Z. Naturforsch. 2018; aop Jean de dieu Dongmo, Carine Mvot Akak, Michel Feussi Tala, Philippe Belle Ebanda Kedi, Anatole Guy Blaise Azebaze*, Juliette Catherine Vardamides* and Hartmut Laatsch Longiflorol, a bergenin α-d-apioside from the stem bark of Diospyros longiflora, and its antioxidant activity https://doi.org/10.1515/znb-2018-0019 Received January 21, 2018; accepted April 11, 2018 1 Introduction Abstract: Phytochemical investigation of the stem bark of Diospyros longiflora yielded longiflorol (1), a new bergenin α-d-apioside, together with bergenin (2) and five known compounds: lupeol (S1), betulin (S2), betulinic acid (S3), stigmasterol (S4) and stigmasterol glucoside (S5). Their structures were determined by one-dimensional (1D) and 2D nuclear magnetic resonance experiments along with electrospray ionization high-resolution mass spectrometry and extended density-functional theory calculations of chiroptical properties. Longiflorol (1) and bergenin (2) were evaluated for their DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant activity, with the crude extract for comparison and ascorbic acid as standard. The results showed that the extract and 2 had good antioxidant activity, whereas 1 showed only moderate activity at high concentration (>2 mg mL−1). The genus Diospyros belongs to the family Ebenaceae and consists of approximately 350 species of trees and shrubs distributed in tropical and subtropical regions of the world [1]. Several Diospyros plants are used in traditional medicine for the treatment of ailments such as cough, fever, diarrhea, dysentery, malaria and skin diseases [2, 3]. Previous phytochemical studies on the genus Diospyros resulted in the isolation of various classes of secondary metabolites including triterpenes, naphthoquinones, coumarins and phenolic glycosides [4–7]. Previous studies on the bark of Diospyros longiflora using chromatographic techniques yielded, by column chromatography or preparative thin layer chromatography over silica gel, diospyrin, lupeol (S1), betulin (S2) and betulinic acid (S3) [8]. We hereby describe the isolation and characterization of longiflorol (1), a new bergenin derivative, together with bergenin (2) [9] and further five known compounds (S1–S5) from the stem bark of D. longiflora. The antioxidant activities of longiflorol (1), bergenin (2) and the methanolic crude extract were also evaluated. Keywords: antioxidant activity; Diospyros longiflora; Ebenaceae; ECD calculations; longiflorol. 2 Results and discussion *Corresponding authors: Anatole Guy Blaise Azebaze and Juliette Catherine Vardamides, Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon, Phone: +237 699 63 76 24 (A.G.B. Azebaze), +237 677 91 96 03 (J.C. Vardamides), E-mail: azebaze@yahoo.com (A.G.B. Azebaze); jucathmas@yahoo.fr (J.C. Vardamides) Jean de dieu Dongmo: Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon Carine Mvot Akak: Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon Michel Feussi Tala and Hartmut Laatsch: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany Philippe Belle Ebanda Kedi: Department of Animal Biology and Physiology, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon By means of AntiBase [10], the known compounds 2 and S1–S5 were identified in the extract of D. longiflora by means of their spectroscopic data as bergenin (2) (Fig. 1) [9], lupeol (S1), betulin (S2), betulinic acid (S3), stigmasterol (S4) and stigmasterol glucoside (S5) (see the Supplementary Information for their structures); they were previously isolated from many other Diospyros species such as D. conocarpa [11], D. glandulosa [12] and D. rubra [13]. A new glycoside of bergenin (2), named longiflorol (Fig. 1), was obtained as a white amorphous solid with the molecular formula C19H24O13, as deduced by electrospray ionization high-resolution mass spectrometry (ESI-HRMS) (m/z = 483.1103 [M + Na]+). The 1H nuclear magnetic resonance (NMR) spectrum exhibited an aromatic singlet at Brought to you by | University of Sussex Library Authenticated Download Date | 7/3/18 6:41 PM 2 J. de dieu Dongmo et al.: Longiflorol and its antioxidant activity 5' OH O 2' 1' 2 O OH H O 12 HO 8 4a 3 O5 6a H OH OH O OH O OH H O HO 6 7 OH 4 10b 10a OH O 11 1 10 OH 3' 4' O O 1 2 H OH Fig. 1: Chemical structure of compounds 1 and 2. δH = 6.99 ppm, a methoxy signal at δH = 3.78 ppm and characteristic resonances of sugar moieties between δH = 3.20 and 5.70 ppm. These data were in accordance with those of the 13C NMR spectrum (see Table 1), which displayed Table 1: 1H NMR (500 MHz) and 13C NMR (125 MHz) data of longiflorol (1) in [D6]DMSO.a Position 1 δC 2 3 3-OH 4 4-OH 4a 6 6a 7 8 8-OH or 10-OH 9 10 10-OH or 8-OH 10a 10b 11 12-OCH3 1′ 2′ 2′-OH 3′ 3′-OH 4′ 5′ 5′-OH 79.0 70.7 73.5 79.2 163.3 118.0 109.4 151.0 140.5 148.0 115.7 72.0 67.8 59.8 109.1 75.9 78.7 73.4 62.9 δH (multiplicity, J in Hz) 3.70–3.76 (m) 3.17–3.25 (m) 5.53 (d, 5.6) 3.66 (dd, 5.4, 3.3) 5.61 (d, 5.3) 4.00 (dd, 10.3, 9.5) – – 6.99 (s) – 8.33 – – 9.77 – 4.99 (d, 10.4) 3.52 (dd, 11.1, 8.2) 3.96 (dd, 11.1, 2.0) 3.78 (s) 4.87 (d, 3.3) 3.80 (m) 5.10 (d br, 6) – 4.53 (s br) 3.62 (d, 9.4) 3.91 (d, 9.4) 3.27–3.39 (m) 4.79 (t, 5.6) HMBC 2, 3 2, 3, 4 4, 10b 6, 6a, 8, 9, 10a 19 carbon signals. The latter were assigned by the heteronuclear single-quantum correlation (HSQC) experiment as six quaternary and one methine sp2 carbons; additionally, 12 oxygenated sp3 carbons were identified, including 1 methoxy, 3 oxymethylene and 8 oxymethine groups. The peak at δC = 163.3 ppm (C-6) was attributed to an ester carbonyl group and further confirmed by the infrared (IR) band at 1718 cm−1 (see Fig. S3, Supporting Information available online). The NMR data showed similarities with those of bergenin (2), also isolated from the same source. Longiflorol was finally confirmed by the heteronuclear multiple bond correlation (HMBC) and correlation spectroscopy (COSY) experiments (Fig. 2) as a bergenin apioside. Related metabolites have been reported recently from the Cissus, Rodgersia and Mollotus genera [14–16]. Among these metabolites, 11-β-d-xylopyranosyl-bergenin [15] had the same molecular formula as longiflorol, but carried two oxymethylenes instead of three found here in the HSQC experiment. The HMBC spectrum showed correlations of H-4′ with C-1′, C-2′ and C-3′, and of H-5′ with C-3′. These data and HMBC correlations between C-11 and H-1′ established an apiofuranosyl moiety at the 11-hydroxy group of 2 (Fig. 2), so that 1 or a stereoisomer thereof resulted (Fig. 3). Apiose [3-C-(hydroxymethyl)-d-glycerotetrose] is a branched sugar with only one stereocenter. It is widespread in plants, where it was found solely as furanosides of d-apiose. Because of the symmetry at C-3′, cyclization of apiose can yield two C-3′-epimers, d-apio-d-furanose and d-apio-l-furanose, both in the α and β forms, respectively; the d-apio-l-furanose [3-C-(hydroxymethyl)-l-threofuranose, found only in the 1′α-form] is, however, very rare in nature [17]. The NOESY spectrum of longiflorol showed correlations of H-2′ (δ = 3.80 ppm) with H-1′, Hb-4′ and with the OH-5′ triplet at δ = 4.79 ppm. The OH-5′ signal coupled additionally with Hb-4′ (3.91 ppm), in agreement with a new bergenin-apiofuranoside. To distinguish between the 2, 4, 10a 1′ 3.62 5' H 9 4' H 3.91 O 2' 1' 4.87 H O 4′ OH 4.79 OH 4.54 OH 5.10 OH O OH H 3.80 O 11 1 2 O OH 1′, 3′, 5′ H 10 O 12 HO 8 4A 3 O5 6A 6 7 O OH 4 10B 10A 3′, 5′ Full assignments of the proton and carbon shifts were accomplished by analysis of COSY, HSQC and HMBC spectra. Chemical shifts are listed in ppm, and coupling constants are in Hertz. OH 3' H OH O OH O OH H O HO H OH O a Fig. 2: Selected NOESY (left), COSY (right; bold bonds) and HMBC correlations (right; arrows) of longiflorol (1). Brought to you by | University of Sussex Library Authenticated Download Date | 7/3/18 6:41 PM J. de dieu Dongmo et al.: Longiflorol and its antioxidant activity ∆ε 3 200 -2 220 240 260 280 300 320 340 360 380 400 λ (nm) -7 -12 Fig. 3: Experimental CD spectrum of longiflorol (1) in methanol (green line) and calculated spectra of 11-(d-apio-α-d-furanosyl)bergenin (1, blue) and 11-(d-apio-α-l-furanosyl)-bergenin (1′,3′-epi-1, red). rare d-apio-α,l-furanoside and the widespread and therefore more plausible d-apio-α,d-furanoside, the chiroptical data, the 1H and 13C NMR shifts and the H-H coupling constants were calculated with the aid of density functional theory (DFT) methods (see Supporting Information). The experimental H1′-H2′ coupling constant (J = 3.3 Hz), the NMR shifts and the electronic circular dichroism (ECD) spectrum agreed better with the DFT-calculated and Boltzmann-averaged values of the d-apio-α-d-furanoside, than with the data calculated for the rare d-apio-α-l-isomer (Fig. 3). For both isomers, negative optical rotations were predicted as were found experimentally. The CD data calculated for the two non-natural d-apio-β-d/l-furanosides were clearly different (see Supplementary Information). The antioxidant activities of the crude extract as well as of compounds 1 and 2 were evaluated by their ability to scavenge free 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and thereby decolorizing the violet solution. The methanolic D. longiflora extract and bergenin (2) showed strong antioxidant activity with IC50 values of 16.86 and 27.08 µg mL−1, respectively; they were, however, less active than ascorbic acid (AA) (IC50 = 6.82 µg mL−1). Surprisingly, longiflorol (1) exhibited no appreciable antioxidant activity. Based on these data, it seems that the strong antioxidant activity of the extract is mainly due to the presence of bergenin (2) [9], which was isolated as the main component. 3 Experimental section 3.1 General experimental procedures Optical rotation (OR) was measured on a Perkin-Elmer polarimeter (model 241) at the sodium D line (λ = 589 nm). 3 UV/Vis spectra were recorded on a JASCO V-650 spectrometer (JASCO Labor- und Datentechnik Deutschland GmbH, Gross-Umstadt, Germany). ECD spectra were recorded on a JASCO J-810 spectrometer equipped with a JASCO ETC-505S/PTC-423S temperature controller. IR spectra were taken on a JASCO FT/IR-4100 type A instrument. The NMR spectra were recorded on a Varian Inova-500 NMR spectrometer at 300.141 MHz (1H) and 125.8 MHz (13C), respectively. Chemical shifts are given as δ values in ppm with tetramethylsilane as internal standard, and coupling constants J are given in hertz (Hz). ESI high-resolution mass spectra were obtained on a Bruker micrOTOF mass spectrometer. Open column chromatography was performed on silica gel (60–200 mesh). Thin layer chromatography (TLC) was carried out on pre-coated silica gel 60F254 plates (Merck), and the TLC spots were visualized under UV light at 254 nm or by spraying with 20% sulfuric acid followed by heating. 3.2 Collection and identification The stem bark of D. longiflora LETOUZEY & F. WHITE was collected at Benakoumbe, a Pygmies village near the Kribi-Campo road in the south region of Cameroon, in November 2015 and identified by Victor Nana of the National Herbarium of Cameroon at Yaoundé, where a voucher specimen (No. 45920 HNC) has been deposited. 3.3 Extraction and isolation The air-dried and powdered stem bark of D. longiflora (3.1 kg) was extracted twice with methanol at room temperature for 48 h. After evaporation under reduced pressure, 453.8 g of crude extract was obtained. A part of the extract was subjected to silica gel column chromatography (216.5 g) and eluted with a gradient of n-hexane-ethyl acetate (1:0 to 0:1) and EtOAc-MeOH (1:0 to 17:3); subfractions of each 200 mL were collected and pooled on the basis of their TLC profile into 12 fractions. Fraction 2 eluted with n-hexane-EtOAc (39:1 to 19:1) yielded S1 (145.6 mg). Fraction 4 eluted with n-hexaneEtOAc (19:1 to 9:1) afforded S4 (31.2 mg). Elution with n-hexane-EtOAc (9:1 to 2:3) led to fraction 6, which yielded S2 (1.32 g) and S3 (5.26 g). Fraction 8 eluted with n-hexane-EtOAc (2:3 to 1:3) yielded S5 (25.6 mg). The elution with EtOAc-MeOH (1:0 to 39:1) gave fraction 10, which afforded 2 (6.01 g) as the major compound. Fraction 11 eluted with EtOAc-MeOH (39:1) gave the new derivative 1 (28.7 mg). Brought to you by | University of Sussex Library Authenticated Download Date | 7/3/18 6:41 PM 4 J. de dieu Dongmo et al.: Longiflorol and its antioxidant activity Longiflorol [11-(d-apio-α-d-furanosyl)-bergenin; 1]: White amorphous solid, Rf = 0.63 with EtOAc-MeOH (17:3), [a ]D20 = –42° (c 1, MeOH). – UV/Vis (MeOH) and ECD (MeOH) spectra: see Fig. S3 (Supporting Information) and Fig. 3. – IR (neat on diamond): 3325 br, 2927, 2855, 1718, 1615, 1595, 1352, 1242, 1017, 960, 875, 828, 770 cm−1 (see also Fig. S4, Supporting Information). – 1H NMR and 13C NMR data see Table 1. – HRMS [(+)-ESI]: m/z = 483.1103 (calcd. 483.1109 for C19H24O13Na, [M + Na]+). 3.4 DPPH antioxidant activity The DPPH antioxidant activities of longiflorol (1), bergenin (2) and the methanolic crude extract of D. longiflora were estimated with the modified method of Kato [18]. Various concentrations of each sample from 0.0125 to 2 mg mL−1 were mixed with 2 mL of 100 µmol DPPH solution in methanol. The mixture was vigorously shaken and left to stand for 30 min in the dark, and its absorbance was measured at 517 nm using a BIOBASE BK-UV-1600 PC spectrophotometer. The concentration of each sample required for scavenging 50% of the free DPPH radicals (IC50) was determined graphically by plotting the percentage of DPPH decrease as a function of the sample concentration: DPPH radical scavenging = ODsample 1 − ODcontrol × 100% As control, a mixture of 1 mL of methanol and 2 mL of DPPH solution in methanol (100 µm) was used; AA served as reference. Four samples were measured: longiflorol (1, IC50 > 2000 µg mL−1), bergenin (2, IC50 = 27.08 µg mL−1), AA (IC50 = 6.82 µg mL−1) and the methanolic crude extract of D. longiflora (IC50 = 16.86 µg mL−1). 3.5 DFT calculations The least energy conformers of longiflorol (1) were determined in a systematic approach using the Merck Molecular Force Field program; 300 000 conformers were analyzed (MMFF, Spartan’14 [19]). Further geometry optimizations with HF/3-21G and then with wB97X-D/631G* afforded nine conformers with Boltzmann factors >0.001. The respective CD spectra were calculated using the RwB97X-D functional and the 6-311G(d,p) basis set with Gaussian 09w [20] and averaged with the Boltzmann factors of the respective conformers. For the OR calculations, RwB97X-D/6-311G(d,p) [polar = optrot CPHF = RdFreq] was applied on geometries optimized as described above. For 1 with a-configured d-apio-l-furanose, an OR of [α]D = –190.5° was calculated (Boltzmannweighted average from nine conformers), while for the d-apio-α-d-furanoside 1, an OR of –228.4° was predicted from six conformers. 1H and 13C NMR data were calculated on the basis of the wB97X-D/6-31G* geometries, using Spartan’s EDF2/6-31G* NMR procedure. The results for all four d-apio-α,β-d/l-furanoside are also listed in the Supplementary Information. Acknowledgments: We thank Dr. H. Frauendorf and Dr. M. 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Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09w (version 7.0), Gaussian, Inc, Wallingford, CT (USA) 2009. Supplemental Material: The online version of this article offers supplementary material (https://doi.org/10.1515/znb-2018-0019). Brought to you by | University of Sussex Library Authenticated Download Date | 7/3/18 6:41 PM Z. Naturforsch. 2018 | Volume x | Issue x(b) Graphical synopsis OH Jean de dieu Dongmo, Carine Mvot Akak, Michel Feussi Tala, Philippe Belle Ebanda Kedi, Anatole Guy Blaise Azebaze, Juliette Catherine Vardamides and Hartmut Laatsch Longiflorol, a bergenin α-d-apioside from the stem bark of Diospyros longiflora, and its antioxidant activity OH O OH O OH O OH H https://doi.org/10.1515/znb-2018-0019 Z. Naturforsch. 2018; x(x)b: xxx–xxx O OH H O HO O Longiflorol Brought to you by | University of Sussex Library Authenticated Download Date | 7/3/18 6:41 PM