Phytochemical study of the stem bark of Tetrorchidium didymostemon (Euphorbiaceae)

This work deals with the phytochemical study of the stem bark of T. didymostemon , a plant used in the Gabonese traditional medicine in the treatment of various ailments. A chromatographic separation of the dichloromethane-methanol (1:1) extract of this plant part led to the isolation of four compounds, that were identified respectively to 1-hentriacontanol 1, stigmasta-5,24,28-trien-3-ol 2, stigmasta-5,24,28-trien-3-O-β-D-glucopyranoside 3 and tithoniamide B 4. Their structures were elucidated on the basis of common spectroscopic analysis techniques and by comparison of their spectral and physical data with those from the literature. From a chemotaxonomic point of view, these compounds are described from T. didymostemon for the first time.


Introduction
Tetrorchidium didymostemon (Baill.) Pax & K. Hoffm.(Euphorbiaceae) is an evergreen shrub or a tree that grows in many parts of the tropical Africa, from Guinea Bissau to Central African Republic, including Cameroon, Equatorial Guinea, Gabon, Congo, DR Congo, Uganda, Tanzania and Angola. The plant can reach up to 25 m tall, medium in size, with hanging branches. The bole can be 60 cm or more in diameter [1,2] . T. didymostemon is widely used in the African folk medicine in the treatment of filariasis, abscesses, leprous sores, glandular swellings, malaria, oedema, purgative enema, rheumatic painful limbs, painful kidney, toothache, and also as snakebites antidote, diuretic, emetic, febrifuge, parasiticide and purgative [3][4][5] . In a previous work [6] , T. didymostemon extracts exhibited good antimicrobial activities and high biomolecules content, including alkaloids, flavonoids, tannins, triterpenes, polyphenols and sterols [6] . In the present paper, we report the isolation and structural determination of four compounds from the stem bark of T. didymostemon, through the chromatographic fractionation of the dichloromethane-methanol extract of this plant part, and the analysis of the spectroscopic data of the biomolecules.

Materials and Method 2.1. Plant material:
Stem bark (2.5 kg) of T. didymostemon was harvested near Franceville (Gabon), and a specimen was kept in our university (N° Td 066/UM).

Extraction and Isolation
The plant material was dried for three weeks at room temperature and finely powdered. Powder obtained was extracted with a dichloromethane-methanol (1:1) mixture for three days and the extract was freeze-dried to yield 150 g of stem bark extract. The extract (12 g) was subjected to repeated flash column chromatography over silica gel (70-230 mesh) columns, and eluted successively with n-hexane and n-hexane-ethylacetate mixtures of increasing polarities. The fractions were checked by TLC and those of similar contents were combined and concentrated.   13

Identification of isolated compounds
Compound 1 was isolated as a white powder soluble in chloroform. Its mass spectrum showed a molecular ion at m/z 452, corresponding to the formula C 34 H 62 O with 0 degree of unsaturation. Its 1 H NMR spectrum showed a peak at δ H 3.62, integrating for two protons, assignable to an oxymethylene group. This spectrum also showed a set of peaks between δ H 1.15 and 1.45 which are those of a long chain of alkylpolymethylenes, and a terminal methyl at δ H 0.86; thus suggesting the lipid nature of 1. The completely decoupled 13 C NMR spectrum of 1, as well as that of DEPT 135 revealed the presence of 1 methyl group and 30 methylenes groups. The 13 C NMR spectrum also showed a peak at δ C 63.12 that could be assigned to a methylene linked to a hydroxyl group.
A comparison between the spectroscopic data of 1 with those found in the literature [7,8] allows us to assign to compound 1 the structure 1 which is that of 1-Hentriacontanol [7,8] (Fig. 1). Compound 2 was obtained as white crystals, with a melting point between 152-154 °C. It was soluble in chloroform and responded positively to the Liebermann-Burchard test, showing a violet colour of sterols. Its 1 H NMR spectrum showed a set of 5 intense peaks between δ H 0.60 and 1.70 assignable to five methyl groups. This suggested the tetracyclic nature of 2. This spectrum also showed five remarkable peaks at δ H 3.50, 4.68, 5.16, 5.22 and 5.32 which are those of oxymethine and unsaturated protons. The 13 C NMR spectrum of compound 2, as well as that of DEPT, revealed the presence of 29 carbon atoms including 5 methyles, 10 methylenes, 10 methines and 4 quaternary carbons. This spectrum also showed a peak at δ C 71.80 assignable to a carbon linked to a hydroxyl group, probably the C-3 carbon of sterols. At δ C 148.62, 140.76, 137.19,  130.34, 121.69 and 109.51, were observed the presence of six double bonded carbons. A comparison of the spectroscopic and physical data of compound 2 with those found in the literature [9][10][11] , allowed us to attribuate to compound 2 the structure 2 which was that of Stigmasta-5,22,25-trien-3-ol [11] (Fig. 1). Compound 3 was obtained as beige powder. It was soluble in pyridine and reacted positively to the Molish test, suggesting that compound 3 was a glycoside. A compared analysis of the [13] C NMR spectra of compound 3 and compound 2 showed that they had almost similar signals. However, compound 3 possessed six additional carbons bonded to oxygen atoms between δ C 62.88 and 102.60, corresponding to the sugar group identified above. In addition, the anomeric carbon appearing at δ C 102.60 indicated that the sugar group was attached to an oxygen atom probably at position 3. This was confirmed by the strong deshielding of the C-3 carbon in compound 3. Thus, it was obvious that compound 3 was the glucoside of compound 2. The comparison of the NMR data of compound 3 with those from the literature [11] , allowed us to identify compound 3 as Stigmasta-5,22,25-trien-3-O-β-D-glucopyranoside [11] (Fig.  1).  (1H, m), and a de-shielded signal at δ H 5.12 (1H, m) that we assigned to the H-2 of sphingosine, characteristic of ceramides [12] . This spectrum also showed signals of two olefinic protons at δ H 5.55 (1H, m) and 5.49 (1H, m) respectively, in addition to the signals of two alkylmethylenes chains, appearing as multiplets at δ H 1.10 -1.50, confirming that compound 4 possessed a ceramide skeleton [13] . The 13 C NMR spectrum of compound 4 showed signals of an amide carbonyl group (NC=O) at δ C 175.5, one methine linked to the amide group at δ C 52.99 and three oxymethines at δ C 76.86 and 73.03 (overlapping). This spectrum also indicated the existence of two olefinic methine carbons respectively at δ C 130.82 and 130.70, suggesting the presence of a double bond. A signal due to the presence of an oxymethylene group appeared at δ C 62.04. The position of the double bonds (C19 / C20) was identified from fragmentations observed on the electron impact mass spectrum, with the presence of fragments at m/z 57, corresponding to a butyl group and at m/z 83, corresponding to a hexenyl group. Moreover, the geometry (Trans) of the double-bond was assigned based on the chemical shifts of allylic carbons (δ C 33.84 and 33.30). Generally, signals of carbon next to a cis double-bond appear at δ C ≈ 27, while those next to trans double-bonds appear at δ C ≈ 32 [14] .
A comparison of spectroscopic data of compound 4 with those from the literature [15] allowed us to assign to compound 4 the structure of tithoniamide B [15] (Fig. 1).

Conclusion
The present phytochemical study of the dichloromethanemethanol extract of the stem bark of Tetrorchidium didymostemon afforded a fatty alcohol: 1-hentriacontanol, two stigmasterol derivatives: stigmasta-5,24,28-trien-3-ol and stigmasta-5,24,28-trien-3-O-β-D-glucopyranoside and one ceramide derivative: tithoniamide B. The structures of the isolated compounds were established through analysis of their spectroscopic data. These compounds are reported from T. didymostemon for the first time. 1-hentriacontanol has been shown to exhibit interesting antiplasmodial activity against Plasmodium berghei and P. vinckei in mice, and devoid of any toxicity [8] . This could justify the use of T. didymostemon in traditional medicine in the treatment of malaria.