Tareciliosides H-M: Further cycloartane glycosides from leaves of Tarenna gracilipes

Article abstract of DOI:10.1248/cpb.59.902

From the 1-BuOH-soluble fraction of a MeOH extract of leaves of Tarenna gracilipes, collected in Okinawa, six further new cycloartane glycosides, named tareciliosides H-M (1-6), were isolated. Their structures were established through a combination of spectroscopic analyses.

Full text of DOI:10.1248/cpb.59.902

902
Note
Chem. Pharm. Bull. 59(7) 902—905 (2011)
Vol. 59, No. 7
Tareciliosides H—M: Further Cycloartane Glycosides from Leaves of
Tarenna gracilipes
a
a
Zhimin ZHAO, Katsuyoshi MATSUNAMI, Hideaki OTSUKA, ,a Takakazu SHINZATO,b and
*
c
Yoshio TAKEDA
a Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-
ku, Hiroshima 734–8553, Japan: b Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1
Senbaru, Nakagami-gun, Okinawa 903–0213, Japan: and c Faculty of Pharmacy, Yasuda Women’s University; 6–13–1
Yasuhigashi, Asaminami-ku, Hiroshima 731–0153, Japan.
Received March 29, 2011; accepted May 4, 2011; published online May 9, 2011
From the 1-BuOH-soluble fraction of a MeOH extract of leaves of Tarenna gracilipes, collected in Okinawa,
six further new cycloartane glycosides, named tareciliosides H—M (1—6), were isolated. Their structures were
established through a combination of spectroscopic analyses.
Key words Tarenna gracilipes; Rubiaceae; tarecilioside; cycloartane glycoside
In a preceding paper,1) we reported the isolation and char- essentially the same as those of tarecilioside A.1) The
acterization of seven cycloartane glycosides, tareciliosides anomeric proton of the glucose unit (dH 4.92, d, Jꢁ8 Hz),
A—G, from the 1-BuOH-soluble fraction of a MeOH extract which showed a long-range correlation with C-3 (dC 88.3) of
of the leaves of Tarenna gracilipes. Further detailed investi- the aglycone moiety, confirmed the position of the glucose
gation of the 1-BuOH-soluble fraction has resulted in the linkage. The anomeric proton of the rhamnose unit (dH 6.50,
isolation of six additional cycloartane glycosides, named tare- s) also exhibited a long-range correlation with C-2ꢂ (dC 77.9)
ciliosides H—M (1—6). In this paper, we report the isola- of the glucose unit, which confirmed the attachment of a
tion and structural elucidation of the six new cycloartane rhamnose unit to the hydroxy group at C-2ꢂ of glucose. The
glycosides.
configuration of the C-24 hydroxy group was indicated to be
24R on comparison with the chemical shift values of cy-
clounifolioside C (24R, C-24: dC 80.3),2) cyclocantogenin
Results and Discussion
The 1-BuOH-soluble fraction of MeOH extract of the (24S, C-24: dC 70.0),2) oleifoliosides A and B (24S, C-24:
leaves of T. gracilipes1) was subjected to a column of Diaion 77.1),3) and tarecilioside A (24R, C-24: dC 80.6).1) Acid hy-
HP-20, using MeOH–H2O mixtures, to give seven fractions. drolysis of 1 gave D-glucose and L-rhamnose. Additionally,
Fractions 14—19 were further subjected to silica gel column the coupling constant (dH 4.92, d, Jꢁ8 Hz) for one (H-1ꢂ) of
chromatography (CC), using a CHCl3–MeOH–H2O solvent the anomeric protons for D-glucopyranose indicated that the
system with increasing polarity. The fractions eluted from mode of linkage was b and that for another anomeric proton
these columns were combined and subjected to octadecyl- (H-1ꢃ) for L-rhamnopyranose at dH 6.50 (s) was indicated to
silanized silica gel (ODS) CC, droplet counter-current chro- be a. Therefore, the structure of tarecilioside H (1) was elu-
matography (DCCC), and HPLC, successively, to afford tare- cidated to be 2ꢂ-O-a-L-rhamnopyranosyl tarecilioside A, as
ciliosides H—M (1—6).
shown in Fig. 1.
Tarecilioside H (1), [a]D25 ꢀ15.5, was isolated as an amor-
Tarecilioside I (2), [a]D25 ꢀ10.3, was isolated as an amor-
phous powder and its molecular formula was determined to phous powder and its molecular formula was determined to
be C42H72O14 on positive-ion high-resolution (HR)-electro- be C47H80O18 on positive-ion HR-ESI-TOF-MS. All of the
spray ionization (ESI)-time-of-flight (TOF)-MS. The strong assignments of the proton and carbon signals for tarecilioside
absorption band at 3408 cmꢀ1 in the IR spectrum indicated I, confirmed by the correlation spectroscopy (COSY),
the presence of sugar moiety. The 1H-NMR spectrum of tare- heteronuclear single quantum coherence (HSQC), and
cilioside H (1) (Table 1) showed two highly shielded signals, heteronulear multiple bond correlation (HMBC) spectra, and
characteristic of methylene protons of a cyclopropane ring as comparison of these data with those for tarecilioside H (1)
an AX system (dH 0.21, 0.77, each d, Jꢁ4 Hz, H2-19), and indicated similarity to 1. A difference between 1 and 2 was
signals due to six singlet methyls (dH 1.12, 1.23, 1.28, 1.46, that the 13C-NMR spectrum of 2 contained five more signals
1.48, 1.50) and two secondary methyl [dH 1.13, (d, Jꢁ7 Hz) than that of 1, which was in good accordance with the pres-
and 1.68 (d, Jꢁ6 Hz)] groups. Additionally, the resonances ence of one xylopyranose unit. In the HMBC spectrum of 2,
for two anomeric protons were observed at dH 4.92 (d, the long range correlation between the anomeric proton of
Jꢁ8 Hz) and 6.50 (s). Thus, tarecilioside H (1) was consid- the xylopyranose unit (dH 5.04, d, Jꢁ8 Hz) and C-25 (dC
ered to be a cycloartane-type triterpene diglycoside analo- 80.9) of the aglycone moiety indicated the position of the
gous to tarecilioside A.1) This assumption was supported by xylopyranose linkage. On acid hydrolysis of 2, D-glucose,
the 13C-NMR spectral data for 1 (Table 2). The 13C-NMR L-rhamnose and D-xylose were obtained. The glucopyranose
spectrum of 1 exhibited the signals of 42 carbons, of which linkage was located at the hydroxy group at C-3 in the b-
30 accounted for an aglycone moiety. The remaining signals mode, since HMBC long-range correlation of the glucose
were in good accordance with the presence of two sugar anomeric proton (dH 4.91, d, Jꢁ8 Hz) with C-3 (dC 88.3)
units. The resonances assigned to the aglycone moiety were was observed. On the other hand, the anomeric proton of the
∗ To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp
© 2011 Pharmaceutical Society of Japan

July 2011
903
Table 1. 1H-NMR Spectral Data for Tareciliosides H—M (1—6) (400 MHz, Pyridine-d5)a)
H
1
2
3
4b)
5
6
1
1.20 m
1.40 m
1.86 m
2.38 m
1.32 m
1.54 m
1.85 m
2.41 m
1.21 m
1.50 m
1.83 m
2.40 m
1.16 m
1.45 m
1.90 m
2.35 m
1.10 m
1.45 m
1.87 m
2.38 m
1.18 m
1.45 m
1.92 m
2.36 m
2
3
5
6
3.47 dd, 11, 4
1.55 dd 10, 2
1.20 m
3.52 dd 12, 4
1.55 m
1.20 m
3.46 dd 12, 4
1.48 m
1.20 m
3.43 dd, 11, 4
1.53 m
1.26 m
3.40 dd 12, 4
1.26 m
0.72 m
3.44 dd 12, 4
1.51 m
1.12 m
2.02 m
1.97 m
1.95 m
2.02 m
1.53 m
1.90 m
7
3.76 d-like 10
3.78 ddd 10, 10, 4
3.75 m
3.77 ddd 9, 9, 4
1.04 m
3.72 ddd 10, 10, 4
1.27 m
8
11
2.04 d 10
1.39 m
2.04 d 10
1.34 m
2.01 d 10
1.23 m
2.01 d 9
1.22 m
1.63 m
1.05 m
1.80 d 10
1.44 m
1.89 m
1.84 m
1.84 m
1.85 m
1.92 m
1.98 m
12
15
1.64 2H m
1.64 2H m
1.62 2H m
1.60 m
1.68 m
1.60 2H m
1.73 2H t 7
2.31 dd, 13, 3
2.72 dd 13, 8
4.74 m
1.81 d-like 10
1.46 s
0.21 d 4
0.77 d 4
2.35 m
1.13 d 7
2.42 m
2.30 dd 14, 4
2.71 dd 14, 8
4.71 m
1.79 dd 11, 8
1.48 s
0.21 d 4
0.77 d 4
2.34 m
1.11 d 6
2.42 m
2.28 dd, 14, 4
2.70 dd 14, 8
4.73 m
1.74 dd 11, 8
1.41 s
0.19 d 4
0.75 d 4
2.18 m
1.02 d 6
1.83 m
2.40 m
3.15 ddd 18, 8, 8
3.28 m
2.31 dd 14, 4
2.74 dd, 14, 8
4.74 m
1.75 dd, 11, 7
1.44 s
0.21 d 4
0.75 d 4
2.20 m
1.05 d 7
1.53 m
2.40 m
3.09 ddd 17, 10, 6
3.16 ddd 18, 8, 8
1.67 m
2.06 dd 13, 8
4.65 m
1.72 dd 11, 7
1.34 s
0.20 d 4
0.52 d 4
2.18 m
1.00 d 6
1.87 m
2.66 d 19
2.72 d 19
16
17
18
19
2.35 d 7
1.21 s
0.28 d 4
0.71 d 4
1.96 m
1.05 d 6
1.90 m
2.52 m
3.04 m
3.11 m
20
21
22
2.51 m
1.81 m
2.10 m
2.49 m
1.62 m
1.98 m
2.38 m
3.12 ddd 18, 8, 8
3.27 ddd 18, 8, 8
23
24
26
3.78 d-like 10
1.50 s
3.86 dd 10, 2
1.49 s
1.54 s
1.50 s
3.95 d 10
4.20 m
1.55 s
27
28
29
30
1.48 s
1.28 s
1.23 s
1.12 s
1.49 s
1.28 s
1.23 s
1.12 s
4.91 d 8
4.26 m
4.24 m
4.12 m
3.91 m
4.33 dd 12, 6
4.51 dd 12, 3
6.50 s
4.81 m
4.62 m
4.26 dd 9, 9
4.77 m
1.68 d 6
1.56 s
1.27 s
1.22 s
1.52 s
1.35 s
1.19 s
1.11 s
4.93 d 8
4.26 m
4.26 m
4.27 m
4.01 m
4.43 dd 11, 4
4.72 dd 11, 2
5.30 d 8
4.06 m
4.15 m
4.16 m
1.49 s
1.32 s
0.16 s
0.88 s
4.89 d 7
4.23 m
4.23 m
4.25 m
3.96 m
4.42 dd 11, 4
4.72 dd 11, 2
5.29 d 8
4.03 m
4.19 m
4.10 m
3.80 m
4.26 m
4.47 dd 11, 4
5.06 d 8
4.00 m
1.57 s
1.34 s
1.20 s
1.20 s
4.93 d 8
4.26 m
4.26 m
4.23 m
4.02 m
4.43 dd 11, 5
4.73 dd 11, 3
5.32 d 8
4.04 m
4.20 m
4.13 m
3.88 m
4.34 m
4.49 dd 11, 3
5.09 d 8
4.03 m
1.09 s
1ꢂ
4.92 d 8
4.27 m
4.25 m
4.10 m
3.90 m
4.33 dd 12, 5
4.50 dd 12, 2
6.50 s
4.80 m
4.62 m
4.30 m
4.76 m
1.68 d 6
4.90b)
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
4.26 m
4.26 m
4.10 m
3.89 m
4.32 dd 12, 5
4.50 dd 12, 2
6.50 s
4.81 m
4.62 dd 7, 3
4.15 m
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
4.79 m
1.68 d 6
3.87 m
4.33 dd 12, 5
4.49 dd 12, 2
5.08 d 8
4.03 m
4.30 m
4.21 m
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
5.04 d 8
3.93 dd 8, 8
4.24 m
4.09 m
4.21 m
4.95 d 8
4.04 m
4.20 m
4.17 m
3.79 m
4.27 m
4.16 m
3.90 m
4.25 m
4.25 m
3.94 m
3.94 m
3.68 dd 10, 10
6ꢄ
4.27 m
4.40 dd 12, 3
4.33 dd 12, 5
4.40 dd 12, 3
4.31 dd 11, 5
4.47 dd 11, 3
4.43 m
4.49 dd 11, 3
a) Assigments were performed by means of COSY, HSQC, HMQC and HMBC experiments. b) In the DHO envelope. m: multiplet or overlapped signal.
rhamnose unit (dH 6.50, s) exhibited a long-range correlation phous powder and its molecular formula was determined to
with C-2ꢂ (dC 77.9) of the glucosee unit, which confirmed be C48H80O19 on positive-ion HR-ESI-TOF-MS. Tarecilioside
the attachment of the rhamnose unit to the hydroxy group at J (3) was an cycloartane triglycoside analogous to tarecilio-
C-2ꢂ of glucose. Therefore, the structure of tarecilioside I (2) side I (2) and tarecilioside F.1) In the downfield region of the
was elucidated to be 25-O-b-D-xylopyranosyl tarecilioside H, 13C-NMR spectrum of 3, a highly deshielded signal at dC
as shown in Fig. 1.
215.4 was expected to represent an isolated ketone functional
Tarecilioside J (3), [a]D25 ꢀ18.8, was isolated as an amor- group, and its location was determined to be C-24, judging

904
Vol. 59, No. 7
Table 2. 13C-NMR Spectral Data for Tareciliosides H—M (1—6)
(100 MHz, Pyridine-d5)
C
1
2
3
4a)
5
6
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
32.0
30.0
88.3
41.0
46.5
32.0
70.3
55.1
20.1
26.9
26.8
33.2
46.1
47.1
50.7
72.2
56.8
18.7
28.5
31.6
18.9
35.0
29.4
80.6
72.7
25.7
26.1
25.9
15.5
20.0
105.2
77.9
79.9
72.0
78.1
62.9
101.7
72.4
72.5
74.1
69.9
18.7
32.0
29.9
88.3
41.0
46.5
32.0
70.3
55.1
20.1
26.9
26.8
33.2
46.1
47.1
50.6
72.2
56.8
18.7
28.5
31.6
18.9
35.0
29.3
79.1
80.9
21.1
24.2
25.7
15.5
20.0
105.2
77.9
79.9
72.0
78.1
63.0
101.7
72.4
72.5
74.1
69.6
18.7
99.4
75.2
78.6
71.1
67.0
30.9
29.9
88.3
40.9
46.5
32.0
70.3
55.1
20.1
26.9
26.8
33.1
46.2
47.0
50.4
72.2
56.9
18.4
28.5
30.5
18.7
30.0
34.4
215.4
82.9
23.7
24.7
25.7
15.5
20.0
105.2
77.9
79.9
72.1
78.1
62.9
101.7
72.4
72.5
74.1
69.6
18.7
99.6
75.3
78.7
71.7
78.2
62.8
31.9
29.8
88.8
41.0
46.5
32.0
70.4
55.2
20.2
27.0
26.8
33.2
46.2
47.1
50.5
72.1
56.8
18.8
28.6
30.5
18.3
30.5
34.1
217.9
76.9
27.2
27.3
26.0
15.4
20.1
104.8
82.9
78.35
71.8
77.7
70.2
105.6
76.8
78.3
71.8
77.9
62.8
105.3
75.3
78.3
71.6
78.3
62.9
32.2
30.4
89.0
41.4
47.7
21.1
26.3
48.1
19.9
26.4
26.5
33.3
45.7
47.2
48.5
71.7
57.4
19.3
29.9
30.4
18.1
30.0
35.7
217.7
80.0
69.2
22.5
26.0
15.5
20.3
104.9
83.0
78.4
71.8
77.1
70.1
105.6
76.7
78.3
71.7
77.9
62.8
105.3
75.3
78.3
71.6
78.0
62.9
31.9
29.7
88.7
41.1
46.7
31.92
70.3
54.7
19.3
27.26
26.2
31.6
45.9
41.9
54.1
219.9
61.2
19.13
29.8
31.7
19.4
30.0
34.5
216.3
76.8
27.26
27.27
25.9
15.5
19.15
104.8
82.9
78.4
71.8
77.1
70.2
105.6
76.5
78.0
71.8
77.97
62.8
105.3
75.3
78.3
71.6
78.0
62.9
Fig. 1. Structures of Isolated Compounds
structure of tarecilioside J (3) was elucidated to be 2ꢂ-O-a-L-
rhamnopyranosyl tarecilioside F, as shown in Fig. 1.
Tarecilioside K (4), [a]D25 ꢀ5.96, was isolated as an amor-
phous powder and its molecular formula was determined to
be C48H80O20 on positive-ion HR-ESI-TOF-MS. Analysis and
comparison of the HMBC and 1H–1H COSY spectra, tarecili-
oside K (4) was also found to be a cycloartane triglycoside
analogous to tarecilioside J (3), except for the sugar varieties
and the positions of attached sugar units. On acid hydrolysis,
only D-glucose was detected as a sugar component. However,
one of the glucoses was not at the hydroxy group at C-25, but
at the 2ꢂ-position of the other glucose moiety, which was at-
tached at C-3 of the aglycone. The third glucose moiety was
attached at C-6ꢂ of the glucose, which attached at the C-3.
All of these attachments were confirmed by the HMBC ex-
periment, since long-range correlations of glucose anomeric
protons H-1ꢂ (dH 4.93) with C-3 (dC 88.8), H-1ꢃ (dH 5.30)
with C-2ꢂ (dC 82.9), and H-1ꢄ (dH 5.08) with C-6 (dC 70.2)
were observed. The glucosylation shift trend also supported
the above observations when the 13C-NMR spectra of tare-
cilioside K (4) and tarecilioside F were compared, the C-2ꢂ
carbon signal being shifted downfield by 7.1 ppm, the C-1ꢂ
anomeric carbon being shifted upfield by 2 ppm and on the
other hand, the C-6ꢂ carbon signal being shifted downfield by
7.1 ppm. Therefore, the structure of tarecilioside K (4) was
elucidated to be 6ꢂ-O-b-D-glucopyranosyl tarecilioside G.
Tarecilioside L (5), [a]D25 ꢅ1.19, was isolated as an amor-
phous powder and its molecular formula was determined to
be C48H80O20 on positive-ion HR-ESI-TOF-MS. The signals
observed in the1H- and 13C-NMR spectra of tarecilioside L
(5) were very similar to those of tarecilioside K (4), except
for the position of one hydroxy functional group. On analysis
of the 13C-NMR spectrum of 5, the methine signal at dC 70.4,
which was observed in the 13C-NMR spectrum of 4, was dis-
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
a) Data for 150 MHz.
from the result of analysis of the HMBC and H–1H COSY placed by a methylene group at dC 26.3 and one oxymethyl-
1
1
spectra of 3 and comparison with tarecilioside F.1) The other ene signal was observed at dC 69.2. In the H–1H COSY
difference between 2 and 3 was expected to be the sugar unit spectrum of 5, proton H-5 (dH 1.26) showed correlation with
linked at C-25. On analysis of the 13C-NMR spectrum of 3, H2-6 (dH 0.72, 1.53), while H2-6 showed cross peaks with
the xylopyranose in 2 was expected to be replaced by glu- H2-7 (dH 1.04, 1.27), thus all of these correlations indicated
copyranose in 3. On acid hydrolysis of 3, only D-glucose and that the C-7 hydroxy functional group was absent in 5. On
L-rhamnose were obtained. The positions of sugar linkages the other hand, in the NMR spectrum of 5, only five tertiary
were also the same as those in 2, and one glucopyranose was methyl carbons were observed with a new primary alcohol
also located at the hydroxy group at C-25 in the b-mode, signal [dC 69.2 with dH 3.95 (d, Jꢁ10 Hz), and dH 4.20 (m)].
since in the HMBC spectrum, long-range correlations of Thus, one of the methyl groups was expected to be modified
anomeric proton H-1ꢂ (dH 4.90) with C-3 (dC 88.3), H-1ꢃ (dH to a primary alcohol. Since the 13C-NMR data for the ring
6.50) with C-2ꢂ (dC 77.9), and H-1ꢄ (dH 4.95) with C-25 (dC portion, and C-20 and 21 of 5 were essentially the same as
82.9) were observed, although H-1ꢂ (dH 4.90) and H-1ꢄ (dH those for 4 (Table 2), one of the germinal methyl groups at
4.95) were overlapped by a huge water signal. Therefore, the C-25 must be oxidized to a carbinol. This was further sup-

July 2011
905
ported by the HMBC experiment for 5, long-range correla-
tions between H-26 (dH 3.95) and C-24 (dC 217.7), H-27 (dH
1.49) and C-24, and C-25 (dC 80.0) and C-26 (dC 69.2) being
observed. The absolute configuration of the newly formed
chiral center remains to be determined. Therefore, the struc-
(Inertsil; GL Science, Tokyo, Japan; Fꢁ20 mm, Lꢁ25 cm, flow rate
4 ml/min) with 70% MeOH to afford 5.83 mg of 5 from the peak at 23.5 min.
The residue (65.8 mg in fractions 88—106) of the eluate obtained on DCCC
was purified by HPLC with 60% MeOH to afford 21.1 mg of 1 from the
peak at 9 min.
Tarecilioside H (1): Amorphous powder; [a]D25 ꢀ15.5 (cꢁ0.73, pyridine);
1
ture of tarecilioside L (5) was tentatively elucidated to be 26- IR nmax (film) cmꢀ1: 3408, 2971, 1457, 1376, 1368, 1068, 1050; H-NMR
(400 MHz, pyridine-d5) and 13C-NMR (100 MHz, pyridine-d5): Tables 1 and
hydroxytarecilioside K.
2, respectively; HR-ESI-MS (positive-ion mode) m/z: 823.4825 [MꢅNa]ꢅ
Tarecilioside M (6), [a]D25 ꢀ32.9, was isolated as an amor-
(Calcd for C42H72O14Na: 823.4814).
phous powder and its molecular formula was determined to
be C48H78O20 on positive-ion HR-ESI-TOF-MS. Tarecilioside
M (6) was also a cycloartane triglycoside analogous to tare-
cilioside K (4). The difference between the 13C-NMR spectra
of 6 and 4 was that one more highly deshielded signal was
observed at dC 219.9 which was expected to represent one
more isolated ketone functional group that was located at C-
16, judging from the cross peaks between H2-15 (dH 2.66,
2.72) and H-17 (dH 2.35), and C-16 (dC 219.9) in the HMBC
spectrum. Therefore the structure of tarecilioside M (6) was
tentatively elucidated to be 16-ketotarecilioside K.
Tarecilioside I (2): Amorphous powder; [a]D25 ꢀ10.3 (cꢁ0.77, pyridine);
1
IR nmax (film) cmꢀ1: 3367, 2937, 1592, 1443, 1380, 1070, 1043; H-NMR
(400 MHz, pyridine-d5) and 13C-NMR (100 MHz, pyridine-d5): Tables 1 and
2, respectively; HR-ESI-MS (positive-mode) m/z: 955.5243 [MꢅNa]ꢅ
(Calcd for C47H80O18Na: 955.5236).
Tarecilioside J (3): Amorphous powder; [a]D25 ꢀ18.8 (cꢁ0.22, pyridine);
1
IR nmax (film) cmꢀ1: 3367, 2936, 1704, 1455, 1380, 1074, 1045; H-NMR
(400 MHz, pyridine-d5) and 13C-NMR (100 MHz, pyridine-d5): Tables 1 and
2, respectively; HR-ESI-MS (positive-mode) m/z: 983.5177 [MꢅNa]ꢅ
(Calcd for C48H80O19Na: 983.5186).
Tarecilioside K (4): Amorphous powder; [a]D25 ꢀ5.96 (cꢁ0.75, pyridine);
1
IR nmax (film) cmꢀ1: 3367, 2937, 1701, 1457, 1377, 1075, 1028; H-NMR
(600 MHz, pyridine-d5) and 13C-NMR (150 MHz, pyridine-d5): Tables 1 and
2, respectively; HR-ESI-MS (positive-mode) m/z: 999.5153 [MꢅNa]ꢅ
(Calcd for C48H80O20Na: 999.5135).
Experimental
Tarecilioside L (5): Amorphous powder; [a]D25 ꢅ1.19 (cꢁ0.17, pyridine);
General Experimental Procedures IR spectra were obtained on a
Horiba Fourier transform infrared spectrophotometer FT-710. Optical rota-
tion data were measured on a JASCO P-1030 polarimeter. 1H- and 13C-NMR
spectra were recorded on a JEOL JNM a-400 spectrometer at 400 MHz and
100 MHz, and a JEOL ECA-600 spectrometer at 600 MHz and 150 MHz, re-
spectively, with tetramethylsilane as an internal standard. Positive-ion HR-
ESI-TOF-MS was performed with an Applied Biosystems QSTAR® XL
NanoSpray System.
1
IR nmax (film) cmꢀ1: 3367, 2935, 1702, 1456, 1380, 1074, 1029; H-NMR
(400 MHz, pyridine-d5) and 13C-NMR (100 MHz, pyridine-d5): Tables 1 and
2, respectively; HR-ESI-MS (positive-ion mode) m/z: 999.5141 [MꢅNa]ꢅ
(Calcd for C48H80O20Na: 999.5135).
Tarecilioside M (6): Amorphous powder; [a]D25 ꢀ32.9 (cꢁ0.26, pyridine);
1
IR nmax (film) cmꢀ1: 3376, 2936, 1716, 1457, 1381, 1075, 1028; H-NMR
(400 MHz, pyridine-d5) and 13C-NMR (100 MHz, pyridine-d5): Tables 1 and
2, respectively; HR-ESI-MS (positive-ion mode) m/z: 997.4966 [MꢅNa]ꢅ
(Calcd for C48H78O20Na: 997.4978).
Highly-porous synthetic resin Diaion HP-20 (Fꢁ60 mm, Lꢁ65 cm) was
purchased from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). Silica gel CC
was performed on silica gel 60 [E. Merck, Darmstadt, Germany, 70—230
mesh]. Reversed-phase [octadecyl silica gel (ODS)] open CC (RPCC) was
performed on Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) [Fꢁ
50 mm, Lꢁ25 cm, linear gradient: MeOH–H2O (1 : 9, 1.5 l)→(7 : 3, 1.5 l),
10 g fractions being collected]. DCCC (Tokyo Rikakikai, Tokyo, Japan) was
equipped with 500 glass columns (Fꢁ2 mm, Lꢁ40 cm), and the lower and
Analyses of the Sugar Moiety About 1 mg each of tareciliosides H—M
(1—6) was hydrolyzed with 1 M HCl (0.1 ml) at 90 °C for 2 h. The reaction
mixtures were partitioned with an equal amount of EtOAc (0.1 ml), and the
water layers were analyzed with a chiral detector (JASCO OR-2090plus) on
an amino column [Asahipak NH2P-504E, CH3CN–H2O (4 : 1), 1 ml/min].
Tareciliosides H (1) and J (3) gave peaks for L-rhamnose and D-glucose at
the retention times of 5.5 min (negative optical rotation sign) and 8.5 min
(positive optical rotation sign), respectively, tarecilioside I (2) gave peaks for
L-rhamnose, D-xylose and D-glucose at the retention times of 5.5 min (nega-
tive optical rotation sign), 6.7 min (positive optical rotation sign), and
8.5 min (positive optical rotation sign), respectively, and tareciliosides K (4),
L (5) and M (6) each gave a peak for D-glucose at the retention time at
8.5 min (positive optical rotation sign), respectively. Peaks were identified by
co-chromatography with anthentic L-rhamnose, D-xylose and D-glucose.
upper layers of
a solvent mixture of CHCl3–MeOH–H2O–1-PrOH
(9 : 12 : 8 : 2) were used as the mobile and stationary phases, respectively.
Five grams fractions were collected and numbered according to their order
of elution with the mobile phase. HPLC was performed on an ODS (Inertsil;
GL Science, Tokyo, Japan; Fꢁ6 mm, Lꢁ25 cm, flow rate 1.6 ml/min) col-
umn. Precoated silica gel 60 F254 TLC plates (E. Merck; 0.25 mm in thick-
ness) were used for identification.
Plant Material Leaves of T. gracilipes (HAYATA) OHWI were collected in
Okinawa, Japan, in July 2002, and a voucher specimen was deposited in the
Herbarium of Pharmaceutical Sciences, Graduate School of Biomedical Sci-
ences, Hiroshima University (02-TG-Okinawa-0705).
Extraction and Isolation The 1-BuOH-soluble fraction (149 g) of a
MeOH extract of dried leaves (9.9 kg) of T. gracilipes1) was subjected to
highly porous synthetic resin (Diaion HP-20) CC (Fꢁ60 mm, Lꢁ65 cm),
using a stepwise-gradient of MeOH–H2O [(1 : 4, 6 l), (2 : 3, 6 l), (3 : 2, 6 l),
(4 : 1, 6 l), and MeOH (6 l)], 500 ml fractions being collected. The residue
eluted with the 60—80% MeOH (29.6 g in fractions 14—19) eluate obtained
on HP-20 CC was subjected to silica gel (500 g) CC using CHCl3 (3 l),
CHCl3–MeOH [(99 : 1, 3 l), (97 : 3, 3 l), (19 : 1, 3 l), (37 : 3, 3 l), (9 : 1, 6 l),
(7 : 17, 3 l), (17 : 3, 3 l), (33 : 7, 3 l), (4 : 1, 3 l), (3 : 1, 3 l) (7 : 3, 3 l)] and
CHCl3–MeOH–H2O (35 : 15 : 2, 3 l), fractions of 500 ml being collected. The
residue (2.51 g in fractions 59—65) of the 25% MeOH in CHCl3 eluate ob-
tained on silica gel CC was subjected to PRCC. The residue (370 mg in frac-
tions 205—219) was separated by DCCC and then the residue (52.4 mg in
fractions 62—72) was separated by HPLC with 65% MeOH to give 7.77 mg
of 6 from the peak at 28 min. The residue (480 mg in fractions 220—240)
was separated by DCCC to give 323 mg of 4 in fractions 51—66. The
residue (302 mg in fractions 241—265) was separated by DCCC. The
residue (44.8 mg in fractions 43—53) of the eluate obtained on DCCC was
purified by HPLC with 60% MeOH to afford 6.30 mg of 3 from the peak at
6 min, and 14.6 mg of 2 from the peak at 22.4 min. The residue (62.9 mg in
fractions 54—74) of the eluate obtained on DCCC was purified by HPLC
Acknowledgements The authors are grateful for access to the supercon-
ducting NMR instrument (JEOL JNM a-400) at the Analytical Center of
Molecular Medicine of the Hiroshima University Faculty of Medicine and
an Applied Biosystem QSTAR XL system ESI (Nano Spray)-TOF-MS at the
Analysis Center of Life Science of the Graduate School of Biomedical Sci-
ences, Hiroshima University. The authors are also grateful for the use of the
NMR instrument (JEOL ECA-600) at the Natural Science Center for Basic
Research and Development, Hiroshima University. This work was supported
in part by Grants-in-Aid from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, the Japan Society for the Promotion of
Science, and the Ministry of Health, Labour and Welfare. Thanks are also
due to the Research Foundation for Pharmaceutical Sciences and the Takeda
Science Foundation for the financial support.
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