Antimicrobial triterpenes from the stem bark of Crossopteryx febrifuga

2.1 Phytochemical investigation

The air-dried, powdered stem bark of C. febrifuga was extracted with MeOH. The dry crude MeOH extract obtained was re-extracted with EtOAc, and the EtOAc soluble fraction subjected to repeated column chromatography over silica gel and/or Sephadex LH20. This process yielded the known compounds monoglyceride of palmitic acid, β-sitosterol and its glucoside, and the epimeric mixtures of the new ursane-type triterpene (1) and its glucoside (2).

Compound 1 was obtained as a brown amorphous solid. It gave a positive reaction in the Lieberman–Burchard test, as usual for a triterpenoid. Its (-)-ESI-HRMS showed a chloride adduct ion peak at m/z 519.28973 [M+Cl]^–, corresponding to the molecular formula C₃₀H₄₄O₅Cl (calcd. 519.28828) containing 9 degrees of unsaturation. The ¹H NMR spectrum of compound 1 exhibited characteristic signals for a pentacyclic triterpene with four methyl singlets at δ 0.60; 0.67; 0.80; 0.88; one methyl group appearing as doublet at δ 0.96 (J = 6.2 Hz), as well as one olefinic methine and one olefinic methylene proton at δ 5.55 and 4.60, 4.70, respectively. The ¹³C NMR data suggested that compound 1 consists of an ursa-12,20-diene [δ 132.2 (C-13); 128.4 (C-12) and δ 105.8 (C-30); 152.3 (C-20)], with two carboxylic acid groups at δ 175.9 (C-27) and δ 177.6 (C-28), respectively [9, 14]. Additional signals of olefinic carbons at δ 132.5 (C-13); 127.9 (C-12) and carboxylic carbons at δ 176.1 (C-27), 178.2 (C-28) were also observed, which was due to the epimerization at C-18. In fact, for an oleanane-type triterpene (which may arise from some ursane-type triterpenes), the 18α and 18β series can be recognized by inspecting the chemical shift of some characteristic carbons. The geometry of the D/E ring junction does not cause a significant alteration in the shielding of carbons in the A and B rings. The chemical shift of C-18 is sensitive to the change in the absolute configuration at C-18, and therefore influencing the chemical shift of neighbouring carbons like C-12, C-13, C-14, C-17, C-19, C-27 and C-28 [14]. The HMBC spectrum of 1 supported the proposed structure, showing correlations from H-18 to C-12, C-13, C-14, C-17, C-19 and C-28; from H-30 to C-19, C-20 and C-21; from H-26 to C-7, C-8, C-9, and C-14; and from H-23, H-24 to C-3. According to DEPT, HMQC and HMBC experiments and literature data, the structure of compound 1 was elucidated as a mixture of 18-epi-3β-urs-12,20(30)-diene-27,28-dioic acid and 3β-urs-12,20(30)-diene-27,28-dioic acid (1:3 ratio) (Figure 1A). The latter had been previously obtained after acid hydrolysis of the 28-O-β-D-glucopyranosyl ester of 3β-[α-L-rhamnopyranosyloxy]-urs-12,20(30)-diene-27,28-dioic acid [9].

Figure 1:

(A) Structures of compounds 1 and 2, (B) selected HMBC correlation in compound 2.

Compound 2 was isolated as a white amorphous solid. It gave a positive reaction in the Lieberman-Burchard test, as usual for a triterpenoid. Its (+)-ESI-HRMS showed a sodium pseudomolecular ion peak at m/z 669.36128 [M+Na]⁺, corresponding to the molecular formula C₃₆H₅₄O₁₀Na (calcd. 669.36092), indicating 10 degrees of unsaturation. The ¹H-NMR spectrum of compound 2 shows signals for a glycosylated pentacyclic triterpene, with four methyl singlets at δ 0.68; 0.86; 0.87; 0.88; one methyl group appearing as doublet at δ 0.96, as well as one olefinic methine and one olefinic methylene proton at δ 5.53 and 4.60; 4.68, respectively. Signals for the β-configurated sugar fragment appear between δ 4.15 and 3.01; with the anomeric proton at δ 4.15 (1H, d, 7.8 Hz, H-1′). These data suggested that compound 2 contains 1 as aglycone. In fact, the ¹³C NMR data of the aglycone moiety was almost identical to that of 1 [δ 128.4/128.1 (C-12); 132.1/132.5 (C-13); 152.2 (C-20); 176.0/176.1 (C-27); 177.6/178.2 (C-28); 105.8 (C-30)]. The structure of the sugar moiety was identified as glucose according to the ¹³C NMR values: 105.3 (C-1′), 76.8 (C-3′), 76.5 (C-2′), 73.9 (C-4′), 70.1 (C-5′), 61.2 (C-6′) [12], and was linked at C-3 of the aglycone according to the HMBC (Figure 1B) correlation from H-3 to C-1′. From the above spectroscopic data, the structure of compound 2 was determined as a mixture of 18-epi-3β-D-glucopyranosyl-urs-12,20(30)-diene-27,28-dioic acid and 3β-D-glucopyranosyl-urs-12,20(30)-diene-27,28-dioic acid (1:3 ratio) (Figure 1A).

3.1 General experimental procedures

The EIMS spectra were recorded on a double focusing mass spectrometer (Varian MAT 311A). ESI-HRMS spectra were recorded on an Apex III (Bruker Daltonik) 7 Tesla (ESI-FT-ICR-MS). Optical rotations were recorded in MeOH solution on a Jasco DIP-360 digital polarimeter. The ¹H NMR spectra and ¹³C NMR spectra were recorded on a Bruker DRX spectrometer operating at 500 MHz and 125 MHz, respectively, with TMS as an internal standard. Silica gel Merck 60 (0.063–0.200 mm) was used for column chromatography. Percolated aluminum backed silica gel 60 F254 sheets were used for TLC. Spots were visualized under UV light (254 nm) and (365 nm) or using a solution of molybdate/Ce⁴⁺ reagent followed by heating.

3.2 Plant collection and identification

The stem bark of C. febrifuga was collected on February 2011 in Foumban (Western Region, Cameroon), and a specimen was identified and deposited in the Cameroon National Herbarium in Yaoundé (ref. Number 41791/HNC).

3.3 Extraction and isolation of compounds

The air dried stem bark of C. febrifuga (3 kg) was ground and exhaustively extracted by maceration in 11 L MeOH at room temperature for 72 h. The filtrate was evaporated to dryness to afford 223 g of extract. The MeOH extract was re-extracted by EtOAc to give a dry residue (50 g) after evaporation. The EtOAc extract (30 g) was subjected to silica gel column chromatography (0.063–0.200 mm, Merck, 206) eluted with a mixture of n-hexane-EtOAc (from 1 to 100 %, v/v) and EtOAc-MeOH (from 1 to 10 %) in increasing polarity, to yield 50 fractions of 150 mL each. Fractions 1 to 10 yielded the monoglyceride of palmitic acid (90 mg). Fractions 15–17 yielded β-sitosterol. Further column chromatography of fractions 29–35, using n-hexane-EtOAc (from 30 to 100 %) as eluent, yielded compound 1 (120 mg). Fractions 39–42 yielded the glucoside of β-sitosterol. Fractions 45–49, after column chromatography on silica gel (EtOAc-MeOH, from 1 to 10 %) followed by column chromatography on Sephadex LH20 yielded compound 2 (78 mg) in the fraction eluting with 4 % MeOH.

Compound 1: [α_D]²⁰ = +106 (c 0.45 mg/mL, MeOH) ¹³C-NMR (DMSO ; 600 MHz): 177.6/178.2 (C-28); 175.9/176.1 (C-27); 152.2 (C-20); 132.1/132.5 (C-13); 128.4/127.9 (C-12); 105.8 (C-30); 76.9 (C-3); 55.5/53.5 (C-18); 54.9.0 (C-5); 55.2/55.2 (C-14); 47.3/47.2 (C-17); 46.0 (C-9); 39.0 (C-1); 38.4 (C-8); 38.3 (C-4); 38.0 (C-22); 36.2 (C-7); 36.2 (C-10); 34.8 (C-19); 31.3 (C-21); 28.2 (Me-23); 26.9 (C-2); 25.0 (C-16); 23.9 (C-15); 22.3 (C-11); 18.1 (Me-25); 17.9 (C-6); 16.0 (Me-26); 15.9 (Me-24); 15.8 (Me-29); ¹H-NMR (DMSO; 600 MHz): 5.53/5.50 (1H, m, H-12); 4.68 (1H, brs, H-30a); 4.60 (1H, brs, H-30b); 2.98 (1H, m, H-3); 2.23/2.15 (1H, brs, H-18); 1.86 (1H, m, H-19); 0.96 (3H, d, 6.2 Hz, H-29); 0.88 (3H, s, H-23); 0.87 (3H, s, H-26); 0.80 (3H, s, H-25); and 0.67 (3H, s, H-24). (-)-ESI-MS: m/z 483[M-H], (-)-ESI-HRMS: m/z 519.28973 [M+Cl]^-, C₃₀H₄₄O₅Cl (calcd. 519.28828), EI-MS: m/z (%):354 (5), 347 (17), 305 (100), 263 (30), and 221 (24).

Compound 2: [α_D]²⁰ = +62 (c 0.6 mg/mL, MeOH) ¹³C-NMR (DMSO ; 600 MHz): 177.6/178.2 (C-28); 176.0/176.1 (C-27); 152.2 (C-20); 132.1/132.5 (C-13); 128.4/128.1 (C-12); 105.3 (C-30); 87.9 (C-3); 55.5/53.5 (C-18); 55.2 (C-5); 55.2/55.2 (C-14); 47.3/47.2 (C-17); 46.0 (C-9); 38.5 (C-1); 38.5 (C-8); 38.4 (C-4); 38.0 (C-22); 36.1 (C-7); 36.1 (C-10); 34.9/36.4 (C-19); 31.3 (C-21); 27.6 (Me-23); 25.5 (C-16); 25.0 (C-2); 23.9 (C-15); 22.3 (C-11); 18.0 (Me-25); 17.7 (C-6); 16.4 (Me-24); 16.0 (Me-26); 15.8 (Me-29); 105.2 (C-1′); 76.8 (C-3′); 76.5 (C-5′); 73.9 (C-4′); 70.1 (C-2′); 61.2 (C-6′). ¹H-NMR (DMSO; 600 MHz): 5.52/5.49 (1H, m, H-12); 4.68 (1H, brs, H-30a); 4.60 (1H, brs, H-30b); 3.00 (1H, m, H-3); 2.23/2.12 (1H, brs, H-18); 0.96 (3H, d, overlaps with H-23, H-29); 0.95 (3H, s, H-23); 0.89 (3H, s, H-26); 0.79 (3H, s, H-25); 0.74 (3H, s, H-24); 4.15 (1H, d, 7.8 Hz, H-1′); 2.94 (1H, m, H-2′); 3.17 (1H, m, H-3′); 3.08 (1H, m, H-4′); 3.04 (1H, m, H-5′); 3.22 (1H, m, H-6′a); 3.01 (1H, m, H-6′b). (+)-ESI-HRMS: m/z 669.36128 [M+Na]⁺, (calcd. 669.36092 for C₃₆H₅₄O₁₀Na).

3.4 In vitro antimicrobial assay