6beta-hydroxymaslinic acid, a triterpene from Vochysia ferruginea

Abstracts

A novel oleane acid was isolated from the leaves and the fruits of Vochysia ferruginea. The structure of the new triterpenoid was elucidated by NMR spectroscopy as 2alpha,3beta,6beta-trihydroxyolean-12-en-28-oic acid (6beta-hydroxymaslinic acid, 1). In addition, beta-sitosterol-glucoside and three mixtures containing known triterpenoids, uvaol and erythrodiol, ursolic and oleanolic acids, 2alpha,3beta-dihydroxyurs-12-en-28-oic acid and its respective oleanolic isomer (maslinic or crategolic acid), were isolated from the leaves and the fruits of Vochysia ferruginea. In the fruits, bellericagenin A and its (28 -> 1) beta-D-glucopyranosyl ester (bellericaside A) were present in high amount.

Vochysia ferruginea; Vochysiaceae; pentacyclic triterpenes; 6beta-hydroxymaslinic acid

Um novo ácido triterpenóide pentacíclico foi isolado a partir de folhas e frutos de Vochysia ferruginea. A estrutura do novo composto foi elucidada por espectroscopia de RMN como sendo o ácido 2alfa,3beta,6beta-triidroxiolean-12-en-28-óico (ácido 6beta-hidroximaslínico, 1). Além do novo triterpenóide, foram isolados das folhas e frutos o glicosídeo de beta-sitosterol e três misturas contendo os triterpenóides conhecidos uvaol e eritrodiol, ácidos ursólico e oleanólico e os ácidos 2alfa,3beta-diidroxiurs-12-en-28-óico e o respectivo isômero oleanólico (ácido maslínico ou cratególico). Nos frutos, a bellericagenina A e o seu éster (28 -> 1) beta-D-glicopiranosílico (bellericasídeo A) estão presentes em elevados teores.

Article

6b-Hydroxymaslinic Acid, a Triterpene from Vochysia ferruginea

Yasmin L. Zucaro Z.^a, Reinaldo S. Compagnone^a, Sonia C. Hessb^* and Franco Delle Monache^c

ªEscuela de Quimica, Universidad Central de Venezuela, Caracas, Venezuela

^bDepartamento de Hidráulica e Transportes/CCET, Universidade Federal de Mato Grosso do Sul, CP 549, 79070-900, Campo Grande - MS, Brazil

^cCentro Chimica dei Recettori del C.N.R., Istituto di Chimica e Chimica Clinica, U.C.S.C., Largo Francesco Vito 1, 00168 , Roma, Italy.

Um novo ácido triterpenóide pentacíclico foi isolado a partir de folhas e frutos de Vochysia ferruginea. A estrutura do novo composto foi elucidada por espectroscopia de RMN como sendo o ácido 2a,3b,6b-triidroxiolean-12-en-28-óico (ácido 6b-hidroximaslínico, 1). Além do novo triterpenóide, foram isolados das folhas e frutos o glicosídeo de b-sitosterol e três misturas contendo os triterpenóides conhecidos uvaol e eritrodiol, ácidos ursólico e oleanólico e os ácidos 2a,3b-diidroxiurs-12-en-28-óico e o respectivo isômero oleanólico (ácido maslínico ou cratególico). Nos frutos, a bellericagenina A e o seu éster (28 ® 1) b-D-glicopiranosílico (bellericasídeo A) estão presentes em elevados teores.

A novel oleane acid was isolated from the leaves and the fruits of Vochysia ferruginea. The structure of the new triterpenoid was elucidated by NMR spectroscopy as 2a,3b,6b-trihydroxyolean-12-en-28-oic acid (6b-hydroxymaslinic acid, 1). In addition, b-sitosterol-glucoside and three mixtures containing known triterpenoids, uvaol and erythrodiol, ursolic and oleanolic acids, 2a,3b-dihydroxyurs-12-en-28-oic acid and its respective oleanolic isomer (maslinic or crategolic acid), were isolated from the leaves and the fruits of Vochysia ferruginea. In the fruits, bellericagenin A and its (28 ® 1) b-D-glucopyranosyl ester (bellericaside A) were present in high amount.

Keywords: Vochysia ferruginea, Vochysiaceae, pentacyclic triterpenes, 6b-hydroxymaslinic acid

Introduction

The genus Vochysia (Vochysiaceae) seems to be an abundant source of triterpenoids. Bartogenic and vismiaefolic acids have been reported from V. vismiaefolia¹; betulinic, 4-epi-vismiaefolic, and 2a,3b,19a-trihydroxy-24-oxo-urs-12-en-28-oic acids from V. piramidalis².

From the stem bark of V. divergens we isolated the terpenoids b-sitosterol, betulinic, sericic, divergioic and 24-hydroxytormentic acids, and the (28 ® 1) b-D-glucopyranosyl ester of later^3-5. Sericic acid (2a,3b,19,24-tetrahydroxyolean-12-en-28-oic acid) presented antifungal³ and antibacterial⁴ activities. 24-Hydroxytormentic acid showed antinociceptive action⁶.

V. ferruginea is a tree commonly found in wet soils of Venezuelan Amazon⁷. In this communication we report the structure elucidation of 6b-hydroxymaslinic acid, 1, a novel triterpenoid of the oleane series obtained from the leaves and the fruits of V. ferruginea. b-sitosterol-glucoside and mixtures of uvaol and erythrodiol, ursolic and oleanolic acids, 2a,3b-dihydroxyurs-12-en-28-oic acid and its respective oleanolic isomer (maslinic or crategolic acid) were also obtained. Bellericagenin A and its (28 ® 1) b-D-glucopyranosyl ester (bellericaside A) were present in high amount in the fruits.

Experimental

General experimental procedures

Melting points were determined using a Kofler hot-stage instrument and were uncorrected. IR spectra were measured on a Perkin-Elmer 1320 spectrometer. ¹H and ¹³C-NMR spectra were recorded in CDCl₃ or in pyridine-d₅, using TMS as internal reference, employing a Varian Gemini 300 (¹H, 300 MHz; ¹³C, 75 MHz) spectrometer. Optical rotations were measured in a Polamat A (Carl Zeiss) polarimeter. Mass spectra were recorded on a HP 5988A apparatus (EI, 70 eV). Isolation procedures were monitored by employing thin-layer chromatography on pre-coated silica gel plates (Merck, Kieselgel 60 F-254).

Plant material

Vochysia ferruginea Mart (Vochysiaceae) was collected in the margins of Cataniapo River near Puerto Ayacucho (Amazona State, Venezuela) and identified by Dr. A. Castillo. A voucher specimen is deposited in the Herbarium of the Escuela de Biologia, Universidad Central de Venezuela, Caracas, under number AC 3301.

Extraction and isolation

Air dried and powdered leaves (400 g) were exhaustively extracted at room temperature with CHCl₃ to give after evaporation a residue (8.0 g), which was purified by CC (SiO₂ ; CHCl₃ with increasing amounts of MeOH). The first three fractions (eluted with CHCl₃ - MeOH, 49:1, 19:1, and 9:1, respectively) gave unseparated mixtures of uvaol and erythrodiol (38 mg), of ursolic and oleanolic acids (280 mg), and of 2a,3b-dihydroxyurs-12-en-28-oic and 2a,3b-dihydroxyolean-12-en-28-oic acids (maslinic or crategolic acid, 220 mg). After repeated chromathography, the fourth fraction also eluted with CHCl₃-MeOH, 9:1, yielded b-sitosterol-glucoside (200 mg) and 6b-hydroxymaslinic acid, 1 (92 mg).

The crushed fruits (196 g) were exhaustively extracted with hot CHCl₃-EtOH,1:1 in Soxlet apparatus. The residue (5.7 g) was suspended in cold MeOH and filtered. By crystallization (MeOH) the solid material gave bellericagenin A (2.8 g). The MeOH soluble portions (1.6 g) were purified by CC (SiO₂ ; EtOAcwith increasing amounts of MeOH). Oleanolic acid (20 mg) and 1 (40 mg) were eluted with EtOAc, further bellericagenin A (1.1 g; total yield, 2% of the fruits) with EtOAc-MeOH, 97:3. The last eluted fraction (EtOAc-MeOH,19:1) yielded bellericaside A after washing with cold acetone (50mg). Methylation of 1 with ethereal diazomethane afforded the monomethyl ester 1a.

6b-Hydroxymaslinic acid, 1

Mp 190-192 ^oC; [a]_D²⁰ +35^o (c = 3, MeOH); ¹H NMR of 1 (300 MHz, C₅D₅N), d1.83 and 1.29 (H-1a and H-1b), 4.28 (H-2, dt, J 9.5 and 4.5 Hz), 3.42 (H-3, d, J 9.5 Hz), 1.18 (H-5), 4.85 (H-6), 1.86 (H-7), 1.99 (H-9), 5.57 (H-12, br t), 2.20 (H-16), 3.33 (H-18, dd, J 14 and 4 Hz), 2.32 and 1.40 (H-19a and H-19b), 1.85 (H-22), 1.46 (H-23, s), 1.78 (H-24, s), 1.71 (H-25, s), 1.61 (H-26, s), 1.30 (H-27, s), 0.96 (H-29), 1.02 (H-30); ¹³C NMR (75MHz) of 1 in C₅D₅N (Table 1); Methylation of 1 with ethereal diazomethane, 0^o C, afforded the mono methyl ester 1a. EIMS of 1a (70 eV), (m/z, %): 502 (M⁺, 5), 484 (M-H₂O, 4), 442 (M-HCOOMe, 6), 262 (77), 249 (12), 203 (100), 189 (40) (Figure 1); ¹H NMR of 1a (300 MHz,CDCl₃) d 5.33 (H-12), 4.53 (H-6), 3.73 (H-2), 3.63 (OMe), 2.95 (H-3), 2.89 (H-18), 1.37, 1.25, 1.20, 1.10, 1.08, 0.93 and 0.90 (7Me); ¹³C NMR of 1a (75 MHz, CDCl₃) d 178.2 (C-28), 142.9 (C-13), 122.5 (C-12), 83.7 (C-3), 68.7 (C-2), 68.4 (C-6), 55.6 (C-5), 51.5 (OMe), ^a48.4 (C-19), 47.8 (C-9), ^a46.6 (C-1), 45.7 (C-17),42.1 (C-14), 41.2 (C-18), 40.6 (C-7), 39.7 (C-8), 38.4, 37.8 (C-10; C-4), 33.8 (C-21), 33.1 (Me-29), 32.3 (C-22), 30.6 (C-20), 28.3 (Me-23), 27.5 (C-15), 25.9 (Me-27), 23.5 (Me-30), 23.3 (C-16), 22.9 (C-11), 18.1 (Me-24, Me-25, Me-26) (^a Interchangeable values).

2a,3b,7a,23-Tetrahydroxyolean-12-en-28-oic acid (Bellericagenin A)

¹H NMR (300 MHz, C₅D₅N) d 5.57 (br s, H-12), 5.10 (br s, H-7), 4.41 (m, H-2, H-23 a), 4.25 (d, J 10.4 Hz, H-3), 4.08 (d, J 11.8 Hz, H-23 b), 3.32 (br dd, H-18), 1.78 (Me-25), 1.74 (Me-24), 1.63 (Me-26), 1.23 (Me-27), 1.00 (Me-30), 0.93 (Me-29) (data not available in the literature, in C₅D₅N); ¹³C NMR (75 MHz, C₅D₅N) d 180.0 (s, C-28), 144.0 (s, C-13), 122.7 (d, C-12), 78.1 (d, C-3), 68.8 (d, C-2), 67.4 (d, C-7), 65.3 (t, C-23), 49.9 (t, C-1), 48.7 (d, C-5), 48.6 (d, C-9), 46.5 (s, C-17), 46.2 (t, C-19), 44.4 (s, C-4), 42.6 (s, C-14), 41.9 (d, C-18), 39.1 (s, C-8), 38.0 (s, C-10), 34.0 (t, C-21), 33.1 (q, Me-29), 33.1 (t, C-22), 30.8 (s, C-20), 28.1 (t; C-6, C-15), 26.1 (q, Me-27), 23.9 (t, C-11), 23.6 (q, Me-30), 23.5 (t, C-16), 18.8 (q, Me-25), 18.4 (q, Me-26), 15.8 (q, Me-24). Some selective INEPTL experiments: irradiation at d 5.57 (H-12) gave response for d 42.6 (C-14), 48.6 (C-9) and 41.9 (C-18); irradiation at d 3.32 (H-18) gave response for d 144.0 (C-13) and 46.5 (C-17); irradiation at d 1.74 (Me-24) gave response for d 44.4 (C-4), 78.0 (C-3) and 65.9 (C-23); irradiation at d 1.63 (Me-26) gave response for d 42.6 (C-14) and 39.1 (C-8); irradiation at d 1.23 (Me-27) gave response for d 144.0 (C-13), 42.6 (C-14) and 39.1 (C-8).

Bellericaside A

¹H NMR (300 MHz, C₅D₅N) d 5.62 (H-12), 5.52 (H-1'), 5.09 (H-7), 4.44-3.95 (H2-23), 3.22 (H-18), 1.80 (Me-25), 1.76 (Me-24), 1.73 (Me-23), 1.19 (Me-27), 0.88, 0.87 (Me-30, Me-29) (data not available in the literature, in C₅D₅N); ¹³C NMR (75 MHz, C₅D₅N) d 176.2 (C-28), 143.3 (C-13), 123.6 (C-12), 95.6 (C-1'), 79.0, 78.5 (C-3', C-5'), 73.9 (C-2'), 70.9 (C-4'), 61.9 (C-6'), 46.8 (C-17), 46.0 (C-19), 41.6 (C-18), 32.3 (C-22) and the other values practically identical to those of bellericagenin A.

Other known compounds

Uvaol and erythrodiol, ursolic and oleanolic acids, and 2a,3b-dihydroxyurs-12-en-28-oic acid and 2a,3b-dihydroxyolean-12-en-28-oic acid (maslinic acid or crategolic acid) were identified by the comparison of ¹³C NMR data with those reported in the literature⁸. Oleanolic acid and b-sitosterol glucoside by comparison with authentic samples available in our laboratory.

Results and Discussion

Upon repeated column chromatography the CHCl₃ extract of the leaves afforded b-sitosterol glucoside and isomeric mixtures of uvaol/erythrodiol, ursolic/oleanolic acids, and 2a,3b-dihydroxyurs-12-en-28-oic/maslinic acids, as well as a single compound characterized as 6b-hydroxymaslinic acid, 1, which was obtained as a dextrorotatory powder. The molecular ion m/z 502 of monomethyl ester 1a and NMR data of 1 are consistent with the molecular formula C₃₀H₄₈O₅. The complete ¹H and ¹³C assignments for 6b-hydroxymaslinic acid, 1, are reported in Experimental and in Table 1 as a result of APT and HETCOR experiments and some INEPT measurements. Preliminary inspection of the data disclosed the presence of seven tertiary methyls, three oxymethine groups (d_C 83.9, 68.2 and 67.4) and a trisubstituted double bond (d_C 144.0 and 122.7; d_H 5.57, br t). These findings clearly suggested a trihydroxyolean-12-en-28-oic gross structure for 6b-hydroxymaslinic acid (1) rather than that of an urs-12-en derivative⁹. The ¹H NMR spectrum disclosed the presence of a doublet (J 9.5 Hz) at d 3.42 (H-3a) suggesting that two of hydroxy groups are in the 2a,3b positions¹⁰. This assumption was confirmed by the comparison of ¹³C NMR parameters for ring A carbons with those of 2a,3b-dihydroxyolean-12-en-28-oic acid¹¹ (maslinic acid; Table 1). Analogously, the third hydroxyl was assigned 6b comparing the ¹³C NMR data for B-E ring carbons with those of 2a,3b,6b,23-tetrahydroxyolean-12-en-28-oic acid¹² (terminolic acid; Table 1) and 2a,3b,6b,19a-tetrahydroxyurs-12-en-28-oic acid¹³ (compound A, Table 1). Signals of carbons 24, 25 and 26 in ¹³C NMR spectrum of 1 are deshielded in respect to maslinic acid (Table 1), which can be justified by d effect of the hydroxyl group on 6b. Therefore, 6b-hydroxymaslinic acid, 1, was characterized as 2a,3b,6b-trihydroxyolean-12-en-28-oic acid.

Most of the assignments of Experimental and Table 1 have been confirmed by selective INEPT experiments (Table 2); in particular, the selective irradiation of the broad singlet at d 4.85 (H-6a) gave a response on the signals at 39.2 and 38.3 ppm, C-4 and C-10, respectively.

Finally, the relative configuration of the molecule was checked by Difference NOE spectra. Mutual enhancement of the signals were observed among H-3, Me-23, H-5 and H-6a, as well as among H-2b, Me-24 and Me-25.

Methylation of 1 with ethereal diazomethane afforded the monomethyl ester 1a. MS spectrum of 1a presented the expected Retro Diels-Alder fragmentation pattern¹⁴ (Figure 1). The ¹H NMR and ¹³C NMR spectra (see Experimental) were also in accordance with the structure proposed for 1a.

Examination of the fruits of Vochysia ferruginea led to the isolation in high yield of bellicagenin A (see Experimental) and its (28 ® 1) b-D-glucopyranosyl ester (bellericaside A) previously found in Terminalia bellerica (Combretaceae)¹⁵. On the basis of HETCOR, APT and INEPTL experiments the signals for C-1, C-9, C-17, C-19, C-4, C-14 and C-18 of bellericagenin A have been reversed with respect to literature¹⁵.

Acknowledgements

The authors are grateful to Dr. A. Castillo for the botanical identification of the plant material.

Received: July 07, 1999

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Publication Dates

• Publication in this collection
  06 Nov 2000
• Date of issue
  June 2000

History

• Received
  07 July 1999