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Plant Material The leaves of C. bojeri SEEM. (Araliaceae) were col-
lected near Andasibe (140 km east of Antananarivo), Madagascar, in March
2000. The plant was identified at the Parc Botanique et Zooligique de Tsim-
bazaza, Antananarivo, Madagascar. A voucher specimen has been deposited
in the Herbarium of the Institute of Pharmaceutical Sciences, Faculty of
remaining 20 (five quaternary, three methines, 10 methyl-
enes, and two methyl carbons) to a diterpenoid aglycone.
Inspection of the 1H- and 13C-NMR spectra of 4, which are
very similar to those of 3 (Table 1), demonstrated that the
aglycone of 4 was also a kaurane-type diterpene with a pri- Medicine, Hiroshima University.
Extraction and Isolation The dried leaves (250 g) of C. bojeri SEEM.
mary hydroxyl group [at dC 66.6, dH 3.78 (1H, d, Jϭ10 Hz),
dH 3.80 (1H, d, Jϭ10 Hz)], a tertiary hydroxyl group (dC
87.0), and a carboxyl (dC 176.9) group. On comparison of
the 13C-NMR spectral data of 4 with those of 3, additional
signals of one b-glucopyranosyl unit, and a downfield shift
of the signal due to C-16 (ϩ5.3) and upfield shifts of the sig-
nals due to C-13 and C-15 (Ϫ5.9 ppm and Ϫ5.3 ppm, respec-
tively) were observed. These suggest that the site of glucosy-
lation was in the hydroxyl group at position 16.6)
The position of the functions as well as the attachments
was confirmed by heteronuclear single quantum coherence
(HSQC) and heteronuclear multiple bond connectivity
(HMBC) experiments. It was observed in the HMBC spec-
trum that the anomeric proton signal of the glucopyranosyl at
d 4.99 showed a long-range correlation with the carbon at
the C-16 position (d 87.0). The ent-nature of (4) was con-
firmed since (6) was obtained from enzymatic hydrolysis.7)
The determination of the absolute configuration of the sugar
were extracted with MeOH. After removal of the solvent by evaporation, the
residue (26.2 g) was suspended in water and extracted with hexane and
EtOAc in succession. The aqueous layer (19.4 g) was subjected to a column
of a highly porous copolymer of styrene and divinylbenzene, and eluted with
H2O, 30% MeOH, 100% MeOH, and Me2CO in succession. The fraction
eluted with MeOH (6.2 g) was chromatographed on a column of silica gel
(system I), affording seven fractions. Fraction 3 yielded compound 4
(34 mg) by precipitation with MeOH. Compounds 2 (137 mg) and 3 (48 mg)
were obtained from fractions 2 and 4, respectively, by ODS-HPLC using
systems III and IV. ODS HPLC of fraction 6 (system IV) afforded com-
pound 5 (24 mg).
The EtOAc extract was subjected to CC on silica gel (system II). Frac-
tions were combined according to their TLC pattern to yield four fractions.
Compounds 1 (15 mg) and 2 (50 mg) were obtained by precipitation with
MeOH of fractions 3 and 4, respectively.
Enzymatic Hydrolysis of Compound (4) An aqueous solution of 4
(20 mg) and crude hesperidinase (ca. 20 mg) was incubated at 37 °C for 72 h.
The solution was extracted with CH2Cl2 and the aglycone produced was
identified as 6 (7 mg) by comparison of its spectral data with those of
16a,17-dihydroxy-kauran-19-oic acid (crystal, mp: 248—255°).7) The aque-
ous layer was examined for identification of the component sugar, and glu-
(D-glucose) was established as follows. Standard samples of cose was identified by direct comparison on silica gel TLC with authentic
sample.
thiazolidine derivatives of D- and L-glucose [methyl 2-(D-glu-
Acid Hydrolysis of Compound (4) Ten milligrams of 4 in 10% H2SO4
(2 ml) was heated under reflux for 2 h. Water was added to the reaction mix-
ture and extracted with EtOAc. The water-soluble fraction was evaporated to
give the sugar fraction.
copentahydroxypentyl)-thiazolidine-4(R)-carboxylate,
methyl 2-(L-glucopentahydroxypentyl)-thiazolidine-4(R)-car-
boxylate] were prepared by the previously reported method.8)
Determination of the Absolute Configuration of the Sugar of (4)
A
Two spots were observed on TLC of the derivative from D-
glucose (0.43, 0.51) due to the C-2 epimers, while only one
single spot (0.46) was shown for the derivative from L-glu-
cose. The water-soluble fraction from acid hydrolysis of (4)
was treated in the same way as the standard samples and ex-
amined by TLC (see Experimental). D-Glucose was deter-
mined by comparison of the Rf values with those of standard
samples. Thus the structure of 4 was determined to be b-D-
glucopyranosyl-17-hydroxy-ent-kauran-19-oate-16-O-b-D-
glucopyranoside.
The anti-HIV activity of 16-hydroxylated ent-kaurane
diterpenes has been reported.3,9) Compound 1 (0.006%) has
been shown to have the most anti-HIV activity (significant
activity against HIV replication in H9 lymphocyte cells, with
an EC50 value of 0.8 mg/ml), while its 16-epimer showed sig-
nificant inhibition of HIV reverse transcriptase. The screen-
ing of the activity of the ent-kaurane diterpenoid isolated
from the genus Cussonia will be the subject of our next in-
vestigation.
mixture of pyridine (0.5 ml), L-cysteine methyl ester hydrocloride (5 mg),
and the sugar fraction was warmed at 60 °C for 1 h in the same way as de-
scribed in the literature.8) After removal of the solvent, the residue was dis-
solved in water and extracted with n-BuOH (1 ml). The organic layer after
evaporation was shown to contain methyl 2-(D-glucopentahydroxypentyl)-
thiazolidine-4(R)-carboxylate, Rf: 0.43, 0.51 (C-2 epimers of thiazolidine)
by TLC on CH2Cl2–MeOH–H2O (25 : 10 : 1.5).
Acknowledgments L. Harinantenaina is grateful to the Japanese Min-
istry of Education, Science and Culture for the award of a scholarship to
study for a Ph.D. at the Institute of Pharmaceutical Sciences, Hiroshima
University, Japan. We also thank the Research Center for Molecular Medi-
cine, Hiroshima University School of Medicine, Japan, for the use of its
NMR facilities.
References
1) Samyn J.-M., “Plantes Utiles des Hautes Terres de Madagascar,”
GRAPHOPRINT, Madagascar, 1999, p. 16.
2) Trotin F., Bézanger-Beaquesne L., Pinkas M., Flavonoids in the leaves
of Cussonia bojeri SEEM. (Araliaceae). Comptes Rendus Hebdo-
madaires Des Seances de l’Academie Des Sciences, Serie D: Sciences
Naturelles, 268, 855 (1969).
3) Cheng D. L., Cao X. P., Wei H. X., He L., Phytochemistry, 33, 1181—
1183 (1993).
4) Yamasaki K., Kohda H., Kobayashi T., Kaneda N., Kasai R., Tanaka
O., Nishi K., Chem. Pharm. Bull., 25, 2895—2899 (1977).
5) Harborne J. B., Marbry T. J., “The Flavonoids: Advances in Research,”
Chapman & Hall, London, 1982.
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175—178 (1977).
7) Jefferies P. R., Payne T. G., Aust. J. Chem., 18, 1441—1450 (1965).
8) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501—506
(1987).
Experimental
General Methods NMR spectra (1H, 13C, HSQC, HMBC) were
recorded in pyridine-d5 using a JEOL JNM A-400 spectrometer (400 MHz
for 1H-NMR and 100 MHz for 13C-NMR). MS were recorded on a JEOL
JMS-SX 102 spectrometer. Optical rotations were measured with Union
PM-1 digital polarimeter. Preparative HPLC was carried out on columns of
octadecyl silica (ODS) (150ϫ20 mm i.d., YMC) with a Tosoh refraction
index (RI-8) detector, flow rate 6 ml/min. For CC, silica gel G 60 (Merck),
RP-18 (50 mm, YMC), and a highly porous copolymer of styrene and di-
vinylbenzene (Mitsubishi Chemical Industries) were used. The solvent sys-
tems were: (I) CH2Cl2–MeOH–H2O (17 : 4 : 0.5 to 17 : 8 : 2), (II)
EtOAc–C6H12 (2 : 8 to 10 : 0), (III) 50% CH3OH, and (IV) 55% CH3OH. The
spray reagent used for TLC was 10% H2SO4 in 50% EtOH.
9) Chang F.-R., Yang P.-Y., Lin J.-Y., Lee K.-H., Wu Y.-C., J. Nat. Prod.,
61, 437—439 (1998).
Harinantenaina, Liva
