Triterpenic acids from Eugenia moraviana

Abstracts

A novel triterpene, characterized as 6alpha-hydroxybetulinic acid, was isolated from the leaves and stems of Eugenia moraviana (Myrtaceae), known in Brazil as Cambuí, together with three known compounds, platanic acid, betulinic acid and beta-sitosterol. Unequivocal ¹H and 13C assignments of 6alpha-hydroxybetulinic acid (3beta,6alpha-dihydroxy-20(29)-lupen-28-oic acid) and platanic acid were undertaken by spectral analysis including NOE and 2 D NMR experiments.

Eugenia moraviana; 6alpha-hydroxybetulinic acid; platanic acid; betulinic acid

Um novo triterpeno caracterizado como ácido 6alfa-hidroxibetulínico foi isolado das folhas e caule da planta, vulgarmente conhecida no Brasil como Cambuí, Eugenia moraviana (Myrtaceae), juntamente com três outras substâncias conhecidas, identificadas como ácido platânico, ácido betulínico e beta-sitosterol. Através da análise de espectros incluindo NOE e experimentos de RMN em duas dimensões foi realizada a atribuição inequívoca dos deslocamentos químicos de ¹H e de 13C do ácido 6alfa-hidroxibetulínico(ácido-3beta,6alfa-diidróxi-20(29)-lupen-28-óico) e do ácido platânico.

Article

Triterpenic Acids from Eugenia moraviana

Inês Lunardi, Juliana L. B. Peixoto, Cleuza C. da Silva, Ivânia T. A. Shuquel, Ernani A. Basso and Gentil J. Vidotti^*

Departamento de Química, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900, Maringá - PR, Brazil.

Um novo triterpeno caracterizado como ácido 6a-hidroxibetulínico foi isolado das folhas e caule da planta, vulgarmente conhecida no Brasil como Cambuí, Eugenia moraviana (Myrtaceae), juntamente com três outras substâncias conhecidas, identificadas como ácido platânico, ácido betulínico e b-sitosterol. Através da análise de espectros incluindo NOE e experimentos de RMN em duas dimensões foi realizada a atribuição inequívoca dos deslocamentos químicos de ¹H e de ¹³C do ácido 6a-hidroxibetulínico(ácido-3b,6a-diidróxi-20(29)-lupen-28-óico) e do ácido platânico.

A novel triterpene, characterized as 6a-hydroxybetulinic acid, was isolated from the leaves and stems of Eugenia moraviana (Myrtaceae), known in Brazil as Cambuí, together with three known compounds, platanic acid, betulinic acid and b-sitosterol. Unequivocal ¹H and ¹³C assignments of 6a-hydroxybetulinic acid (3b,6a-dihydroxy-20(29)-lupen-28-oic acid) and platanic acid were undertaken by spectral analysis including NOE and 2 D NMR experiments.

Keywords: Eugenia moraviana, 6a-hydroxybetulinic acid, platanic acid, betulinic acid

Introduction

Eugenia moraviana Berg. (Myrtaceae) is a tree that occurs in South America, mainly in Brazil, Argentina and Paraguai¹. The leaves of some Eugenia species are used in folk medicine for several therapeutics finalities^2,3. Compounds such as flavonoids, triterpenes, tannins and especially essential oils constituted of monoterpenes and sesquiterpenes have already been isolated from the genus Eugenia^4-6. However, no reports were found on the Eugenia moraviana constituents. Our phytochemical studies on the leaves and stems of E. moraviana resulted in the isolation of a new lupane-type triterpenoid, which was characterized as 6a-hydroxybetulinic acid (1) together with three known compounds identified as platanic acid (2), betulinic acid and b-sitosterol⁷. Compound 2 and betulinic acid have been attracting much attention in natural products chemistry because they present biological activities, e.g. anti-HIV⁸, antitumor promoter⁹, antimalarial and anti-inflammatory activities^10,11. Previous assignments of ¹³C signals ^8,12,13 led to divergence between the C-18 and C-19 chemical shifts of compounds 1 and 2 and also between the C-15 and C-21 chemical shifts of 2. In the present study we used techniques like COSY, HETCOR, HETCORLR and NOE difference, acquired at 300 MHz (¹H), to report an unequivocal and complete ¹H and ¹³C assignment of compounds 1 and 2. Peng and cols ¹⁴ reported the betulinic acid resonance assignment through a combination of high resolution NMR spectroscopy and a computer-assisted structure elucidation expert system. The authors run ¹³C/DEPT, DQF COSY, HMBC, HMQC and NOESY spectra, acquired with a 720 MHz (¹H) spectrometer.

Results and Discussion

Structural elucidation and NMR signal assignments of 6a-hydroxybetulinic acid (1)

Compound 1, mp 285-288 °C; was isolated as white crystals. Its EIMS spectrum showed a molecular peak at m/z 472 corresponding to the formula C₃₀H₄₈O₄ and other peaks at m/z 248, 203, 187 and 175, which are characteristic for a pentacyclic triterpene skeleton of the lupane series¹⁵. The IR spectrum showed absorptions due to hydroxyl (3454 cm^-1), carboxyl (1685 cm^-1) and exo-methylene (1639 and 879 cm^-1) groups. The complete and unequivocal ¹H and ¹³C chemical shifts assignments of 1 were assisted by DEPT, COSY (¹H x ¹H), HETCOR (¹³C x ¹H) and HETCORLR (¹³C x ¹H) spectra (Table 1). The ¹³C NMR spectral data of 1 were compared with those from 6b-hydroxybetulinic acid¹⁶. According to our results the C-17 was erroneously assigned and the C-15/C-21 and C-18/C-19 were interchanged. The antiperiplanar coupling (J_6,7a and J_6,5a) of 10.4 Hz and the synclinal coupling (J_6,7e) of 3.8 Hz for H-6 proton at d 4.35, indicated that the hydroxyl group at C-6 is a-orientation. The NOE difference NMR experiment was also performed to confirm the hydroxyl group orientation at C-6. Irradiation of the H-6 signal at d 4.35 produced an enhancement in the methyl protons resonances at d 1.45 (H-24, 5.0%), 0.96 (H-25, 6.5%) and 1.19 (H-26, 5.9%), which reveals a coaxial relationship between H-6, H-24, H-25 and H-26 thus confirming the equatorial position of the hydroxyl group at C-6. According to the observed NOE enhancements the structure of compound 1 should be as shown in Figure 1.

Assignments of C-15, C-18, C-19 and C-21 in the platanic acid (2)

To unequivocally assign carbons 15, 18, 19 and 21 in compound 2 the NOE difference spectra and a HETCOR spectrum were obtained. Initially the proton at d 3.71 was irradiated and a NOE enhancement was observed at d 2.24 (H-29). Irradiation of the signal at d 2.40 provided NOE enhancements at d 1.61(Ha-16) and 1.10 (H-27).

Interpretation of the observed NOE enhancements suggests a structure for 2 as shown in Figure 2 that shows all protons mentioned above in 1,3-diaxial interaction.

After the interpretation of the NOE enhancements we were able to identify carbons 18 and 19 in the HETCOR spectrum. The proton at d 2.40 (H-18) correlates with the carbon at d 49.9, and the proton at d 3.71 (H-19) correlates the carbon at d 52.1. To assign carbons C-15 and C-21, the proton at d 2.53 (H-13) was irradiated and the NOE enhancement was observed on the protons at d 1.57 (Ha-15) and 1.03 (H-26). Those protons are on 1,3-diaxial positions with respect to H-13 (see Figure 2). The HETCOR spectrum indicated the correlation between the proton at d 1.57 (H-15) and the carbon at d 28.8. The carbon at d 30.3, that showed no correlation, must be C-21.

The complete assignments for the ¹H and ¹³C NMR signals, as well the ¹H x ¹H and ¹³C x ¹H correlations of 2 are reported in Table 2.

Experimental

General

Mps: uncorr. IR: KBr pellet; EIMS: 70 eV; ¹H (300 MHz) and ¹³C (75,5 MHz) NMR spectra were obtained in pyridine-d₅ with TMS as internal reference; CC: silica gel 60 (70-230 mesh) and silica gel 60 (230-400 mesh); TLC: silica gel F254 (0.25 mm in thickness).

Plant material

Leaves and stems of the plant were collected in September 1996, at the edge of Paraná river, Porto Rico city, State of Paraná, Brazil and identified by Dr. Graziela Maciel Barroso, Jardim Botânico do Rio de Janeiro and Dr. Lucia Elena Soares e Silva, Universidade Federal de Brasilia. A voucher specimen (HUM 2163) was kept at the herbarium of the Biological Department of the Universidade Estadual de Maringá.

Isolation

Air-dried and powdered leaves and stems (920 g) of E. moraviana Berg., were extracted with 95% aq. EtOH (18 L) at room temp. The combined EtOH extracts were concd in vacuum and after lyophilization yielded 84g of crude ethanolic extract. The crude ethanolic extract was partitioned with n-hexane (900 mL), chloroform (500 mL), ethyl acetate (500 mL) and n-butanol(500 mL), yielding 11.7g (32%), 27.3g (32.5%), 8.2g (9.8%) and 20.4g (24.3%) respectively. Part of the chloroformic fraction (6.0 g) was subjected to CC on silica gel (101 g) and eluted with different ratios of n-hexane and EtOAc. The appropriate frs (monitored by TLC analysis) were combined resulting in 11 frs. Frs 6-9, eluted with n-hexane-EtOAc (4:1, 2:3) afforded ppts, which were purified by recristalization on MeOH to give betulinic acid (603 mg –10%). Fr. 10 (763.4mg) was subjected to CC on silica gel, eluted with n-hexane, n-hexane-CHCl₃ (1:1), CHCl₃, CHCl₃-EtOAc (1:1), EtOAc, EtOAc-MeOH (1:1) and MeOH. The frs CHCl₃-EtOAc (1:1) afforded ppts, which by recristalization on CHCl₃-MeOH gave 6a-hydroxybetulinic acid (1) (30 mg-3.9%). The combined frs n-hexane and n-hexane-CHCl₃ (1:1) (571mg) were subjected to repeated CC on silica gel, eluted with n-hexane-CHCl₃ mixts, and flash CC on silica gel, eluted with CHCl₃-MeOH mixts of increasing polarity to give platanic acid (2) (25 mg-4.3%). The n-hexane fraction (4.4 g) was refractionated successively by CC on silica gel, eluted with n-hexane-EtOAc mixts of increasing polarity which afforded b-sitosterol (20.5mg – 0.5%) and betulinic acid (180mg – 4.0%).

6a-hydroxybetulinic acid (1): White crystals, mp 285-288°. IR n_max /cm^-1: 3454, 1685, 1639, 1038 and 879 (KBr). EIMS m/z (rel. int.): 472 (0.5), 454 (2.9), 436 (3.7), 248 (10.9), 220 (3.9), 219 (5.6), 207 (11.3), 203 (14.9), 189 (25.8), 187 (66.9), 175 (17.5) and 43 (100). ¹H NMR (300 MHz, pyridine-d₅): d 0.96 (3H, s, H-25), 1.11 (3H, s, H-27), 1.19 (3H, s, H-26), 1.45 (3H, s, H-24), 1.80 (3H, s, H-30), 2.01 (3H, s, H-23), 3.60 (1H, m, H-3), 4.35 (1H, dt, J 10.4 and 3.8 Hz, H-6), 4.79 (1H, brs, H-29), and 4.96 (1H, d, J 2,3 Hz, H-29).

Acknowledgements

The authors thanks CAPES and CNPq for schoolarships (I. L. and J. L. B. P.). We also thank Dr. M. C. de Souza for support in the plant collection, Dr. G. M. Barroso (Jardim Botânico do Rio de Janeiro) and Dr. L. E. Soares e Silva (Universidade Federal de Brasilia) for the identification of the plant material.

Received: July 11, 2000

Published on the web: February 15, 2001

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Publication Dates

• Publication in this collection
  02 July 2001
• Date of issue
  Apr 2001

History

• Received
  11 July 2000