Triterpenoid saponins from stem bark of Pentaclethra macroloba

Abstracts

Two new and two known triterpenoid saponins were isolated from the stem bark of Pentaclethra macroloba. Their structures were determined using a combination of homo- (1D ¹H NMR, 13C NMR-HBBD and 13C NMR-DEPT) and heteronuclear 2D NMR techniques (¹H-¹H-COSY, ¹H-¹H-TOCSY, ¹H-¹H-NOESY, HMQC and HMBC), ESIMS and chemical methods. The structures of the two new pentacyclic triterpenoid saponins were established as 3beta-O-{[beta-D-glucopyranosyl-(1->3)-alpha-L-rhamnopyranosyl-(1->2)],beta-D- glucopyranosyl-(1->4)}-alpha-L-arabinopyranosylhederagenin (3) and 3beta-O-{[beta-D-glucopyranosyl-(1->4)-beta-D-glucopyranosyl-(1->3)-alpha-L-rhamnopyranosyl- (1->2)],beta-D- glucopyranosyl(1->4)}-alpha-L-arabinopyranosyloleanolic acid (4).

Pentaclethra macroloba; Mimosoideae; pentacyclic triterpenoid saponins

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: RMN¹H, RMN13C-{¹H}, RMN13C-DEPT135; 2D: ¹H-¹H-COSY, ¹H-¹H-TOCSY, ¹H-¹H-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 3beta-O-{[beta-D-glicopiranosil-(1->3)-alfa-L-ramnopiranosil-(1->2)],beta-D-glicopiranosil- (1->4)}-alfa-L-arabinopiranosilhederagenina (3) e ácido 3beta-O-{[beta-D-glicopiranosil-(1->4)-beta-D-glicopiranosil-(1->3)-alfa-L-ramnopiranosil- (1->2)],beta-D-glicopiranosil(1->4)}-alfa-L-arabinopiranosiloleanólico (4).

SHORT REPORT

Triterpenoid saponins from stem bark of Pentaclethra macroloba

Francisco Arnaldo Viana^I; Raimundo Braz-Filho^II,*; Yvone B. M. Pouliquen^III; Manoel Andrade Neto^III; Gilvandete M. P. Santiago^III; Edson Rodrigues-Filho^IV

^IDepartamento de Química, Universidade do Estado do Rio Grande do Norte, 59610-210 Mossoró - RN, Brazil

^IISetor de Química de Produtos Naturais - LCQUI - CCT, Universidade Estadual do Norte Fluminense, 28013-600 Campos dos Goytacazes - RJ, Brazil

^IIIDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-970 Fortaleza - CE, Brazil

^IVDepartamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos - SP, Brazil

ABSTRACT

Two new and two known triterpenoid saponins were isolated from the stem bark of Pentaclethra macroloba. Their structures were determined using a combination of homo- (1D ¹H NMR, ¹³C NMR-HBBD and ¹³C NMR-DEPT) and heteronuclear 2D NMR techniques (¹H-¹H-COSY, ¹H-¹H-TOCSY, ¹H-¹H-NOESY, HMQC and HMBC), ESIMS and chemical methods. The structures of the two new pentacyclic triterpenoid saponins were established as 3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)],b-D- glucopyranosyl-(1®4)}-a-L-arabinopyranosylhederagenin (3) and 3b-O-{[b-D-glucopyranosyl-(1®4)-b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl- (1®2)],b-D- glucopyranosyl(1®4)}-a-L-arabinopyranosyloleanolic acid (4).

Keywords:Pentaclethra macroloba, Mimosoideae, pentacyclic triterpenoid saponins

RESUMO

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: RMN¹H, RMN¹³C-{¹H}, RMN¹³C-DEPT135; 2D: ¹H-¹H-COSY, ¹H-¹H-TOCSY, ¹H-¹H-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 3b-O-{[b-D-glicopiranosil-(1®3)-a-L-ramnopiranosil-(1®2)],b-D-glicopiranosil- (1®4)}-a-L-arabinopiranosilhederagenina (3) e ácido 3b-O-{[b-D-glicopiranosil-(1®4)-b-D-glicopiranosil-(1®3)-a-L-ramnopiranosil- (1®2)],b-D-glicopiranosil(1®4)}-a-L-arabinopiranosiloleanólico (4).

Introduction

Pentaclethra macroloba (Willd.) Kuntze, of the family Mimosoideae,^1,2 occurs in the northern region of Brazil, where it is known as "pracaxi" and is used by the local population to treat several afflictions, including snakebites.³ The first chemical and biological investigations on P. macroloba has shown the presence of steroids (b-sitosterol, stigmasterol and campesterol)⁴ and insecticidal properties.^5-7

In this paper we report the isolation and characterization of two known, 3b-O-[b-D-glucopyranosyl-(1®4),a-L-rhamnopyranosyl- (1®2)]-a-L-arabinopyranosyloleanolic acid (1)^8,9 and 3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl- (1®2)],b-D-glucopyranosyl-(1®4)}-a-L-arabinopyranosyloleanolic acid (2),⁸ and two new pentacyclic triterpenoid saponins, 3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl- (1®2)],b-D-glucopyranosyl-(1®4)}-a-L-arabinopyranosylhederagenin (3) and 3b-O-{[b-D-glucopyranosyl-(1®4)-b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl- (1®2)], b-D-glucopyranosyl (1®4)}-a-L-arabinopyranosyloleanolic acid (4). The two known saponins (1 and 2) are described for the first time from this species.

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 n_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 ¹³C NMR-HBBD and ¹³C 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).

The known triterpene saponins 1 and 2 were identified as 3b-O-[b-D-glucopyranosyl-(1®4),a-L-rhamnopyranosyl-(1®2)]-a-L-arabinopyranosyloleanolic acid (1)^8,9 and 3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)], b-D-glucopyranosyl-(1®4)}-a-L-arabinopyranosyloleanolic acid (2)⁸ on the basis of spectral data, especially 1D and 2D NMR and ESIMS, and through comparison with the data available in the literature.^8,9

Compound 3 was obtained as an amorphous powder. The ESIMS (negative ion mode) showed a quasimolecular ion peak at m/z 1073 ([M-H]^-), indicating a molecular weight of 1074 Da consistent with a molecular formula C₅₃H₈₆O_22. Other significant negative ion peaks were observed at m/z 911 ([M-H-162]^-, 3a and 3b), 765 ([M-H-162-146]^-, 3c), 749 ([M-H-162-162]^-, 3d), 603 ([M-H-162-162-146]^- and/or [M-H-162-146-162]^-, 3e) and 471 ([M-H-162-162-162-146-132]^-, 3f), Scheme 1 (A). These peaks were attributed to fragments corresponding to the loss of one hexosyl (m/z 911), one hexosyl and one deoxyhexosyl (m/z 765), two hexosyl (m/z 749), two hexosyl and one deoxyhexosyl (m/z 603) and two hexosyl, one deoxyhexosyl and one pentosyl (m/z 471) radicals (Scheme 1). Based on these data we postulated the elimination of two glucopyranosyl, one rhamnopyranosyl and one arabinopyranosyl moieties to yield the aglycone ion at m/z 471 (hederagenin - H^- = [C₃₀H₄₈O₄ - H]^-= [C₃₀H₄₇O₄]^- , 3f), in accordance with the sugars arabinose, rhamnose and glucose obtained by the acid hydrolysis experiment. The peak at m/z 911 ([M-H-glucopyranosyl]^-, 3a and/or 3b) suggested a terminal glucopyranosyl unit. Additional peaks at 765 ([M-H-glucopyranosyl-rhamnopyranosyl]^- = m/z 911 - rhamnopyranosyl, 3c) and 749 ([M-H-glucopyranosyl-glucopyranosyl, 3d]^- = m/z 911-glucopyranosyl) revealed a branched sugar chain involving a disubstituted arabinopyranosyl moiety (Schemes 1 and 2).^9,10

In fact, the hydrogen broad band decoupled (HBBD) ¹³C NMR spectrum of 3 (Tables 1 and 2), showed 53 signals. Comparative analysis of the HBBD and DEPT ¹³C NMR spectra allowed the identification of signals corresponding to eight quaternary [(C)_8: six sp³ and two sp² of an olefinic double bond at d_C 144.95 and carboxyl group at d_C 180.42 = (C)₇(COOH)], twenty four methine [(CH)₂₄: one sp² and twenty three sp³, including twenty oxygenated = (CH)₄(O-CH)₂₀], fourteen methylene [(CH₂)₁₄: all sp³, including four oxygenated = (CH₂)₁₀(CH₂-O) ₄] and seven methyl [(CH₃)₇] carbon atoms. Consequently, expanded formula (C)₇(COOH)(CH)₄(O-CH)₂₀ (CH₂)₁₀(CH₂-O) ₄[(CH₃)₇ = C₅₃H₇₄O₂₆ was deduced, which after considering the presence of twelve hydroxyl groups and four ether functions was established as C₅₃H₈₆O₂₂ in accordance with the ESMS. The presence of four sugar moieties was confirmed by the HMQC spectrum, which showed the anomeric ¹H NMR signals at d_H 4.90 (d, J 7.1 Hz, H-1A), 6.17 (br s, H-1R), 5.08 (d, J 7.9 Hz, H-1G1) and 5.46 (d, J 7.8 Hz, H-1G2) correlated with ¹³C NMR signals at d_C 105.09, 101.67, 106.97 and 106.90, respectively.¹¹ Complete ¹H and ¹³C chemical shift assignments of each sugar unit was achieved by the ¹H-¹H-COSY, TOCSY, HMQC and the HMBC spectra (Table 2). The ¹H NMR spectrum of 3 displayed six singlet signals of seven methyl groups at d_H 0.91, 0.91, 0.98, 1.00, 1.09 and 1.23, one doublet at d_H 1.56 (J 6.2 Hz), one of olefinic hydrogen in a trisubstituted double bond (d_H 5.45, br s) and one double-doublet (J 13.5, 3.8 Hz) at d_H 3.20 attributed to the carbinolic hydrogen H-3 located at an axial position with a coupling constant value J=13.5 Hz (axial-axial interaction). These data suggested a pentacyclic triterpenoid skeleton type olean-12-ene as aglycone,¹² in accordance with the signals at d_C 122.76 (CH-12) and 144.95 (C-13) observed in the ¹³C NMR spectra (HBBD and DEPT). The ¹H and ¹³C chemical shift assignments of CH-12 and CH-3 were unambiguously made by the cross peaks observed in the HMQC spectrum revealing correlation of the signals at d_H 5.45 (H-12) with d_C 122.76 (CH-12) and d_H 3.20 (H-3) with d_C 81.47 (CH-3), which were confirmed by heteronuclear connectivity to long-range coupling revealed by HMBC spectrum (Table 1). The lower ¹³C chemical shifts of the CH-3 (d_C 81.47), CH-5 (d_C 48.31) and CH₃-24 (d_C 14.26) when compared with those of 1, 2 and 4 [e. g. d_C 90.02 (CH-3), 57.37 (CH-5) and 18.43 (CH₃-24, Table 1] revealed the g-effects,^13,14 of the hydroxy function presence in the hydroxymethylene group HOCH₂-23 [d_C 64.11, d_H 4.30 (d, J 11.7 Hz) and 3.89 (d, J 11.7 Hz)]. This result was confirmed by the HMQC and HMBC spectra (Table 2). The characterization of the [b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)], b-D-glucopyranosyl-(1®4)}-a-L-arabinopyranosyl moiety and its placement at CH-3 were mostly based on the HMBC spectrum, which revealed cross peaks corresponding to heteronuclear spin-spin interaction between (Table 2): a) CH-3 (d_C 81.47) and H-1A (4.90) and CH-1A (d_C 105.09) and H-3 (d_H 3.20); b) CH-1R (d_C 101.67) and H-2A (d_H 4.40); c) CH-4A (d_C 80.79) and H-1G1 (d_H 5.08); d) CH-3R (d_C 83.00) and both H-1R (d_H 6.17) and H-1G2 (d_H 5.46); e) CH-1G2 (d_C 106.90) and H-3R (d_H 4.83). In addition, we observed heteronuclear correlation of methyl signal at d_H 1.57 (d, J 6.2 Hz) with d_C 18.74 in the HMQC and both CH-4R (d_C 73.05, ³J_CH) and CH-5R (d_C 70.02, ²J_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 2.

Therefore, the structure of the new pentacyclic triterpenoid glycoside was characterized as 3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1 ®2)],b-D-glucopyranosyl-(1®4)}-a-L-arabinopyranosyl hederagenin (3) on the basis of spectral data (including evaluation of spin-spin coupling deduced by 1D ¹H NMR analysis involving confirmation by ¹H-¹H-COSY, TOCSY and ¹H-¹H-NOESY) together with the identification of the aglycone hederagenin and sugars obtained (arabinose, rhamnose and glucose) from saponin hydrolysates.

Comparison of the ¹H and ¹³C spectral data of the saponin 4 (amorphous colorless solid) with those of 3 (Tables 1 and 2) indicated that the CH₂OH group [d_C 64.11; d_H 4.30 (d, J 11.7 Hz) and 3.89 (d, J 11.7 Hz)] in 3 was replaced by a methyl group (d_H 1.39 and d_C 29.64) in 4. This hypothesis was confirmed by the ¹³C chemical shifts of the CH-3 at d_C 90.02 (d_C 81.47 in 3), CH-5 at d_C 57.37 (d_C 48.31 in 3) and CH₃-24 at d_C 18.43 (d_C 14.26 in 3), which indicated absence of the g-effects of a hydroxy function of hydroxymethylene group HOCH₂-23 in a 3b-O-glycosylated aglycone.^13,14 Significant additional differences observed in the comparative analysis of the NMR spectra of 3 and 4 were justified by oligosaccharide moiety, containing the triterpenoid 4 the same type and sequence the sugars chain as saponin 3 and one additional glucopyranosyl group (Table 2). These deductions were confirmed by ESIMS (negative ion mode) of 4, which showed a molecular ion peak at m/z 1220 ([M]^-) compatible with a molecular formula C₅₉H₉₆O₂₆ (_.molecular weight 1220 Da). Other significant negative ion peaks were observed at m/z 1057 ([M-163]^-, 4a and/or 4b), 895 ([M-163-162]^-, 4c and/or 4d), 749 ([M-163-162-146]^-, 4e), 733 ([M-163-162-162]^-, 4f), 587 ([M-163-162-162-146]^-, 4g) and 455 ([M-163-162-162-146-132]^-, 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-¹³C 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-¹³C 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 ¹H-¹H-COSY, TOCSY and NOESY). The single hydrogen spin systems for each sugar residue were delineated by ¹H-¹H-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 (³J_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 b for glucose and a for arabinose.

Thus, the structure 3-O-{b-D-glucopyranosyl-(1®4)-b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)]-[ b-D-glucopyranosyl-(1®4)}-a-L-arabinopyranosyloleanolic acid (4) was established.

Experimental

General experimental procedures

NMR spectra were run on a Bruker Advance 500 (500 MHz for ¹H and 125 MHz for ¹³C) in pyridine-d₅ (C₅D₅N) and residual C₅D₅N used as internal references (CH-2/CH-6: d_H 8.64 and d_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 mL/min to the mass spectrometer. The desolvation and ion source block temperatures were set, respectively, at 350 and 140 ºC. Gaseous N₂ 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.

Extraction and isolation of constituents

The dried and powdered stem bark of Pentaclethra macroloba (800 g) was defatted with n-hexane and extracted with ethanol at room temperature. After removal of the solvent by evaporation under reduced pressure, the EtOH extract (26 g) was obtained. This extract was submitted to column chromatography on silica gel eluted with mixtures of CHCl₃-CH₃OH-H₂O (9:1:0.1) increasing the quantity of MeOH, yielding 126 fractions. Fraction 28/36 (2.4 g) was rechromatographed on silica gel column cromatography eluted with CHCl₃-CH₃OH (8:2), yielding saponin 1 (55 mg); fraction 41/52 (430 mg) was solubilized in MeOH (1.5 mL) and precipitated in Me₂CO (3 x 30 mL), yielding 295 mg of a crude saponin mixture, which was chromatographed on a Sephadex LH-20 column eluting by MeOH to yield a white power (160 mg); further separation of this fraction (150 mg) was performed by successive HPLC on a WATERS RP-8 semipreparative (7 mm, 7.8 x 150 mm) eluted with CH₃CN-H₂O [linear gradient 35:65 (v/v) to 65:35 (v/v) for time 25 min] to furnish compounds 2 (47.5mg), 3 (17.5 mg) and 4 (15.1 mg).

3b-O-[b-D-glucopyranosyl-(1®4)-a-L-rhamnopyranosyl-(1®2)]-a-L-arabinopyranosyloleanolic acid (1). Spectral data in agreement with literature values.^8,9

3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)]-b-D-glucopyranosyl-(1®4)}- a-L-arabinopyranosyloleanolic acid (2). Spectral data in agreement with literature values. ⁸

3b-O-{[b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)],b-D-glucopyranosyl-(1®4)}- a-L-arabinopyranosylhederagenin (3). Amorphous colorless solid, mp 261-268 ºC; [a]_D - 3.0º (c 0.5, MeOH); ESIMS: Schemes 1 and 2; ¹H and ¹³C NMR spectral data: Tables 1 and 2.

3b-O-{[b-D-glucopyranosyl-(1®4)-b-D-glucopyranosyl-(1®3)-a-L-rhamnopyranosyl-(1®2)], b-D-glucopyranosyl (1®4)}-a-L-arabinopyranosyloleanolic acid (4). Amorphous colorless solid, mp 245-247 ºC; [a]_D - 0.004º (MeOH); ESIMS: Schemes 1 and 2; ¹H and ¹³C NMR spectral data: Tables 1 and 2.

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 H₂O (20 mL) and extracted with EtOAc. The combined EtOAc extracts were washed with H₂O, dried over anhydrous Na₂SO₄ 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 CH₂Cl₂-MeOH-H₂O(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% CH₃CN, 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.

Acknowledgements

The authors are grateful to CAPES, BNB (Banco do Nordeste do Brasil), CNPq and FAPERJ for grants, to CNPq for fellowship research, Prof. Afrânio Gomes Fernandes for botanical identification of the plant and to the Instituto de Pesquisa do Estado do Amapá-AP (Brazil).

Received: November 3, 2003

Published on the web: May 28, 2004

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  15 Sept 2004
• Date of issue
  Aug 2004

History

• Received
  03 Nov 2003
• Accepted
  28 May 2004