Lupane Type Triterpene Isolated from the Leaves of Thecacoris Annobonea (Euphorbiaceae)

a Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon b Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan * Corresponding author: E-mail: poumale@yahoo.fr ABSTRAT A new lupane type triterpene (1), together with betulinic acid (2), friedelin (3), aristolochic acid I (4), alpinumisoflavone (5) and 4’-O-methylepinumisoflavone (6) have been isolated from the leaves of Thecacoris annobonea. The structure of the new compound was elucidated on the basis of 1 and 2D NMR experiments. The isolated compounds were evaluated for their phytotoxicity and antimicrobial activity. 1 exhibited significant antimicrobial activity at 30 μg/ml and compounds 1, 2, 3, 4, 5 and 6 inhibited root growth lettuce at 100 μg/ml.


INTRODUCTION
Thecacoris annobonea (Euphorbiacea) is a small tree or shrub growing in South and South-West provinces of Cameroon. It is widely used in traditional Cameroonian folk medicine. The leaf decoction of T. batesii is used as purgative and antirheumatic remedies in the medicinal plant therapy in Cameroon [1]. In our previous study on the genus Thecacoris, aristolochic acid, vanillic acid and friedelin which showed good antimicrobial activities were isolated from the stem bark of T. annobonea [2] and, diterpenoids and triterpenoids were isolated from the twigs of T. batesii [1]. As a continuation of our search for biologically active compounds in the genus Thecacoris, one new compound together with five known compounds were isolated from the leaves of T. annobonea. The known compounds were identified as betulinic acid (2), friedelin (3), aristolochic acid I (4), alpinumisoflavone (5) and 4'-O-methylepinumisoflavone (6). The present paper deals with the isolation and structural elucidation of one new lupane type triterpene and also demonstrates its phytotoxicity and antimicrobial activity together with the other isolated known compounds.

RESULTS AND DISCUSSION
The leaves of T. annobonea were extracted with CHCl3/acetone (1:1) at room temperature during 24 hours. The extract was submitted to repeated column chromatography and preparative TLC to afford betulinic acid, friedelin, aristolochic acid I, alpinumisoflavone, 4'-O-methylepinumisoflavone as well as one new lupane type triterpene (1). The 1 H and 13 C NMR, and MS of the known compounds were consistent with those reported in the literature.

1
The 1 H NMR and DEPT spectra (Table 1) indicated that compound 1 is a pentacyclic triterpenoid [6] with six methyl groups at δ 0.86, 0.89, 0.92, 0.99, 1.00 and 3.63 (3H each, s), six methine and twelve methylene groups. The signal at δ 3.63 (3H, s) was attributed to a methyl connected to an oxygen which showed a correlation with the carbon C-28 (δ 174.1). The olefinic methyl which normally appears in lupeol/lupane type triterpene at δ 1.70 was absent suggesting the absence of C-30 in the molecule. The methine signals at δ 2.30 (2H) were attributed to the protons at positions C-18 and C-19 according to the

Experimental General
ESI mass spectra were recorded on a Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument). HRESI mass spectra were recorded on a Bruker FTICR 4.7 T mass spectrometer. EI mass spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV) with perfluorkerosene as a reference substance for HREI-MS.
The 1 H and 13 C NMR spectra were acquired with a Jeol EX-400 spectrometer. Chemical shifts are given on a δ (ppm) scale with TMS as an internal standard. Melting point is uncorrected and was obtained with a micro melting point apparatus (Yanaco, Tokyo-Japan). Optical rotation values were measured with a Horiba SEPA-300 polarimeter, and IR spectra were recorded on a Perkin-Elmer 1600 Series FT-IR spectrometer from films. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan) and Sephadex LH-20 (Pharmacia, Sweden). TLC analysis was carried out using precoated silica gel plates (Merck), and spots were detected by spraying with H2SO4/10% vanillin and then heating. Flash chromatography was carried out on silica gel (230-400 mesh). Rf values were measured on Polygram SIL G/F254 (Macherey-Nagel & Co.) and melting and decomposition points were measured by the melting point apparatus of Electrothermal and were not corrected.

Plant Material
The leaves of T. annobonea were collected in April 2008 from Kumba, South-West Cameroon and were identified by Mr. Victor Nana of the Cameroon National Herbarium (Yaoundé), where a voucher specimen was deposited (Ref. N° 38569/HNC).

Extraction and Isolation
The leaf powder of T. annobonea (500 g) was extracted with CHCl3/acetone (1:1) at room temperature for 24 hours. After removing the solvents by evaporation under reduced pressure, the crude extract (40 g) was chromatographed on silica gel. Using hexane/ethyl acetate of increasing polarity, a total of 105 sub-fractions (ca. 250 ml each) were collected and combined on the basis of TLC analysis leading to two main fractions A and B.

Phytotoxic Assay
Lettuce seeds (Lactuca sativa L.) were used for the bioassay. Nine seeds were deposited on filter paper containing a defined concentration of the test compound in a Petri dish (4 cm id.). Distilled water (1 ml, containing 100 ppm (w/v) Tween 80) was added to the Petri dish, and incubation was done at 25 °C under continuous light for 7 days. The control experiments were conducted with distilled water alone. The elongation of the roots and shoots was measured and compared with those of the control.

Antimicrobial assay
Agar diffusion tests were performed in the usual manner [15] with Bacillus subtilis and Escherichia coli (on peptone agar), Staphylococcus aureus (Bacto nutrient broth), Streptomyces viridochromogenes (M Test agar), the fungi Mucor miehei and Candida albicans (Sabouraud agar), and three microalgae (Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus subspicatus). N o v e m b e r 25, 2 0 1 3 Compounds were dissolved in an azeotrope chloroform/MeOH (87:13) and 30 g pro paper disks (Ø 8 mm) were impregnated with each using a 100 l syringe, dried for 1 h under sterile conditions and placed on the pre-made agar test plates. Bacteria and fungi plates were kept in an incubator at 37 °C for 12 h, micro algae plates for three days at room temperature in a day light incubator. The diameter of inhibition zones was measured.

Conclusion
In this study, we focused on the secondary metabolites of CHCl3/acetone (1:1) extract. One new compound, 30-norlup-20en-28-oic acid methyl ester (1) with five known compounds, betulinic acid (2), friedelin (3), aristolochic acid I (4), alpinumisoflavone (5) and 4'-O-methylepinumisoflavone (6) were isolated from the leaves of T. annobonea. Using 1 and 2D NMR, the new compound was fully characterized. All the isolated compounds were evaluated for their phytotoxic and antimicrobial activity. At a concentration of 100 μg/mL and 30 μg/mL, the new isolated (1) was the most active compound compare to the control for phytotoxicity and antimicrobial activity, respectively. Friedelin (3) and aristolochic acid I (4) were also isolated from the stem bark of T. annobonea. 2 It still difficult to predict the biological activities as well as the chemical constituents from Thecacoris species. The few differences between the secondary metabolites isolated from the leaves of T. annobonea and other Thecacoris species [1,2] are may be related to the real specific differences or, more probably to a geographic or environmental influence on biosynthesis [16]. In conclusion, more studies are required to determine properly the chemical constituents of the Thecacoris species as well as a relationship between their biological activities.