New triterpenoids from Gentiana lutea.

Article abstract of DOI:10.1248/cpb.51.89

Three new triterpenoids, 2,3-seco-3-oxours-12-en-2-oic acid, 2,3-seco-3-oxoolean-12-en-2-oic acid, and betulin 3-O-palmitate, have been isolated from the rhizomes and roots of Gentiana lutea, together with five known ones. The structures of the new compou

Full text of DOI:10.1248/cpb.51.89

January 2003
Notes
Chem. Pharm. Bull. 51(1) 89—91 (2003)
89
New Triterpenoids from Gentiana lutea
Yoshitaka TORIUMI, Rie KAKUDA, Masafumi KIKUCHI, Yasunori YAOITA, and Masao KIKUCHI*
Tohoku Pharmaceutical University, 4–4–1 Komatsushima, Aoba-ku, Sendai, Miyagi 981–8558, Japan.
Received August 30, 2002; accepted October 8, 2002
Three new triterpenoids, 2,3-seco-3-oxours-12-en-2-oic acid, 2,3-seco-3-oxoolean-12-en-2-oic acid, and be-
tulin 3-O-palmitate, have been isolated from the rhizomes and roots of Gentiana lutea, together with five known
ones. The structures of the new compounds were determined by spectral and chemical methods.
Key words Gentiana lutea; Gentianaceae; triterpenoid
203, and 189, diagnostic of triterpenoids having a D12-double
bond.8) The 1H-NMR spectrum (Table 1) showed signals due
to six tertiary methyl groups [dH 0.80 (3H, H3-28), 1.01 (3H,
H3-25), 1.03 (3H, H3-26), 1.05 (3H, H3-27), 1.11 (3H, H3-
24), 1.17 (3H, H3-23)], two secondary methyl groups [dH
0.77 (3H, H3-29), 0.92 (3H, H3-30)], a methylene group [dH
2.30 (1H, H-1a), 2.51 (1H, H-1b)], a trisubstituted olefinic
proton [dH 5.16 (1H, H-12)], and an aldehyde proton [dH
9.78 (1H, H-3)]. The 13C-NMR spectrum (Table 2) revealed
30 carbon signals that included two olefinic carbons [dC
124.2 (C-12), 139.4 (C-13)], a carboxyl carbon [dC 176.3 (C-
The rhizomes and roots of Gentiana lutea L. (Gen-
tianaceae) are the crude drug Gentianae Radix, used as an
appetite stimulant.1) The constituents of this crude drug have
been previously investigated and shown to contain secoiri-
doid glucosides2) and xanthones.3) In this paper, we describe
the isolation and structural elucidation of three new triter-
penoids, 2,3-seco-3-oxours-12-en-2-oic acid (1), 2,3-seco-3-
oxoolean-12-en-2-oic acid (2) and betulin 3-O-palmitate (3),
together with five known ones from the rhizomes and roots
of G. lutea. The known compounds were identified as a-
amyrin (4),4) b-amyrin (5),4) lupeol (6),5) uvaol 3-O-palmitate
(7)6) and erythrodiol 3-O-palmitate (8),7) respectively, by
comparison of their spectroscopic data with those previously
described in the literature. To the best of our knowledge, this
is the first report of the latter of these compounds from G.
lutea. Extraction and isolation were carried out as described
in the Experimental section.
1
2)], and an aldehyde carbon [dC 207.9 (C-3)]. The H- and
13C-NMR data for 1 were very similar to those of a-amyrin
1
(4), except for the signals ascribed to ring A. The H-de-
tected heteronuclear multiple bond connectivity (HMBC)
correlations of H2-1 to a carboxyl carbon (C-2) revealed that
1
the carboxyl group was attached to C-2. The H–13C long-
Compound 1 was isolated as an amorphous powder, [a]D
ꢀ68.1°. The IR spectrum suggested the presence of a car-
bonyl group (1713 cmꢁ1). The molecular formula was deter-
mined to be C30H48O3 by high-resolution (HR)-MS and elec-
tron impact (EI)-MS gave fragment ion peaks at m/z 218,
range correlations of H3-23 and H3-24 methyl groups to an
aldehyde carbon (C-3) revealed that the aldehyde group was
attached to C-3. In the nuclear Overhauser effect (NOE) dif-
ference spectra, NOEs were detected between H-5 and H3-23
and between H3-24 and H3-25. These NOEs implied that H-5
Chart 1
∗ To whom correspondence should be addressed. e-mail: mkikuchi@tohoku-pharm.ac.jp
© 2003 Pharmaceutical Society of Japan

90
Vol. 51, No. 1
Table 1. 1H-NMR Chemical Shifts of Compounds 1, 2, and 4 (600 MHz, instead of two secondary methyl groups in 1. The HMBC
CDCl3)
correlations of H3-29 and H3-30 to C-19, C-20 and C-21 re-
vealed that the H3-29 and H3-30 methyl groups were attached
to C-20. Thus 2 was deduced to be 2,3-seco-3-oxoolean-12-
en-2-oic acid.
Hydrogen
1
2
4
1a
1b
3
2.30 d (16.5)
2.51 d (16.5)
9.78 s
2.28 d (16.5)
2.49 d (16.5)
9.76 s
Compound 3 was isolated as an amorphous powder, [a]D
ꢀ34.9°. The molecular formula was determined to be
C46H80O3 by HR-MS. The 1H-NMR spectral data of 3 closely
resembled those of betulin (9), except for the presence of a
saturated long-chain fatty acid ester group [dH 0.88 (3H, H3-
16ꢂ), 1.25 (CH2), 2.28 (2H, H2-2ꢂ)]. The alkaline hydrolysis
of 3 in methanolic KOH yielded 9 and methyl palmitate. The
1H-NMR chemical shift at H-3 of 3 was shifted downfield by
ꢀ1.28 ppm compared with that of 9, indicating that the
palmitoyl group is located at the C-3 hydroxyl group. Thus 3
was determined to be betulin 3-O-palmitate.
3.23 dd (9.9, 5.1)
12
23
24
25
26
27
28
29
30
5.16 dd (4.4, 2.9) 5.20 dd (3.7, 3.7) 5.13 dd (3.6, 3.6)
1.17 s
1.11 s
1.01 s
1.03 s
1.05 s
0.80 s
1.16 s
1.10 s
1.00 s
0.99 s
1.12 s
0.83 s
1.00 s
0.79 s
0.96 s
1.01 s
1.07 s
0.80 s
0.77 d (5.9)
0.92 d (6.2)
0.869 sa)
0.872 sa)
0.79 d (5.6)
0.92 d (5.9)
Coupling constants (J in Hz) are given in parentheses. a) Assignments may be in-
terchangeable.
Although most of the naturally occurring seco-triter-
penoids are 3,4-seco-compounds,9) there are some 2,3-seco-
derivatives [e.g., 2,3-seco-olean-12-ene-2,3,28-trioic acid from
Bursera graveolens (Burseraceae),10) 19a-hydroxy-2,3-seco-
urs-12-ene-2,3,28-trioic acid,11) 15a,19a-dihydroxy-2,3-seco-
urs-12-ene-2,3,28-trioic acid and 19a,22a-dihydroxy-2,3-
seco-urs-12-ene-2,3,28-trioic acid from Musanga cecropi-
oides (Cecropiaceae),12) and 2,3-seco-2-oxoolean-12-en-3-
methylester-30-oic acid from Dillenia papuana (Dilleni-
aceae)13)]. Compounds 1 and 2 are, to the best of our knowl-
edge, the first 2,3-seco-triterpenoids from the genus Gen-
tiana and the first 2,3-seco-triterpenoids with a C-2 carboxyl
group and C-3 aldehyde group isolated from natural sources.
Table 2. 13C-NMR Chemical Shifts of Compounds 1, 2, and 4 (150 MHz,
CDCl3)
Carbon
1
2
4
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
42.8
176.3
207.9
50.7
47.8
20.4
32.1
39.9
40.5
42.3
23.8
124.2
139.4
43.2
26.5
28.1
33.8
59.1
39.8
39.5
31.3
41.5
24.0
19.4a)
19.5a)
17.0
22.8
28.8
17.5
21.4
42.6
174.4
207.9
50.7
47.6
20.4
31.8
39.8
40.4
42.3
23.8
121.4
144.9
42.3
26.1
26.9
32.5
47.2
46.6
31.1
34.7
37.1
24.0
19.3b)
19.4b)
17.0
25.5
28.4
33.3
23.6
38.7c)
27.2
78.3
38.7c)
55.2
18.3
32.9
40.0
47.7
36.9
23.3
124.3
139.3
42.0
26.6
28.1
33.7
58.9
39.6d)
39.6d)
31.2
41.5
28.1
15.6e)
15.6e)
16.8
23.3
28.7
17.4
21.3
Experimental
General Procedures Optical rotations were determined using a JASCO
DIP-360 digital polarimeter. IR spectra were recorded with a Perkin-Elmer
FT-IR 1725X IR spectrophotometer. 1H- and 13C-NMR spectra were
recorded using a JEOL JNM-LA 600 (600 and 150 MHz, respectively) spec-
trometer. Chemical shifts are given on a d (ppm) scale, with tetramethylsi-
lane as an internal standard. EI- and HR-MS were recorded on a JEOL JMS-
DX 303 mass spectrometer. Column chromatography was carried out on
Kieselgel 60 (Merck; 230—400 mesh). Preparative HPLC was carried out
on a Tosoh HPLC system (pump, CCPS; detector, RI-8020) using a TSK gel
ODS-120T (7.8 mm i.d.ꢃ30 cm) column (Tosoh). HPLC conditions: mobile
phase, MeOH; flow rate, 1.0 ml/min; column temperature, 40 °C. GC was
carried out on a Shimadzu GC-7A gas chromatograph.
Plant Material The dried and powdered rhizomes and roots of G. lutea
(from France) were purchased from Uchida Wakanyaku Co., Ltd., Tokyo,
Japan, in 2001.
Extraction and Isolation The dried and powdered rhizomes and roots
of G. lutea (1.5 kg) were extracted with MeOH at room temperature. The
MeOH extract was concentrated under reduced pressure and the residue
(160.0 g) was suspended in a small amount of water. This suspension was
extracted with CHCl3. The CHCl3-soluble fraction was concentrated under
reduced pressure to affored a residue (67.0 g). Part of this residue (44.0 g)
was chromatographed on a silica gel column using CHCl3–MeOH–H2O
(30 : 10 : 1), and the eluate was separated into 35 fractions (frs. 1—35). Frac-
tion 3 was purified by preparative HPLC to give 3 (1.1 mg), 7 (1.8 mg), and
8 (3.0 mg). Fraction 4 was purified by preparative HPLC to give 4 (4.1 mg),
5 (5.0 mg), and 6 (2.0 mg). Fraction 5 was purified by preparative HPLC to
give 1 (7.9 mg) and 2 (3.6 mg).
a, b) Assignments may be interchangeble. c—e) Signals were overlapped.
and H3-23 methyl group occurred on the same face (a) of the
molecule and H3-24 and H3-25 methyl groups occurred on
the same face (b) of the molecule. Therefore 1 was deter-
mined to be 2,3-seco-3-oxours-12-en-2-oic acid.
2,3-seco-3-Oxours-12-en-2-oic Acid (1): Amorphous powder. [a]D23 ꢀ68.1°
(cꢄ0.8, CHCl3). IR nmax CHCl3 cmꢁ1: 3030, 2927, 2858, 1713, 1457, 1380.
HR-MS m/z: 456.3600 (Mꢀ, Calcd for C30H48O3: 456.3604). EI-MS m/z (rel.
int): 456 (Mꢀ, 7), 218 (100), 203 (27), 189 (21). 1H-NMR (600 MHz,
CDCl3): see Table 1. 13C-NMR (150 MHz, CDCl3): see Table 2.
Compound 2 was isolated as an amorphous powder, [a]D
ꢀ52.7°. The IR spectrum suggested the presence of a car-
bonyl group (1724 cmꢁ1). The molecular formula was deter-
1
mined to be C30H48O3 by HR-MS. The H- and 13C-NMR
2,3-seco-3-Oxoolean-12-en-2-oic Acid (2): Amorphous powder. [a]D23
ꢀ52.7° (cꢄ0.4, CHCl3). IR nmax CHCl3 cmꢁ1: 3029, 2930, 2857, 1724,
1466, 1381. HR-MS m/z: 456.3625 (Mꢀ, Calcd for C30H48O3: 456.3604). EI-
spectral data of 2 closely resembled those of 1, except for the
appearance of two tertiary methyl groups [dH 0.869 and
0.872 (each 3H, H3-29, H3-30); dC 23.6 (C-30), 33.3 (C-29)] MS m/z (rel. int): 456 (Mꢀ, 7), 218 (100), 203 (39), 189 (12). 1H-NMR

January 2003
91
(600 MHz, CDCl3): see Table 1. 13C-NMR (150 MHz, CDCl3): see Table 2.
Betulin 3-O-Palmitate (3): Amorphous powder. [a]D23 ꢀ34.9° (cꢄ0.1,
CHCl3). HR-MS m/z: 680.6125 (Mꢀ, Calcd for C46H80O3: 680.6107). EI-MS
m/z (rel. int): 680 (Mꢀ, 5), 662 (MꢀꢁH2O, 20), 647 (MꢀꢁH2OꢁCH3, 16),
424 (MꢀꢁC16H32O2, 8), 234 (10), 218 (18), 203 (27), 189 (21). 1H-NMR
(600 MHz, CDCl3) d: 0.836 (3H, s, H3-24), 0.841 (3H, s, H3-23), 0.85 (3H,
s, H3-25), 0.88 (3H, t, Jꢄ7.3 Hz, H3-16ꢂ), 0.98 (3H, s, H3-27), 1.02 (3H, s,
H3-26), 1.25 (CH2), 1.69 (3H, br s, H3-30), 2.28 (2H, t, Jꢄ7.3 Hz, H2-2ꢂ),
3.34 (1H, d, Jꢄ11.4 Hz, H-28a), 3.80 (1H, d, Jꢄ11.4 Hz, H-28b), 4.47 (1H,
dd, Jꢄ11.0, 5.1 Hz, H-3), 4.58 (1H, d, Jꢄ1.5 Hz, H-29a), 4.68 (1H, d,
Jꢄ2.2 Hz, H-29b).
Hydrolysis of 3 Compound 3 was refluxed with 5% methanolic KOH
for 3 h. The reaction mixture was extracted with CHCl3, and the CHCl3 layer
was concentrated under reduced pressure to yield betulin and methyl palmi-
tate. Each compound was identified by HPLC comparison with authentic
samples. HPLC conditions: column, TSK gel ODS-120T (7.8 mm i.d.ꢃ
30 cm, Tosoh); mobile phase, MeOH; flow rate, 1.0 ml/min; column temper-
ature, 40 °C. Betulin, tR 14.0 min. Methyl palmitate, tR 21.0 min. Methyl
palmitate was also identified by GC comparison with authentic sample. GC
conditions: column, 3% SE-52 on Chromosorb W (AW) (60—80 mesh),
3 mm i.d.ꢃ2 m; carrier gas, N2; flow rate, 1.0 kg/cm2; detector, FID; column
temperature, 190 °C. Methyl palmitate, tR 6.2 min.
this university for measurement of the mass and NMR spectra.
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Acknowledgments We are grateful to Mr. S. Sato and Mr. T. Matsuki of