ABSTRACT
A new C-glycosylflavone, apigenin 7-methyl ether 6-C-[β-xylopyranosyl-(1→3)-β-glucopyranoside] named distemonanthoside was isolated from the stem bark of Distemonanthus benthamianus Baill., Fabaceae, along with six known compounds, sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid, syringic acid, quercetin, 6"-O-acetylvitexin, quercetin 3-O-β-D-glucopyranoside. The structures of those compounds and others were determined through spectral analyses. Compounds distemonanthoside, sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid and quercetin were tested against a clinical isolate strain of Mycobacterium tuberculosis AC 45; they exhibited good to moderate antitubercular activities with MIC values ranged from 31.25 to 125 µg/ml.
Keywords:
Fabaceae; Antitubercular activity; Distemonanthoside; Flavonoids; Phenolic acids
Introduction
Tuberculosis (TB) is a chronic contagious disease caused by several species of Mycobacterium. Due to the fact that there is a doubt nowadays on the efficiency of current antibiotics for the treatment of tuberculosis, micro-organisms developed resistance inducing an increase of the number of patients with the disease in worldwide (WHO, 2016WHO, 2016. Global Tuberculosis Report. Global and Investments Fall Far Short of Those Needed to End the Global TB Epidemic. World Health Organisation, Geneva, Switzerland, http://www.who.int/tb/publications/globalreport/gtbr2016maintext.pdf [accessed 03.05.17].
http://www.who.int/tb/publications/globa...
). This increasing of MDR-TB incidence has led to an urgent need for the discovery of new plant natural products that may potentially eradicate TB. Several in vitro growth inhibition of different strains of M. tuberculosis by plant extracts have been reported (Okunade et al., 2004Okunade, L., Elvin-Lewis, M.P., Lewis, W.H., 2004. Natural antimycobacterial metabolites: current status. Phytochemistry 65, 1017-1032.; Copp and Norrie Pearce, 2007Copp, B.R., Norrie Pearce, A., 2007. Natural product growth inhibitors of Mycobacterium tuberculosis. Nat. Prod. Rep. 24, 278-297.; Gautam et al., 2007Gautam, R., saklani, A., Jachak, S.M., 2007. Indian medicinal plants as a source of antimycobacterial agents. J. Ethnopharmacol. 110, 200-234.; Mc Gaw et al., 2008Mc Gaw, L.J., Lall, N., Meyer, J.J.M., Eloff, J.N., 2008. The potential of South African plants against Mycobacterium infections. J. Ethnopharmacol. 119, 482-500.). The Cameroonian medicinal plant Distemonanthus benthamianus Baill., Fabaceae, is a large rainforest tree widely distributed in Africa, especially in equatorial region. This essence is highly appreciated industrially for heavy construction and some countries use to export it as “Movingi”. In Mayombé region (Congo), traditional healers employ the stem bark in the treatment of several diseases as: parasitic, dermatitis, furuncles, aces and chancres. In the Chaillu region (Congo), that plant is used to cure bronchitis affections and children fever (Bouquet, 1969Bouquet, A., 1969. Fetish and traditional medicine of Congo (Brazzaville). O.R.S.T.O.M. Paris 36, 177-178.). In previous works carried on D. benthamianus, mainly methoxylated flavonols and flavones were isolated (King et al., 1952King, F.E., King, T.J., Sellars, K., 1952. The chemistry of extractives from hardwoods. Part V. The isolation of 3,7,4'-trimethylquercetin (ayanin) from the heartwood of Distemonanthus benthamianus. J. Chem. Soc. 0, 92-95.; King et al., 1954King, F.E., King, T.J., Stokes, P.J., 1954. The chemistry of extractives from hardwoods. Parts XIX. The structures of further new flavones occurring in ayan (Distemonanthus benthamianus). J. Chem. Soc. 0, 4587-4594.; Malan and Roux, 1979Malan, E., Roux, D.G., 1979. Flavonoids from Distemonanthus benthamianus Baillon. Methoxylated flavones and inter-relationships of benthamianin, a [2]-benzopyrano-[4,3-b] [I]-benzopyran. J. Chem. Soc. 1, 2696-2703.; Happi and Mpondo, 1994Happi, E.N., Mpondo, T.N., 1994. Two polymethoxylated flavones from Distemonanthus benthamianus. J. Nat. Prod. 57, 291-293.); this paper describes the isolation and structure elucidation of constituents from stem bark of D. benthamianus. The evaluation of antitubercular activities of compounds distemonanthoside (1), sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid (2) and quercetin against resistant strain of M. tuberculosis was also examined.
Material and methods
General procedures
Melting points were uncorrected and were measured on a Mettler Toledo instrument. IR spectra were recorded on an Alpha FT-IR Spectrometer from Bruker, while 1D and 2D NMR spectra were obtained on a Bruker DRX 500 (500 MHz for 1H and 125 MHz for 13C spectra) spectrometer (Bruker, Rheinstetten, Germany) with chemical shifts reported in δ (ppm) using TMS (δH) as an internal standard. The HR-ESI-MS was obtained on LTQ-FT instrument (Thermo Scientific). UPLC–MS was measured by a Shimadzu UPLC-MS system. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. Silica gel 60 (230–400 mesh E. Merck, Darmstadt, Germany) was employed for column chromatography, the solvent mixing systems for elution were mainly CH2Cl2/MeOH with increasing polarity each.
Plant material
Stem bark of Distemonanthus benthamianus Baill., Fabaceae, were collected at Eséka (Koumoul) near Yaoundé (3º 38' 60.00" N, 10º 46' 0.00" E) in the Centre Region of Cameroon in March 2014 and identified by Victor Nana. A voucher specimen (No. 45488 HCN) was deposited at the National Herbarium in Yaoundé, Cameroon.
Extraction and isolation
Dried and powdered stem bark of D. benthamianus (254 g) were extracted for 48 h with MeOH (3 × 1l) at room temperature. After filtration and evaporation of solvent, the crude MeOH extract (16 g) was subjected to CC (150 × 3 cm) [(SiO2), eluting with a gradient solvent system (CH2Cl2/MeOH)] giving four main fractions: I (1.9 g), II (3.8 g), III (3.6 g) and IV (6.7 g). Fractions (100 ml) were collected and grouped on the basis of TLC analysis. Fraction II (3.8 g) was submitted to CC (SiO2, 100 × 1 cm) using solvent system CH2Cl2/MeOH (50/1) to give sitosterol 3-O-β-D-glucopyranoside (65 mg). Fraction III (3.6 g) was submitted to CC (SiO2, 100 × 1 cm) using solvent system CH2Cl2/MeOH (60/1 to 5/1) to give four sub-fractions (IIIa, IIIb, IIIc and IIId). Sub-fraction IIIc (1 g) was chromatographed (SiO2, 50 × 1 cm) using CH2Cl2/MeOH (40/1–15/1) to afford compound 2 (480 mg) and compound 3 (3 mg). Sub-fraction IIId (0.65 g) was subjected to a silica gel column in gradient elution mixture solvent composed of CH2Cl2/MeOH (25/1–5/1) to afford quercetin (8 mg) and quercetin 3-O-β-D-glucopyranoside (11 mg). Using the same process, fraction IV (6.7 g) gave three sub-fractions (IVa, IVb and Vc). Sub-fraction IVa (0.98 g) was further chromatographed on a silica gel column (100 × 1 cm) using CH2Cl2/MeOH (10/1) to afford compound 4 (4 mg). Sub-fraction IVb (2.8 g) was purified by repeated CC on silica gel (100 × 1 cm) with the solvent system CH2Cl2/MeOH (10/1–1/1) to provide compound 1 (28 mg).
Structural characterization of distemonanthoside (1)
Yellow solid; [α]D25= −54º (c 0.05, MeOH); m.p. 285–287 ºC; IRυmaxKBr cm−1: 3267, 2923, 2853, 1595, 1512, 1226, 1159; TLC, Rf: 0.28 (CH2Cl2/MeOH: 90/10); ESI-MS m/z: ESI-MS: 577.4 [M−H]−•, LC–MS: m/z 579 [M+H]+ and ESI-MS: m/z 601.5 [M+Na] + (Calcd for C27H30O14Na = 601.5); 1H NMR (500 MHz, DMSO-d6), δH: 8.09 (2H, d, J = 8.8 Hz, H-2' and H-6'), 6.96 (2H, d, J = 8.8 Hz, H-3' and 5'), 6.82 (1H, s, H-3), 6.44 (1H, s, H-8), 4.81 (1H, d, J = 10.0 Hz, H-1"), 4.01b (1H, H-3"), 3.86 (3H, s, –OCH3), 3.82 (1H, d, J = 7.0 Hz, H-1'''), 3.72 (1H, dd, J = 11.3; 2.4 Hz, H-6"), 3.42b (1H, H-6"), 3.39b (1H, H-2"), 3.36b (1H, H-4"), 3.21b (1H, H-5"), 2.83 (1H, dd, J = 11.5; 4.0 Hz, H-5'''), 2.81b (1H, H-3'''), 2.78b (1H, H-2'''), 2.39b (1H, H-5'''); 13C NMR (125 MHz, DMSO-d6): δC: 182.1 (C-4), 164.1 (C-2), 163.4 (C-7), 161.4 (C-9), 161.2 (C-4'), 155.5 (C-5), 128.9 (C-2' and C-6') 121.3 (C-1'), 115.8 (C-3' and C-5'), 105.3 (C-1'''), 104.7 (C-6), 104.1 (C-10), 102.3 (C-3), 94.8 (C-8), 81.7 (C-5"), 80.4 (C-3"), 78.1 (C-2"), 76.4 (C-3'''), 75.6 (C-2'''), 73.5 (C-1"), 71.3 (C-4"), 69.2 (C-4'''), 65.3 (C-5'''), 60.8 (C-6"), 56.5 (–OCH3).
bSignal patterns are unclear due to overlap.
Antitubercular activity
MIC values were determined for the extract against M. tuberculosis strain AC 45 (clinical isolate obtained from Sangmelima district's Hospital in South Region of Cameroon) employing the microplate Alamar Blue assay, using Rifampicin as reference. The 96 wells plate received 100 µl of Middlebrook 7H9 medium supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) 2% glycerol and 0.05% v/v of tween 80. Broth and serial dilution of compounds were made directly on the plate with drug concentrations of 0.244 to 250 µg/ml. Plates were covered and sealed with parafilm and incubated at 37 ºC for 14 days. Then, 40 µl Alamar Blue solution was added to the plate and incubated for 24 h. A blue colour in the well was interpreted as no bacterial growth and pink colour was scored as growth. The MIC was defined as the lowest drug concentration, which prevented colour change from blue to pink. The result of antitubercular activity depicted in Table 1. The MIC and MBC were determined according to the guidelines of CLSI (2011)CLSI, 2011. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes, 2nd ed. Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, USA, Approved Standard M24-A2.. Each experiment was performed at least twice according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2011CLSI, 2011. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes, 2nd ed. Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, USA, Approved Standard M24-A2.).
MIC and MBC values of the methanol extract and the isolated compounds against clinical isolate strain of Mycobacterium tuberculosis (AC 45).
Acid hydrolysis of 1
Compound 1 (8 mg) was dissolved in7% H2SO4 (0.5 ml) and heated on an aqueous bath at 100 ºC for 4 h. The reaction mixture was diluted with H2O and extracted with CH2Cl2. The CH2Cl2 layer was evaporated to dryness and purified by preparative TLC over silica gel with CH2Cl2–MeOH (5/1) as eluent. Apigenin-7-methyl ether 6-C-glucoside (3 mg) was isolated and identified through direct comparison with authentic samples (TLC, MP, and IR). The neutralized and lyophilized aqueuous hydrosylates of the aqueous solution gave only xylose. GC-MS (Column: 5% phenyl and 95% methyl silicone on ultra 2, 0.2 × 46 m, column temp.: 250 ºC, carrier gas: He 0.8 ml/min, sample: trimethylsilyl derivatives: tR (min) xylose (19.29 for 1).
Results and discussion
The detailed investigation of methanol extract of the stem bark of D. benthamianus led to the isolation of seven compounds. Six of them were identified as the known sitosterol 3-O-β-D-glucopyranoside (Ngono Bikobo et al., 2014Ngono Bikobo, D.S., Mosset, P., Abouem, A., Zintchem, A., Atchadé, A.T., Balemaken Missi, M., Mbabi Nyemeck, N., Pegnyemb, D.E., 2014. Campylospermine, an N-hydroxy alkaloid from the leaves of Campylospermum densiflorum (Ochnaceae). Int. J. Pharm. Phytopharmacol. Res. 6, 719-728.), 4-methoxygallic acid (2) (Ouyang et al., 2007Ouyang, M.A., Wein, Y.S., Su, R.K., Kuo, Y.H., 2007. Rhusemialins A-C, new cyclolignan esters from the roots of Rhus javanica var. roxburghiana. Chem. Pharm. Bull. 55, 804-807.), syringic acid (3) (Bayiha Ba Njock et al., 2011Bayiha Ba Njock, G., Bartholomeusz, T.A., Foroozandeh, M., Pegnyemb, D.E., Christens, P., Jeannerat, D., 2011. NASCA-HMBC, a new NMR methodology for the resolution of severely overlapping signals: application to the study of agathisflavone. Phytochem. Anal. 23, 126-130.), quercetin (Güvenalp and Demirezer, 2005Güvenalp, Z., Demirezer, O., 2005. Flavonol glycosides from Asperula arvensis L.. Turk. J. Chem. 29, 163-169.), 6"-O-acetylvitexin (4) (Bayiha Ba Njock et al., 2011Bayiha Ba Njock, G., Bartholomeusz, T.A., Foroozandeh, M., Pegnyemb, D.E., Christens, P., Jeannerat, D., 2011. NASCA-HMBC, a new NMR methodology for the resolution of severely overlapping signals: application to the study of agathisflavone. Phytochem. Anal. 23, 126-130.), quercetin 3-O-β-D-glucopyranoside (Murai et al., 2014Murai, Y., Kitajima, J., Iwashina, T., 2014. Flavonoids from two alpine Campanula species in Japan. Bull. Natl. Mus. Nat. Sci. 40, 113-118.). The structures of these compounds were elucidated by NMR spectroscopy analysis, including 1D and 2D techniques and also by comparing experimental data with respective literature data
Compound 1 was obtained as yellow amorphous powder, [α]D25=−54º (c = 0.05, MeOH). Its molecular formula, C27H30O14 was established by negative-ion HR-ESI-MS (Fig. S4). The spectrum displayed the deprotonated molecule peak [M−H]− at m/z = 577.4 in agreement with the above formula (calcd, 577.44). The IR spectrum of 1 showed absorption bands characteristic of hydroxyl groups (3219 cm−1), conjugated carbonyl groups (1652 cm−1) and aromatic rings (1603 and 1572 cm−1). UV spectral properties of 1 showed absorption maxima at λmax 340 nm and 268 nm in MeOH, characteristic for a substituted flavone (Mabry et al., 1970Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer, 354 p.). In addition, acid hydrolysis of 1 gave apigenin 7-methyl ether 6-C-glucoside and β-xylose which were identified by TLC analysis and comparison with authentic samples (GC; tR 19.29 min). In the 1H NMR spectrum (Table 2) the set of ortho-coupled AA'BB' type protons at δH 8.04 (2H, d, J = 8.8 Hz) and 6.96 (2H, d, J = 8.8 Hz), was respectively assigned to H-2'/6' and H-3'/5' protons of the B-ring of the molecule, while an isolated aromatic proton appeared at δH 6.44 (s, H-8) from A ring. The spectrum also revealed the presence of a methoxyl group at δH 3.86 and two signals assignable to anomeric sugar protons, which were identified to be an inner β-glucopyranose and a terminal β-pyranose structure of xylose. This was strengthened by the observation in 13C NMR and DEPT spectra of eleven carbon signals (Table 2) among which two are anomeric carbon signals at δC 73.5 and 105.3, seven methine carbon signals, two oxymethylene carbons at δC 60.8 and 65.3. Since the anomeric protons of glucose and xylose at δH 4.81 and 3.82 exhibited large coupling constants (J = 10.0 and 7.0 Hz), the sugars were considered of the β-pyranose type. The HMBC spectrum of compound 1 revealed correlations of the anomeric proton at δH 4.81 (H-1") and carbons at δC 161.3 (C-7), 155.6 (C-5) 104.7 (C-6) and 81.7 (C-5") (Fig. 1), indicating the C-C bond between the inner β-glucopyranosyl moiety and the aglycone at 6-position. In addition, H-1''' at δH 3.82 correlates to both C-3" (δC 80.4) and C-5''' (δC 65.3) revealing that the β-xylopyranosyl moiety was linked to C-3" at δC 80.4, showing that glucose and xylose are linked through a 1→3 type. This was strengthened by the NOESY crosspeaks of the protons H-3" (δH 4.01) with H-1''' (δH 3.82) confirming the aforementioned bonding. The attachment of a methoxyl group to the 7-position was shown by the observation of the crosspeaks at δH 3.86 (3H, s, OMe) and δC 164.1 (C-7) in the long-range HMBC spectrum. this position through the correlation between H-8 (δH 6.44) and the methoxyl proton signals at δH 3.86. The complete assignment of all proton and carbon resonances was achieved after careful analysis of COSY, HSQC and HMBC techniques.
Some significant HMBC correlations are shown in Fig. 1 and in Table 2. Compound 1 is closely related to the previously reported swertisin 2"-O-arabinoside from the tall bearded iris (Takayuki et al., 2012Takayuki, M., Tsutomu, Y., Nobuhiro, S., Tsukasa, I., 2012. Phenolic compounds, including novel C-glycosylflavone, from the flowers of the tall bearded Iris cultivar ‘Victoria Falls’. Nat. Prod. Commun. 7, 1591-1594.); meanwhile, differences occur in the sequence of sugar moieties and this is exemplified by the values of the retention times of xylose [which is close to reported data (Liu et al., 2009Liu, R., Ma, S., YU, S., Pei, Y., Zhang, S., Chen, X., Zhang, J., 2009. Cytotoxic oleanane triterpene saponins from Albizia chinensis. J. Nat. Prod. 72, 632-639.)]. This assertion is also strengthened by the upper chemical shift values of protons of the xylose moieties compared to those of arabinose (Gu et al., 2011Gu, X., Lee, S.G., Bar-Peled, M., 2011. Biosynthesis of UDP-xylose and UDP-arabinose in Sinorhizobium meliloti 1021: first characterization of a bacterial UDP-xylose synthase, and UDP-xylose 4-epimerase. Microbiology 157, 260-269.).
Accordingly, 1 was defined as apigenin 7-methyl ether 6-C-[β-xylopyranosyl-(1→3)-β-glucopyranoside] named distemonanthoside. To the best of our knowledge, this is the first report of the isolation of this compound and others (phenolic acids and sterols) from D. benthamianus.
According to Cantrell et al. (2001)Cantrell, C.L., Franzblau, S.G., Fischer, N.H., 2001. Antimycobacterial plant terpenoids. Planta Med. 67, 685-694. isolated compounds that exhibit a MIC of 64 µg/ml or lower are considered promising. For crude extracts, the MIC should be equal to or lower than 125 µg/ml (Gu et al., 2004Gu, J.Q., Wang, Y., Franzblau, S.G., Montenegro, G., Yang, D., Timmermann, B.N., 2004. Antitubercular constituents of Valeriana laxiflora. Planta Med. 70, 509-514.). Thus, the values of 125, 62.5, 31.25 and 62.5 µg/ml for 1, sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid and quercetin, respectively obtained here, are as good as a promising isolated compounds except for compound 1 (Table 1). According to Gu et al. the methanol extract of D. benthamianus showed poor inhibitory activity against M. tuberculosis, exhibiting a MIC and MBC of 1250 and 2500 µg/ml respectively, suggesting the low lipophilicity of its constituents (more polar compounds) when they act mutually in synergy. According to Peterson and Shanholtzer (1992)Peterson, L.R., Shanholtzer, C.J., 1992. Tests for bactericidal effects of antibacterial agents: technical performance and clinical relevance. Clin. Microbiol. Rev. 5, 420-432. bacteriostatic activity has been defined as a ratio of MBC to MIC of >4. Thus, all tested compounds exhibited bactericidal activity. The results of the present study are in accordance with previous report regarding the values of MIC of isolated compounds (Gu et al., 2004Gu, J.Q., Wang, Y., Franzblau, S.G., Montenegro, G., Yang, D., Timmermann, B.N., 2004. Antitubercular constituents of Valeriana laxiflora. Planta Med. 70, 509-514.; Jiménez-Alleranes et al., 2007Jiménez-Alleranes, A., Meckes, M., Torres, J., Herrera-Luna, J., 2007. Antimycobacterial triterpenoids from Lantana hispida (verbenaceae). J. Ethnopharmacol. 111, 202-205.).
Conclusion
The species D. benthamianus, is known as abundant sources of flavonoids. Compounds 1, sitosterol 3-O-β-D-glucopyranoside and 4-methoxygallic acid were isolated for the first time from this species. The bioactivity study of the isolated compounds indicated that three compounds (sitosterol 3-O-β-D-glucopyranoside, 4-methoxygallic acid and quercetin) exhibited interesting antitubercular activity.
Acknowledgements
The authors gratefully acknowledge financial support from the Swiss National Science Foundation (SNSF) (No: IZK0Z2-157272) for research fellowships in Switzerland to D. S. Ngono Bikobo. We thank Mr V. Nana for the collection and identification of plant material. We thank Mr Felix Fehr of Department of Chemistry of University of Fribourg and Koert's team, particularly Mr Oliver Born of Philipps-Universität Marburg for spectral analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2017.09.006.
References
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Publication Dates
-
Publication in this collection
Nov-Dec 2017
History
-
Received
10 May 2017 -
Accepted
12 Sept 2017