Abstract
Two new 24-nor-ursane-type triterpenoids, 2α,3β,19α-trihydroxy-24-norurs-4(23),12-dien-28-oic acid (1) and 3β-acetoxy-2α,19α-dihydroxy-24-norurs-4(23),12-dien-28-oic acid (2), along with 15 known compounds were isolated from the methanol extract of the twigs of Mostuea hirsuta. While 2-hydroxymethylbenzamide (13) was isolated for the first time from the natural source, compounds 3–12 are reported here for the first time from the genus Mostuea. Their structures were elucidated by means of spectroscopic analyses including 1D- and 2D-NMR spectroscopy, high-resolution mass spectrometric data as well as comparison with data from the literature. Compounds 1, 2, 4–9 and 13 were tested against bacteria, fungi and plant pathogen oomycetes by the paper disk agar diffusion assay resulting in missing to low activities corresponding with minimum inhibitory concentrations (MICs) > 1 mg mL–1. However, the respective compounds 1, 2, 8, 9 and 13 exhibited moderate cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC50 10.6–16.5 μm compared to the standard doxorubicin with IC50 0.9 μm.
1 Introduction
In a continuing search for bioactive molecules from Cameroonian rainforest medicinal plants, Mostuea hirsuta (T. Anderson ex Benth. & Hook. f.) Baill. ex Baker (Gelsemiaceae) was studied. The genus Mostuea belongs to the Gelsemiaceae family, and had been previously attributed to the family of Loganiaceae [1]. Members of the genus are tropical and subtropical shrubs or vines [2]. Mostuea is a small genus with nine recognized species, seven thereof in Africa and two disjoints in northern South America [3]. Mostuea hirsuta is used in traditional medicine to treat pains, lower heart action and stimulate respiration in low dosage, for the treatment of colds, to dispel sleep, to cure umbilical hernia in infants and as an aphrodisiac [4]. Until now, no investigation on chemical constituents and biological activity of this plant has been carried out. However, previous research on Mostuea brunonis reported the isolation of various quinoline and indole alkaloids with potent cytotoxicity [5–7]. In this report, we describe the isolation and structural elucidation of two new 24-nor-ursane-type triterpenoids, 2α,3β,19α-trihydroxy-24-norurs-4(23),12-dien-28-oic acid (1) and 3β-acetoxy-2α,19α-dihydroxy-24-norurs-4(23),12-dien-28-oic acid (2), together with the cytotoxicity and antimicrobial properties of isolated compounds 1–13.
2 Results and discussion
The methanolic extract of the twigs of M. hirusta was separated by repeated column chromatography and preparative thin-layer chromatography (PTLC) to afford 2 new and 15 known compounds (Fig. 1). The known compounds were identified as α-amyrin (3), ursonic acid (4), oleanolic acid (5), 3β-acetoxyoleanolic acid (6), β-amyrin (7), betulinic acid (8), 3β-acetoxybetulinic acid (9), lupeol (10), 3β-acetoxylupeol (11), lupeone (12), 2-hydroxymethylbenzamide (13), stigmasterol-3-O-β-d-glucopyranoside, stigmasterol, β-sitosterol-3-O-β-d-glucopyranoside and β-sitosterol. The structures were confirmed by spectra comparison with authentic and published values [8, 9].
Structures of the isolated compounds.
Compound 1 was obtained as a white powder which gave a positive Liebermann–Burchard test for triterpenes. The molecular composition was found to be C29H44O5Na by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) ([M+Na]+ at m/z= 495.3080, calcd. 495.3080). Its IR spectrum showed the presence of carboxyl (1695 cm–1), hydroxyl (3523 cm–1) and exocyclic methylene (3100, 1655 cm–1) absorptions [10].
The 1H NMR spectra (Table 1) of compound 1 revealed the presence of four methyl signals as singlets at δ = 0.79, 0.86, 1.22 and 1.39 ppm, one methyl as a doublet at δ= 0.92 (d, J= 6.6 Hz), an allylic proton at δ = 2.53 ppm (s, H-18), two oxymethine protons at δ = 3.45 (ddd, J= 11.5, 8.5 Hz, H-2) and 3.75 ppm (d, J = 8.5 Hz, H-3), an AB system of two exocyclic methylene groups at δ = 5.14 (d, J = 2.0 Hz, H-23a) and 4.74 ppm (d, J = 2.0 Hz, H-23b), and one olefinic proton at δ = 5.33 ppm (t, J = 3.5 Hz, H-12).
1H (500 MHz) and 13C (125 MHz) NMR assignments for 1 and 2 in MeOD.a
| Attribution | 1 | 2 | ||
|---|---|---|---|---|
| 13C | 1H (m, J in Hz) | 13C | 1H (m, J in Hz) | |
| 1 | 46.9 | 1.10 (dd, 12.0, 10.3) | 47.0 | 1.11 (dd, 11.5, 10.3) |
| 2.05 (dd, 10.3, 6.5) | 2.06 (dd, 10.3, 6.8) | |||
| 2 | 72.1 | 3.45 (ddd, 11.5, 8.5, 4.2) | 74.3 | 3.60 (ddd, 11.5, 8.9, 4.3) |
| 3 | 78.2 | 3.75 (d, 8.5) | 82.9 | 4.70 (d, 8.9) |
| 4 | 150.4 | – | 150.1 | – |
| 5 | 49.9 | 1.73 (br d, 11.0) | 50.0 | 1.74 (br d, 11.0) |
| 6 | 20.9 | 1.47 (m) | 20.8 | 1.50 (m) |
| 1.56 (m) | 1.57 (m) | |||
| 7 | 31.3 | 1.36 (m) | 31.4 | 1.38 (m) |
| 1.39 (m) | 1.40 (m) | |||
| 8 | 39.8 | – | 39.7 | – |
| 9 | 44.5 | 1.89 (m) | 44.4 | 1.90 (m) |
| 10 | 37.9 | – | 38.0 | – |
| 11 | 24.2 | 2.02 (m) | 25.0 | 2.02 (m) |
| 2.13 (m) | 2.13 (m) | |||
| 12 | 128.0 | 5.33 (t, 3.5) | 128.0 | 5.34 (t, 3.5) |
| 13 | 138.9 | – | 139.0 | – |
| 14 | 41.5 | – | 41.6 | – |
| 15 | 28.3 | 1.85 (m) | 28.2 | 1.87 (m) |
| 1.07 (m) | 1.08 (m) | |||
| 16 | 25.2 | 2.60 (m) | 28.2 | 2.60 (m) |
| 1.60 (m) | 1.58 (m) | |||
| 17 | 47.5 | – | 47.5 | – |
| 18 | 53.8 | 2.53 (s) | 53.9 | 2.54 (s) |
| 19 | 72.5 | – | 73.1 | – |
| 20 | 41.7 | 1.36 (m) | 41.8 | 1.36 (m) |
| 21 | 25.8 | – | 25.9 | – |
| 22 | 37.6 | 1.65 (m) | 37.7 | 1.66 (m) |
| 1.76 (m) | 1.77 (m) | |||
| 23 | 103.8 | 5.19 (d, 2.0) | 103.9 | 5.19 (d, 1.9) |
| 4.74 (d, 2.0) | 4.74 (d, 1.9) | |||
| 24 | – | – | – | – |
| 25 | 13.8 | 0.79 (s) | 14.0 | 0.80 (s) |
| 26 | 16.0 | 0.86 (s) | 16.1 | 0.86 (s) |
| 27 | 23.5 | 1.39 (s) | 23.4 | 1.40 (s) |
| 28 | 180.8 | – | 180.9 | – |
| 29 | 25.6 | 1.22 (s) | 25.6 | 1.24 (s) |
| 30 | 15.2 | 0.94 (d, 6.6) | 15.1 | 0.95 (d, 6.6) |
| C=O | – | – | 170.3 | – |
| CH3 | – | – | 21.1 | 2.02 (s) |
aAssignments were based on HMQC, HMBC, COSY and NOESY experiments.
The occurrence of H-18 (δ = 2.53 ppm) as a singlet signal is caused by the presence of a 19α-hydroxyl group. The above information suggests compound 1 to be an urs-12-ene derivative possessing an α-hydroxyl group at C-19 [11].
The 13C NMR spectrum (Table 1) displayed 29 carbon atoms instead of 30 carbons as usual in pentacyclic triterpenes showing that compound 1 is a nor-ursane derivative. The distortionless enhancement by polarization transfer (DEPT) experiment discriminated into five methyl, nine methylene, seven methine groups including an olefinic carbon at δ = 128.0 ppm, and two oxymethine groups at δ = 78.2 and 72.1 ppm. In addition, the 13C NMR confirmed the presence of the exocyclic methylene at δ = 103.7 and 150.4 ppm, and the carboxylic acid group at δ = 180.8 ppm.
The complete assignment of compound 1 was based on EI-MS, correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) experiments. In the HMBC spectrum (Fig. 2), correlations between H-1 (δ = 2.05 ppm) and C-2 (δ = 72.1 ppm), C-3 (δ = 78.2 ppm), C-5 (δ= 49.9 ppm) and C-25 (δ = 13.8 ppm), and between H-3 (δ = 3.75 ppm) and C-23 (δ = 103.8 ppm), C-1 (δ = 46.9 ppm), C-5 (δ = 49.9 ppm), C-2 (δ = 72.1 ppm) and C-4 (δ = 150.4 ppm) indicate the presence of one hydroxyl group at C-2 and the exocyclic methylene group at C-4 thereby confirming the absence of methyl-24 [12]. This assignment was in agreement with the EI-MS, which revealed three prominent ion fragments at m/z = 264 (C16H24O3), 246 (264–H2O) and 201 (246–COOH) usually resulting from the typical retro-Diels–Alder cleavage of urs-12-enes with a C-17 carboxyl group and a hydroxyl group on ring D or E [12]. The relative trans-configuration of H-2 and H-3 protons in compound 1 was in agreement with the coupling constant (J = 8.5 Hz) between OH-2α and OH-3β compared to that observed in the case of OH-2α and OH-3α (J = 2.4 Hz) [12, 13]. The trans-configuration was further supported by the cross peaks between H-3 and H-5, H-5 and H-9, H-9 and Me-27, Me-27 and H-18, H-18 and Me-30, and between H-2 and Me-25, Me-25 and Me-26, Me-26 and Me-29 observed in the NOESY spectrum (Fig. 3). This information indicates that OH-2 and OH-3 are in trans (e,e) configuration, stabilises the molecule. From the above spectroscopic studies, the structure of compound 1 was determined as 2α,3β,19α-trihydroxy-24-norurs-4(23),12-dien-28-oic acid.
Selected correlations observed in the HMBC spectrum of compound 1.
Selected correlations observed in the NOESY spectrum of compound 1.
Compound 2 was obtained as a white powder which gave a positive Liebermann–Burchard test for triterpenes. The molecular composition was found to be C31H46O6Na by HR-ESI-MS ([M]+ at m/z = 537.3185, calcd. 537.3192). The additional 42 mass units compared to compound 1 suggested here the presence of an acetyl group. The EI-MS showed significant fragments at m/z = 264 (C16H24O3), 246 (264–H2O) and 201 (246–COOH) usually resulting from the typical retro-Diels–Alder cleavage of urs-12-enes possessing a C-17 carboxyl group and a hydroxyl group on ring D or E. These ion fragments showed the acetyl group to be on ring A or B.
The IR, 1H and 13C NMR spectra of 2 (Table 1) suggested the same skeleton as compound 1 [12]. The 1H NMR spectrum also revealed the presence of an acetyl group by one additional methyl at δ = 2.02 ppm (s, CH3), supported by signals in the 13C NMR spectrum at δ = 21.1 (CH3) and 170.3 ppm (C=O) [14].
In addition, the 1H and 13C NMR spectra of 2 showed the downfield chemical shift of H-3 at δ = 4.70 (d, J = 8.9 Hz) and C-3 at δ = 82.9 ppm, compared with those of compound 1, suggesting the presence of an acetyl group at C-3 [14]. This assignment was in agreement with the correlations displayed in the HMBC spectrum between H-3 (δ = 4.70 ppm) and C-1 (δ = 47.0 ppm), C-5 (δ = 50.0 ppm), C-23 (δ = 103.9 ppm) and C=O (δ = 170.3 ppm), indicating as well the position of the acetyl group at C-3. The relative trans-configuration of H-2 and H-3 protons in compound 2 was confirmed by the coupling constant (J = 8.9 Hz) between OH-2α and OH-3β. From the above spectroscopic studies, the structure of compound 2 was determined as 3β-acetoxy-2α,19α-dihydroxy-24-norurs-4(23),12-dien-28-oic acid (2).
Tests of pure compounds 1, 2, 4–9 and 13 by paper disk diffusion assay against the bacteria Bacillus subtilis, Staphylococcus aureus, Escherichia coli, the fungi Mucor miehei and Candida albicans, and the plant pathogen oomycetes Aphanomycescochlioides, Pythium ultimum and Rhizoctonia solani resulted in missing or low activities corresponding with MIC values > 1 mg mL–1. However, compounds 1–2, 8–9 and 13 displayed moderate cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3 with IC50 10.6 and 16.5 μm compared to the standard doxorubicin with IC50 0.9 μm (Table 2).
Cytotoxicity against the human prostate adenocarcinoma cell line PC-3.
| Compounds | IC50 ± S.E.M. (μg mL–1) |
|---|---|
| 1 | 16.5 ± 0.9 |
| 2 | 14.8 ± 1.5 |
| 4 | 21.5 ± 2.6 |
| 5 | 45.2 ± 2.5 |
| 6 | 61.5 ± 9.4 |
| 7 | 75.3 ± 5.8 |
| 8 | 10.6 ± 0.9 |
| 9 | 11.4 ± 0.5 |
| 13 | 15.1 ± 0.3 |
| Doxorubicina | 0.9 ± 0.1 |
aStandard used in the assay.
3 Experimental section
3.1 General
Optical rotation [α]D (in MeOH, c in g mL–1) was determined by using a JASCO digital polarimeter (model DIP-3600). Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer in MeOH. Infrared spectra were recorded on a JASCO 302-A spectrophotometer. ESI-HR mass spectra were recorded on a Bruker FTICR 4.7 T mass spectrometer. EI-MS were recorded on a Finnigan MAT 95 spectrometer (70 eV) with perfluorokerosene as reference substance for HR-EI-MS. The 1H and 13C NMR spectra were recorded at 500 MHz and 125 MHz, respectively, on Bruker DRX 500 NMR spectrometers. Methyl, methylene and methine carbons were distinguished by DEPT experiments. Homonuclear 1H connectivities were determined by using the COSY experiment. One-bond 1H–13C connectivities were determined with HMQC gradient pulse factor selection. Two- and three-bond 1H-13C connectivities were determined by HMBC experiments. Chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as an internal standard and coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel (70–230 mesh, Merck). TLC was performed on Merck percolated silica gel 60 F254 aluminum foil, and spots were detected using a ceric sulfate spray reagent. The purity of the compounds was investigated by means of 1H NMR and ESI-MS. The degree of purity of the positive control compounds was ≥98 %, while that of the isolated compounds was >95 %. Doxorubicin was purchased from Sigma-Aldrich (Germany), nystatin was purchased from Maneesh Pharmaceuticals Pvt. Ltd. (Govandi, Mumbai, India) and gentamicin from Jinling Pharmaceutic (Group) Corp., Zhejang Tieng Feng Pharmaceutic Factory (Huzhou City, Zhejang, P. R. China). The Caucasian prostate adenocarcinoma cell line PC-3 was purchased from the American Type Culture Collection (Manassas, VA, USA). All reagents used were of analytical grade.
3.2 Collection and identification
Fresh twigs of M. hirsuta were collected from Batouri Road, East Region, Cameroon, and identified by Victor Nana, a botanist at the National Herbarium, Yaoundé, Cameroon, where a voucher specimen was deposited under ref. 9061 SRF/CAM.
3.3 Extraction and isolation
After evaporation under reduced pressure, 31.6 g of crude extract was obtained. The extract was purified by column chromatography over silica gel 60 (230–400 mesh) and PTLC using a gradient system of petroleum ether, CH2Cl2 and MeOH. A total of 75 sub-fractions (ca. 200 mL each) were collected and pooled on the basis of TLC analysis leading to three main fractions A–C.
Fraction A (8.5 g, combined from sub-fractions 1–25) was chromatographed over a silica gel 60C column with a petroleum ether–CH2Cl2 gradient. A total of 30 fractions of ca. 100 mL each were collected and combined on the basis of TLC. Fractions 4–10 were further chromatographed on silica gel 60H with a mixture of petroleum ether–CH2Cl2 (5:1) for elution to yield lupeone (12) (8.5 mg), 3-acetyllupeol (11) (5.2 mg), stigmasterol (12.5 mg) and β-sitosterol (12.5 mg). Fractions 11–25 were chromatographed on silica gel 60H with a mixture of petroleum ether–CH2Cl2 (4:1) for elution to yield lupeol (10) (14.5 mg), β-amyrin (7) (9.5 mg) and α-amyrin (3) (10.6 mg). Fraction B (9.0 g, combined from sub-fractions 26–52) was chromatographed over a silica gel 60C column with petroleum ether–CH2Cl2 gradient. A total of 25 fractions of ca. 100 mL each were collected and combined on the basis of TLC. Fractions 1–12 were further chromatographed over a silica gel 60H column with a mixture of petroleum ether–CH2Cl2 (4:2) to yield 3β-acetoxy-2α,19α-dihydroxy-24-norurs-4(23),12-dien-28-oic acid (2) (5.5 mg), 3-acetyloleanolic acid (6) (6.8 mg), 3-acetylbetulinic acid (9) (5.5 mg) and 2-hydroxymethylbenzamide (13) (7.2 mg). Similarly, fraction C (12.5 g, combined from sub-fractions 53–75) was chromatographed over a silica gel 60C column with a petroleum ether–CH2Cl2 (1:5), CH2Cl2 and CH2Cl2–methanol (9:1) gradient. The resulting 20 fractions of ca. 100 mL each were collected and combined on the basis of TLC. Fractions 1–15 were further chromatographed over a silica gel 60H column with CH2Cl2 and CH2Cl2–methanol (9:1) to yield oleanolic acid (5) (11.5 mg), ursonic acid (4) (8.5 mg), betulinic acid (8) (15.5 mg), 2α,3β,19α-trihydroxy-24-norurs-4(23),12-dien-28-oic acid (1) (10.5 mg), stigmasterol-3-O-β-d-glucopyranoside (15.6 mg) and β-sitosterol-3-O-β-d-glucopyranoside (15.6 mg).
3.4 2α,3β,19α-Trihydroxy-24-norurs-4(23),12-dien-28-oic acid (1)
White powder (MeOH); m.p. 230–234 °C; Rf = 0.75, silica gel 60 F254, CH2Cl2–methanol (9.5:0.5). –
3.5 3β-Acetoxy-2α,19α-dihydroxy-24-norurs-4(23),12-dien-28-oic acid (2)
White powder (MeOH); m.p. 255–260 °C; Rf = 0.55, silica gel 60 F254, CH2Cl2. –
3.6 Biological activities
3.6.1 Antimicrobial assays
Agar diffusion test plates with the bacteria B. Subtilis, E. coli (on peptone agar) and S. aureus (Bacto nutrient agar) and the fungi M. miehei and C. albicans (Sabouraud agar) as test strains were performed as previously described [15]. For the plant pathogen oomycetes A.cochlioides, P. ultimum and R. solani, squares of 0.5 × 0.5 cm2 were cut with a microbiological hook from the growth margins of mycelial mats grown on potatoes destrose agar (PDA) plates, inoculated onto the centers of fresh plates and cultivated for 24 h at 28 °C to initiate radial growth.
Compounds 1–2, 4–9 and 13 were dissolved in CH2Cl2–MeOH (9:1) and paper disks (Ø 9 mm) were impregnated with 40.0 μg each, dried for 1 h under sterile conditions and arranged evenly on the pre-made agar test plates, while for oomycete plates, the disks were placed around the mycelia squares at a distance of 30 mm. Bacteria and fungi plates were kept in an incubator at 37 °C for 15 h, and oomycetes at 28 °C for 48 h. The diameter of inhibition zones (in mm) was measured directly or calculated from the radius. Nystatin was used as positive control for fungi and gentamycin for bacteria. Bacillus subtilis, E. coli, S. aureus and C. albicans are clinical isolates received from the Centre Pasteur du Cameroun (Yaoundé, Cameroon) and were carefully purified. M. miehei and the plant pathogen oomycetes A.cochlioides, P. ultimum and R. solani originate from the Institute of Organic and Biomolecular Chemistry, Georg-August University Göttingen (Tammannstraße 2, Göttingen, Germany).
3.6.2 Cytotoxicity assay
Cytotoxic activities of compounds 1–2, 4–9 and 13 were evaluated against the human Caucasian prostate adenocarcinoma cell line PC-3 by the 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method according to a reported protocol [16]. Freshly trypsinized cell suspensions were seeded into 96-well microtiter plates at densities of 1 × 104 cells per well, and test compounds were added from dimethylsulfoxyde (DMSO)-diluted stock. After 3 days, the attached cells were incubated with MTT and subsequently solubilized in DMSO. The absorbance at 550 nm was measured by using a microplate reader. The IC50 is the concentration of the agent that reduced cell growth under experimental conditions by 50 %, with doxorubicin as positive control (IC50 = 0.9 μm).
Acknowledgments
J.D.W. wishes to thank the Alexander von Humboldt Foundation (AvH, Bonn-Bad Godesberg, Germany) for the generous support with laboratory equipment and the German Academic Exchange Service (DAAD, Bonn-Bad Godesberg, Germany) for financial support of a 3-month research period at Bielefeld University.
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