An iridoid glucoside dimer and a non-glycosidic iridoid from the leaves of Lasianthus wallichii.

Article abstract of DOI:10.1248/cpb.50.1395

A new iridoid glucoside dimer (1) and a non-glycosidic iridoid (2) was isolated together with the known compounds, asperuloside (3), paederoside (4), daphylloside (5), citroside A (6) and benzyl 6-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranoside (7), from the leaves of Lasianthus wallichii. The structures of the new compounds were elucidated by spectroscopic and chemical evidence.

Full text of DOI:10.1248/cpb.50.1395

October 2002
Notes
Chem. Pharm. Bull. 50(10) 1395—1397 (2002)
1395
An Iridoid Glucoside Dimer and a Non-glycosidic Iridoid from the Leaves
of Lasianthus wallichii
Yoshio TAKEDA, ,a Hideko SHIMIDZU,a Kenta MIZUNO,a Sachiyo INOUCHI,a Toshiya MASUDA,a
*
e
Eiji HIRATA,b Takakazu SHINZATO,c Mitsunori ARAMOTO,d and Hideaki OTSUKA
a Faculty of Integrated Arts and Sciences, The University of Tokushima; 1–1 Minamijosanjima-cho, Tokushima 770–8502,
Japan: b Faculty of Agriculture, University of the Ryukyus; 1 Senbaru, Nishihara-cho, Nakagami-gun, Okinawa 903–0213,
Japan: c Yona Field, Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 685 Yona,
Kunigami-son, Kunigami-gun, Okinawa 905–1427, Japan: d Tropical Biosphere Research Center, University of the
Ryukyus; 870 Uehara, Taketomi-cho, Yaeyama-gun, Okinawa 907–1541, Japan: and e Department of Pharmacognosy,
Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima 734–8551, Japan.
Received May 13, 2002; accepted June 26, 2002
A new iridoid glucoside dimer (1) and a non-glycosidic iridoid (2) was isolated together with the known
compounds, asperuloside (3), paederoside (4), daphylloside (5), citroside A (6) and benzyl 6-O-a-L-rhamnopyra-
nosyl-b-D-glucopyranoside (7), from the leaves of Lasianthus wallichii. The structures of the new compounds
were elucidated by spectroscopic and chemical evidence.
Key words Lasianthus wallichii; Rubiaceae; iridoid glucoside dimer; non-glycosidic iridoid
Genus Lasianthus is classified in the tribe Morindeae, sub-
family Rubioideae (Rubiaceae). Kooiman1) pointed out the
presence of so called asperulosidic acid in almost all Ru-
bioideae plants except for some exceptions based on the test
of the extracts with the Trim and Hill reaction.2) Although
the presence of asperuloside (3) in some of plants of the
genus Lasianthus was reported3) based on the results of TLC
and GC of the glycosidic fraction, few reports dealing with
the constituents have appeared. In the course of studies on
the constituents of the plants which are grown under a sub-
tropical climate, we investigated the constituents of the
leaves of Lasianthus wallichii (WIGHT & ARN.) Wight (Rubi-
aceae) and isolated two new compounds, one of which is bis-
iridoid glucoside (1) and the other non-glycosidic iridoid
(2), together with five known compounds: asperuloside (3),4)
paederoside (4),5,6) daphylloside (5),7) citroside A (6),8) ben-
zyl 6-O-a-L-rhamnopyranosyl-b-D-glucopyranoside (7).9)
This paper deals with the isolation and structure elucidation
of the new compounds.
lowed to d 3.23 (H-9) and 5.83 (H-1), successively. The
structure of the aglycone of the other unit was also elucidated
to be the same as that of 10-O-acetylasperulosidate (partial
1
structure B) based on the results of H–1H COSY spectrum.
Thus, starting from the signal at d 7.70, cross peaks were fol-
lowed to the signals at d 3.05 (H-5), 4.84 (H-6), 6.02 (H-7),
and 4.80 and 4.94 (H2-10), successively. The cross peaks
were further followed to the signals at d 2.66 (H-9) and 5.07
(H-1) starting from the signal at d 3.05. The 13C-NMR data
shown in Table 1 clearly demonstrated that compound 1 is
constituted from 3 unit and 10-O-acetylasperulosidate unit.
Since the C-11 signal of unit B resonated in almost the same
region as in 5 (Table 1), the carboxyl group at C-4 in the par-
tial structure unit B formed an ester linkage with one of the
hydroxyl group in the glucose moiety in the partial structure
of unit A. Alkaline hydrolysis of compound 1 gave deacety-
lasperulosidic acid (8).11) The location of the ester linkage
was inferred at O-6Ј in unit A, because the signal assigned to
H2-6Ј of that unit suffered downfield shift to d 4.22 and 4.63
and 13C-NMR signals of sugar portion for unit A well corre-
spond to those of 6-O-acylated glucose derivatives.10) This
Compound 1 (1) was isolated as an amorphous powder,
[a]D Ϫ58.4° (MeOH) and the molecular formula was as-
signed as C36H44O22, based on its negative ion high resolu-
tion (HR)-FAB-MS. It showed an absorption maximum at
1
228 nm (e 14120) in the UV spectrum. The H-NMR spec-
trum showed, in addition to two singlets at d 2.05 and 2.08
(each 3H), two signals at d 7.30 (1H, d, Jϭ2.2 Hz) and 7.70
(1H, dd, Jϭ1.7, 0.6 Hz), which are characteristic of the pro-
ton on C-3 of iridoid glucosides having a carbonyl group on
C-4, two signals at d 5.07 (1H, dd, Jϭ9.0, 0.6 Hz) and 5.83
(1H, dd, Jϭ0.6, 0.6 Hz) assigned to acetalic protons and two
anomeric protons at d 4.73 (2H, d, Jϭ7.9 Hz). Thus com-
pound 1 was presumed to be composed of two iridoid glyco-
side units. The structure of the aglycone portion of one unit
was shown to be the same as that of 3 (partial structure A) on
1
the results of H–1H correlation spectroscopy (COSY) spec-
trum. Thus, starting from the signal at d 7.30 assignable to
the proton on C-3, cross peaks were followed to d 3.67 (H-
5), 5.56 (H-6), 5.73 (H-7), and 4.63 and 4.72 (H2-10). In con-
trast, starting from the signal at d 3.67, cross peaks were fol-
* To whom correspondence should be addressed. e-mail: takeda@ias.tokushima-u. ac.jp
© 2002 Pharmaceutical Society of Japan

1396
Vol. 50, No. 10
Table 1. 13C-NMR Data (d, ppm)a) of Compound 1 (1), Asperuloside (3) Table 2. 1H- and 13C-NMR Data (d) of Compound 2 (2) (in CD3OD)
and Daphylloside (5) (in CD3OD)
Atom
dC
dH
1
Carbon
3
5
1
3
58.5
63.5
4.19 (2H, br s)
Unit A
Unit B
3.86 (dd, Jϭ10.6, 3.7 Hz)
3.93 (dd, Jϭ10.6, 4.1 Hz)
2.83 (ddd, Jϭ6.4, 4.1, 3.7 Hz)
3.67 (m)
5.11 (td, Jϭ6.6, 1.1 Hz)
2.71 (d, Jϭ18.1 Hz)
1
3
4
93.4
150.2
106.4
37.5
101.5
155.8
108.2
42.6
93.3
150.3
106.2
37.5
101.3
155.4
108.1
42.4
4
5
6
7
48.7
53.2
83.2
41.9
5
6
86.3
75.6
86.3
75.4
2.91 (ddd, Jϭ18.1, 6.6, 1.1 Hz)
7
8
9
129.3
144.2
45.4
132.0
146.1
46.4
128.9
144.2
45.2
131.8
146.0
46.2
8
9
10
138.5
137.4
57.1
4.15 (br d, Jϭ13.2 Hz)
4.29 (br d, Jϭ13.2 Hz)
10
11
Ac
61.9
172.1
20.8
63.7
168.7
20.8
60.9
172.2
20.7
63.8
169.3
20.8
11
180.8
172.5
100.1
74.7
172.5
100.7
75.0
172.5
100.0
74.6
78.3
71.5
172.5
100.6
74.9
78.6
71.5
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
78.0
78.6
71.7b)
76.0
71.8b)
77.9
77.8
62.8
77.9
63.0
64.4
63.1
a) The assignments are based on 1H-COSY, HSQC, HMBC and comparisons of
spectra of closely related compounds. b) May be interchanged.
Fig. 2. The Results of HMBC Spectrum of Compound 2 (2) (Jϭ8 Hz)
Fig. 3. The Results of NOE Experiments for Compound 2 (2)
hydroxyl group; three methine groups, one of which bears an
carbonyloxy group; a tetrasubstituted double bond and a lac-
tonic carbon atom. Analysis of one-dimensional spectra and
1H-COSY, heteronuclear single quantum coherence (HSQC)
and HMBC (Fig. 2) spectra led to the proposed structure for
Fig. 1. The Results of HMBC Spectrum of Compound 1 (1) (Jϭ8 Hz)
1
compound 2 and enabled the complete assignments of H-
and 13C-NMR signals. The observed coupling constant in the
1H-NMR spectra and the co-occurrence of 3 which might be
a possible biogenetic precursor strongly suggested that chiral
centers at C-5 and 6 are R and S-configurations. The results
of differential nuclear Overhauser effect (NOE) experiments
shown in Fig. 3 and CD spectrum support the above pre-
sumption and the stereochemistry at C-4 is R-configura-
tion.12) Thus, the structure of compound 2 was elucidated as
shown in Fig. 2.
presumption was confirmed by the facts that the proton sig-
nals of H2-6Ј of unit A crossed peaks with the ester carbonyl
signal at d 168.7 (C-11 in unit B) which crossed peaks with
the signal due to H-3B and H-5B in the unit B in the het-
eroatom multiple bond correlation (HMBC) spectrum, the re-
sults of which are shown in Fig. 1. Thus, the structure of
compound 1 was elucidated as shown in Fig. 1.
Compound 2 (2) was isolated as a syrup, [a]D Ϫ30.6°
(MeOH). The molecular formula was assigned as C10H14O5
based on its negative ion HR-FAB-MS. The compound was
transparent above 220 nm in the UV spectrum and showed
absorptions of hydroxyl groups (3332 cmϪ1) and a five-mem-
bered lactone group (1747 cmϪ1) in the IR spectrum. The
13C-NMR spectrum (Table 2) showed only ten carbon signals
composed of four methylene groups, three of which bear a
Experimental
Optical rotations were measured on a JASCO DIP-360 digital polarimeter.
IR spectra were measured on a Perkin-Elmer IR spectrometer and UV spec-
tra on a JASCO V-530 SR spectrophotometer. 1H- and 13C-NMR spectra
were taken on a JEOL JNM EX-400 or a-400 spectrometer at 400 and
100 MHz, respectively, with tetramethylsilane as an internal standard. HR-

October 2002
1397
FAB-MS were performed on a JEOL JMS SX-102 spectrometer with poly- (45 mm in diameter and 470 mm in length). Adsorbed material was eluted
ethylene glycol-400 as a matrix. Column chromatography was performed on with H2O–MeOH with a stepwise increase of 50% MeOH content (4.5 l) and
Diaion HP-20 (Mitsubishi Kagaku Co., Ltd., Tokyo), silica gel 60 (230— 60% MeOH (4.5 l) eluted successively, collecting 100 ml fractions. Fractions
400 mesh, Merck), and octadecyl silica (ODS) gel (Cosmosil 75C18-OPN, 6—7 gave a residue (1.07 g) which was further separated by silica gel (55 g)
Nacalai Tesque, Kyoto), and TLC was performed on precoated silica gel column chromatography with CHCl3–MeOH as eluent with an increasing
plates 60 F254 (0.25 mm in thickness, Merck). HPLC was performed on ODS amount of MeOH in CHCl3 and finally by HPLC (solvent: MeOH–H2O
gel (Cosmosil 10C18, Nacalai Tesque, Kyoto, fϭ20 mm, Lϭ250 mm) with a 7 : 13, detection 230 nm) to give compound 1 (1) (36.3 mg).
mixture of H2O and MeOH at the flow rate of 6 ml minϪ1 and the eluate was
monitored by UV.
The known compounds isolated, asperuloside (3), paederoside (4), da-
phylloside (5), citroside A (6) and benzyl 6-O-a-L-rhamnopyranosyl-b-D-
Plant Material Plant material was collected in Kunigami-son, Ku- glucopyranoside (7) were identified with authentic samples by direct com-
nigami-gun and Taketomi-cho, Yaeyama-gun, Okinawa Prefecture in July, parison or by comparison of their spectral data with those reported. The
1998 and in October, 2000, respectively, and identified as Lasianthus wal- physical properties of the new compounds are as follows.
lichii (Wight & Arn.) Wight by one (T.S.) of the authors . Voucher speci-
Compound 1 (1): an amorphous powder, [a]D27 Ϫ58.4° (cϭ0.66, MeOH).
mens (98-LW-Okinawa-0709 and 00-LW-Okinawa-1004) were deposited in UV lmax (MeOH) nm (e): 228 (14120). IR nmax (film) cmϪ1: 3382, 1732,
1
the Herbarium of the Department of Pharmacognosy, Division of Medicinal 1658, 1633, 1257, 1053, 1026. H-NMR (CD3OD): 2.05, 2.08 (each 3H, s,
Chemistry, Graduate School of Biomedical Sciences, Hiroshima University. 2ϫOAc), 2.66 (1H, ddd, Jϭ8.2, 8.2, 0.6 Hz, H-9B), 3.05 (1H, ddd, Jϭ8.2,
In this report, we used the sample collected in Kunigami-gun, since the TLC 6.0, 1.7 Hz, H-5B), ca. 3.23 (H-9A), 3.61 (1H, dd, Jϭ11.7, 4.0 Hz, H1-6Ј),
B
pattern of n-BuOH soluble fractions obtained from both samples is the 3.60—3.66 (2H, m, H-5Ј , H-5Ј), 3.67 (1H, ddd, Jϭ6.5, 6.5, 2.2 Hz, H-5A),
A
B
same.
3.85 (1H, dd, Jϭ11.9, 1.8 Hz, H1-6Ј), 4.22 (1H, dd, 11.9, 6.2 Hz, H1-6Ј ),
B
A
Extraction and Isolation Dried leaves (3.9 kg) of L. wallichii were ex- 4.63 (1H, dd, Jϭ11.9, 2.2 Hz, H1-6Ј ), 4.63 (1H, dd, Jϭ14.1, 1.2 Hz, H1-
A
tracted with MeOH (72 l) at room temperature for 2 weeks. Extraction was 10A), 4.72 (1H, dd, Jϭ14.1, 1.2 Hz, H1-10A), 4.73 (2H, d, Jϭ7.9 Hz, H-1Ј
A
repeated once in the same manner. The combined methanolic extract was and H-1Ј), 4.80 (1H, dd, Jϭ15.0, 1.8 Hz, H1-10B), 4.84 (1H, dd, Jϭ6.0,
B
concentrated in vacuo. The residue was dissolved in 90% MeOH (1.1 l) and 2.6 Hz, H-6B), 4.94 (1H, dd, Jϭ15.0, 1.8 Hz, H1-10B), 5.07 (1H, dd, Jϭ9.0,
the solution was washed with n-hexane (1 lϫ3). The 90% MeOH layer was
concentrated in vacuo. The residue was suspended in H2O and the suspen-
0.6 Hz, H-1B), 5.56 (1H, dt, Jϭ6.5, 1.7 Hz, H-6A), 5.73 (1H, m, H-7A), 5.83
(1H, d, Jϭ0.6 Hz, H-1A), 6.02 (1H, br d, Jϭ1.8 Hz, H-7B), 7.30 (1H, d,
sion was extracted with EtOAc (1 lϫ3). The aqueous layer was extracted Jϭ2.2 Hz, H-3A), 7.70 (1H, dd, Jϭ1.7, 0.6 Hz, H-3B). 13C-NMR: see Table
with n-BuOH (1 lϫ3). The n-BuOH extract was evaporated in vacuo to give 1. HR-FAB-MS (negative) m/z: 827.2258 [MϪH]Ϫ (Calcd for C36H43O22:
a residue (70.5 g).
827.2246).
The residue was chromatographed on Diaion HP-20 (70 mm in diameter
and 445 mm in length). Adsorbed material was eluted successively with
H2O–MeOH with a stepwise increase of MeOH content. Three and one-half
Compound 2 (2): A viscous syrup, [a]D28 Ϫ30.6° (cϭ0.72, MeOH). IR
n
max (film) cmϪ1: 3332, 1747, 1362, 1192, 1061, 991. 1H- and 13C-NMR: see
Table 2. CD lmax (MeOH) nm (De): 286 (Ϫ0.013), 218 (ϩ1.61). HR-FAB-
liters of 0%, 20%, 40% and 60% MeOH in H2O and MeOH were eluted suc- MS (negative) m/z: 213.0783 [MϪH]Ϫ (Calcd for C10H13O5: 213.0763).
cessively, and 500 ml fractions were collected. The residue (2.62 g) of frac-
tions 11—14 was subjected to silica gel (55 g) column chromatography. Five 0.5 N NaOH aqueous solution (1 ml) and the solution was stirred for 4.5 h at
hundred milliliters of each of CHCl3 and CHCl3–MeOH (97 : 3, 19 : 1, 9 : 1, room temperature. The solution was neutralized with an ion-exchange resin,
Alkaline Hydrolysis of Compound 1 (1) 1 (13.1 mg) was dissolved in
22 : 3, 17 : 3, 4 : 1, 3 : 1, 7 : 3) were eluted successively. The eluate (520 mg) Amberlite IR-120B (H-form). The ion exchange resin was filtered off and
from 5—10% MeOH–CHCl3 which contained a spot (Rf 0.30, solvent: the filtrate was concentrated in vacuo to give 8 (10.5 mg).11)
CHCl3–MeOH–H2O 15 : 6 : 1) on TLC was separated by HPLC (solvent:
MeOH–H2O 1 : 9, detection 210 nm) to give compound 2 (94.7 mg) as a vis-
cous gum.
Acknowledgements The authors are grateful to the Cooperative Center
of the University of Tokushima and the Analytical Center of Molecular
The residue (10.1 g) of fractions 15—24 of Diaion HP-20 column was Medicine, Graduate School of Biomedical Sciences, Hiroshima University
chromatographed over silica gel (500 g). Three liters of CHCl3 and CHCl3–
MeOH (97 : 3, 19 : 1, 93 : 7, 9 : 1, 22 : 3, 17 : 3, 4 : 1, 3 : 1, 7 : 3) were eluted
for providing the facilities for recording the NMR spectra.
successively. From the eluate of CHCl3–MeOH (9 : 1), 300 ml fractions were References
collected. An aliquot (300 mg) of the residue (863 mg) from fractions 11—
16 was separated by HPLC (solvent: MeOH–H2O 3 : 7; detection 210 nm) to
give asperuloside (3) (23.2 mg) and citroside A (6) (14.4 mg). Fractions
17—21 gave a residue (742 mg), an aliquot (152 mg) of which was separated
by HPLC (solvent: MeOH–H2O 7 : 13; detection 210 nm) to give benzyl al-
cohol 6Ј-a-L-rhamnopyranosyl-b-D-glucopyranoside (7) (19.6 mg). An
aliquot (199 mg) of the residue (578 mg) was separated by HPLC (solvent:
MeOH–H2O 3 : 7; detection 210 nm) to give another aliquot of asperuloside
(3) (18.6 mg) and benzyl 6-O-a-L-rhamnopyranosyl-b-D-glucopyranoside
(7) (23.3 mg).
1) Kooiman P., Acta Bot. Neerl., 18, 124—137 (1969).
2) Trim A. R., Hill R., Biochem. J., 50, 310—319 (1952).
3) Inouye H., Takeda Y., Nishimura H., Kanomi A., Okuda T., Puff C.,
Phytochemistry, 27, 2591—2598 (1988).
4) Briggs L. H., Cain B. F., Le Quesne P. W., Shoolery J. N., Tetrahedron
Lett., 1963, 69—74 (1963).
5) Inouye H., Inouye S., Shimokawa N., Okigawa M., Tetrahedron Lett.,
1968, 683—688 (1968).
6) Kapadia G. J., Shukla Y. N., Bose A. K., Fujiwara H., Lloyd H. A.,
Tetrahedron Lett., 1979, 1937—1938 (1979).
Fractions 25—32 of Diaion HP-20 column chromatography gave a
residue (33.2 g) which was chromatographed over silica gel (1 kg) with
CHCl3–MeOH as eluent with increasing amounts of MeOH content CHCl3
(4.5 l), CHCl3–MeOH (9 : 1, 4.5 l), CHCl3–MeOH (22 : 3, 4.5 l), CHCl3–
MeOH (17 : 3, 6.5 l), and CHCl3–MeOH (4 : 1, 5.5 l) were eluted succes-
sively, 500 ml fractions were collected. Fractions 19—22 gave a residue
7) Inouye H., Ueda S., Hirabayashi M., Shimokawa N., Yakugaku Zasshi,
86, 943—947 (1966).
8) Umehara K., Hattori I., Miyase T., Ueno A., Hara S., Kageyama C.,
Chem. Pharm. Bull., 36, 5004—5008 (1988).
9) De Tommasi N., Rastrelli L., Cumanda J., Speranza G., Pizza C., Phy-
tochemistry, 42, 163—167 (1996).
(698 mg), an aliquot (110 mg) of which was separated by HPLC (solvent: 10) Garcia J., Lavaitte S., Gey C., Phytochemistry, 28, 2199—2201
MeOH–H2O 7 : 13, detection 230 nm) to give paederoside (4) (19.7 mg) and (1989).
daphylloside (5) (6.3 mg). Fractions 23—25 gave a residue (615 mg). An 11) Inouye H., Okigawa M., Shimokawa N., Chem. Pharm. Bull., 17,
aliquot (100 mg) was separated by HPLC (solvent: MeOH–H2O 7 : 13, de- 1949—1954 (1969).
tection 230 nm) to give 3 (12.4 mg), 4 (11.1 mg) and 5 (8.1 mg). Fractions 12) Jennings J. P., Klyne W., Scopes P. M., J. Chem. Soc., 1965, 7211—
35—40 gave a residue (4.20 g) which was chromatographed over ODS gel 7229 (1965).