Chemical Constituents from Terminalia glabrescens

Das folhas de Terminalia glabrescens foram obtidos um novo triterpeno pentacíclico (3β,6β,23,28tetraidroxiolean-12-eno), além dos ácidos ursólico, 2α-hidroxiursólico, oleanólico, maslínico, arjunólico, sumaresinólico e asiático, esqualeno, fitol, sitosterol-3-O-β-D-glucopiranosídeo e nalcanos. Da casca do caule foram obtidos friedelina, taraxerol, lupeol, lupenona, betulina, betulona, ácido betulínico, arjunglucosídeo I, estigmastano-3β,6α-diol, β-sitosterol, (-) catequina, β-Dpiranotagatose, β-D-furanofrutose e α-D-furanofrutose.

The structures of the known and new compounds were established on the basis of spectral data, mainly 1 H and 13 C (1D and 2D) NMR spectra and by comparison with authentic samples.

Results and Discussion
The hexane and CHCl 3 solubles obtained from partition of the ethanol extract of leaves were subjected to a series of normal and reversed phase silica gel column chromatography, gel filtration and preparative TLC on silica gel separations to yield the new pentacyclic triterpene 3β,6β,23,28-tetrahydroxyolean-12-ene (1) in addition to ursolic, 9 oleanolic, 9 2α-hydroxyursolic, 9 maslinic, 9 sumaresinolic (2), 9 asiatic (3) 9 and arjunolic (4) 9 acids, squalene, 10 phytol, sitosterol-3-O-β-D-glucopyranoside and long chain hydrocarbons. These were characterized as n-alkanes in the range between C 18 and C 33 , with a large predominance of chains with odd numbers of carbon atoms, where C 29 and C 31 were found as the main homologues. The isolation of squalene and of the triterpene 2 in the J. Braz. Chem. Soc.
genus Terminalia is being reported for the first time. The known compounds were identified by their 1 H and 13 C NMR spectral data, by comparison with literature values and/or with authentic samples. Identification of 2 as well as the isomeric triterpenes maslinic / 2α-hydroxyursolic acids and 3 / 4 was supported by conversion into their corresponding C-28 methyl ester derivatives whose 1 H and 13 C NMR resonances were in accordance with reported data. 9,11 The alkane composition was determined on the basis of GC-FID retention times and by comparison with authentic standards.
Compound 1 was obtained as an amorphous solid and its HBBD 13 C NMR spectrum displayed signals for 30 carbon atoms. With the aid of information afforded by the DEPT spectra these signals could be attributed to seven quaternary, six methine, eleven methylene and six methyl carbon atoms. The presence of a trisubstituted double bond was inferred by the signals of a methine carbon at δ 123.0 and a quaternary carbon at δ 144.4. In the HMQC spectrum a cross-peak correlation was observed between the former carbon signal and the broad hydrogen singlet at δ 5.57, which was assigned to the vinylic hydrogen. In the 1 H NMR spectrum, the signals at δ 5.04 (br s) and 4.26 (dd, J 11.4 and 4.1 Hz) which showed connectivities in the HMQC spectrum with the carbon signals at δ 67.7 and 73.4, respectively, were attributed to two carbinolic hydrogens. Similarly, the broad singlet at δ H 4.06 (2H) and the two doublets at δ H 4.38 (1H, J 10.5 Hz) and 4.03 (1H, J 10.5 Hz), which showed cross-peak correlations with the carbon signals at δ 64.5 and 67.2, respectively, were assigned to hydroxymethylene hydrogens. These information, along with the absorption at ν max 3429 cm -1 observed in the IR spectrum, led to the assumption that 1 was an olean-12-ene-type triterpene with two hydroxymethylene and two secondary hydroxyl groups and its molecular formula established as C 30 H 50 O 4 . The aforementioned data when compared with those of other known structurally related compounds suggested that 1 would have the same functionality on rings A and B as 3β, 6β, 23trihydroxyolean-12-en-28-oic acid (5) previously isolated from Timonius timon (Rubiaceae). 12 Indeed, the 1 H and 13 C NMR spectra of 1 showed close resemblance with those of 5, except for the signals due to the carboxylic group at C-28 observed in the spectra of the latter, which were replaced by a singlet at δ H 4.06 (2H), indicative of a hydroxymethylene group at C-28. Unambiguous assignments of the hydroxymethine carbons C-3 and C-6 (δ 73.4 and 67.7, respectively) were established on the basis of connectivities observed from an HMBC experiment (Table 1). Accordingly, cross-peak correlations between the carbon signals of C-23 and C-24 and H-3 resonance at δ 4.26, which in turn displayed one-bond 1 H-13 C connectivity with the carbon signal at δ 73.4 allowed the assignments of C-3/H-3. In a similar fashion, H-6 (δ 5.04) presented long-range correlations with C-7, C-8 and C-10. The appearance of H-6 as a broad singlet in the 1 H NMR spectrum indicated its α-equatorial orientation. A similar feature was also observed for the signal of H-6 in sumaresinolic acid methyl ester 2a [δ H 4.53 (br s)] and in 6β-hydroxymaslinic acid [δ H 4.85 (s)] 13 which bear the same stereochemistry as H-6 in 1 and 5. The β-hydroxyl substitution at C-3 was inferred by the chemical shift and multiplicity of the axial H-3 observed as a double doublet at δ 4.26 (J 11.4 and 4.1 Hz). Thus, compound 1 was characterized as 3β,6β,23,28-tetrahydroxyolean-12-ene. Further evidence for the structure of 1 was provided by additional two-and three-bond correlations discernible in the HMBC spectrum (Table 1). After acquisition of its spectroscopic data and storage at room temperature, however, compound 1 was decomposed to a mixture of oxidation products, as revealed by TLC and IR spectroscopy. This fact prevented further analysis of 1 by ESIMS. After a series of column chromatography separations on silica gel of the hexane and CHCl 3 solubles, obtained from partition of the ethanol extract from the trunk bark, seven triterpenes were isolated, together with stigmastane-3β-6α-diol (7), β-sitosterol, (-) catechin, β-D-tagatose 14 and αand β-D-fructose. 14 The structures of these triterpenes have been established as friedelin, 9 lupenone, 9 lupeol, 9 betulone, 15 betulin, 9 betulinic acid 9 and taraxerol, 9,16 on the basis of spectral analyses and by comparison with previously reported data. In spite of the wide distribution of these compounds in other plant genera, only few records are available for the presence of friedelan-and lupanetype triterpenoids in Terminalia, which is well known for the occurrence of triterpenes with oleanane and ursane skeletons. 8,9 On the other hand, no records related to the isolation of taraxarane-type triterpenes, e.g. taraxerol, have hitherto been reported in this genus.
Compound 6 was identified by means of 1 H and 13 C NMR as arjunglucoside I, a triterpene glucoside previously characterized in several species of Terminalia (e.g., T. arjuna 17 and T. bellerica 18 ).
The structure of 7 was shown to be of stigmastane-3β-6α-diol on the basis of its 1 H and 13 C NMR spectral data, which were in accordance with those reported for the same steroid previously isolated from Trichosantes kirilowii (Cucurbitaceae), 19 Spatholobus suberetus (Leguminosae) 20 and Urtica dioica (Urticaceae) 21 and until now, not yet described in Combretaceae.

Plant material
The leaves and trunk bark of Terminalia glabrescens Mart. were collected in Campo Grande, Mato Grosso do Sul, Brazil, in June, 1996. The plant was identified by Dr. Nilda Marquette (Jardim Botânico do Rio de Janeiro, RJ, Brazil) and a voucher specimen, 11264, is deposited in the Herbarium of the Universidade Federal de Mato Grosso do Sul.

Extraction and isolation of chemical constituents
Air-dried and powdered leaves (1.9 kg) were extracted at room temperature with EtOH. The residue obtained from the EtOH extract was subsequently partitioned between EtOH-H 2 O (9:1) and hexane and EtOH-H 2 O (1:1) and CHCl 3 . The hexane phase (11.9 g) was subjected to CC on silica gel (70-230 mesh), eluted with a gradient of hexane-CHCl 3 -EtOAc-MeOH resulting in 43 frs. of 125 mL each. Fraction 1 consisted of a mixture (38 mg) of C 18 -C 33 nalkanes, while fraction 2 yielded squalene (48 mg). Air-dried and powdered trunk bark (1 kg) was extracted with EtOH at room temperature to obtain an EtOH extract which, after concentration under reduced pressure, was partitioned successively between MeOH-H 2 O (9:1) and hexane and between MeOH-H 2 O (1:1) and CHCl 3 .
The hexane phase (4.5 g) was chromatographed on a silica gel (70-230 mesh) column eluted with increasing amounts of EtOAc in hexane to obtain 35 fractions of 100 mL each.
Fraction 7 was treated with hot benzene. The insoluble residue was then washed with acetone to yield a precipitate and a supernatant fraction. The former was further chromatographed on a silica gel (230-400 mesh) column, with a gradient of hexane-EtOAc to give friedelin (25 mg) and taraxerol (29 mg). In a similar fashion, the acetonesoluble fraction afforded lupeol (10 mg) and 7 ( 8 mg).