Steroids isolated from Millettia versicolor Baker ( Fabaceae )

1 Département des Sciences Exactes, Ecole Normale Supérieure, Université Marien Ngouabi, BP 69, Brazzaville – Congo. 2 Unité de Chimie du Végétal et de la Vie, Faculté des Sciences, Université Marien Ngouabi, BP 69, Brazzaville – Congo. 3 Institut de Chimie des Substances Naturelles (CNRS), 1, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex – France. 4 Centre d’Etudes et de Recherches Médecins d’Afrique (CERMA), BP 45, Brazzaville – Congo. 5 Université d’Abomey Calavi, 01 BP 526, Cotonou – Bénin.


INTRODUCTION
Millettia versicolor Baker (Fabaceae) is a medicinal plant used in African traditional medicine (Angola Congo, D.R. Congo and Gabon) to relieve pain and cure parasitosis.An aqueous decoction of stem bark is employed in Congo for intestinal parasitoses, kidney pains, cough, female sterility, senile impotence of men.An infusion is used in DR Congo to rub the syphilitic wounds.The aqueous decoction of leaves is taken against feverish rheumatisms, headache, kidney pains, intestinal parasitoses, and cough (Congo).It is also used in bath against syphilis (Gabon).The trunk bark has anthelminthic applications (Angola, Congo, and Gabon) (Bouquet, 1969;Adjanohoun et al., 1988).
Pharmacological studies confirmed the anthelminthic potential of the plant roots and leaves (Kasonia et al., 1989;Ongoka et al., 2004) but the active compounds res-*Corresponding author.E-mail: ongokapascal@yahoo.fr.ponsible for this activity have not yet been determined.The stem bark has reported anti-inflammatory properties, attributed to a furoquinone (Fotsing et al., 2003).From the leaves, which are the major plant part used for relieving pain, only lupeol had yet been isolated (Ekouya et al., 1990).However, our preliminary chemical screening (Ongoka et al., 2004) indicated the presence of numerous secondary metabolites in the aqueous and alcoholic fractions: flavonoids, tanins, polyphenols, saponins, terpenes and steroids.The present study aims to separate and identify the chemical constituents of the leaves of M. versicolor, to try and support its traditional use.Physiology of Université Marien Ngouabi (Brazzaville, Congo) where a voucher specimen has been deposited under number OP2004-1.The leaves were dried under shed then ground to powder prior to extraction.

Preparation of the extracts
70 g of dried leaves powder were extracted with methanol (soxhlet).
The solvent was evaporated in vacuo and yielded 10 g of crude extract.
Isolation and determination of the steroids and triterpenes 5 g of the crude extract were separated on Sephadex LH20 column (200 g, methanol) and 5 fractions were obtained.Fraction 3 was chromatographed on silica gel column (200-300 mesh) with the following solvent systems: heptane-EtOAc (50:50), EtOAc (100) and EtOAc-MeOH (80:20), then by preparative silica TLC (Merck).Isolated compounds were analyzed by HPLC/MS (Symmetry column (C18), isocratic: H2O-ACN (60:40), flow rate 1 ml/min) and GC/MS (GC TRACE Thermo 2000, solvent: CH2Cl2; Supelco Equity 5 silica capillary column 28089-U (30 m x 0.25 mm x 0.25 m), carrier gas: helium, at a flow rate of 2 ml/min, column held initially at 160°C for 2 min and then increased to 280°C with a 5°C/min heating ramp, injection performed in split mode (50:1) at 280°C) to assess their purity.The chemical structures were elucidated on the basis of IR, RMN 1 H, 13 C spectral studies and confirmed by comparison with published data.

RESULTS AND DISCUSSION
The spectral analysis enabled us to identify 4 known compounds: 2 phytosterols: stigmasterol (1), 24methylenecycloartan-3β-ol (2), 22,23-dihydrostigmasterol (3) and a phytostanol, stigmastan-3-ol (4) (Figure 1).Three triterpenes were also identified: lupeol, taraxasterol and β-amyrin.We have isolated these triterpenes from extracts of M. versicolor in previous studies (Ekouya et al., 1990;Alphonse et al., 2006).Compound 1 is the major composite of the leaves methanol extract.The structure of these compounds was confirmed by comparison with published data (Toshihiro et al., 1988;Kojima et al., 1990) and by the use of authentic samples.With the exception of lupeol, all these compounds are isolated from M. versicolor for the first time and compounds 1, 2, 3, 4, had not yet been isolated in the Milletia genus, though all are already known from the Fabaceae family.

Conclusion
This study permitted the isolation of seven known compounds from the methanolic extract of the leaves of M. versicolor Bak, six of them being new for the species.A majority of them has reported analgesic or antiinflammatory activities, which support the traditional use of the plant for pain relief.Some of the compounds have other interesting biological effects, for which the leaves of M. versicolor could be investigated.

Figure 1 .
Figure 1.Structures of the compounds isolated from the leaves of Millettia versicolor.