SECONDARY METABOLITES FROM CAESALPINIA PLUVIOSA

Yonny Flores, Jose Vila, Giovanna R. Almanza*

Laboratorio de Productos Naturales, Instituto de Investigaciones Químicas, Carrera de Ciencias Químicas, Universidad Mayor de San Andrés, Campus Universitario de Cota Cota c. 27, C. P. 303 La Paz, Bolivia
*Autor corresponsal: giovyalmanza@yahoo.com.ar

Two phenolic compounds, ethyl gallate and rhuschalcone VI, together with lupeol, betulinic acid and stigmasterol were isolated from the stem bark of Caesalpinia pluviosa D.C. Their structures were determined by spectroscopic means mainly by NMR experiments, completing all the NMR assignments of phenolic compounds.

In addition, the extracts and pure compounds were evaluated against the bacteria Staphylococcus aureus, HPIA test and the antimalarial in vitro assay against Plasmodium falciparum, determining that CH₂Cl₂ extract and rhuschalcone VI showed good activity in the antibacterial and HPIA tests.

Key words: Caesalpinia pluviosa, rhuschalcone VI, ethyl gallate, triterpenes, antibacterial, antimalarial.

RESUMEN

Dos compuestos fenólicos, galato de etilo y rhuschalcona VI, junto con lupeol, ácido betulínico y estigmasterol fueron aislados de la corteza de Caesalpinia pluviosa DC. Sus estructuras fueron determinadas por medios espectroscópicos, completando todos los asignamientos de RMN de los compuestos fenólicos.

Además, los extractos y compuestos puros fueron evaluados contra la bacteria Staphylococcus aureus, la prueba HPIA y el ensayo antimalarico in vitro contra Plasmodium falciparum determinando que el extracto CH₂Cl₂ y rhuschalcone VI muestran buena actividad en las pruebas antibacterianas y HPIA.

Palabras claves: Caesalpinia pluviosa, rhuschalcona VI, galato de etilo, triterpenos, antibacteriano, antimalárico

Caesalpiniaceae is a large family distributed throughout Bolivia; it includes 26 genus with approximately 76 species ^[1]. Caesalpinia pluviosa D.C. is a tree that can grow until 20 meters tall, that is found in the Amazonian part of Bolivia. The decoction of its stem bark is used in the Bolivian folk medicine as a remedy for diarrhea ^[2]. According to literature, several Caesalpinia species have been studied phytochemically and pharmacologically, yielding cassane type diterpenes ^{[3],[4],[5],[6]} flavonoids ^[7],[8], biflavonoids ^[9] and tannins.^[10]

In this paper, we report the isolation and structural elucidation of two phenolic compounds, ethyl gallate (1) and rhuschalcone VI (2) from the stem bark of C. pluviosa, completing all their NMR assignments. In addition, we determine the presence of three triterpenic compounds: lupeol (3), betulinic acid (4) and stigmasterol (5), metabolites widely distributed in the Plant Kingdom. Finally, continuing the research of antibacterial and antimalarial natural products from Bolivian Plants, the extracts and pure compounds were evaluated against Staphylococcus aureus, Plasmodium falciparum and HPIA test.

RESULTS AND DISCUSSION

1.67 kg of milled dried stem bark of C. pluviosa was extracted successively with petroleum ether and ethanol. The concentrated EtOH extract was further extracted with CH₂Cl₂: MeOH (9:1). Ethyl gallate (1) and rhuschalcone VI (2) were isolated from the CH₂Cl₂:MeOH fraction following diverse chromatographic techniques for instance flash chromatography, preparative chromatography and gel filtration. Lupeol (3), betulinic acid (4) and stigmasterol (5), were isolated from the petroleum ether extract. The structures of all compounds were established by spectroscopic means. For the triterpenoids and sterol the spectroscopic data were compared with those reported in the literature.

Compound (2) was isolated as a yellow powder, the HREIMS showed a molecular ion at m/z 511.1342 [M-H⁺]; calc. for 511.1387 which was consistent with the molecular formula C₃₀H₂₂O₁₀. The ¹³C NMR spectrum exhibited 30 signals: two carbonyls, and 28 olefinic and aromatic carbons, suggesting a biflavonoid structure ^[11]. The ¹H NMR spectrum together with the ¹H-¹H COSY experiment allowed the identification of two set of trans-alkenes [δ 7.83 (1H, d, 15.5 Hz) and 7.62 (1H, d, 15.5 Hz)] and [δ 7.78 (1H, d, 15.7 Hz) and 7.58 (1H, d, 15.7 Hz)], one 4-oxyphenyl groups [δ 7.56 (2H, d, 8.7 Hz), 6.78 (2H, d, 8.7 Hz)]; one 2,4-dioxyphenyl group [δ 7.94 (1H, d, 9.0 Hz), 6.37 (1H, dd, 9.0, 2.4 Hz.), 6.27 (1H, d, 2.4 Hz)]; one tri-substituted aromatic rings [δ 6.94 (1H, d, 8.5 Hz), 7.60 (1H, dd, 8.5, 2.1 Hz), 7.67 (1H, d, 2.1 Hz)]; and one tetra-substituted aromatic ring [δ 7.95 (1H, s), 6.41 (1H, s)], the ¹H and ¹³C NMR spectra are showed in Figure 3. Full assignments of the proton and carbon NMR signals were made by use of HMQC and HMBC spectra to confirm the structure as rhuschalcone VI, previously reported by Mdee et al. ^[12], Some HMBC correlation are showed in the Figure 3.

The comparison of physical and spectroscopic data for (3), (4), and (5) with those from literature made possible their identification as lupeol, betulinic acid and stigmasterol. Table No. 3 shows the comparison of the ¹³ C NMR data with those of the literature.

The antibacterial activity for dichloromethane extract and pure compounds was evaluated against Staphylococcus aureus (Table No. 4). The results show that the compound 4 showed some bacteriostatic activity against S. aureus.

In addition, in a previous study ^[16] the stem bark ethanolic extract of Caesalpinia pluviosa D.C. showed activity on Chloroquine-resistant D2 strain of P. falciparum (IC₅₀ 15 μg:ml against F32 strain and IC₅₀ 8.3 μg:ml against D2 strain).

Finally based on the HPIA results the dichloromethane extract and compound 5 were tested against Plasmodium falciparum in vitro. Unfortunately the results show that the compound 5 is inactive as well as the extract. (Table No. 6)

EXPERIMENTAL

The melting points (uncorrected) were recorded on a Sanyo Gallenkamp Melting Point Apparatus. Mass spectra (HREIMS) were measured in a Waters Micromass Q-TOF apparatus. ¹H and ¹³C NMR spectra were recorded with a Varian 300 and two-dimensional experiments were done in a Bruker 500 using as solvents CDCl₃ and MD₃OD; chemical shifts are reported in δ units (ppm) and coupling constants (J) in Hz. Sephadex LH-20 was used for gel filtration; Silica gel (E.M. Merck,70-230 mesh) and silica gel G-60 (E.M. Merck) were used for Chromatography Column (CC) and Vacuum Liquid Chromatography (VLC), respectively, while aluminum plates impregnated with silica gel 60 F₂₅₄ (E.M. Merck) were used for analytical (0.25 mm) Thin Layer Chromatography (TLC) analyses. Spots on chromatograms were detected under UV light (254 and 365 nm) and by spraying the plates with 10% H₂SO₄, followed by heating.

Extraction and Isolation. The dry and ground plant material (1667 g) was first extracted with 3 L of petroleum ether for 72 h at room temperature, the solution was filtered off and evaporated to yield the petroleum ether extract. The solid residue was macerated for 72 hours with 3 L ethanol, which after filtering and evaporation of the solvent gave the ethanolic extract. Finally, the ethanolic extract was extracted with 150 mL of a mixture (CH₂Cl₂:MeOH 9:1) this process was done for 4 times giving the CH₂Cl₂ extract. The petroleum ether extract (6,7 g) was subjected to VLC on silica gel, eluting with increasing amounts of CH₂Cl₂ in petroleum ether, followed by increased amounts of MeOH in CH₂Cl₂ finalizing with MeOH, to give nine main fractions. Fractions 3, 4 and 5 where submitted to other chromatography techniques followed by recrystallization with MeOH giving the compounds 1 (22.3 mg), 2 (17.9 mg) and 3 (37.2 mg). The CH₂Cl₂ extract was also submitted to a chromatographic analysis by VLC on silica gel followed by gel filtration on Sephadex LH-20 (CH₂Cl₂:MeOH 50% as eluent) giving two compounds in pure form, compound 4 (20 mg) and compound 5 (45 mg).

Compound 1 (Ethyl galleate) White crystals of mp. 213-215ºC, ¹H and ¹³ C NMR see Table No. 1.

Compound 2 (Rhuschalcone VI) Amorphous yellow solid, mp 184-186°C, ¹H and ¹³ C NMR see Table No. 2. HREIMS m/z 511.1342, calc. for C₃₀H₂₂O₉+H, 511.1387.

Compound 3 (Lupeol) White crystals of mp 214-216°C, ¹H NMR (CDCl₃, 500 MHz) δ 4.57 d & 4.69 d (H-29_A and H-29_B); 3.19 dd (H-3); 2.35 ddd (H-19); 1.89 m (H-13); 1.68 s (H-30); 1.03 s (H-26); 0.97 s (H-23); 0.94 s (H-27); 0.83 s (H-25); 0.79 s (H-24); 0.76 s (H-28) and 0.68 t (H-5). C NMR see Table No. 3. ¹³

Compound 4 (Betulinic acid) White crystals of mp. 316-318ºC, ¹H NMR (CDCl₃, 500 MHz) δ 3.17 dd (H-3); 3.01 m (H-19); 0.94 s (H-23); 0.75 s (H-24); 0.85 s (H-25); 0.96 s (H-26); 1.00 s (H-27); 4.73 d (H-29_A); 4.60 d (H-29_B) and 1.69 s (H-30). C NMR see Table No. 3. ¹³

Compound 5 (Stigmasterol) White crystals of mp. 165-167ºC, ¹H NMR (CDCl₃, 500 MHz) δ 3.52 m (H-3); 5.35 d (H-6); 0.67 s (H-18); 1.00 s (H-19); 0.92 d (H-21); 5.15 m (H-22); 5.01 m (H-23); 1.47 m (H-25); 0.86 d (H-26); 0.79 d (H-27) and 0.84 d (H-29). ¹³ C NMR see Table No. 3.

Parasites

For the antimalarial in vitro evaluation, two strains of Plasmodium falciparum, F32 (sensible to chloroquine) and D2 (resistant to chloroquine), were used.

Microorganisms

Biological tests

Methodologies employed for in vitro assays against protozoa are given in full details in a previous paper ^[16]. The protocol for HPIA test is described in ^[17]. The bacterial assays was done by dilution and the protocol is described in Gimenez et al., 1996 ^[18].

Minimal Inhibitory Concentration (MIC)

The MIC was determined by a microdilution technique using the Mueller Hinton (MHB-DIFCO) broth. The dilutions were prepared from a solution of 2 mg/ml where we added a bacterial population of 6x10⁶ ufc/ml of each microorganism, placing in the microplate controls of bacterial growth (solvent and antibiotic). The plates were incubates at 37 °C for16-18 hrs, after this period the plates was examined visually. The MIC is considered the lowest concentration of the sample that prevents visible growth.

Minimal Bactericidal Concentration (MBC) and Minimal Bacteriostatic Concentration (MbtaticaC) were determined by sub-culturing the negative samples of the previous technique. Since the bactericidal substances not always sterilize totally a bacterial population, the minimal concentration of the bacteriological agent that allows surviving less than 0.1% of original inocule is denominates Minimal Bactericidal Concentration (MBC). MBstaticC is defined as the lowest concentration that avoid the grow of the bacteria without sterilize the bacterial population [18].

ACKNOWLEDGEMENTS

The authors are gratefully acknowledged to the Swedish Agency for International Development Cooperation (SIDA/Sarec) for the financial support of phytochemical studies; FONAMA (Fondo Nacional para el Medio Ambiente) for the financial support of the field trip and biological assays; Barcelona University and Lund University for the NMR spectra; IBBA (Instituto Boliviano de Biología de la Altura) for the antibacterial assays; IIFB (Instituto de Investigaciones Químicas) for the antibacterial assays; LPB (Herbario Nacional de Bolivia) for the identification of plant material and IRD (Francoise Institute of Research for the Development) for the HPIA evaluation and recollection of plant material.

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