Triterpenoid Saponins from Stem Bark of Pentaclethra macroloba

Duas novas e duas conhecidas saponinas triterpênicas foram isoladas da casca do caule de Pentaclethra macroloba. As estruturas foram determinadas usando uma combinação de técnicas de RMN homo(1D: RMNH, RMNC-{H}, RMNC-DEPT135; 2D: H-H-COSY, H-HTOCSY, H-H-NOESY) and heteronuclear 2D (HMQC and HMBC), espectros de massas obtidos com ionização por pulverização eletrônica (ESIMS) e métodos químicos. As estruturas das duas novas saponinas triterpênicas foram estabelecidas como 3 -O-{[ -D-glicopiranosil-(1 3)-Lramnopiranosil-(1 2)], -D-glicopiranosil-(1 4)}-L-arabinopiranosilhederagenina (3) e ácido 3 -O-{[ -D-glicopiranosil-(1 4)-D-glicopiranosil-(1 3)-L-ramnopiranosil-(1 2)], -Dglicopiranosil(1 4)}-L-arabinopiranosiloleanólico (4).


Results and Discussion
Two known (1 and 2) and two new triterpenoid saponins (3 and 4) were isolated from an ethanolic extract of the stem bark of Pentaclethra macroloba (Leguminosae-Mimosoideae) using a combination of silica gel, Sephadex LH-20 and reverse-phase chromatography (HPLC).The structures were established on the basis of chemical methods and spectral data.The IR spectra of the four compounds showed bands at max 3400 (strong and broad, suggesting several OH groups) and 1700 cm -1 (carboxylic group).Additional spectral data, mainly NMR and ESMS spectra, led us to postulate the structures 1 -4.Oleanolic acid was identified as aglycone of saponins 1, 2 and 3 after acid hydrolysis while hederagenin identified as aglycone of saponin 3 along with the carbohydrates arabinose, rhamnose and glucose for all compounds.
Comparative analysis of the 13 C NMR-HBBD and 13 C NMR-DEPT spectra of the four pentacyclic triterpenoid saponins was used to identify the number of signals attributed to quaternary, methine, methylene and methyl carbon atoms (Tables 1 and 2).
Comparative analysis of the HBBD and DEPT 13 2. Therefore, the structure of the new pentacyclic triterpenoid glycoside was characterized as 3 3) on the basis of spectral data (including evaluation of spin-spin coupling deduced by 1D 1 H NMR analysis involving confirmation by 1 H-1 H-COSY, TOCSY and 1 H-1 H-NOESY) together with the identification of the aglycone hederagenin and sugars obtained (arabinose, rhamnose and glucose) from saponin hydrolysates.
Comparison of the 1 H and 13 C spectral data of the saponin 4 (amorphous colorless solid) with those of 3 (Tables 1 and 2) indicated that the CH 2 OH group [ C 64.11;  , 4h), corresponding to the loss of one hexosyl, two hexosyl, three hexosyl, three hexosyl and one deoxyhexosyl and three hexosyl, one deoxyhexosyl and one pentosyl moieties, respectively, as revealed by the arithmetical analysis summarized in Scheme 1(B).
The hydrogen broad band decoupled HBBD-13 C NMR spectrum of 4 (Tables 1 and 2) showed 59 signals, which were identified as corresponding to quaternary, methine, methylene and methyl carbon atoms by comparative analysis involving the DEPT-13 C NMR spectrum (Tables 1  and 2).The presence of the additional sugar moiety bonded to carbon CH-4 of the glucopyranosyl G2 was deduced from the 2D NMR experiments [heteronuclear HMQC and HMBC (Table 2) and homonuclear 1 H-1 H-COSY, TOCSY and NOESY).The single hydrogen spin systems for each sugar residue were delineated by 1 H-1 H-COSY, TOCSY and HMQC experiments and comparison with those of the depicted methyl pyranosides, 15 taking into account the known effects of glycosidation (Table 2).
The values corresponding to vicinal spin-spin interaction ( 3 J H,H ) between the anomeric hydrogens of arabinopyranosyl [J 7.1 (3) and 6.7 Hz (4)] and glucopyranosyl (J 7.8 to 7.9 Hz) moieties are consistent with axial-axial couplings and, consequently, the configuration of the anomeric carbons was defined as for glucose and for arabinose.

General experimental procedures
NMR spectra were run on a Bruker Advance 500 (500 MHz for 1 H and 125 MHz for 13 C) in pyridine-d 5 (C 5 D 5 N) and residual C 5 D 5 N used as internal references (CH-2/CH-6: H 8.64 and C 149.80).ESI-MSMS data were collected in a triple quadrupole Micromass QuattroLC instrument equipped with a "Z-spray" ion source (Micromass, Wythenshawe, Manchester, UK).A Shimadzu LC10AD HPLC pump was used to deliver methanol-water [7:3]  solutions at 70 L/min to the mass spectrometer.The desolvation and ion source block temperatures were set, respectively, at 350 and 140 o C. Gaseous N 2 was used as nebulizer (80 L/h) and desolvation (450 L/h).The optimal voltages found for the probe and ion source components to produce maximum intensity of the ions [M-H]-were 3.2 kV for the stainless steel capillary, 39 V for the sample cone, and 9 V for the extractor cone.The tandem mass spectrometry experiments were performed by adding Ar in the collision cell to produce a pressure of 2 x 10 -3 mBarr for CAD.The optimal collisional energies (CE) used for decomposition of the ions [M-H] -generated from saponins 1-4 were 35 eV.

Plant material
The stem bark of Pentaclethra macroloba was collected in September 1997 in Macapá, Amapá, Brazil.The plant was identified by Dr. Afrânio G. Fernandes and a voucher specimen (no.EAC-25947) is deposited in the Herbarium Prisco Bezerra of the Departamento de Biologia, Universidade Federal do Ceará, Brazil.

Acid hydrolysis
A solution of the isolated saponin (5 mg) in 2N HCl -MeOH (8 mL) was refluxed for 3 h, the reaction mixture was cooled to room temperature, diluted with H 2 O (20 mL) and extracted with EtOAc.The combined EtOAc extracts were washed with H 2 O, dried over anhydrous Na 2 SO 4 and then evaporated to dryness in vacuo.The aqueous layer was neutralized with aqueous NaOH 2% and concentrated under reduced pressure; the residue was compared with a standard mixture of the sugars arabinose, glucose and rhamnose using silica gel TLC and CH 2 Cl 2 -MeOH-H 2 O(6:4:0.5) as solvent.Furthermore, the mole ratio of each sugar was determined using RI detection in HPLC (Shodex RS pak DC-613, 75% CH 3 CN, 1 mL min -1 , 50 °C) by comparison with authentic samples of the sugars (10 mmol L -1 each of Ara, Glc and Rha).The retention time of each sugar was as follows: Ara 6.0 min; Glc 7.4 min and Rha 4.8 min.

Table 1 .
13, H-1G2) correlated with13C NMR signals at C 105.09, 101.67, 106.97 and 106.90, respectively.11Complete 1 H and13C chemical shift assignments of each sugar unit was achieved by the 1 H-1 H-COSY, TOCSY, 1 H (500 MHz) and13C (125 MHz) spectral data for aglycones of the compounds 3 and 4 including results obtained by heteronuclear 2D shift-correlated HMQC and HMBC spectra, in C 5 D 5 N and TMS as internal standard.Chemical shifts ( , ppm) and coupling constants (J in Hz, in parenthesis) a CH 2 and CH 3 deduced by comparative analysis of HBBD-and DEPT-13 C NMR spectra.Superimposed 1 H signals are described without multiplicity and chemical shifts deduced by HMQC, HMBC and 1 H-1 H-COSY.

Table 2 .
13 (500 MHz) and13C (125 MHz) for the carbohydrate moieties of the compounds 3 and 4 including results obtained by heteronuclear 2D shift-correlated HMQC and HMBC spectra, in C 5 D 5 N as solvent.Chemical shifts ( , ppm) and coupling constants (J, Hz, in parenthesis) a 33Homonuclear 1 H-1 H-COSY,1H-1 H-TOCSY and 1 H-13C-COSY-1 J CH (Table1) spectra were also used for these assignments.Chemical shifts of hydrogen atoms obtained from 1D 1 H NMR spectra.Crbon signals corresponding to C, CH, CH 2 and CH 3 deduced by comparative analysis of HBBD-and DEPT-13 C NMR spectra.Superimposed 1 H signals are described without multiplicity and chemical shifts deduced by HMQC, HMBC, 1 H-1 H-COSY and 1 H-1 H-TOCSY.H 1.57 (d, J 6.2 Hz) with d C 18.74 in the HMQC and both CH-4R ( C 73.05,3J CH ) and CH-5R ( C 70.02, 2 J CH ) in the HMBC, in accordance with the presence of the rhamnose.Other heteronuclear long-range couplings observed in the HMBC spectrum of 3 are summarized in Table