A new phenyl alkyl ester and a new combretin triterpene derivative from Combretum fragrans F. Hoffm (Combretaceae) and antiproliferative activity

2.1 General experimental procedure

The melting point of the new compounds was recorded in an open capillary using Electrothermal 9100 and is uncorrected. IR spectra were recorded on FT-IR spectrometer (Perkin-Elmer Spectrum 100). The proton and ¹³carbon nuclear magnetic resonance (NMR) data were recorded on spectrometer Bruker Avance AV-500 and 600, and tetramethylsilane (TMS) was used as standard. Chemical shifts are given in ppm (δ) and coupling constant (J) in Hz. CDCl₃, MeOD, and DMSO-d₆ were used as NMR solvents. Electrospray ionization-mass spectrometry (ESI-MS) spectra were registered on a QTOF Spectrometer (Bruker, Germany) equipped with a ZQ F1 source. The spectrometer was operated in positive mode (scan: 150–1,500; centroid CV = 20). The high resolution mass spectrum was registered on LC-MS-QTOF Spectrometer (Bruker, Germany) equipped with ESI source operating in positive mode. Column chromatography (CC) was performed on silica gel 60 (70–230 mesh, Merck), and thin layer chromatography was performed on silica gel precoated plates F-254 Merk (20 × 20 cm). Spots were visualized under UV light (254 and 365 nm), sprayed with 5% of phosphomolybdic acid prepared in ethanol, then heated. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) reagent from Promega was used to evaluate antiproliferatve activity.

2.2 Plant material

Leaves and stem bark of C. fragrans F. Hoffm were collected during the month of May 2017 at Padarmé located in Bibémi subdivision, in the north region of Cameroon. The sample was identified at the National Herbarium of Cameroon, where a voucher specimen is deposited under the number 39,753/HNC.

2.3 Extraction and isolation

The powders of 1 kg of leaves and 1 kg of stem bark of C. fragrans F. Hoffm were extracted separately by maceration at room temperature with 5 L of dichloromethane/methanol (CH₂Cl₂/MeOH) (1:1) and methanol (MeOH) respectively for 48 h with intermittent stirring. After filtration on Whatman filter paper, the resulting solutions were concentrated using a rotary evaporator. The procedure was repeated thrice to optimize extraction. We obtained 55 g of crude extract from the leaves and 40 g from the stem bark. Then, a quantity of 30 g of each crude extract was taken differently and each subjected to CC separation on silica gel with the gradient eluting system hexane–ethyl acetate (100:0 → 0:100), followed by ethyl acetate–methanol (100:0 → 0:100). The fractions of 100 mL each were collected and concentrated on rotary evaporator. From the extract of leaves, two new compounds, fragransinate (1, 5.1 mg) and combretin C (2, 215 mg), alongside four known compounds combretin A (3, 46 mg), belamcanidin (4, 12.3 mg), cirsilineol (5, 64 mg), and velutin (6, 60 mg), and a mixture of β-sitosterol-3-O-β-d-glupyranoside and stigmasterol-3-O-β-d-glucopyranoside (10a,b, 9 mg) were isolated. From the methanol crude extract of the stem bark, three known compounds: betulinic acid (7, 4.3 mg), bellericagenin B (8, 13 mg), and a mixture of β-sitosterol and stigmasterol (9a, b, 15.6 mg) were isolated (Figure 1).

Figure 1

Chemical structures of compounds isolated from Combretum fragrans F. Hoffm.

2.4 Biological evaluation

3.1 Structural elucidation of compounds

Chromatographic separation on silica gel of the CH₂Cl₂/MeOH (1:1) crude extract of leaves of C. fragrans F. Hoffm led to two new compounds (1 and 2) and five known compounds (3, 4, 5, 6, and 10), whereas the methanol crude extract from the stem bark yielded three known compounds (7, 8, and 9). The structures of the compounds were elucidated by spectral analysis using ESI-MS and 1D, 2D NMR data.

Compound 1 was isolated as a whitish powder and melted between 66.5 and 67.5°C. Its molecular formula, C₂₇H₄₆O₅, implying five degrees of unsaturation, was deduced from its ESI-MS spectrum, which showed a pseudomolecular ion peak [M + 2Na]⁺ at m/z 496 in positive mode. Its IR spectrum showed broad absorption bands at 3,400 cm⁻¹, indicating the presence of hydroxyl group at 1,735 cm⁻¹ characteristic of a carbonyl of an ester moiety, and of aromatic ring between 1,450 and 1,600 cm⁻¹, and C–O bonds (1,100 and 1,150 cm⁻¹). The ¹H NMR analysis revealed in up-field region the presence of a terminal methyl signal at δ_H 0.90 (3H, t, j = 6.9 Hz), with aliphatic methylene (–CH₂–) signals ranging from δ_H 1.37 to 1.22 ppm, corresponding to an aliphatic chain. The spectrum also displayed resonances of α and β protons adjacent to the ester function of an aliphatic chain, respectively, at δ_H 2.33 (2H, t, j = 7.5 Hz) [12] and 1.64 (2H, dd, j = 7.5; 14.2 Hz), as well as the signal of an oxymethylene proton appearing at 4.10 (2H, t, j = 6.5 Hz). On the same spectrum, the signals of aromatics protons appeared as singlets at δ_H 6.45 (1H, s) and 6.31 ppm (1H, s) suggesting the presence of phenyl ring, one methoxy proton signal at δ_H 3.89 (3H, s), and a broad signal of free hydroxyl protons at δ_H 5.30 ppm as singlet. All the above information suggests an alkylphenyl skeleton. The ¹³C NMR broad-band showed 16 carbon signals, and together with HSQC and DEPT spectra, these signals were unambiguously assigned as: a carbonyl of ester at δ_C 174.0 (C-1), six tertiary and quaternary aromatic signals at δ_C 103.2, 108.5, 130.5, 133.1, 143.7, and 146.7, one oxymethylene carbon at δ_C 63.5, one methoxy carbon at δ_C 56.1, and one primary carbon at δ_C 14.1 corresponding to the terminal methyl of aliphatic chain. The signals of carbon atoms of the aliphatic chain at α and β positions to the carbonyl of the ester function appear at δ_C 34.4 and 25.0 respectively, and methylene carbon signals ranging from δ_C 29.2 to 29.7 correspond to a long aliphatic chain. In addition, two methylene carbon signals at δ_C 32.1 and 30.3 were attributed to the two methylenes between the phenyl group and the ester function. The two signals at δ_C 143.7 and 146.7 indicate that two of the oxygenated quaternary carbons are in ortho position. The ¹H–¹H COSY experiment shows correlations between the oxymethylene proton at δ_H 4.10 and the methylene proton at δ_H 1.93, which in turn correlates with another methylene proton at δ_H 2.59. These correlations suggest a propane chain substituted on position C-1ʹ and C-3ʹ enabling the assignment of chemical shifts at 4.10 to H-1ʹ, 1.93 to H-2ʹ, and 2.59 to H-3ʹ. The spectrum also shows correlations between the α and β protons to a carbonyl (ester) respectively at 2.31 (H-2) and 1.63 (H-3). The proton in β correlates in turn with the proton at δ_H 1.33 (H-4). The heteronuclear multiple bond correlation (HMBC) experiment shows correlations between the proton at 4.10 (H-1ʹ) and carbons at δ_C 32.0 (C-3ʹ), 30.5 (C-2ʹ) and a carbonyl signal at 173.9 (C-1). The spectrum also displays correlation between the proton at δ_H 1.93 (H-2ʹ) and carbons at δ_C 31.6 (C-3ʹ), 63.3 (C-1ʹ) as well as with an aromatic carbon signal at 133.2 (C-1″). Cross-peak correlations were also observed between the proton at δ_H 2.59 (H-3ʹ) and aromatic carbons at 108.3 (C-6″), 103.1 (C-2″), and 133.2 (C-1″) as well as carbons at δ_C 30.2 (C-2ʹ), 63.4 (C-1ʹ). Proton H-3 is the only proton that has several correlations with carbons from phenyl ring, indicating that C-3 is directly attached to the phenyl ring on C-1″ position. This result suggests that the aliphatic chain, which is a fatty ester, is located on C-1ʹ position of the propyl chain. In addition, the HMBC spectrum shows correlations of an aromatic proton at δ_H 6.45 (C-2″) with carbons at δ_C 31.9 (C-3ʹ), 108.6 (C-6″), 130.2 (C-4″), and 143 (C-3″). The aromatic proton at δ_H 6.30 (H-6″) correlates with different carbons at δ_C 31.9 (C-3ʹ), 103.2 (C-2″), 130.3 (C-4″), and 146.7 (C-5″). Proton of the methoxy group (δ_H 3.89) correlates with the carbon at δ_C 146.7, indicating that the methoxy group is attached on C-5″ position of the aromatic ring. Important HMBC correlations were also observed between the proton at δ_H 2.31 (H-2) from the long chain and carbons at δ_C 29.02 (C-4), 25.05 (C-3), and the carbonyl function at δ_C 173.98 (C-1). The length of the aliphatic chain was determined in conjunction with its ¹H NMR spectrum and different fragmentation patterns observed on its mass spectrum. Characteristic fragments appeared at m/z 311, 253, and 181 (Figure S1). All these spectral data and some information reported in literature enabled us to characterize compound 1 as 3ʹ-(3″,4″-dihydroxy-5-methoxyphenyl)propyl heptadecanoate to which the trivial name fragransinate has been given. To the best of our knowledge, this compound is isolated for the first time from natural sources.

Compound 2 was isolated as a white powder and melted between 239.1 and 240.1°C. Its HRESI-MS mass spectrum in positive mode shows the pseudomolecular ion peak at m/z 643.4149 [M + Na]⁺ (calculated for C₃₆H₆₀O₈, 643.4288) corresponding to the molecular formula C₃₆H₆₀O₈ accounting seven degrees of unsaturation. The IR spectrum exhibits absorption bands ν_max at 3,400 cm⁻¹, 1,700 cm⁻¹, and 1,000–1,025 cm⁻¹, which were respectively attributable to a hydroxyl group, a carbonyl group, and C–O bonds. The analysis of its ¹H NMR spectrum in conjunction with HSQC spectrum shows in upfield region the signals of two protons as doublet at δ_H 0.35 (1H, d, j = 4.0 Hz) and δ_H 0.54 (1H, d, j = 4.0 Hz) corresponding respectively to proton H-19b and H-19a of a cyclopropane from a cycloartane-type triterpene [13]. The spectrum also revealed the presence of six methyl groups at δ_H 0.80, 0.86, 0.92, 1.00, 1.01, and 1.03, and two oxymethine proton signals appearing at δ_H 3.24 (1H, m) and 3.96 (1H, dd, j = 11.0; 4.7 Hz). The spectrum also displays signals of osidic proton indicating the presence of a sugar moiety. Signals of oxymethine protons of a sugar moiety appeared at δ_H 3.67 (1H, dd, j = 11.3; 5.3 Hz), 3.24 (1H, m), 3.02; 2.97 (1H, m), 2.89 (1H, m) as well as the anomeric proton at 4.14 (1H, d, j = 7.5 Hz). The larger coupling constant (j = 7.5 Hz) of the anomeric proton suggests the β-configuration of the sugar moiety. The ¹H NMR spectrum of compound 2 was very similar to that of Combretin B [9], except the missing signal of an olefinic proton in 2. Combretin B has been previously isolated from the same plant. The sugar moiety was identified as xylose. The ¹³C NMR spectrum confirmed the absence of olefinic function compared with that of Combretin B. The spectrum shows signals of 36 carbons including three methyl group signals as singlet at δ_C 18.9 (C-18), 10.0 (C-29), and 14.3 (C-30); four methyl group signals as doublet at δ_C 18.6 (C-21), 25.7 (C-26), 25.0 (C-27), and 18.6 (C-31); two oxymethine signals at δ_C 82.4 (C-3) and 71.2 (C-15); one carbonyl function at δ_C 177.5 (C-28). The signals of sugar moiety appear at δ_C 73.4 (C-2ʹ), 76.3 (C-3ʹ), 69.4 (C-4ʹ), and 65.5 (C-5ʹ) together with the anomeric carbon at δ_C 104.0 attributable to C-1ʹ. The HMBC experiment shows important correlation between the anomeric proton at δ_H 4.14 and the carbon at δ_C 82.43 (C-3), indicating that the sugar moiety is attached to the aglycone moiety at the C-3 position. Other correlations were also observed between the proton at δ_H 1.03 (H-29) and carbons at δ_C 177.59 (C-28), 82.43 (C-3), and 53.54 (C-4), indicating that the carbonyl group is located on C-28 position. According to the great similarity observed between compound 2 and Combretin B, the structure of compound 2 has been proposed as 3-(β-d-xylopyranosyl)-15α-hydroxycycloartan-28-oic acid for which the trivial name Combretin C has been given. The compound is isolated for the first time as a new derivative combretin triterpene.

The known compounds were identified from their NMR spectroscopic data and comparisons made with those reported in the literature: combretin A (3) [9]; belamcanidin (4), cirsilineol (5), velutin (6) [14]; betulinic acid (7) [15], bellericagenin B (8) [16], a mixture of β-sitosterol (9a) and stigmasterol (9b) [17]; and a mixture of β-sitosterol-3-O-β-d-glucopyranoside (10a) and stigmasterol-3-O-β-d-glucopyranoside (10b) [18]. Compounds 7, 8, 10a, and 10b are reported for the first time from this plant species.

3.2 Antiproliferative activity

The antiproliferative activity of extracts and compounds 2, 3, 5, and 8 of C. fragrans F. Hoffm on U87 human GBM cells, C6 rat GBM cells, and PC-3 prostate cancer was evaluated by XTT test. From the results presented in Table 3, it is deduced that the MeOH crude extract of the stem bark of C. fragrans F. Hoffm was found to be more active on all tested cells than the CH₂Cl₂/MeOH (1:1) crude extract from the leaves. The MeOH extract powerfully inhibits the proliferation of U87, C6, and PC-3 cells after 72 h treatment compared to untreated control cells. The extract was most active on PC-3 prostate cancer cells (IC₅₀ = 11.24 µg/mL) than C6 (IC₅₀ = 12.17 µg/mL) and U87 (IC₅₀ = 20.13 µg/mL) cancer cells. On the contrary, the CH₂Cl₂/MeOH extract from the leaves also showed important inhibition of C6 cells proliferation (IC₅₀ = 14.19 µg/mL) and moderate cytotoxicity effect on U87 (IC₅₀ = 31 µg/mL) and PC-3 (36.26 µg/mL) cells. The literature mentions that many species from the combretum genus, similar to the specie studied here, are known to possess antiproliferative activity [8].

Among the tested compounds, combretin A (3) was the most active followed by compound cirsilineol (5), whereas combretin C (2) and bellericagenin B (8) were not active. Compound 3 exhibited very good inhibition on PC-3 cells proliferation (IC₅₀ = 22.57 µM), as well as on U87 (IC₅₀ = 29.30 µM) and C6 cells (58.93 µM) which is moderated. Compound 5 also showed important inhibition on PC-3 (IC₅₀ = 30.46 µM) and C6 (IC₅₀ = 31.61 µM) cells’ proliferation and weak effect on U87 cells growth (IC₅₀ = 65.46 µM). Compounds 3 and 5 isolated from the leaves could be responsible for the antiproliferative activity obtained with the related extract.

Combretin A (3) and C (2) are cycloartane-type triterpenes. The second is different from the first just by the presence in its structure of a sugar moiety on carbon C-3 and by the absence of a double bond on C-24 and C-31 carbons. However, combretin A showed good activity on all tested cells, whereas combretin C was inactive. The loss of the activity in combretin C could be attributable to the presence in its structure of the sugar moiety and the lack of the double bonds which negatively influences its activity. Previous study concerning structure–activity relationship showed that sugar moiety in gensenoside decreases its anticancer potential, whereas its aglycone presents good anticancer activity [19]. This study could explain our obtained results. Cycloartane triterpene types are also known for their anticancer activity [20,21], and this corroborates with the result obtained with combretin A. This is the first time that Combretin A is evaluated for its antiproliferative activity and it could be a good candidate for developping an anticancer agent in a pharmaceutical areas.

Compound (5) or cirsilineol is a flavonoid from flavonone type. Its activity was moderated on C6 and PC-3 cells but weak on U87MG cells. Its antiproliferative activity has already been documented for the first time on a large panel of cancer cell lines [22], and the results are similar with those obtained here with PC-3 cells. Flavonoids are known to possess anticancer activity, and this activity correlates positively with the presence of methoxyl group, carbonyl on carbon C-4, and the double bond on carbon C-2 and C-3 of flavonoids [23].

Compound (8) or bellericagenin B is an oleanane-12-ene triterpene type polyhydroxylated and showed no activity on all tested cancer cells. It is known that hydroxyl group correlates negatively with the anticancer activity in flavonoids [23] and other compounds [24]. Thus, the number of hydroxyl groups in compound 8 could explain its inactivity. In addition, compound 8 could be an oleanolic acid derivative and is different from this later with four hydroxyl groups. However, it is known that oleanolic acid, which possesses one hydroxyl group, has very good anticancer activity [25,26].

Betulinic acid (7) and stigmasterol (9b) were isolated from the bark and are known for their anticancer activity on many types of cancers [27,28]. Thus, the higher antiproliferative activity observed with the MeOH crude extract of the stem bark could be attributable in part to the presence of compounds 7 and 9b. The results of the antiproliferative activity of both extracts and some compounds of C. fragrans support with scientific evidences the traditional use of this plant in the treatment of cancer.