieties and coincided with those of the sugar
drug’s constituents in conjunction with studies of the aerial moieties in BFA-22 (2), as listed in Table 1. Therefore, the
parts of B. rotundifolium, which is cultivated for cut flowers, structure of 6 was characterized as b-D-glucopyranosyl-
and the seeds of B. falcatum. We also examined the liver-pro- (1→16)-[a-L-rhamnopyranosyl-(1→4)]-b-D-glucopyranosyl
tecting functions of the obtained specimens and elucidated 3b,16a,23,28-tetrahydroxyolean-11,13(18)-diene.4)
the relationship between their structure and activity.
BFA-17 (7) was obtained as an amorphous powder having
Harvested aerial parts (2.6 kg) of B. falcatum were ex- [a]D ꢃ45.9° (MeOH). Positive HR-FAB-MS revealed a mo-
tracted by refluxing with MeOH to give an extract (262.2 g), lecular formula of C36H58O9Na at m/z 657.3982 [MꢃNa]ꢃ.
1
which was then separated by using the solvent partition The H-NMR spectrum displayed signals due to six tertiary
method. Various chromatographies on Diaion HP-20, methyl groups at d 0.81, 0.97, 1.10, 1.21, 1.28, and 1.35
Sephadex LH-20, silica gel, and Chromatorex octadecylsilyl (each 3H, s); one olefinic proton at d 5.44; and one hexosyl
(ODS) silica were applied to provide BFA-5 (108 mg), anomeric proton at d 6.33 (1H, d, Jꢀ7.9 Hz). Since the 13C-
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: none@ph.sojo-u.ac.jp

1330
Vol. 59, No. 11
queretaroic acid.14) HMBC from the glucosyl anomeric pro-
ton was observed at C-28 at d 176.5. This glycoside was rec-
ognized as a new one.
Table 1. 13C-Data for Compounds 1—7
1
2
3
4
5
6
7
Next, the seeds (2.6 kg) of B. falcatum were crushed with a
mixer and extracted by refluxing with MeOH; the resulting
extract (84.7 g) was then separated by using various chro-
matographies on Diaion HP-20, Sephadex LH-20, silica gel,
and preparative thin layer chromatography to provide BFS-1
(30 mg), BFS-8 (8 mg), BFS-9 (13 mg), BFS-10 (31 mg),
BFS-11 (34 mg), BFS-13 (28 mg), BFS-14 (15 mg), and
BFS-16 (15 mg).
C-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38.6
26.0
81.6
43.7
47.3
17.2
31.5
42.2
53.0
36.2
132.2
131.1
84.0
45.6
36.1
64.0
46.9
52.1
37.7
31.5
34.6
25.7
64.0
13.0
18.7
20.0
20.8
73.0
33.6
23.8
38.4
26.4
89.0
39.6
55.2
18.4
31.8
42.1
52.8
36.3
132.1
131.1
84.0
45.6
36.0
64.0
47.0
52.1
37.8
31.6
34.7
25.7
27.9
16.3
18.1
19.9
20.9
72.6
33.7
23.8
38.8
26.5
89.0
39.5
55.7
18.5
33.4
40.1
47.0
36.7
24.1
122.6
143.9
43.8
36.7
66.6
41.0
44.4
47.0
31.1
34.3
26.5
28.2
17.0
15.6
17.0
27.1
69.0
32.9
24.1
38.4
26.1
81.7
43.7
47.3
18.8
31.9
41.1
54.0
36.5
126.2
126.2
136.0
41.8
32.3
67.6
45.3
133.0
39.0
32.6
35.4
24.4
64.1
13.1
18.2
17.2
21.9
64.7
25.1
32.5
38.4
26.1
81.7
43.7
47.4
18.3
31.9
41.9
54.0
36.5
126.3
126.2
136.0
41.1
32.3
67.7
45.3
133.1
39.0
32.6
35.5
24.4
64.8
24.1
18.9
17.3
21.9
64.7
25.1
32.6
38.3
25.9
82.1
43.6
47.3
18.3
31.9
41.1
53.9
36.3
126.2
126.2
136.1
41.9
32.3
67.6
45.3
133.0
39.0
32.6
35.5
24.4
64.3
13.1
18.5
17.2
21.9
38.6
26.3
78.9
39.4
55.7
18.5
32.4
39.9
48.0
36.0
23.7
123.0
144.0
42.2
33.1
23.7
47.0
41.3
41.7
35.7
29.5
32.4
28.2
17.0
15.5
17.5
26.2
BFS-9 (1), BFS-1 (2), BFS-16 (8), BFS-11 (10), and BFS-
13 (4) were identified as saikosaponin a,3) saikosaponin c,3)
bupleuroside IX,9) saikosaponin b1,2) and saikosaponin b2, re-
spectively, by FAB-MS and 1H- and 13C-NMR spectra.
BFS-10 (12) was obtained as an amorphous powder hav-
ing [a]D ꢃ78.1° (MeOH). Positive HR-FAB-MS revealed
a molecular formula of C42H68O13Na at m/z 803.4563
1
[MꢃNa]ꢃ. The H-NMR spectrum displayed signals due to
six tertiary methyl groups at d 0.89, 0.97, 1.00, 1.28, 1.29,
and 1.30 (each 3H, s); one methyl pentosyl methyl group at d
1.44 (3H, d, Jꢀ6.1 Hz, H3-6); two anomeric protons at d 4.97
(1H, d, Jꢀ7.9 Hz) and 5.33 (1H, d, Jꢀ7.9 Hz); and two
olefinic protons at d 5.65 (1H, d, Jꢀ6.1 Hz) and 5.76 (1H, d,
Jꢀ6.1 Hz). The 13C-NMR spectrum (Table 3) showed the
presence of two trisubstituted double bonds at d 116.0,
121.1, 145.3, and 154.9; their carbon chemical shifts and
proton coupling constants indicated that they were located at
the C-ring and homoannular diene, respectively. The respec-
tive carbon signals at d 81.5, 66.8, 64.2, and 69.3 could be
assigned to C-3, C-16, C-23, and C-28, respectively, and a b
configuration was estimated for the hydroxyl group at C-16
by comparing its chemical shift with the reported one.3)
Therefore, sapogenol 27 was determined to be saikogenin
H,4) which was derived from saikosaponin a by treatment
with 1 N H2SO4 followed by acid hydrolysis. The sugar moi-
ety was assignable to the glucosyl-(1→3)-fucosyl residue.
Consequently, the structure of BFS-10 (12) was determined
to be 3-O-b-D-glucopyranosyl-(1→3)-b-D-fucopyranosyl
3b,16b,23,28-tetrahydroxyolean-9(11),12(13)-diene; this is
identical to that of saikosaponin g, which has been artificially
derived from saikosaponin a by treatment with 1 N H2SO4.4)
BFS-8 (9) was obtained as an amorphous powder having
[a]D ꢂ15.0° (MeOH). Positive HR-FAB-MS revealed a mo-
lecular formula of C30H48O4Na at m/z 495.3446 [MꢃNa]ꢃ.
64.7 1765.5
25.1
64.0
28.4
65.4
fuc-1 105.9
105.9 106.0
2
3
4
5
6
71.5
85.1
71.7
70.9
17.2
71.5
85.2
71.8
71.0
17.2
71.6
85.4
72.1
71.5
17.4
glcI-1
106.5
75.7
78.7
72.1
78.4
62.7
106.7
75.1
76.7
79.8
75.5
69.0
106.7
75.1
76.7
79.8
75.5
68.8
106.5
75.7
78.7
72.1
78.3
62.7
20.1
170.8
106.4
75.3
78.1
75.5
78.1
64.2
106.7
75.1
76.7
79.8
75.5
69.0
95.7
74.1
79.3
71.1
78.9
62.2
2
3
4
5
6
OCOC
H3
OCOCH3
glcII-1
105.0 105.0
105.0
74.7
78.4
71.4
78.3
62.5
2
3
4
5
6
74.7
78.4
71.4
78.3
62.5
74.8
78.4
71.4
78.4
62.6
1
The H-NMR spectrum displayed signals due to six tertiary
methyl groups at d 0.93, 1.01, 1.05, 1.07, 1.08, and 1.68
(each 3H, s), and two olefinic protons at d 5.77 (1H, d,
Jꢀ10.7 Hz) and 6.74 (1H, dd, Jꢀ2.8, 10.7 Hz). The 13C-
NMR spectrum (Table 3) showed a total of 30 carbon signals,
including those of a disubstituted double bond and a tetra-
substituted double bond, and four oxygen-bearing carbon sig-
nals at d 73.2, 67.6, 67.7, and 64.7, ascribable to C-3, C-16,
rha-1
102.9 102.9
102.9
72.5
72.5
73.8
70.5
18.1
2
3
4
5
6
72.5
72.5
73.8
70.5
18.1
72.6
72.5
73.8
70.4
18.5
NMR spectrum exhibited a total of 36 carbon signals that C-23, and C-28, respectively. The configuration at C-16 in 9
suggested the presence of one b-D-glucopyranosyl moiety, was revealed to be identical with that of saikogenin D, which
the remaining 30 carbon signals were regarded as attributable has been artificially derived from saikosaponin d by treat-
to a sapogenol moiety. Heteronuclear multiple bond coher- ment with 1 N H2SO4 followed by acid hydrolysis with 2 N
ence (HMBC) analysis identified six tertiary methyl groups H2SO4.4)
and correlated an oxygen-bearing methylene at d 65.4 with
BFS-14 (11) was obtained as an amorphous powder hav-
H3-29; therefore, CH2O is present at C-30. This sapogenol is ing [a]D ꢂ37.5° (MeOH). Positive HR-FAB-MS revealed a
identified as 3b,30-dihydroxyolean-12-ene-28-oic acid, or molecular formula of C48H78O17Na at m/z 949.5133

November 2011
1331
Table 2. Oleanene Glycosides Obtained from the Aerial Parts of Bupleurum falcatum
13,28-Epoxy type
12-Ene type
11,13(18)-Diene type
28-Acid type
1
[MꢃNa]ꢃ. The H-NMR spectrum displayed signals due to spectrum (Table 1) suggested the presence of a heteroannular
seven tertiary methyl groups at d 0.83, 0.86, 0.90, 0.98, 0.99, diene and saikogenin C as a sapogenol, which is produced
1.14, and 1.32 (each 3H, s); one methyl pentosyl methyl from saikosaponin c by treatment with intestinal bacteria.
group (3H, d, Jꢀ6.1 Hz, H3-6) at d 1.67; three anomeric pro- The sugar moiety was identical with glucosyl-(1→6)-[rham-
tons at d 4.85 (1H, d, Jꢀ7.3 Hz), 4.98 (1H, d, Jꢀ7.9 Hz), and nosyl-(1→4)]-glucosyl. HMBC analysis indicated that the
5.87 (1H, br s); and two olefinic protons at d 5.64 (1H, d, sugar chain bonding connected with the C-3 of the sa-
Jꢀ10.7 Hz) and 6.52 (1H, br d, Jꢀ10.7 Hz). The 13C-NMR pogenol. Therefore, BFS-14 (11) was characterized as

1332
Vol. 59, No. 11
saikosaponin h, naturally isolated as a genuine saponin for (12 mg), BRA-17 (14 mg), BRA-22 (22 mg), BRA-18
the first time.4)
(38 mg), BRA-19 (6 mg), and BRA-20 (5 mg).
The aerial parts (1.4 kg) of B. rotundifolium L. were ex-
BRA-3 (13), BRA-5 (14), BRA-2 (15), BRA-4 (16), and
tracted by refluxing with MeOH; the resulting extract (152 g) BRA-7 (17) were identified as rotundifolioside J,11,12) rotun-
was then separated by using the solvent partition method. difolioside I,11,12) rotundioside E,15,16) and rotundioside
Various chromatographies on Diaion HP-20, silica gel, and W, 11,12) and rotundioside V,11,12) respectively.
Chromatorex ODS were applied to yield BRA-2 (19 mg),
BRA-22 (18) was obtained as an amorphous powder hav-
BRA-3 (19 mg), BRA-4 (35 mg), BRA-5 (60 mg), BRA-7 ing [a]D ꢂ22.5° (MeOH). Positive HR-FAB-MS revealed
(14 mg), BRA-11 (36 mg), BRA-14 (69 mg), BRA-16 a molecular formula of C48H78O17Na at m/z 949.5143
[MꢃNa]ꢃ. The H-NMR spectrum displayed signals due to
six tertiary methyl groups at d 0.87, 0.91, 1.09, 1.25, 1.37,
and 1.77 (each 3H, s); two methyl pentosyl methyl groups at
1
Table 3. 13C-Data for Compounds 8—12
8
9
10
11
12
d 1.52 (3H, d, Jꢀ6.7 Hz, H3-6) and 1.86 (3H, d, Jꢀ6.1 Hz,
H3-6); three anomeric protons at d 4.79 (1H, d, Jꢀ7.3 Hz),
5.69 (1H, d, Jꢀ7.3 Hz), and 6.44 (1H, br s); and two olefinic
protons at d 5.70 (1H, d, Jꢀ7.9 Hz) and 6.77 (1H, br d,
Jꢀ7.9 Hz). The 13C-NMR spectrum (Table 5) showed a total
of 48 carbon signals, of which 18 indicated the presence of a
rhamnosyl-(1→2)-glucosyl-(1→2)-fucosyl moiety identical
to that of BRA-3 (13). The remaining signals showed good
agreement with those of rotundiogenin B in addition to those
of C-20, C-29, and C-30. This means that in 18, a hydrox-
ymethyl is present at d 73.6 at C-29 or C-30. HMBC analysis
also supported this conclusion. By comparison with the
3b,16a,28,30-tetrahydroxyolean-11,13(18)-dine derivative17)
isolated from B. spinosum and the 30-CH2OH-isomer18) iso-
lated from B. rigidum, 18 was determined to be a C-30-hy-
droxymethyl derivative. Therefore, its structure was charac-
terized as 3-O-a-L-rhamnopyranosyl-(1→2)-b-D-glucopyra-
nosyl-(1→2)-b-D-fucopyranosyl 3b,16,28,30-tetrahydroxy-
olean-11,13(18)-diene.
C-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OCH3
40.1
27.0
88.7
43.9
55.9
18.6
33.5
41.0
52.1
38.2
76.0
122.5
148.2
43.6
36.8
66.3
39.9
44.1
47.0
31.2
34.3
26.0
28.2
17.4
18.3
17.3
26.3
68.6
33.1
24.0
54.1
38.4
27.6
73.2
43.0
48.4
18.6
31.9
41.0
53.9
36.8
126.2
126.2
136.0
41.9
32.3
67.6
45.3
133.0
39.0
32.6
35.4
24.4
67.7
12.6
18.7
17.2
21.9
64.7
25.1
32.5
38.4
26.0
81.6
43.6
47.2
18.1
32.3
40.4
54.4
36.4
127.0
125.6
136.4
44.2
34.8
76.5
44.3
133.3
38.3
32.6
35.1
29.9
63.9
13.0
18.7
17.2
21.9
63.9
24.7
32.2
38.4
26.4
89.0
39.6
55.2
18.4
32.6
40.4
54.2
36.5
126.9
125.7
136.3
44.2
34.8
76.5
44.4
133.3
38.1
32.6
35.1
29.9
27.9
16.4
18.4
16.9
21.9
63.9
24.8
32.2
37.6
26.8
81.5
43.7
43.5
17.9
32.1
43.1
154.9
38.7
116.0
121.1
145.3
43.3
36.1
66.8
40.5
133.4
46.9
31.0
34.1
26.1
64.2
13.7
21.0
21.2
26.1
69.3
33.1
24.0
BRA-11 (19) was obtained as an amorphous powder hav-
ing [a]D ꢂ41.8° (MeOH). Positive HR-FAB-MS revealed a
molecular formula of C48H78O17Na at m/z 949.5139
1
[MꢃNa]ꢃ. The H-NMR spectrum displayed signals due to
seven tertiary methyl groups at d 0.87, 0.92, 1.09, 1.30, 1.34,
1.38, and 1.77 (each 3H, s); two methyl pentosyl methyl
groups at d 1.53 (3H, d, Jꢀ6.1 Hz, H3-6) and 1.87 (3H, d,
Jꢀ6.1 Hz, H3-6); one oxygen-bearing methine proton at d
3.33 (1H, br d, Jꢀ8.0 Hz); one oxygen-bearing methylene
proton at d 3.85 (1H, d, Jꢀ10.9 Hz) and 4.33 (overlapped);
three anomeric protons at d 4.80 (1H, d, Jꢀ6.7 Hz), 5.70
(1H, d, Jꢀ7.3 Hz), and 6.44 (1H, br s); and two olefinic pro-
tons at d 5.73 (1H, d, Jꢀ10.4 Hz) and 6.77 (1H, br d,
Jꢀ10.4 Hz). The 13C-NMR spectrum (Table 5) showed a
total of 48 carbon signals, of which 18 revealed the presence
of a rhamnosyl-(1→2)-glucosyl-(1→2)-fucosyl moiety, as in
BRA-22 (18). In the remaining signals due to the sapogenol
fuc-1
106.9
71.6
85.3
72.2
71.0
17.0
105.9
71.5
85.1
72.1
70.1
17.0
105.9
71.5
85.2
72.0
70.9
17.2
2
3
4
5
6
glcI-1
106.7
75.9
78.7
71.4
78.1
62.7
106.4
75.7
78.6
71.7
78.3
62.6
106.7
75.2
76.7
79.8
75.5
69.0
106.5
75.7
78.7
71.7
78.3
62.6
2
3
4
5
6
glcII-1
105.0
74.8
78.4
71.5
78.3
62.6
2
3
4
5
6
rha-1
102.9
72.5
72.6
73.8
70.5
18.2
2
3
4
5
6
Fig. 1. HMBC of BRA-11 (19)

November 2011
1333
Table 4. Oleanene Glycosides Obtained from the Seeds of Bupleurum falcatum
13,28-Epoxy type
12-Ene type
9(11),12-Diene type
28-Acid type
moiety, three oxygen-bearing carbon signals were observed; the structure of BRA-11 (19) was characterized as 3-O-a-L-
the signals at d 67.3 and 89.5 were assigned to carbons hav- rhamnopyranosyl-(1→2)-b-D-glucopyranosyl-(1→2)-b-D-
ing an a-hydroxyl group at C-16 and a b-hydroxyl group at fucopyranosyl 3b,16a,21b,28-tetrahydroxyolean-11,13(18)-
C-3, respectively. The remaining signal, at d 73.7, was as- diene, which indicates a new sapogenol.
signed to C-21 because HMBC analysis exhibited correla-
BRA-16 (20) was obtained as an amorphous powder hav-
tions from H3-29 and H3-30 to C-22, and from H2-22 to C-22 ing [a]D ꢂ1.5° (MeOH). Positive HR-FAB-MS revealed a
(Fig. 1). Here, the configuration at the C-21 hydroxyl group molecular formula of C47H76O17Na at m/z 935.4973
1
was deduced to be b-equatorial because the signals due to [MꢃNa]ꢃ. The H-NMR spectrum showed signals due to
H3-29 and H3-30 in BRA-11 (19) were shifted upward rela- seven tertiary methyl groups at d 0.88, 0.94, 1.11, 1.30, 1.31,
tive to those of rotundiogenin C, which has a C-21 hydroxyl 1.34, and 1.76 (each 3H, s); one methyl pentosyl methyl
group,19) by the influence of the hydroxyl group. Therefore, groups at d 1.59 (3H, d, Jꢀ6.1 Hz, H3-6) and 1.87; one oxy-

1334
Vol. 59, No. 11
Table 5. 13C-Data for Compounds 13—19
Table 6. 13C-Data for Compounds 20—25
20
21
22
23
24
25
13
14
15
16
17
18
19
C-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38.4
26.6
88.8
39.8
55.5
18.6
32.6
41.1
53.8
36.6
126.4
126.4
136.3
41.9
31.7
67.3
47.8
132.2
39.4
38.0
73.7
33.2
28.0
16.4
18.4
17.2
21.9
65.8
29.3
18.4
38.5
26.6
89.5
39.9
55.6
18.6
32.4
42.3
54.0
36.7
126.0
128.0
137.0
40.9
32.3
68.4
46.0
131.0
39.3
32.6
217.1
44.3
28.2
16.3
18.4
17.4
22.2
69.5
26.1
24.6
38.7
26.4
89.8
39.6
56.0
18.5
32.3
40.0
48.0
36.9
23.8
122.5
144.6
42.1
29.3
23.0
47.0
41.7
40.2
30.7
34.0
33.2
28.6
16.8
15.4
17.4
26.2
176.5
23.7
33.1
38.9
26.4
89.9
40.0
56.2
18.4
32.3
40.1
48.2
37.0
23.8
122.6
144.5
42.1
29.4
22.9
47.0
41.9
46.2
30.7
34.0
33.5
28.6
16.7
15.5
17.5
26.2
176.5
23.7
33.2
38.8
26.4
89.7
39.6
56.0
18.5
32.1
40.0
47.1
37.0
23.8
122.6
144.4
42.0
35.9
74.1
49.1
41.3
47.2
30.8
36.1
33.4
28.3
16.8
15.6
17.5
27.2
175.5
24.6
33.2
38.8
26.4
89.7
39.6
56.0
18.5
31.6
40.1
47.1
36.9
23.8
122.1
145.0
41.9
36.0
73.9
49.1
41.3
47.3
30.8
36.6
33.5
28.5
16.8
15.6
17.5
27.2
175.9
24.6
33.2
C-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38.7
26.6
89.5
39.9
55.6
18.0
32.0
42.0
52.7
36.4
134.2
129.1
85.1
44.1
35.6
77.1
46.4
61.5
38.5
41.7
33.7
35.4
28.1
16.2
17.4
19.0
16.4
77.7
18.9
19.8
38.7
26.6
88.8
39.8
55.5
18.0
32.0
42.0
52.6
36.3
134.1
129.1
85.1
44.1
35.5
76.9
46.4
61.5
38.3
41.7
33.7
35.4
17.9
16.3
18.3
19.4
16.4
77.7
18.9
19.8
38.4
26.6
89.5
40.0
55.6
18.6
32.6
42.0
53.8
36.6
126.3
126.2
136.0
41.1
31.9
67.7
45.3
133.1
39.0
32.6
35.5
24.5
28.1
16.3
18.3
17.4
21.9
64.8
25.1
32.6
38.5
26.6
88.8
39.8
55.5
18.6
32.6
41.1
53.8
36.6
126.2
126.3
135.1
41.9
31.9
67.7
45.3
133.1
39.1
32.6
35.5
24.5
28.0
16.4
18.4
17.3
21.9
64.8
25.1
32.6
38.4
26.0
84.6
43.7
48.4
18.5
32.4
41.9
54.0
36.6
126.3
126.2
136.1
41.1
31.9
67.7
45.3
133.1
39.1
32.6
35.5
24.5
65.6
12.9
18.9
17.3
21.9
64.8
25.1
32.6
38.4
26.6
89.6
39.9
55.6
18.7
32.6
42.0
53.9
36.6
126.4
126.1
136.4
41.1
32.1
68.0
45.9
133.0
34.0
38.3
30.3
24.3
28.2
16.3
18.3
17.4
22.0
65.1
21.1
73.6
38.4
26.6
89.5
39.9
55.5
18.6
32.5
41.1
53.8
36.6
126.5
126.4
136.3
31.7
67.3
47.7
47.8
132.2
39.3
38.0
73.7
33.1
28.1
16.3
18.3
17.2
21.9
65.7
29.2
18.4
fuc-1
105.0
80.7
75.5
72.6
71.2
17.3
105.3
77.4
76.3
73.0
70.9
17.3
2
3
4
5
6
fuc-1
105.3
77.2
76.3
72.8
70.9
19.4
105.3
80.6
75.5
72.6
71.2
17.3
105.3
77.4
76.2
72.9
70.8
17.2
105.2
80.6
75.5
72.6
71.2
17.2
103.7
81.2
75.6
72.6
71.2
17.3
105.3
77.6
76.3
72.9
70.9
17.2
105.3
77.3
76.2
72.9
70.8
17.4
glcI-1
103.2
84.8
78.0
71.8
77.7
62.8
102.2
78.1
79.5
72.8
77.0
63.3
105.2
78.5
79.5
70.1
77.9
62.8
105.2
78.5
78.8
70.1
77.9
62.8
105.0
78.3
78.9
72.5
77.8
62.6
105.2
78.4
78.8
72.6
77.8
62.8
2
3
4
5
6
2
3
4
5
6
glcII-1
93.4
78.4
79.4
78.8
79.2
61.9
93.5
78.4
79.2
79.5
79.4
61.9
95.8
74.2
79.4
78.8
79.3
62.1
93.8
78.2
79.3
79.9
79.3
61.8
glc-1
102.2
78.1
79.5
73.0
76.9
63.3
103.1
84.7
77.7
71.8
77.5
62.8
102.2
78.1
79.5
72.8
77.0
63.3
103.2
84.8
78.0
71.8
77.7
62.8
103.4
84.6
77.9
71.1
77.7
62.4
102.2
78.1
79.5
73.0
77.3
63.3
102.2
78.1
79.5
72.9
77.1
63.3
2
3
4
5
6
2
3
4
5
6
glcIII-1
101.9
75.9
77.9
72.8
77.5
63.9
102.0
75.9
77.9
72.8
77.5
63.4
101.9
76.0
78.3
72.8
77.4
63.4
101.9
75.9
77.9
72.8
77.5
63.3
2
3
4
5
6
rha-1
101.9
72.5
72.8
74.4
69.5
18.2
101.9
72.5
72.8
74.3
69.5
19.0
101.9
72.5
72.8
74.4
69.5
19.0
101.9
72.8
72.5
74.3
69.5
19.0
2
3
4
5
6
glcIV-1
104.6
75.9
79.5
72.0
78.2
62.8
105.2
76.7
79.6
72.1
78.6
62.8
105.1
75.8
79.4
71.5
78.2
62.7
2
3
4
5
6
xyl-1
106.5
75.8
78.0
70.8
67.4
106.6
75.9
77.5
70.8
67.4
106.5
75.9
77.6
70.8
67.5
2
3
4
5
glcV-1
102.4
78.6
79.2
72.4
77.1
63.7
2
3
4
5
6
gen-bearing methine proton at d 3.33 (1H, br d, Jꢀ8.0 Hz);
one oxygenated methylene proton at d 3.85 (1H, d,
Jꢀ10.9 Hz), three anomeric protons at d 4.83 (1H, d,
Jꢀ7.9 Hz), 5.37 (1H, d, Jꢀ7.0 Hz), and 5.42 (1H, d,
Jꢀ7.3 Hz); and two olefinic protons at d 5.73 (1H, d,
Jꢀ10.4 Hz) and 6.78 (1H, br d, Jꢀ10.4 Hz). The 13C-NMR
spectrum (Table 5) showed a total of 47 carbon signals,
which indicated the presence of 3b,16a,21b,28-tetrahydrox-
yolean-11,13(18)-diene, as in BRA-11 (19), and a xylosyl-
(1→2)-glucosyl-(1→2)-fucosyl moiety, as in BRA-5 (14),
xyl-1
106.6
75.9
77.4
70.8
67.4
2
3
4
5
rha-1
101.9
72.6
72.8
74.3
69.6
19.1
101.8
72.4
72.6
74.4
69.5
19.1
101.8
71.7
72.7
74.4
69.5
19.1
101.9
71.8
72.3
74.4
69.5
18.9
101.8
71.9
72.8
74.3
69.5
19.0
2
3
4
5
6

November 2011
1335
Table 7. Oleanene Glycosides Obtained from the Aerial Parts of Bupleurum rotundifolium
13,28-Epoxy type
11,13(18)-Diene type
28-Acid type

1336
Vol. 59, No. 11
BRA-4 (16), and BRA-7 (17). Therefore, the structure of group at d 1.79 (3H, d, Jꢀ6.1 Hz, H3-6); one olefinic proton
BRA-16 (20) was characterized as 3-O-b-D-xylopyranosyl- at d 5.40 (1H, br s); and five anomeric protons at d 5.01 (1H,
(1→2)-b -D-glucopyranosyl-(1→2)-b -D-fucopyranosyl d, Jꢀ6.1 Hz), 5.75 (1H, d, Jꢀ7.9 Hz), 5.85 (1H, d,
3b,16a,21b,28-tetrahydroxyolean-11,13(18)-diene,
indicates a new sapogenol.
which Jꢀ7.3 Hz), 6.21 (1H, Jꢀ7.9 Hz), and 6.40 (1H, br s). HMBC
was observed from the terminal rhamnosyl anomeric proton
BRA-17 (21) was obtained as an amorphous powder hav- at d 6.40 to the inner glucosyl C-2 at d 78.5; from its inner
ing [a]D ꢃ11.6° (MeOH). Positive HR-FAB-MS revealed a glucosyl anomeric proton at d 5.01 to the sapogenol C-3 at d
molecular formula of C48H76O17Na at m/z 947.4987 89.8; from the terminal glucosyl anomeric proton at d 5.85
1
[MꢃNa]ꢃ. The H-NMR spectrum showed signals due to to the inner glucosyl C-2 at d 78.4; from another terminal
seven tertiary methyl groups at d 0.83, 0.90, 1.10, 1.25, 1.32, glucosyl anomeric proton at d 5.75 to the inner glucosyl C-4
1.39, and 1.72 (each 3H, s); two methyl pentosyl methyl at d 78.8; and from its inner glucosyl anomeric proton at d
groups at d 1.52 (3H, d, Jꢀ6.1 Hz, H3-6) and 1.88 (3H, d, 6.21 to the sapogenol C-28 at d 176.5. Consequently, the
Jꢀ6.1 Hz, H3-6); two methylene protons at d 2.67 and 2.96 structure of BRA-20 (22) was characterized as 3-O-a-L-
(2H, ABq, Jꢀ14.0 Hz) and 3.24 and 3.49 (2H, ABq, rhamnopyranosyl-(1→2)-b-D-glucopyranosyl oleanolic acid
Jꢀ5.3 Hz); one oxygen-bearing methine proton at d 3.34 28-O-b-D-glucopyranosyl-(1→2)-[b-D-glucopyranosyl-
(1H, m); one oxygen-bearing methylene proton at d 3.88, (1→4)]-b-D-glucopyranoside.
4.04 (2H, ABq, Jꢀ11.0 Hz); two olefinic protons at d 5.77
BRA-19 (23) was obtained as an amorphous powder hav-
(1H, d, Jꢀ11.0 Hz) and 6.71 (1H, br d, Jꢀ11.0 Hz); and three ing [a]D ꢃ23.7° (MeOH). Positive HR-FAB-MS revealed a
anomeric protons at d 4.81 (1H, d, Jꢀ7.3 Hz), 5.72 (1H, d, molecular formula of C66H108O32Na at m/z 1435.6725
Jꢀ7.9 Hz), and 6.44 (1H, br s).
[MꢃNa]ꢃ, which corresponds to one mole of hexose more
The 13C-NMR spectrum (Table 5) showed the presence of than BRA-20 (22). The 13C-NMR spectrum showed the pres-
the same sugar moiety as in BRA-11 (19) and the occurrence ence of oleanolic acid as a sapogenol, as in BRA-20 (22).
of a carbonyl group at d 217.1 in the sapogenol moiety. HMBC similar to that of 22 revealed connectivity between
HMBC appeared from H3-29, H3-30, and H2-22 to the carbon the rhamnosyl-(1→2)-glucosyl-(1→2)-glucosyl-(1→C-3 of
at d 217.1; therefore, its carbonyl carbon should be located at sapogenol) and glucosyl-(1→2)-[glucosyl(1→4)-glucosyl-
C-21. Consequently, the structure of BRA-17 (21) was char- (1→C-28 of sapogenol). Therefore, the structure of BRA-19
acterized as 3-O-a-L-rhamnopyranosyl-(1→2)-b-D-glucopy- (23) was characterized as 3-O-a-L-rhamnopyranosyl-(1→2)-
ranosyl-(1→2)-b-D-fucopyranosyl 3b,16a,28-trihydroxy- b-D-glucopyranosyl-b-D-glucopyranosyl-(1→2)-oleanolic
olean-11,13(18)-diene-21-one.
acid 28-O-b-D-glucopyranosyl-(1→2)-[b-D-glucopyranosyl-
BRA-20 (22) was obtained as an amorphous powder hav- (1→4)]-b-D-glucopyranoside.
ing [a]D ꢃ34.0° (MeOH). Positive HR-FAB-MS revealed a
BRA-14 (24) was obtained as an amorphous powder hav-
molecular formula of C60H98O27Na at m/z 1273.6198 ing [a]D ꢃ3.2° (MeOH). Positive HR-FAB-MS revealed a
1
[MꢃNa]ꢃ. The H-NMR spectrum showed signals due to molecular formula of C54H88O23Na at m/z 1127.5669
seven tertiary methyl groups at d 0.83, 0.87, 0.89, 1.00, 1.24, [MꢃNa]ꢃ. The 13C-NMR spectrum showed a total of 54 car-
1.28, and 1.31 (each 3H, s); one methyl pentosyl methyl bon signals, of which 24 were assignable to the sugar moi-
eties rhamnopyranosyl-(1→2)-glucosyl-(1→the C-3 of the
sapogenol) and glucosyl-(1→4)-glucosyl-(1→the C-28 of sa-
Table 8. Hepatoprotective and Hepatotoxic Activity of 13,28-Epoxy Type
pogenol). The other 30 carbon signals include one hydroxy
Saponins
carbon at d 74.1, which was identified as an a-hydroxy-bear-
ing carbon at C-16 on oleanolic acid by HMBC. This sa-
pogenol was identified as echinocystic acid.20) Thus, the
structure of BRA-14 (24) was characterized as 3-O-a-L-
rhamnopyranosyl-(1→2)-b-D-glucopyranosyl echinocystic
acid b-D-glucopyranosyl-(1→2)-b-D-glucopyranoside.
Substances
Dose (mM)
Protection (%) Toxicity (%)
1 (Saikosaponin a)
10
30
13
ꢂ8
96
317†
492†
455†
390†
ND
90
ꢂ33†
ꢂ30†
ꢂ27†
13*
2
200
500
10
BRA-18 (25) was obtained as an amorphous powder hav-
2 (Saikosaponin c)
30
90
ND
Table 9. Hepatoprotective Activity of 12-Ene Type Saponins
13*
ND
200
500
10
30
90
200
500
10
30
90
20***
ND
ND
110
Substances
Dose (mM)
Protection (%) Toxicity (%)
42***
0
13
14
ꢂ50†
605†
705†
647†
638†
101
3 (Saikosaponin f)
10
30
90
200
500
10
30
90
200
500
ꢂ11
11
7
111
105
114
105
129
118
136
128†
124†
138†
ꢂ49†
ꢂ62†
ꢂ72†
5
11
45***
8 (Bupleuroside IX)
2
8
0
120
ꢂ67†
ꢂ77†
ꢂ93†
504†
644†
699†
10
13
27
200
500
Hepatoprotective effects of compounds 1, 2, 13 and 14 toward in vitro immunologi-
cal liver injury on primary cultured rat hepatocytes. Significantly different from ref.,
effective ∗ pꢄ0.05, ∗∗∗ pꢄ0.001. Hepatotoxicity of compounds 1, 2, 13 and 14 in
primary cultured rat hepatocytes. Significantly different from ref., toxic † pꢄ0.001.
Hepatoprotective effects of compounds 3 and 8 toward in vitro immunological liver
injury on primary cultured rat hepatocytes. Significantly different from ref., effective
∗∗∗ pꢄ0.001. Significantly different from ref., toxic † pꢄ0.001.

November 2011
1337
Table 10. Hepatoprotective and Hepatotoxic Activity of 11,13(18)-Diene
Type Saponins
Table 11. Hepatoprotective and Hepatotoxic Activity of 28-Acid Type
Saponins
Substances
Dose (mM)
Protection (%) Toxicity (%)
Substances
Dose (mM)
Protection (%)
Toxicity (%)
4 (Saikosaponin b2)
10
30
90
200
500
10
30
90
200
500
10
30
90
200
500
10
30
90
200
500
10
30
90
200
500
10
30
90
200
500
10
30
90
200
500
10
30
90
200
500
10
ꢂ74
ꢂ59
ꢂ50
ꢂ28
ꢂ108
ꢂ10
ꢂ7
94
88
98
24
10
30
90
200
500
10
30
90
200
500
15
33
99
98
83
79***
55***
54**
ꢂ6
6
4
0
22
94
89
154
117
110
144
144
153
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
124
123
103
118
135
ND
ND
ND
ND
ND
103
91
114
133
125
95
112
107
10 (Saikosaponin b1)
25
ꢂ23
ꢂ3
ꢂ269†
ꢂ43
ꢂ18
ꢂ10
8
11 (Saikosaponin h)
Hepatoprotective effects of compounds 24 and 25 toward in vitro immunological
liver injury on primary cultured rat hepatocytes. Significantly different from ref., effec-
tive ∗∗ pꢄ0.01, ∗∗∗ pꢄ0.001. Hepatotoxicity of compounds 24 and 25 in primary
cultured rat hepatocytes.
ꢂ10
22
25
ꢂ4
ꢂ19
5
ꢂ13
ꢂ44
ꢂ17
40
ꢂ21
ꢂ31
ꢂ37
ꢂ44
ꢂ16
ꢂ23
ꢂ58
ꢂ41
ꢂ28
ꢂ10
41
15 (Rotundioside E)
falcatum. They are divided into four groups: the 13,28-epoxy
type, the 12-ene type, the 9(11),12-diene type, and the 28-
acid type, according to their structural characteristics.
Next, some compounds obtained from them were investi-
gated for their hepatoprotective activity and hepatotoxicity.
At the 13,28-epoxy type saponins, 2 (saikosaponin c) had
weak hepatoprotectivity at 500 mM, but 1 (saikosaponin a)
showed hapatotoxity from 30 mM. It was suggested that
methyl group of the 23 position of 1 influenced activity.21)
But compound 3 of which an epoxy ring cleaved at C-13,28
of 1 has hepatoprotective activity at 500 mM. Ursane type (13,
14) showed hepatotoxity from middle concentration. It was
guessed that conformation of 29 and 30 position methyl
group in ursane skelton damaged against cell membranes. On
the other hands, 11,13(18)-diene type saponins did not ex-
press hepatoprotective activity and 4 and 10 showed the toxi-
city from middle concentration among them. The 28-acid
type saponin (24, 25) which has a glucosyl carboxy group
showed hepatoprotective action. This result accorded with
our previous paper.22)
16
17
18
19
20
21
100
97
93
0
14
9
21
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
32
ꢂ43
ꢂ28
ꢂ21
ꢂ12
43
ꢂ38
ꢂ48
ꢂ31
ꢂ3
30
90
200
500
10
30
90
200
500
Experimental
General Procedure Optical rotations were measured with a JASCO P-
1020 (lꢀ0.5) automatic digital polarimeter. FAB-MS were obtained with a
glycerol matrix in the positive ion mode using a JEOL JMS-DX300 and a
JMS-DX 303 HF spectrometer. The H- and 13C-NMR spectra were meas-
1
ured in pyridine-d5 with JOEL a-500 spectrometer, and chemical shifts are
given on a d (ppm) scale with tetramethylsilane (TMS) as the internal stan-
dard. Column chromatographies were carried out on a Diaion HP-20 (Mit-
subishi Chemical Ind.), and silica gel 60 (230—400 mesh, Merck). TLC was
performed on silica gel plates (Kieselgel 60 F254, Merck) and PR C18 silica
gel plates (Merck). The spots on TLC were visualized by UV light
(254/366 nm) and sprayed with 10% H2SO4, followed by heating.
27
Hepatoprotective effects of compounds 4, 10, 11 and 15—21 toward in vitro im-
munological liver injury on primary cultured rat hepatocytes. Significantly different
from ref. Hepatotoxicity of compounds 4, 10, 11 and 15—21 in primary cultured rat
†
hepatocytes. Significantly different from ref., toxic pꢄ0.001.
Extraction and Isolation of Respective Plants The aerial parts (2.6 kg)
of Bupleurum falcatum L. cultivated at Kikka-cho, Kumamoto prefecture,
was cutted and refluxed with MeOH for 10 h. Evaporation of MeOH gave
the extractive (262.2 g), which was partitioned between n-hexane and 80%
MeOH, and the methanolic layer was evaporated to give the residue. It was
then chromatographed on Diaion HP-20 eluted with firstly water and gradi-
ently mixed with MeOH to provide five fractions, fr. 1 (132.0 g), fr. 2
(48.7 g), fr.3 (25.6 g), fr. 4 (14.6 g), and fr. 5 (12.4 g). The fr. 4 eluted by
80% MeOH was subjected Sephadex LH-20 with MeOH to afford flavonoid
and saponin faractions, from the latter of which was chromatographed on
Chromatorex ODS with 60—100% MeOH to give BFA-5 (3, 108 mg), BFA-
6 (4, 264 mg), BFA-8 (1, 45 mg), BFA-11 (21 mg), BFA-13 (110 mg), BFA-
17 (7, 21 mg), BFA-20 (6, 20 mg), BFA-22 (2, 72 mg) . The fr. 5 eluate by
100% MeOH was also subjected to silica gel chromatography with CHCl3–
ing [a]D ꢃ2.7° (MeOH). Positive HR-FAB-MS revealed a
molecular formula of C60H98O28Na at m/z 1289.6140
[MꢃNa]ꢃ. The 13C-NMR spectrum showed 60 carbon sig-
nals consisting of echinocystic acid and the same sugar moi-
eties as in BRA-20 (22). Therefore, the structure of BRA-18
(25) was characterized as 3-O-a-L-rhamnopyranosyl-(1→2)-
b-D-glucopyranosyl echinocystic acid b-D-glucopyranosyl-
(1→2)-[b-D-glucopyranosyl-(1→4)]-b-D-glucopyranoside.
We isolated a total of 25 oleanene saponins from the aerial
parts of B. falcatum and B. rotundifolium and the seeds of B. MeOHꢀ15 : 1—CHCl3–MeOH–waterꢀ9 : 1 : 0.1 to give BFA-25 (5, 17 mg).

1338
The seeds (1.2 kg) of Bupleurum falcatum L. used for cultivating at
Vol. 59, No. 11
(Ref.), 10, 30, 90, 200, 500 mM were 31.50ꢆ0.6, 33.00ꢆ0.5, 30.00ꢆ1.4,
30.50ꢆ1.0, 30.00ꢆ, 25.25ꢆ0.5 (IU/l), respectively. The value of 8 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 33.25ꢆ2.1, 33.00ꢆ2.1, 32.00ꢆ0.8,
31.75ꢆ1.5, 31.25ꢆ2.0, 29.00ꢆ1.2 (IU/l), respectively. The control value 4,
10, 11, 15, 16, 17, 18, 19, 20 and 21 was 9.17ꢆ1.4 (IU/l). The value of 4 at
0 (Ref.), 10, 30, 90, 200, 500 mM were 17.25ꢆ1.4, 23.25ꢆ2.9, 22.00ꢆ1.7,
21.25ꢆ1.8, 19.50ꢆ3.3, 26.00ꢆ1.0 (IU/l), respectively. The value of 10 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 26.25ꢆ2.7, 28.00ꢆ15, 27.50ꢆ1.7,
30.25ꢆ3.3, 26.75ꢆ2.1, 72.25ꢆ3.0 (IU/l), respectively. The value of 11 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 19.00ꢆ2.4, 23.25ꢆ1.3, 20.75ꢆ2.6,
20.00ꢆ3.8, 18.25ꢆ1.5, 20.00ꢆ2.8 (IU/l), respectively. The value of 15 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 33.25ꢆ3.0, 28.00ꢆ6.1, 27.25ꢆ1.9,
34.25ꢆ5.3, 37.75ꢆ4.1, 32.00ꢆ1.8 (IU/l), respectively. The value of 16 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 21.00ꢆ2.2, 22.50ꢆ1.0, 26.25ꢆ4.2,
23.00ꢆ1.4, 16.25ꢆ0.5, 23.50ꢆ1.3 (IU/l), respectively. The value of 17 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 18.75ꢆ2.2, 21.75ꢆ1.7, 22.25ꢆ3.2,
23.00ꢆ1.8, 20.25ꢆ0.9, 21.00ꢆ1.8 (IU/l), respectively. The value of 18 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 19.00ꢆ1.8, 24.75ꢆ4.3, 23.00ꢆ3.4,
21.75ꢆ3.3, 20.00ꢆ1.2, 15.00ꢆ1.8 (IU/l), respectively. The value of 19 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 23.2ꢆ1.0, 23.25ꢆ0.5, 21.25ꢆ2.1,
22.00ꢆ1.2, 20.25ꢆ1.3, 18.75ꢆ3.1 (IU/l), respectively. The value of 20 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 19.75ꢆ3.0, 24.25ꢆ2.5, 22.75ꢆ2.2,
22.00ꢆ1.4, 21.00ꢆ3.0, 15.25ꢆ3.0 (IU/l), respectively. The value of 21 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 16.50ꢆ3.0, 19.25ꢆ1.7, 20.00ꢆ2.7,
18.75ꢆ1.3, 16.75ꢆ1.7, 14.50ꢆ1.7 (IU/l), respectively. The control value 24
and 25 was 17.54ꢆ1.4 (IU/l). The value of 24 at 0 (Ref.), 10, 30, 90, 200,
500 mM were 38.75ꢆ2.5, 35.50ꢆ1.3, 31.75ꢆ1.7, 22.00ꢆ3.2, 27.00ꢆ2.2,
27.25ꢆ5.0 (IU/l), respectively. The value of 25 at 0 (Ref.), 10, 30, 90, 200,
500 mM were 30.25ꢆ1.5, 29.50ꢆ0.6, 29.50ꢆ1.3, 29.75ꢆ1.3, 30.25ꢆ2.2,
27.50ꢆ1.3 (IU/l), respectively. Reference (ref.) value treated with the anti-
serum and not treated with the tested sample. The percent of protection is
calculated as {1ꢂ(substanceꢂcontrol)/(ref.ꢂcontrol)}ꢅ100. The percent of
protection of glycyrrhizin (positive control) was 32% at 500 mM.
Determination of Hepatotoxicity of Saponins toward Hepatocytes
(without Anitiserum) In the same way as above, the cultured cells were
exposed to the above-prepared medium (300 ml) containing the DMSO solu-
tion (4 ml) of the test samples [final concentration 0 (Ref.), 10, 30, 90, 200,
500 mM]. Forty minute after the test samples were administered, the medium
was withdrawn for determination of ALT. The value of 1 at 0 (Ref.), 10, 30,
90, 200, 500 mM were 24.80ꢆ2.1, 23.80ꢆ5.5, 78.50ꢆ5.0, 122.00ꢆ3.7,
112.80ꢆ1.9, 96.75ꢆ3.9 (IU/l), respectively. The value of 13 at 0 (Ref.), 10,
30, 90, 200, 500 mM were 19.25ꢆ5.7, 21.25ꢆ4.8, 116.5ꢆ4.4, 135.80ꢆ13.7,
124.80ꢆ4.8, 122.80ꢆ5.3 (IU/l), respectively. The value of 14 at 0 (Ref.), 10,
30, 90, 200, 500 mM were 20.50ꢆ3.7, 20.75ꢆ2.2, 24.50ꢆ0.6, 103.30ꢆ13.3,
132.00ꢆ7.0, 143.30ꢆ12.0 (IU/l), respectively. The value of 3 at 0 (Ref.), 10,
30, 90, 200, 500 mM were 14.00ꢆ2.0, 15.50ꢆ0.6, 14.75ꢆ2.4, 16.00ꢆ0.9,
14.75ꢆ1.5, 18.00ꢆ1.6 (IU/l), respectively. The value of 8 at 0 (Ref.), 10, 30,
90, 200, 500 mM were 12.50ꢆ2.1, 14.75ꢆ1.0, 17.00ꢆ2.8, 13.00ꢆ1.7,
15.50ꢆ1.3, 17.25ꢆ2.1 (IU/l), respectively. The value of 4 at 0 (Ref.), 10, 30,
90, 200, 500 mM were 17.25ꢆ1.4, 23.25ꢆ2.9, 22.00ꢆ1.7, 21.25ꢆ1.8,
19.50ꢆ3.3, 26.00ꢆ1.0 (IU/l), respectively. The value of 10 at 0 (Ref.), 10,
30, 90, 200, 500 mM were 26.25ꢆ2.7, 28.00ꢆ1.5, 27.50ꢆ1.7, 30.25ꢆ3.3,
26.75ꢆ3.0, 27.25ꢆ1.7 (IU/l), respectively. The value of 16 at 0 (Ref.), 10,
30, 90, 200, 500 mM were 21.00ꢆ2.2, 22.50ꢆ1.0, 26.25ꢆ4.2, 23.00ꢆ1.4,
16.25ꢆ0.5, 23.5ꢆ1.7 (IU/l), respectively. The value of 18 at 0 (Ref.), 10, 30,
90, 200, 500 mM were 19.00ꢆ1.8, 24.75ꢆ4.3, 23.00ꢆ3.4, 21.75ꢆ3.3,
20.00ꢆ1.2, 15.00ꢆ0.8 (IU/l), respectively. The percent of cytotoxicity is
calculated as (sample/reference)ꢅ100. Reference is the value of hepatocytes
which were not treated with the tested samples. The value of 24 at 0 (Ref.),
10, 30, 90, 200, 500 mM were 20.75ꢆ2.5, 20.50ꢆ1.3, 20.25ꢆ1.7,
17.25ꢆ3.2, 18.50ꢆ2.2, 23.75ꢆ5.0 (IU/l), respectively. The value of 25 at 0
(Ref.), 10, 30, 90, 200, 500 mM were 19.00ꢆ1.5, 25.25ꢆ0.6, 23.75ꢆ1.3,
18.00ꢆ1.3, 21.25ꢆ2.2, 20.25ꢆ1.3 (IU/l), respectively. The percent of cyto-
toxicity is calculated as (sample/reference)ꢅ100. Reference is the value of
hepatocytes which were not treated with the tested samples. The percent of
cytotoxicity is calculated as (sample/reference)ꢅ100. Reference is the value
of hepatocytes which were not treated with the tested samples.
Kikka-cho were smashed and extracted with MeOH for 1 h, and usual work-
up gave the extractive (84.7 g), which was subjected to Diaion HP-20 with
water–MeOH, gradiently, finally recovered with acetone) to provide seven
fractions. The fr. 5 (23.2 g) eluted by 80% MeOH was chromatographed on
silica gel with CHCl3–MeOH–waterꢀ9 : 1 : 0.1—7 : 3 : 0.5 to afford fr. 5-1—
fr. 5-8. The fr. 5-1 (3.0 g) was further purified by silicagel with CHCl3–
MeOHꢀ50 : 1 and prepaartive TLC with CHCl3–MeOHꢀ15 : 1 to give
BFS-8 (9, 8 mg). The fr. 5-4 (1.5 g) was chromatographed by HPLC to give
BAS-9 (1, 13 mg), BAS-10 (12, 31 mg), and BAS-11 (10, 34 mg). From fr.
5-5 (14.6 g), fr. 5-6 (1.3 g), and fr. 5-8 (1.4 g), the respective BAS-13 (4,
28 mg), BAS-14 (11, 15 mg), and BAS-1 (2, 30 mg) were obtained by using
HPLC with 80% MeOH. The fr. 6 (9.5 g) eluted by MeOH was chro-
matographed on silica gel with CHCl3–MeOH–waterꢀ8 : 2 : 0.2 to give
BAS-16 (8, 15 mg).
The aerial parts (1.4 kg) of Bupleurum rotundifolium L. cultivated at
Oyano, Kumamoto prefecture, was cutted and refluxed with MeOH for 7 h.
Evaporation of MeOH gave the extractive (152.1 g), which was defatted with
n-hexane. The insoluble residue was passed through Diaion HP-20 eluted
with water–MeOH, gradiently, to give five fractions. A part (2.3 g) of the fr.
4 (6.6 g) eluted by 80% MeOH was various chromatographed on silica gel
with CHCl3–MeOH–waterꢀ8 : 2 : 0.2, Chromatorex ODS with 50—100%
MeOH, gradiently, and HPLC with 60% MeOH to give BRA-11 (19,
36 mg), BRA-14 (24, 69 mg), BRA-16 (20, 12 mg), BRA-17 (21, 11 mg),
BRA-22 (18, 22 mg), BRA-18 (25, 38 mg), BRA-19 (23, 6 mg), and BRA-
20 (22, 5 mg). From a part (6.5 g) of the fr. 5 (22.3 g) eluted by MeOH,
BRA-2 (15, 19 mg), BRA-3 (13, 19 mg), BRA-4 (16, 35 mg), BRA-5 (14,
60 mg), BRA-7 (17, 14 mg) by using silica gel and ODS column chro-
matographies with CHCl3–MeOH–waterꢀ8 : 2 : 0.2—7 : 3 : 0.5, and ODS
with 60—100% MeOH, gradiently.
Identification of Component Monosaccharide of the Glycosides
A
glycoside (5 mg) was dissolved in 1 N HCl–MeOH (0.5 ml) and heated at
90 °C for 1 h. The acidic solution was neutralized with an ion exchanger
resin (Amberlite IR-410) and concentrated in vacuo. The residue was
trimethylsilyalted and checked by gas–liquid chromatography (GLC). Au-
thentic sugar samples were treated in the same manner and tR values were
compared with those of the tetramethylsilyl derivatives of the metahnolysate
of the glycoside.
The absolute configurations of the component monosaccharides were de-
termined according to the method reported by Hara et al. Thus, a glycoside
(5 mg) was hydrolyzed with 1 N HCl. After neutralization with Amberliet IR-
410, the free sugars in the hydrolysate were converted into the thiazolidine
derivatives and checked by GLC after trimethylsilylation. Authentic sugar
samples were treated in the same manner and an unknown sugar was identi-
fied by comparison of its tR value with those of the authentic sugar deriav-
tives.
Animals Male Wistar rats (6 weeks old, body weight 150—160 g) and a
male New Zealand white rabbit (body weight 3 kg) were used.
Preparation of Primary Cultured Rat Hepatocytes Liver cells were
isolated to a procedure developed by Berry and Friend.23)
Preparation of Antiserum against Rat Hepatocytes The antiserum
was prepared according to the method of Shiki et al.24) An antibody to rat
hepatocytes was raised in rabbits, first by injection of 1ꢅ108 cells, followed
by four injections of 5ꢅ107 cells over a period of 4 weeks. The antiserum to
the rat hepatocytes was prepared by the method of Harboe and Ingild.25)
Determination of Hepatoprotective Activity of Compounds toward in
Vitro Immunological Liver Injury One day after the isolated rat hepato-
cytes were plated, the cultured cells were exposed to the above medium
(300 ml) containing the antiserum against rat plasma membranes (80 ml/ml)
and a dimethyl sulfoxide (DMSO) solution (4 ml) of the test samples [final
concentration 0 (reference; Ref.), 10, 30, 90, 200, 500 mM]. Forty min after
the antiserum was administered, the medium was withdrawn for determina-
tion of alanine aminotransferase (ALT). Control is the value of hepatocytes
which were not administered the antiserum. The control was hepatocytes not
treated with antiserum. The control value 1, 2, 13 and 14 was 6.48ꢆ1.4
(IU/l). The value of 1 at 0 (Ref.), 10, 30, 90, 200, 500 mM were 27.50ꢆ4.1,
24.75ꢆ2.9, 29.25ꢆ3.0, 34.50ꢆ2.2, 33.75ꢆ1.0, 33.25ꢆ1.7 (IU/l), respec-
tively. The value of 2 at 0 (ref.), 10, 30, 90, 200, 500 mM were 27.50ꢆ0.6,
24.75ꢆ1.7, 27.00ꢆ0.8, 24.75ꢆ1.0, 23.25ꢆ1.0, 18.75ꢆ0.5 (IU/l), respec-
tively. The value of 13 at 0 (Ref.), 10, 30, 90, 200, 500 mM were 26.25ꢆ4.9,
26.50ꢆ, 36.50ꢆ10.2, 36.25ꢆ3.1, 39.00ꢆ5.1, 41.00ꢆ1.5 (IU/l), respec-
tively. The value of 14 at 0 (Ref.), 10, 30, 90, 200, 500 mM were 26.00ꢆ4.9,
25.00ꢆ4.5, 26.00ꢆ1.0, 39.00ꢆ3.1, 41.00ꢆ5.1, 44.25ꢆ1.5 (IU/l), respec-
tively. The control value 3 and 8 was 17.54ꢆ1.4 (IU/l). The value of 3 at 0
Instrument and Assay Method The activities of ALT were assayed by
autoanalyzer COBAS MIRA (Roche) using commercial kits based on the
ALT assay method.26)
Statistical Analysis The data are shown as the meanꢆS.D. (nꢀ4). After
analysis of variances, Sheffe’s test was employed to determine the signifi-
cance of differences between reference and experimental samples.

November 2011
1339
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