Accepted Article Article Type: Full Paper Tricalycoside, a New Cerebroside from Tricalysia coriacea (Rubiaceae) a Maurice D. Awouafack, *,a,b Pierre Tane, a and Hiroyuki Morita *,b Laboratory of Natural Products Chemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon, e-mail: ducret.awouafack@univ-dschang.org b Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan, email: hmorita@inm.u-toyama.ac.jp A new cerebroside, named as tricalycoside (1), was isolated from the CH2Cl2 / MeOH (1:1) extract of twigs and leaves of Tricalysia coriacea using repeated silica gel open column chromatography followed by Prep. TLC and Sephadex LH-20, together with six known compounds (2 - 7). The structure of the new compound was determined by analysis of 1D and 2D NMR, MS data, chemical conversion, and by comparison of these data with those from the literature. Tricalycoside (1) possessed a weak antibacterial activity against Klebsiella pneumoniae (MIC = 75 µg/mL). Keywords: Tricalysia coriacea, Rubiaceae, Cerebroside, Tricalycoside, Antibacterial Introduction Tricalysia coriacea (Benth.) Hiern., a member of Rubiaceae family, is a shrub or subshrub of 3 m height with elliptical and rectangular leaves and yellow fruits at mature stage. [1] It is found generally in tropical regions of Africa and usually at higher altitude in Cameroon such as Mount Manengouba, Mount Koupé, Mount Bakossi, and their neighborhoods. [2] This plant species is used in traditional medicine as sedative and emetic, and for the treatment of malaria, jaundice, and skin diseases. [3] Previous phytochemical studies of some plant species of the genus Tricalysia have reported the isolation of steroids, [4] terpenoids, [5 – 7] diterpenoid alkaloids, [8] fatty acid esters, and glycosides. [4] However, to the best of our knowledge, T. coriacea has not been investigated so far. As a part of our ongoing search for bioactive constituents from medicinal plants of Cameroon, [9 – 12] we isolated a new cerebroside, tricalycoside (1), along with six known compounds (2 – 7) from the CH2Cl2 / MeOH (1:1) extract of twigs and leaves of T. coriacea. In this paper, we report the isolation, the structural determination, and the antibacterial activity of 1. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/cbdv.201700472 This article is protected by copyright. All rights reserved. Results and Discussion Accepted Article The CH2Cl2 / MeOH (1:1) extract from twigs and leaves of T. coriacea was subjected to repeated silica gel open column chromatography, Prep. TLC, and Sephadex LH-20 to afford a new cerebroside named as tricalycoside (1), together with six known compounds, isoquercitrin (2), [13] astragalin (3), [14] rutin (4), [13] oleanolic acid (5), [15] ursolic acid (6), [15] and β-sitosterol-3-O-β-D-glucopyranoside (7, Figure 1). [16] The structures of the known compounds were identified by comparison of their spectroscopic data with those reported in the literature. Tricalycoside (1) was obtained as white powder and its molecular formula, C49H93NO11, was deduced from NMR data and HR-ESI-MS, from which a pseudo-molecular ion peak was obtained at m/z 894.6781(calc. for C49H93NO11Na 894.6747). Its IR spectrum displayed characteristic absorption bands, corresponding to hydroxyls and amide NH (3428 cm-1), amide carbonyl and non-conjugated C=C (1654 cm-1), and C-O (1027 cm-1) groups. The structure of 1 was assigned by analyses of 1D (1H-, 13C-) and 2D (HMQC, HMBC, 1H – 1H COSY, and ROESY) NMR and MS data. The 1H-NMR spectrum (Table 1) had signals of an amide proton (δ(H) 7.54 (d, J = 9.2)), long-chain aliphatic methylene protons (δ(H) 1.35 – 1.20 (br. s)), two terminal methyl groups (each δ(H) 0.85 (t, J = 6.3)), and an anomeric proton (δ(H) 4.13 (d, J = 7.5)), which are the characteristic signals for cerebroside skeleton. [10][17 - 19] Further signals were observed in the 1HNMR spectrum at δ(H) 5.34 – 5.29 (m, H-C(10) / H-C(11) / H-C(16) / H-C(17)), 3.10 – 3.06 (m, H-C(3)), 3.06 – 3.00 (m, H-C(4)), 3.80 (q, J = 8.0, 4.0, H-C(5)), and 3.37 – 3.30 (m, H-C(2')), suggesting that the two long-chain moieties of 1 had two olefinic, and four oxymethin groups. Moreover, the 1H-NMR spectrum revealed the presence of one glycosyl moiety in 1, which is characterized by the signals at δ(H) 2.93 (t, J = 8.0, H-C(2")), 3.10 – 3.06 (m, H-C(3")), 3.85 (br. s, H-C(4")), 3.17 – 3.14 (m, H-C(5")), 3.65 (br. d, J = 11.5, Ha-C(6")), and 3.45 – 3.40 (m, Hb-C(6")). This article is protected by copyright. All rights reserved. Accepted Article Figure 1. Chemical structures of compounds (1 - 7) isolated from T. coriacea The 13C-NMR spectrum (Table 1) of 1 displayed carbon signals at δ(C) 174.3 (C(1')), 69.4 (C(1)), 50.4 (C(2)), 29.6 – 29.5 (C(7)/ C(20) – C(22) / C(4') – C(15')), and 14.5 (C(25) / C(18')), corresponding to the typical signals for sphingolipids. [10][17 - 19] Moreover, the presence of signals on the 13C-NMR spectrum at δ(C) 104.0 (C(1")), 74.1 (C(2")), 77.0 (C(3")), 71.4 (C(4")), 77.4 (C(5")), and 61.6 (C(6")) confirmed the existence of a glycosyl moiety in 1, as suggested by 1H-NMR data. Its nature was identified as D-glucose by HPLC analysis after hydrolysis of 1 and conversion of the released sugar to o-tolylthiocarbamoyl thiazolidine derivative. The o-tolylthiocarbamoyl thiazolidine sugar derivative from 1 showed the same retention time as that of the standard reference of D-glucose. [20] The large coupling constant value (3J = 7.5) of the anomeric proton (H-C(1")) suggested the β-configuration of the glucose. The HMBC correlations (Table 1) from the proton at δ(H) 4.13 (H-C(1")) to the carbon at δ(C) 69.4 (C(1)) and from 3.64 (Hb-C(1)) to 104.0 (C(1")) allowed us to attach the glucose moiety at C(1). Further carbon signals observed at δ(C) 130.8 (C(10)), 130.4 (C(11)), 130.2 (C(16)), 129.9 (C(17)), 77.0 (C(3)), 70.5 (C(4)), 74.7 (C(5)), and 71.1 (C(2')) also confirmed the presence of two olefinic and four oxymethin groups in the two long-chain moieties of 1. Further important HMBC correlations observed from the proton at δ(H) 3.82 (Ha-C(1)) to the carbon at δ(C) 50.4 (C(2)), from 4.10 (H-C(2)) to 69.4 (C(1)), from 3.10 – 3.06 (H-C(3)) to 70.5 (C(4)), from 3.06 – 3.00 (H-C(4)) to 50.4 (C(2)) and 74.7 (C(5)), and from 3.80 (H-C(5)) to 70.5 (C(4)) allowed us to locate three hydroxyl groups at C(3), C(4), and C(5) of the sphingosine moiety. Another hydroxyl group was located at This article is protected by copyright. All rights reserved. Accepted Article position C(2') of the fatty acid moiety based on the carbon chemical shift in comparison with similar related compounds, laportoside A, and politamide. [21][22] The NMR data of 1 were similar to those of previous reported politamide isolated from Ficus polita. [22] The significant differences between 1 and politamide were the lengths of the fatty acid (C18 in 1, while C15 for politamide) and sphingosine (C25 for 1, whereas C29 in politamide) moieties, as well as the location, number, and configuration of the double bonds (two double bonds with Econfigurations on the sphingosine moiety in 1, while only one with Z-configuration on the fatty acid chain in politamide). The chain lengths of the fatty acid and sphingosine, and the position of the double bonds in 1 were determined by characteristic ion-fragment peaks obtained from ESI-MS experiment. Important ion-fragments observed on the ESI-MS spectra (Fig. 2) of 1 at m/z 616 ([M C16H33CHOH]-), 607 ([(M+H) - C16H33CHOHCO + H2O]+), and 573 ([M - C16H33CHOHCONH]+) allowed us to assign the fatty acid and the long-chain base lengths to be C18 for the fatty acid moiety and C25 for sphingosine in 1, respectively. [21][23][24] Furthermore, ion-fragments (Fig. 2) at m/z 651 ([(M + H) – C16H29]+), 695 ([(M + H) – C14H28 + H2O]+), 775 ([(M + K) – C10H20 + 3H]+), and 794 ([(M + Cl) – C8H18]-) suggested that the olefinic groups were located at C(10) and C(16) in 1, respectively. Their Econfigurations were assigned from the 13C-NMR chemical shift values of 31.8 ppm of their allylic carbons (C(9) / C(12) / C(15) / C(18)). [23][25] The geometry (E or Z-configuration) of the double bond in the long-chain alkene is mainly identified by coupling constant values (16 Hz for trans and 8 Hz for cis) of olefinic protons, however in the case that the olefinic protons are observed as multiplet, this could also be assigned from the 13C-NMR chemical shifts of allylic methylenes (δ(C) more than 30 for E-configuration, and δ(C) at 26 for Z-configuration). [23][25-27] Table 1. H- and C-NMR data (500 and 125 MHz, resp.) for 1 in (D6) DMSO. δ in ppm, J in Hz 1 13 Position δ(H) δ(C) HMBC (H to C) 50.4 C-1 77.0 C-4 70.5 C-2, C-5 74.7 C-4 31.8 C-8 29.6 – 29.5 - 29.2 C-10 31.8 C-10, C-11 130.8 C-9, C-12 5.34 – 5.29 (m) 130.4 C-9, C-12 1.94 – 1.89 (m) 31.8 C-10, C-11 This article is protected by copyright. All rights reserved. Accepted Article 1.94 – 1.89 (m) 29.2 C-11 29.2 C-16 31.8 C-16, C-17 130.2 C-15, C-18 129.9 C-15, C-18 31.8 C-16, C-17 29.2 C-17 29.6 – 29.5 - 31.8 - 22.6 C-22, C-25 14.5 C-1', C-2 ' 174.3 ' 71.1 - ' 31.8 - 29.6 – 29.5 - ' 31.8 - ' 22.6 C-15', C-18' ' 14.5 - ' " 4.13 (d, J = 7.5) 104.0 C-1 " 2.93 (t, J = 8.0) 74.1 C-1" 3.10 – 3.06 (m) 77.0 3.85 (br. s) 71.4 " 3.17 – 3.14 (m) 77.4 " 3.65 (br. d, J = 11.5), 3.45 – 3.40 (m) 61.6 " " a ' Overlapped signal with that of residual H 2O from (D6) DMSO This article is protected by copyright. All rights reserved. Accepted Article Figure 2. Important mass fragmentation pattern of tricalycoside (1). The relative configurations were assigned by the ROESY experiment. The ROESY correlations (Fig. 3) of Hb-C(1), H-C(2), H-C(4), and H-C(5) allowed us to assign α-configurations of HO-C(4) and HO-C(5), respectively. Furthermore, lack of the ROESY correlations for H-C(2)' and H-C(3), and the comparison of the chemical shifts of carbons C(2') and C(3) with those of politamide [22] as well as considering the steric hindrance of sphingolipids, [23] suggested the α- and β-configurations of HOC(2') and HO-C(3), respectively. In fact, considering the structural aspect of sphingolipids, even though C-C single bonds of carbon chain could rotate freely, it is possible that from the steric hindrance HN-C(2), HO-C(3), HO-C(4), and HO-C(5) would not be oriented in the same side of the molecule. The absolute configuration of this type of compounds has been theoretically assigned on the basis of the spatial orientation of the hydroxyl groups. Thus, the absolute configurations of C(2), C(2'), C(3), C(4), and (5) were assigned to be (2S), (2'R), (3S), (4R), and (5S), based on the spatial orientations of their substituents and by the fact that their NMR data were close to those of politamide. [22] Hence, compound 1 was elucidated as (2R)-N-[(2S,3S,4R,5S,10E,16E)-1-(β-Dglucopyranosyloxy)-3,4,5-trihydroxypentacosa-10,16-dien-2-yl]-2-hydroxyoctadecanamide, and named tricalycoside. Figure 3. Key ROESY correlations of tricalycoside (1). This article is protected by copyright. All rights reserved. Antibacterial activity Accepted Article The antibacterial activity of 1 was assessed using a broth microdilution method [11][28] to determine its minimum inhibitory concentration (MIC) against four bacteria strains (two Grampositive bacteria: Bacillus subtilis NBRC 13719 and Staphylococcus aureus NBRC 100910, and two Gram-negative bacteria: Klebsiella pneumoniae NBRC 14940 and Escherichia coli NBRC 102203). Tricalycoside (1) had weak activity against K. pneumoniae (MIC = 75 µg/mL), and was not active against other bacteria strains. Ampicillin (Nacalai Tesque) and kanamycin (Nacalai Tesque) were used as positive controls, while the negative control was DMSO (solvent used to dissolve the sample, concentration less than 5% which did not affect the results). Conclusions A phytochemical investigation of the CH2Cl2 / MeOH (1:1) extract of twigs and leaves of T. coriacea gave a new cerebroside, tricalycoside (1), together with six known compounds (2 - 7). Tricalycoside (1) had a weak antibacterial activity against K. pneumoniae (MIC = 75 µg/mL). Isolation of further secondary metabolites with possibly strong antibacterial activity in the plant species are currently under investigation. Experimental Section General IR spectra were recorded on JASCO FT-IR 460 spectrometer (Kyoto, Japan). Alpha D were measured on JASCO P-2100 polarimeter. MS data were measured on JEOL MS Station JMS-700 and SHIMADZU LCMS-IT-TOF spectrometers. NMR spectra were recorded on JEOL 500 spectrometer. Chemical shift values were expressed in δ (ppm) downfield from tetramethylsilane (TMS), as an internal standard. Column chromatography was run on silica gel 60N spherical, neutral, 40−50 μm, (Kanto, Japan), Cosmosil 75C18-silica gel (Kyoto, Japan), and on Sephadex LH-20. TLC was carried out on silica gel GF254 pre-coated (MERCK) plates with detection accomplished by visualizing with UV lights (254 and 365 nm), and followed by spraying with 1% Ce(SO4)2 -10% aqueous H2SO4 and then heating up to 150°C. Plant materials Twigs and leaves of T. coriacea (Benth.) Hiern. were collected in Dschang, West Region of Cameroon, on February 2016 and were identified by Mr. Victor Nana, a botanist at the Cameroon National Herbarium in Yaoundé where our specimen was deposited under a voucher number 46470/HNC. Extraction and isolation Dried and powdered twigs and leaves of T. coriacea (1.5 kg) were macerated in (5 L) CH2Cl2 / MeOH (1;1, v/v) for 24 h and repeated 3 times to afford a crude extract (142 g, 9.5% yield) after filtration and removal of the solvents using rotary evaporator. Part of this extract (45.8 g) was This article is protected by copyright. All rights reserved. Accepted Article subjected to HP-20 Diaion using water and methanol to furnish H2O (5.58 g), H2O / MeOH (1:1, v/v, 27 g), and MeOH (1.12 g) fractions, respectively. The H2O / MeOH (1:1, 27 g) fraction was subjected to open column chromatography (CC) over silica gel using CHCl3, and MeOH in increasing polarity to give 65 fractions of 500 mL each. Combined fraction 25-30 (617.4 mg) obtained from CHCl3 / MeOH (22:3) precipitated partially in acetone to give a powder made by a mixture of two major compounds that was further purified by preparative TLC using EtOAc / MeOH / H2O (9: 0.5: 0.5, v/v/v) to afford 1 (6.7 mg), and 2 (2.8 mg). The residue from the combined fraction 25-30 after the filtration was subjected to Sephadex LH-20 using CH2Cl2 / MeOH (1:1, v/v) to afford 3 (8.5 mg). Combined fraction from 45-50 [1.5 g, CHCl3 / MeOH (7:3)] was subjected to reverse phase CC over cosmil 75C18-silica gel using H2O / MeOH (3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, and 0:1) as mobile phase to give 46 sub-fractions of 100 mL each. Sub-fraction 5-6 [120.5 mg, H2O / MeOH (3:1)] was further subjected to Sephadex LH-20 using CH2Cl2 / MeOH (1:1, v/v) to afford 4 (37.8 mg). Combined fraction from 17-18 (17.3 mg) obtained from CHCl3 / MeOH (9:1) was purified by repeated Sephadex LH-20 to afford 5 (5. 5 mg), and 6 (6. 4 mg), whereas combined fraction from 21 -24 (174.2 mg) obtained from the same polarity precipitated partially in acetone to afford 7 (22.5 mg) after filtration. Tricalycoside (1): white powder. [α]22D – 57 (c 0.1, DMSO). IR (KBr): υmax 3428, 2923, 1654, 1072, 826, 766 cm-1. 1H-(500 MHz) and 13C-(125 MHz) NMR ((D6) DMSO): see Table 1. ESIMS (+): m/z 894.7 ([M + Na]+), 852.6, 775.9, 747.0, 695.2, 651.2, 628.9, 607.1, 573.4. ESIMS (-): m/z 906.7 ([M + Cl]-), 878.6, 794.6, 616.2. HRESIMS (+): m/z 894.6781 (calc. for C49H93NO11Na 894.6747). Acid hydrolysis of 1 Compound 1 (3 mg) was dissolved in MeOH (1 mL), to which 10% HCl (3 mL) was added and refluxed for 4 h at 105°C. The extraction of the reaction mixture with EtOAc gave the aqueous layer that was neutralized by Amberlite IRA-400 (Sigma-Aldrich, France), concentrated to dryness, and incubated at 60°C for 1 h in a solution of L-cysteine methyl ester hydrochloride (0.5 mg) / pyridine (100 µL). Thereafter, 100 µL of pyridine / o-tolylisothiocyanate (1:1, v / v) was added to the reaction mixture and incubated for further 1 h at 60°C. The o-tolylthiocarbamoyl thiazolidine derivative of Dglucose (Isotec, Miamisburg, USA) was also prepared separately as described above and all derivatives were analyzed by reversed-phase HPLC using a Cosmosil 5C18-AR-II column (Nacalai Tesque, 250 x 4.6 mm, temperature: 35°C) with isocratic mobile phase of MeCN (0.1% TFA)-H2O (0.1% TFA) (75:25, v / v), and a flow rate of 0.8 mL / min with a UV detection at 250 nm. [20] Supplementary Material Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number. This article is protected by copyright. All rights reserved. Acknowledgements Accepted Article We are grateful to the Japan Society for the Promotion of Science for Postdoctoral Fellowship awarded to Dr. M.D. Awouafack as Overseas Researcher (P16411) to work at the Institute of Natural Medicine, University of Toyama. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants JP15H05138, JP17H02203, JP17H05435). Author Contribution Statement MDA carried out the experimental part and wrote the manuscript. PT and HM supervised the work and edited the final version of the manuscript. Disclosure statement The authors declare no conflict of interest. References [1] D. M. Bridson, B. Verdcourt, 'Rubiaceae, Part 3', Flora Zambesiaca 2003, 5, 483 - 484. [2] M. Cheek, B. J. Pollard, I. Darbyshire, J. M. Onana, W. 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All rights reserved. Tricalycoside (1)