Accepted Manuscript Chemical Constituents from Sonneratia ovata Backer and their in vitro Cytotoxicity and Acetylcholinesterase Inhibitory Activities Thi-Hoai-Thu Nguyen, Huu-Viet-Thong Pham, Nguyen-Kim-Tuyen Pham, Ngo-Diem-Phuong Quach, Khanitha Pudhom, Poul Erik Hansen, Kim-PhiPhung Nguyen PII: DOI: Reference:
S0960-894X(15)00328-5 http://dx.doi.org/10.1016/j.bmcl.2015.04.017 BMCL 22600
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
10 August 2014 30 March 2015 6 April 2015
Please cite this article as: Nguyen, T-H., Pham, H-V., Pham, N-K., Quach, N-D., Pudhom, K., Hansen, P.E., Nguyen, K-P., Chemical Constituents from Sonneratia ovata Backer and their in vitro Cytotoxicity and Acetylcholinesterase Inhibitory Activities, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl. 2015.04.017
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Chemical Constituents from Sonneratia ovata Backer and their in vitro Cytotoxicity and Acetylcholinesterase Inhibitory Activities Thi-Hoai-Thu Nguyena, Huu-Viet-Thong Pham b, Nguyen-Kim-Tuyen Phamc, Ngo-Diem-Phuong Quachd, Khanitha Pudhome, Poul Erik Hansenf, Kim-Phi-Phung Nguyenb,* a
Department of Basic Science, University of Medicine and Pharmacy – Ho Chi Minh City, Vietnam. Department of Organic Chemistry, VNUHCM - University of Science, Ho Chi Minh City, Vietnam. c Department of Environmental Science, Saigon University, Ho Chi Minh City, Vietnam. d Department of Biology, VNUHCM - University of Science, Ho Chi Minh City, Vietnam. e Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. f Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark. * Correspondence to: Nguyen Kim Phi Phung, Department of Organic Chemistry, Vietnam National University - University of Science, 227 Nguyen Van Cu Str., Dist. 5, Ho Chi Minh City, Vietnam. E-mail: [email protected]. Tel.: +84-1226 966 660 b
Abstract Sonneratia ovata Backer, Sonneratiaceae, is a widespread plant in mangrove forests in Vietnam, Cambodia, Thailand, Indonesia,… Sonneratia ovata’s chemical composition remains mostly unknown. Therefore, we now report on the structural elucidation of three new phenolics, sonnerphenolic A (1), sonnerphenolic B (2), and sonnerphenolic C (23), a new cerebroside, sonnercerebroside (3) together with nineteen known compounds, including nine lignans (5− −13), two steroids (14, 15), two triterpenoids (16, 17), three gallic acid derivatives (18− −20), two phenolic derivatives (4, 22) and a 1-O-benzyl-β-D-glucopyranose (21) isolated from the leaves of Sonneratia ovata. Their chemical structures were established by spectroscopic data, as well as high resolution mass spectra and comparison with literature data. The in vitro acetylcholinesterase (AChE) inhibition and cytotoxic activities against HeLa (human epithelial carcinoma), NCI-H460 (human lung cancer), MCF–7 (human breast cancer) cancer cell lines and PHF (primary human fibroblast) cell were evaluated on some extracts and purified compounds at a concentration of 100 µg/mL. Compounds (5, 6, 23) exhibited cytotoxicity against the MCF-7 cell line with the IC50 values of 146.9±9.0, 114.5±7.2, and 112.8±9.4 µM, respectively, while they showed nontoxic with the normal cell (PHF) with IC50s > 277 µM. Among 15 tested compounds, (S)-rhodolatouchol (22) showed inhibition against AChE with an IC50 value of 96.1±14.5 µM. Key words: Sonneratiaceae, Sonneratia ovata, phenolics, cerebroside, in vitro cytotoxicity and acetylcholinesterase inhibition. Abbreviations: AChE: acetylcholinesterase HeLa: human epithelial carcinoma NCI-H460: human lung cancer 1
MCF–7: human breast cancer PHF: primary human fibroblast 1D/2D-NMR: One/Two Dimensional –Nuclear Magnetic Resonance COSY: Correlation Spectroscopy HSQC: Heteronuclear Single Quantum Correlation HMBC: Heteronuclear Multiple-Bond Correlation NOESY: Nuclear Overhauser Effect Spectroscopy HR-ESI-MS: High Resolution – Electrospray Ionization – Mass Spectrometry
The Sonneratia species growing widely in mangrove forests in tropical areas possess a huge biomass that promises a potent resource for human use. Indeed, for a long time, the fruits, bark, and leaves of some Sonneratia plants have been used in folk medicine to treat different diseases such as asthma, febrifuge, ulcers, hepatitis, piles, sprain, and hemorrhages.1 Therefore, this genus is attractive for pharmacological and chemical studies. Previously pharmaceutical investigations showed that the methanol extract of S. caseolaris seeds was a potential AChE inhibitor with the IC50 value of 10.52 µg/mL.2 Isoorientin exhibited promising inhibition against both AChE and BChE with IC50 of 26.8 and 31.5 µM, respectively, while isovitexin exhibited acetylcholinesterase inhibition with IC50 of 36.4 µM.3 These two compounds were previously reported from leaves of S. ovata. Luteolin isolated from stems and twigs of S. caseolaris exhibited significant cytotoxicity against SMMC–7721 with IC50 value of 2.8 µg/mL.4 From the fruits of S. ovata, three compounds (–)-(R)-nyasol, (–)-(R)-4’-O-methylnyasol and maslinic acid were isolated. Determination by the MTT assay method, these three compounds showed moderate cytotoxic activities against the rat glioma C-6 cell line with IC50 values of 19.02, 20.21, and 31.77 µg/mL, respectively.5 Previous chemical studies on this plant reported a number of compounds isolated from S. ovata.5,6 Based on this we would like to study the chemical constituents of leaves of S. ovata, and test in vitro cytotoxicity and AChE inhibition of extracts and some isolated compounds. The chemical investigation on the S. ovata leaves collected at the Can Gio mangrove forest led to the isolation of twenty three compounds by the use of efficient separation techniques (See Fig. 1) including four new compounds, sonnerphenolic A (1), sonnerphenolic B (2), sonnerphenolic C (23) and sonnercerebroside (3). The other compounds were identified as (-)-(R)-nyasol (4),7 (7S,8R)-dehydroconiferyl alcohol (5),8 (7S,8R)-5-methoxydehydroconiferyl alcohol (6),8 (7S,8R)urolignoside (7),9 lingueresinol (8),10 (+)-isolariciresinol (9),11 (+)-isolariciresinol 9’-O-β-Dglucopyranoside (10),12 (–)-isolariciresinol 9’-O-β-D-glucopyranoside (11),12 (–)-episyringaresinol (12),13 (+)-syringaresinol (13),13 β-sitosterol (14),4 3-O-palmitoyl-β-sitosterol (15),4 corosolic acid (16),14 3-O-acetylursolic acid (17),15 6-O-galloyl-D-glucopyranose (18),16 gallic acid 3-O-β-Dglucopyranoside (19),17 1-O-benzyl-6-O-galloyl-β-D-glucopyranose (20),18 1-O-benzyl-β-Dglucopyranose (21),12 and (−)-rhodolatouchol (22)19 via spectroscopic data analysis and comparison with those reported in the literature. In this paper, the chemical structure elucidation of four new compounds was described. The bioactive assays of the AChE inhibition and the cytotoxic activities 2
against the HeLa, NCI-H460, MCF–7 cell lines and the PHF cell were evaluated on some extracts and purified compounds (with purities better than 95% as determined by proton NMR data). Compound 1 was obtained from an ethyl acetate fraction as a colorless wax, ( [α ]D = +3.8, c 25
0.37, MeOH), with a molecular formula of C17 H18O4 determined by the pseudo-molecular ion peak in the HR-ESI-MS at m/z 309.1117 [M+Na]+ (Calcd. for C17H18O4Na, 309.1097). The 13C-NMR data (Table 1) displayed 13 carbon signals, including four signals at δC 131.5, 129.9, 116.1, and 115.8 appearing as double intensity. Additionally, the 1 H-NMR spectrum confirmed the presence of four doublet proton signals of eight aromatic protons at δH 7.07 (2H, d, 8.5 Hz, H-2’’, H-6’’), 7.03 (2H, d, 9.0 Hz, H-2’, H-6’), 6.65 (2H, d, 9.0 Hz, H-3’, H-5’) and 6.62 (2H, d, 8.5 Hz, H-3’’, H-5’’). These observations suggested the presence of two para-disubstituted benzene groups in the structure of the compound. At the lower frequency range from 1.70 to 4.30 ppm, the 1H-NMR spectrum displayed two oxygenated methine protons, two methine protons and two methylene protons corresponding to five carbon signals from 44.0 to 79.6 ppm in the 13C-NMR spectrum. These proton and carbon signals belonged to a cyclopentane ring which was confirmed through the COSY correlations of H-1/H-2/H-3/H-4/H-5/H-1 (See Fig. 2). The HMBC correlations of H-2 with C-1’, C-2’, C-6’, of H-2’ with C-2, of H-3 with C-2’’, C-6’’ and of H-2’’ with C-3 suggested the attachment of two 4-hydroxyphenyl groups to the cyclopentane ring at its C-2 and C-3. On the basis of the above analysis, the gross structure of 1 was 1,4-dihydroxy-2,3-di-(4hydroxyphenyl)cyclopentane. The relative configuration of 1 was further proven by the NOESY spectrum. The NOESY spectrum exhibited cross peaks between H-3 and H-4, between H-4 and H-5b, between H-5b and H1, between H-1 and H-3 (See Fig. 3), therefore these four hydrogen atoms were on the same side of the ring. The NOESY correlation of H-5a with H-2 proved that these two hydrogen atoms were on the same side of the plane, and on the opposite side comparing to the four mentioned above atoms. Therefore, 1 was thus determined to be (1β,2α,3β,4β)-1,4-dihydroxy-2,3-di-(4-hydroxyphenyl) cyclopentane and was named sonnerphenolic A. Compound 2 was obtained from an ethyl acetate fraction as a yellowish oil, ( [α ]D = -282.8, c 25
0.18, MeOH) with a molecular formula of C18H18O3 determined by the pseudo-molecular ion peak in the HR-ESI-MS at m/z 305.1160 [M+Na]+ (Calcd. for C18H18O3Na, 305.1154). The 1H-NMR spectrum displayed the presence of seven aromatic protons including three signals at δH 6.90 (1H, brs, H-2’), 6.81 (1H, d, 8.0 Hz, H-5’) and 6.80 (1H, brd, 8.0 Hz, H-6’) of an ABX system of an Abenzene ring and two signals at δH 7.09 (2H, d, 8.4 Hz, H-2’’ and H-6’’) and 6.78 (2H, d, 8.4 Hz, H-3’’ and H-5’’) of an AA'BB' system of a B-benzene ring. The 1 H-NMR and HSQC spectra also showed the presence of two olefinic methylene protons, three olefinic methine protons of two double bonds in the zone from 5.15 to 6.50 ppm, a down field shifted saturated methine proton at δH 4.54 (1H, dd, 9.6, 6.4 Hz, H-3) and a signal at δH 3.89 (3H, s) of a methoxy group. These corresponded to the observation of sixteen carbon signals including two signals appearing as double intensity at δC 129.0 and 115.6 belonging to an AA'BB' system in the 13C-NMR spectrum. Moreover, the COSY spectrum revealed the correlations of the proton H-1 at δH 6.49 and proton H2 at δH 5.67, of proton H-2 and proton H-3 at δH 4.54, of proton H-3 and proton H-4 at δH 6.01, and of H-4 and protons H-5 at δH 5.20 and 5.17 (See Fig. 2). These findings confirmed the presence of a 3
1,3-diarylpenta-1,4-diene moiety in the structure. The coupling constant of 11.6 Hz assigned the 1,2-double bond as cis. HMBC correlations of H-1 at δH 6.49 to the three aromatic carbons at δC 130.9 (C-1’), 115.0 (C-2’), 120.8 (C-6’) suggested the linkage of an aryl group to this penta-1,4-diene moiety at its C-1. The 1 H-NMR and HSQC spectra showed that H-2’ at δH 6.90 was a broad singlet and H-6’ at δH 6.80 was a broad doublet with the coupling constant of 8.0 Hz. These signals suggested that these two protons were meta to each other. Additionally, H-6’ had a COSY cross peak with a doublet proton signal at δH 6.81 (1H, d, 8.0 Hz, H-5’). This implied that the A-benzene ring possessing the ABX system with two other substituents at C-3’ and C-4’ was attached to the penta-1,4-diene moiety at its C-1. The HMBC correlations of the hydroxyl proton at δH 5.59 with carbons C-2’ and C-3’, and of methoxy protons at δH 3.89 with C-4’ indicated that the hydroxyl group and the methoxy group linked to the A-benzene ring at its C-3’ and C-4’, respectively. The hydroxyl group at C-4’’ of the B-benzene ring was determined by the HMBC correlations of the hydroxyl proton at δH 4.80 with carbons C-3’’ (C-5’’) and C-4’’. The attachment of this 4’’-hydroxyphenyl group to C3 of the penta-1,4-diene moiety was confirmed via the HMBC correlations of H-2, H-3, and H-4 with C-1’’, and vice versa the correlations of the aromatic proton signals of the B-ring at δH 7.09 (2H, d, 8.4 Hz, H-2’’ and H-6’’) with C-3. The absolute configuration of C-3 was not determined. However, 2 was levorotatory, therefore it was proposed to be (–)-1-(3-hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)penta-1,4diene and was named sonnerphenolic B. Compound 23 was obtained from an ethyl acetate fraction as a colorless oil, ( [α]D = +33.4, c 25
1.8, MeOH), with a molecular formula of C16H24 O8 determined by the pseudo-molecular ion peak in the HR-ESI-MS at m/z 367.1344 [M+Na]+ (Calcd. for C16H24O8Na, 367.1369). The 1H-NMR spectrum of 23 displayed three typical signals of an ABX spin system at δH 6.88 (1H, d, 1.5 Hz, H2’), 6.91 (1H, d, 8.0 Hz, H-5’), and 6.78 (1H, dd, 8.0, 2.0 Hz, H-6’). It corresponded to the presence of six aromatic carbon signals from 116.0 to 144.0 ppm in the 13C-NMR spectrum. The 13C-NMR spectrum also showed the presence of a glucopyranosyl moiety with carbon signals at δC 101.5 (C1’’), 73.3 (C-2’’), 75.9 (C-3’’), 69.6 (C-4’’), 75.8 (C-5’’), and 60.7 (C-6’’) which, in turn, had the HSQC correlations with proton signals at δH 4.50 (H-1’’), 3.30 (H-2’’), 3.53 (H-3’’), 3.43 (H-4’’), 3.42 (H-5’’), 3.75 (H-6’’a) and 3.91 (H-6’’b). The large coupling constant of the anomeric proton at δH 4.50 (d, 8.0 Hz) indicated the β-configuration of the glucose unit. At the lower frequency range, the 1 H-NMR spectrum displayed a three-proton doublet signal at δH 1.32 (H-1) of a methyl group which showed the COSY cross peak with an oxygenated proton at δH 3.95 (H-2) (See Fig. 2). This proton H-2 revealed the COSY cross peak with both proton signals at δH 1.91 (H-3a) and 1.81 (H-3b) of a methylene group. The COSY correlations of protons H-3a and H-3b with a two-proton signal at δH 2.65 (H-4) were observed. It corresponded to the presence of the four remaining carbon signals at δC 20.5 (C-1), 76.7 (C-2), 37.5 (C-3), and 29.9 (C4) in the 13C-NMR spectrum. These findings suggested the presence of a 3-oxygenated butyl moiety. The down field shifted of methylene protons H-4 appeared as a triplet signal at δH 2.65 indicating that this methylene group was adjacent to a benzene ring. This was confirmed via the HMBC correlations of H-4 with both carbons C-2’ and C-6’, vice versa the HMBC correlations of H-2’ (δH 6.88) and H-6’ (δH 6.78) with carbon C-4 at δC 29.9. Furthermore, the correlations of the 4
proton signal H-6’ at δH 6.78 with 13C signal at δC 141.8 confirmed that this signal belonged to C-4’ and therefore, carbon C-3’ resonated at δC 143.8. The attachment of the β-D-glucose at C-2 was assigned by the HMBC correlations of H-2 and C-1’’, as well as of H-1’’ and C-2. From the above NMR analysis, the gross chemical structure of 23 was determined as 4-(3,4dihydroxyphenyl)butane-2-ol 2-O-β-D-glucopyranoside. Compound (2R)-4-(3,4-dihydroxyphenyl)butane-2-ol 2-O-β-D-glucopyranoside or 20 tripodantoside was isolated from Tripodanthus acutifolius. It possessed a negative specific rotation with the value of –47.1 (c 0.232, CH3OH), and the aglycone of tripodantoside was also levorotatory ( [α ]D = –18.1, c 0.312, CH3OH). Compound 23 possessed a positive specific rotation 25
with the value of +33.4 (c 1.8, CH3OH), and its aglycone was dextrorotatory ( [α ]D = +13.1, c 0.13, 25
CH3OH). Therefore, 23 was an epimer at C-2 of tripodantoside. This was supported by the different chemical shift values in the same deuterated solvent of carbons of 23 with the corresponding ones of tripodantoside (Table 2). 23 was identified as (2S)-4-(3,4-dihydroxyphenyl)butane-2-ol 2-O-β-Dglucopyranoside and was named sonnerphenolic C. Compound 3 was obtained from a petroleum ether fraction as a white amorphous powder, ( [α ] = +8.8, c 0.5, CH3OH), with a molecular formula of C40H75O9N determined by the pseudo25 D
molecular ion peak in the HR-ESI-MS at m/z 736.5346 (Calcd. for C40H75O9NNa, 736.5340). The 1 H-, and HSQC-NMR spectra showed that 3 possessed four olefinic protons at δH 5.72 (H-5), 5.48 (H-4), 5.43 (2H, H-13, H-14), seven protons of a sugar unit, a methine bearing a nitrogen atom, two oxygenated methines and an oxygenated methylene group at δH 4.20−3.10, some methylene groups in the high field zone at δH 2.20−1.20 and a six-proton triplet signal at δH 0.90 of two terminal methyl groups. In the 13C-NMR spectrum, an amide carbon signal at δC 177.2, four olefinic carbon signals at δC 134.4, 132.0, 131.2 and 130.7, one anomeric carbon at δC 104.7, some oxygenated carbon signals at δC 78.0–62.7 and one nitrogenated methine carbon at δC 54.7, some aliphatic methylene carbon signals at δC 36.0−23.0 and one signal for two terminal methyl groups at δC 14.4. The presence of a β-D-glucopyranosyl moiety was demonstrated by the doublet signal of an anomeric proton at δH 4.27 (d, J=8.0 Hz, H-1”) which had COSY correlations with H-2”/H-3”/H4”/H-5”/H-6” in the range from 3.18 ppm to 3.86 ppm. The linkage of this sugar to the aglycone at C-1 was confirmed by HMBC correlations of the anomeric proton H-1’’ with a carbon signal at δC 69.8 (C-1) and of the methylene protons H-1 at δH 4.12 and 3.72 with carbon C-1’’ at δC 104.7. Up to this point, 3 was a cerebroside composing of a D-glucose, a fatty acid and a fatty amino-alcohol. The acid hydrolysis in methanol of 3 yielded a mixture of a fatty acid methyl ester (3A), sphingosine (3B), and a methyl D-glucopyranoside. The fragment 3A was determined as methyl 2-hydroxyhexadecanoate by GC-MS analysis. The HR-ESI-MS spectrum of 3B proved that this fragment was an aliphatic 18-carbon chain containing two hydroxyl groups, one amino group and two double bonds. The positions of the two hydroxyl groups, one amino group and the first double bond could be determined on the analysis of the 2D-NMR data of 3. The COSY spectrum showed the correlations of two oxygenated methylene protons at δH 4.12 (H-1a) and 3.72 (H-1b) with the methine proton bearing nitrogen at δH 3.99 (H-2). This methine proton had COSY cross peak with the methine proton at δH 4.14 (H-3) which correlated with the olefinic proton at δH 5.48 (H-4). All these COSY correlations suggested the presence of two hydroxyl groups at C-1 and C-3, 5
an amino group at C-2 and a double bond at C-4 in 3B. HMBC experiments well supported these assignments. The position of the second double bond of 3B could be proposed at C-13 based on the result of MS/MS on 3 with a fragment at m/z = 667 [M−C5H10 +Na]+ (Fig. 4). The 2D-NMR data of 3 also supported these assignments. The double bond at C-4 with the E-configuration was evidenced by the large vicinal coupling constant (J4,5 =15.5 Hz) and by the chemical shift of the adjacent carbon at δC 33.6 (C-6).21 The protons of olefinic carbons C-13 and C-14 resonated at the same value at δH 5.43 but as evidenced by the chemical shifts of their adjacent carbons at δC 33.3 (C-12) and 33.7 (C-15), the E-configuration was assigned for the double bond at C-13. All data suggested that 3 was a cerebroside with two straight long chains as shown in Fig. 4. By considering biogenetic relationships steric factors,22 the chemical shift values of H-2, C-1, C-2, C-3 and C-2’ could be utilized to determine the absolute stereochemistry of glucosphingolipids and sphingolipids.23,24 The chemical shifts values of proton H-2 at δH 3.99, and of carbons at δC 69.8 (C-1), 54.7 (C-2), 72.9 (C-3), 73.1 (C-2’) of 3 were in good agreement with those reported for glycosphingosine (as model structure) with 2S, 3R, 2’R configurations. With these considerations, the structure of 3 was proposed as 1-O-β-D-glucopyranosyl-(2S,3R,2’R,4E,13E)-2-N-(2’hydroxyhexadecanoylamino)octadeca-4,13-dien-3-ol and was named Sonnercerebroside. In the in vitro cytotoxic assay using the Sulforhodamine B method (SRB), described by Skehan25 with camptothecin as the positive control, compounds (5, 6, 23) exhibited cytotoxicities against only the MCF-7 cancer cell line with IC50 values of 146.9±9.0, 114.5±7.2, and 112.8±9.4 µM, respectively (see Table 3), while they showed non toxic with the normal cell (PHF) with IC50s > 277 µM. Compound (22) showed inhibitory activity against MCF-7 and NCI-H460 cancer cell lines with IC50 values of 222.2±10.8, and 286.6±8.5 µM, respectively. However this compound also showed toxic with the normal cell (PHF) with IC50 of 185.7±4.7 µM. The AChE inhibitory activity of 4 extracts and 15 isolated compounds from the S. ovata leaves was tested in vitro by Ellman’s method26 with galanthamine as the positive control. Among the tested samples, only (S)-rhodolatouchol (22) showed inhibition against AChE with an IC50 value of 96.1±14.5 µM (Table 3). The results showed that the above compounds are less active than positive controls (Table S1 and S2 in Supplementary material), and also less active than extracts of other Sonneratia species as well as some other compounds isolated from Sonneratia species in previous reports. However, in our studies, some of the isolated compounds have the potential cytotoxicity and AChE inhibition and represent a rich bio-resource which could be easily collected in mangrove forests, so S. ovata deserves to be more thoroughly studied to obtain valuable pharmaceutical products. Acknowledgements This research was supported by the Department of Science and Technology – Ho Chi Minh city, grant #1058/QĐ-SKHCN. N.T.H.T would like to thank Vietnamese government for a scholarship at Roskilde University under the 322 projects, to thank Chulalongkorn University for a scholarship at Chulalongkorn University under the One Semester Scholarships Program for ASEAN Countries. Supplementary data Supplementary data associated with this article can be found in the online version (The preliminary screening results of bioassay, HR-ESI-MS, 1D, 2D-NMR spectra of 1-3 and 23, 6
detailed experimental procedures including general experimental procedures, plant material, extraction and isolation procedure for compounds 1–23, cytotoxicity assay and AChE inhibition assay) References 1. Bandaranayake, W. M. Wetl. Ecol. Manag. 2002, 10, 421. 2. Wetwitayaklung, P.; Limmatvapirat, C.; Phaechamud, T. Indian J. Pharm. Sci. 2013, 75, 649. 3. Conforti, F.; Rigano, D.; Menichini, F.; Loizzo, M.R.; Senatore, F. Fitoterapia 2009, 80, 62. 4. Tian, M.; Dai, H.; Li, X.; Wang, B. Chinese J. Oceanol Limnol 2009, 27, 288. 5. Wu, S. B.; Wen, Y.; Li, X. W.; Zhao, Y.; Zhao, Z.; Hu, J. F. Biochem. Syst. Ecol. 2009, 37, 1. 6. Zheng, Z.; Pei, Y. H. Journal of Shenyang Pharmaceutical University 2008, 25, 35. 7. Iida, Y.; Oh, K. B.; Saito, M.; Matsuoka, H.; Kurata, H.; Natsume, M.; Abe, H. J. Agric. Food Chem. 1999, 47, 584. 8. Li, L.; Seeram, N. P. J. Agric. Food Chem. 2010, 58, 11673. 9. Kuang, H. X.; Xia, Y. G.; Yang, B. Y.; Wang, Q. H.; Lü, S. W. Arch. Pharm. Res. 2009, 32, 329. 10. Wu, Q. L.; Wang, M.; Simon, J. E.; Yu, S. C.; Xiao, P. G.; Ho, C. T. Acs. Sym. Ser. 2003, 859, 292. 11. Jutiviboonsuk, A.; Zhang, H.; Tan, G. T.; Nguyen, V. H.; Nguyen, M. C.; Ma, C.; Bunyapraphatsara, N.; Soejarto, D. D.; Fong, H. H. Phytochemistry 2005, 66, 2745. 12. Wen, Q.; Lin, X.; Liu, Y.; Xu, X.; Liang, T.; Zheng, N.; Kintoko, K.; Huang, R. Molecules 2012, 17, 12330. 13. Yan, L. H.; Xu, L. Z.; Lin, J.; Zou, Z. M.; Zhao, B. H.; Yang, S. L. J. Chin. Mater. Med. 2008, 33, 1839. 14. Lemes, G. F.; Ferri, P. H.; Lopes, M. N. Quim. Nova. 2011, 34, 39. 15. Gnoatto, S. C.; Dassonville-Klimpt, A.; Nascimento, D. S.; Galera, P.; Boumediene, K.; Gosmann, G.; Sonnet, P.; Moslemi, S. Eur. J. Med. Chem. 2008, 43, 1865. 16. Hsu, F. L.; Lee, Y. Y.; Cheng, J. T. J. Nat. Prod. 1994, 57, 308. 17. Lu, Y.; Foo, L. Y. Food Chem. 1999, 65, 1. 18. Isaza, J. H.; Ito, H.; Yoshida, T. Phytochemistry 2001, 58, 321. 19. Li, H. Z.; Song, H. J.; Li, H. M.; Pan, Y. Y.; Li, R. T. Arch. Pharm. Res. 2012, 35, 1887. 20. Soberon, J. R.; Sgariglia, M. A.; Sampietro, D. A.; Quiroga, E. N.; Sierra, M. G.; Vattuone, M. A. J. Appl. Microbiol. 2010, 108, 1757. 21. Yang, N. Y.; Ren, D. C.; Duan, J. A.; Xu, X. H.; Xie, N. Helv. Chim. Acta. 2009, 92, 291. 22. Thomas, K.; Konrad, S. Angew. Chem. Int. Ed. 1999, 38, 1532. 23. Kang, S. S.; Kim, J. S.; Xu, Y. N.; Kim, Y. H. J. Nat. Prod. 1999, 62, 1059. 24. Tang, J.; Meng, X.; Liu, H.; Zhao, J.; Zhou, L.; Qiu, M.; Zhang, X.; Yu, Z.; Yang, F. Molecules 2010, 15, 9288. 25. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107. 26. Ellman, G. L.; Courtney, D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
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Table 1. NMR spectral data of compounds of 1 and 2 Table 2. NMR spectral data of compounds 3 and 23 Table 3. The IC50 values of cell growth inhibitory effects and AChE inhibitory activity of some isolated compounds
Figure 1. Structures of compounds isolated from the leaves of S. ovata Figure 2. Selected COSY and HMBC correlations of 1, 2, and 23 Figure 3. Selected NOESY correlations of 1 Figure 4. Selected COSY and HMBC correlations and ESI-MS/MS fragment analysis of 3
8
Figure 1. Structures of compounds isolated from the leaves of S. ovata
9
Figure 2. Selected COSY and HMBC correlations of 1, 2, and 23
Figure 3. Selected NOESY correlations of 1
Figure 4. Selected COSY and HMBC correlations and ESI-MS/MS fragment analysis of 3
10
Table 1. NMR spectral data of compounds of 1 and 2 Position
Compound 1a δH (J in Hz) 4.10 (ddd, 8.5, 8.5, 6.0) 3.34 (dd, 13.0, 8.5) 3.14 (dd, 13.0, 4.5) 4.21 (ddd, 6.5, 4.5, 2.0) 1.74 (ddd, 14.5, 6.0, 2.0) 2.63 (ddd, 14.5, 8.5, 6.0)
Compound 2b δH (J in Hz) 6.49 (d, 11.6) 5.67 (dd, 10.8, 10.4) 4.54 (dd, 9.6, 6.4) 6.01 (ddd, 16.8, 10.4, 6.0) 5.20 (brd, 18.4) 5.17 (brd, 9.2)
δC 1 79.6 2 55.8 3 56.7 4 74.5 5a 44.0 5b 1’ 134.0 2’ 7.03 (d, 9.0) 129.9 6.90 (brs) 3’ 6.65 (d, 9.0) 116.1* 4’ 156.6 5’ 6.65 (d, 9.0) 116.1* 6.81 (d, 8.0) 6’ 7.03 (d, 9.0) 129.9 6.80 (brd, 8.0) 1’’ 130.8 2’’, 6’’ 7.07 (d, 8.5) 131.5 7.09 (d, 8.4) 3’’, 5’’ 6.62 (d, 8.5) 115.8* 6.78 (d, 8.4) 4’’ 156.6 3’-OH 5.59 (s) 4’-OMe 3.89 (s) 4’’-OH 4.80 (s) 1 1 Assignments were done by H- H COSY, HSQC, HMBC experiments a Measured at 500 MHz for 1H and 125 MHz for 13C using methanol-d4 as solvent b Measured at 400 MHz for 1H and 100 MHz for 13C using chloroform-d as solvent * The assignments interchangeable in the vertical column
11
δC 128.8 132.3 47.0 140.8 115.2 130.9 115.0 145.5 145.8 110.6 120.8 135.8 129.0 115.6 154.2 56.1
Table 2. NMR spectral data of compounds 3 and 23
2
Compound 3a δH (J in Hz) δC 4.12 (m) 69.8 3.72 (dd, 3.5, 10.5) 3.99 (m) 54.7
3
4.14 (m)
72.9
4 5 6 7−11 12 13 14 15 16 17 18 1’ 2’
5.48 (dd, 15.5, 7.5) 5.72 (dt, 15.5, 6.5) 2.07 (m) 1.29−1.33 (m) 1.98−2.07 (m) 5.43 (m) 5.43 (m) 2.07 (m) 1.29−1.33 (m) 1.29−1.33 (m) 0.90 (t, 7.0)
131.2 134.4 33.6 30.2−30.8 33.3* 130.7 132.0 33.7* 33.1 23.7 14.4 177.2 73.1
Position 1
Compound 23b δH (J in Hz)
δC
Tri ** δC
1.32 (d, 6.5)
20.5
18.2
3.95 (m) 1.91 (m) 1.81 (m) 2.65 (t, 7.5)
76.7
75.0
37.5
37.2
29.9
29.2
135.4 143.0 3.99 (m) 6.88 (d, 1.5) 116.2 115.3 1.71 (m) 3’ 35.9 143.8 141.0 1.55 (m) 4’ 1.42 (m) 26.2 141.8*** 134.5 5’ 6.91 (d, 8.0) 116.2 115.5 1.29−1.33 (m) 30.2−30.8 6’ 6.78 (dd, 8.0, 2.0) 120.6 119.9 1.29−1.33 (m) 30.2−30.8 7’−13’ 1.29−1.33 (m) 30.2−30.8 14’ 33.1 1.29−1.33 (m) 15’ 23.7 1.29−1.33 (m) 16’ 0.90 (t, 7.0) 14.4 1’’ 4.27 (d, 8.0) 104.7 4.50 (d, 8.0) 101.5 101.8 2’’ 3.20 (t, 9.0, 8.0) 75.0 3.30 (dd, 9.0, 8.0) 73.3 72.3 3’’ 3.36 (dd, 9.0, 9.0) 78.0 3.53 (dd, 9.0, 9.0) 75.9 75.1 4’’ 3.28 (m) 71.6 3.43 (m) 69.6 74.9 5’’ 3.28 (m) 77.9 3.42 (m) 75.8 68.8 3.86 (dd, 11.5, 1.5) 3.75 (dd, 12.0, 5.0) 6’’ 62.7 60.7 61.8 3.68 (dd, 11.5, 5.0) 3.91 (brd, 12.0) 1 1 Assignments were done by H- H COSY, HSQC, HMBC experiments a Measured at 500 MHz for 1H and 125 MHz for 13C using methanol-d4 as solvent b Measured at 500 MHz for 1H and 125 MHz for 13C using deuterium oxide-d2 as solvent * The assignments interchangeable in the vertical column ** Tri: Tripodantoside (using deuterium oxide-d2 as solvent)20 *** In the HMBC spectrum, the proton signal of H-6’ at δH 6.78 correlated with 13C signal at δC 141.8 (C-4’) and did not with 13C signal at δC 143.8 (C3’)
12
Table 3. The IC50 values (µM) of cell growth inhibitory effects and AChE inhibitory activity of some isolated compounds IC50 values (µM)
Compounds MCF-7
NCI-H460
PHF
AChE
5
146.9±9.0
-
>277.8
-
6
114.5±7.2
-
297.7±17.8
-
22
222.2±10.8
286.6±8.5
185.7±4.7
96.1±14.5
23
112.8±9.4
-
>290.7
-
Camptothecin
0.108±0.039
0.022±0.002
4.772±2.414
-
Galanthamine
-
-
-
0.141±0.024
13
Graphical abstract
14
S0960-894X(15)00328-5 http://dx.doi.org/10.1016/j.bmcl.2015.04.017 BMCL 22600
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
10 August 2014 30 March 2015 6 April 2015
Please cite this article as: Nguyen, T-H., Pham, H-V., Pham, N-K., Quach, N-D., Pudhom, K., Hansen, P.E., Nguyen, K-P., Chemical Constituents from Sonneratia ovata Backer and their in vitro Cytotoxicity and Acetylcholinesterase Inhibitory Activities, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl. 2015.04.017
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Chemical Constituents from Sonneratia ovata Backer and their in vitro Cytotoxicity and Acetylcholinesterase Inhibitory Activities Thi-Hoai-Thu Nguyena, Huu-Viet-Thong Pham b, Nguyen-Kim-Tuyen Phamc, Ngo-Diem-Phuong Quachd, Khanitha Pudhome, Poul Erik Hansenf, Kim-Phi-Phung Nguyenb,* a
Department of Basic Science, University of Medicine and Pharmacy – Ho Chi Minh City, Vietnam. Department of Organic Chemistry, VNUHCM - University of Science, Ho Chi Minh City, Vietnam. c Department of Environmental Science, Saigon University, Ho Chi Minh City, Vietnam. d Department of Biology, VNUHCM - University of Science, Ho Chi Minh City, Vietnam. e Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. f Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark. * Correspondence to: Nguyen Kim Phi Phung, Department of Organic Chemistry, Vietnam National University - University of Science, 227 Nguyen Van Cu Str., Dist. 5, Ho Chi Minh City, Vietnam. E-mail: [email protected]. Tel.: +84-1226 966 660 b
Abstract Sonneratia ovata Backer, Sonneratiaceae, is a widespread plant in mangrove forests in Vietnam, Cambodia, Thailand, Indonesia,… Sonneratia ovata’s chemical composition remains mostly unknown. Therefore, we now report on the structural elucidation of three new phenolics, sonnerphenolic A (1), sonnerphenolic B (2), and sonnerphenolic C (23), a new cerebroside, sonnercerebroside (3) together with nineteen known compounds, including nine lignans (5− −13), two steroids (14, 15), two triterpenoids (16, 17), three gallic acid derivatives (18− −20), two phenolic derivatives (4, 22) and a 1-O-benzyl-β-D-glucopyranose (21) isolated from the leaves of Sonneratia ovata. Their chemical structures were established by spectroscopic data, as well as high resolution mass spectra and comparison with literature data. The in vitro acetylcholinesterase (AChE) inhibition and cytotoxic activities against HeLa (human epithelial carcinoma), NCI-H460 (human lung cancer), MCF–7 (human breast cancer) cancer cell lines and PHF (primary human fibroblast) cell were evaluated on some extracts and purified compounds at a concentration of 100 µg/mL. Compounds (5, 6, 23) exhibited cytotoxicity against the MCF-7 cell line with the IC50 values of 146.9±9.0, 114.5±7.2, and 112.8±9.4 µM, respectively, while they showed nontoxic with the normal cell (PHF) with IC50s > 277 µM. Among 15 tested compounds, (S)-rhodolatouchol (22) showed inhibition against AChE with an IC50 value of 96.1±14.5 µM. Key words: Sonneratiaceae, Sonneratia ovata, phenolics, cerebroside, in vitro cytotoxicity and acetylcholinesterase inhibition. Abbreviations: AChE: acetylcholinesterase HeLa: human epithelial carcinoma NCI-H460: human lung cancer 1
MCF–7: human breast cancer PHF: primary human fibroblast 1D/2D-NMR: One/Two Dimensional –Nuclear Magnetic Resonance COSY: Correlation Spectroscopy HSQC: Heteronuclear Single Quantum Correlation HMBC: Heteronuclear Multiple-Bond Correlation NOESY: Nuclear Overhauser Effect Spectroscopy HR-ESI-MS: High Resolution – Electrospray Ionization – Mass Spectrometry
The Sonneratia species growing widely in mangrove forests in tropical areas possess a huge biomass that promises a potent resource for human use. Indeed, for a long time, the fruits, bark, and leaves of some Sonneratia plants have been used in folk medicine to treat different diseases such as asthma, febrifuge, ulcers, hepatitis, piles, sprain, and hemorrhages.1 Therefore, this genus is attractive for pharmacological and chemical studies. Previously pharmaceutical investigations showed that the methanol extract of S. caseolaris seeds was a potential AChE inhibitor with the IC50 value of 10.52 µg/mL.2 Isoorientin exhibited promising inhibition against both AChE and BChE with IC50 of 26.8 and 31.5 µM, respectively, while isovitexin exhibited acetylcholinesterase inhibition with IC50 of 36.4 µM.3 These two compounds were previously reported from leaves of S. ovata. Luteolin isolated from stems and twigs of S. caseolaris exhibited significant cytotoxicity against SMMC–7721 with IC50 value of 2.8 µg/mL.4 From the fruits of S. ovata, three compounds (–)-(R)-nyasol, (–)-(R)-4’-O-methylnyasol and maslinic acid were isolated. Determination by the MTT assay method, these three compounds showed moderate cytotoxic activities against the rat glioma C-6 cell line with IC50 values of 19.02, 20.21, and 31.77 µg/mL, respectively.5 Previous chemical studies on this plant reported a number of compounds isolated from S. ovata.5,6 Based on this we would like to study the chemical constituents of leaves of S. ovata, and test in vitro cytotoxicity and AChE inhibition of extracts and some isolated compounds. The chemical investigation on the S. ovata leaves collected at the Can Gio mangrove forest led to the isolation of twenty three compounds by the use of efficient separation techniques (See Fig. 1) including four new compounds, sonnerphenolic A (1), sonnerphenolic B (2), sonnerphenolic C (23) and sonnercerebroside (3). The other compounds were identified as (-)-(R)-nyasol (4),7 (7S,8R)-dehydroconiferyl alcohol (5),8 (7S,8R)-5-methoxydehydroconiferyl alcohol (6),8 (7S,8R)urolignoside (7),9 lingueresinol (8),10 (+)-isolariciresinol (9),11 (+)-isolariciresinol 9’-O-β-Dglucopyranoside (10),12 (–)-isolariciresinol 9’-O-β-D-glucopyranoside (11),12 (–)-episyringaresinol (12),13 (+)-syringaresinol (13),13 β-sitosterol (14),4 3-O-palmitoyl-β-sitosterol (15),4 corosolic acid (16),14 3-O-acetylursolic acid (17),15 6-O-galloyl-D-glucopyranose (18),16 gallic acid 3-O-β-Dglucopyranoside (19),17 1-O-benzyl-6-O-galloyl-β-D-glucopyranose (20),18 1-O-benzyl-β-Dglucopyranose (21),12 and (−)-rhodolatouchol (22)19 via spectroscopic data analysis and comparison with those reported in the literature. In this paper, the chemical structure elucidation of four new compounds was described. The bioactive assays of the AChE inhibition and the cytotoxic activities 2
against the HeLa, NCI-H460, MCF–7 cell lines and the PHF cell were evaluated on some extracts and purified compounds (with purities better than 95% as determined by proton NMR data). Compound 1 was obtained from an ethyl acetate fraction as a colorless wax, ( [α ]D = +3.8, c 25
0.37, MeOH), with a molecular formula of C17 H18O4 determined by the pseudo-molecular ion peak in the HR-ESI-MS at m/z 309.1117 [M+Na]+ (Calcd. for C17H18O4Na, 309.1097). The 13C-NMR data (Table 1) displayed 13 carbon signals, including four signals at δC 131.5, 129.9, 116.1, and 115.8 appearing as double intensity. Additionally, the 1 H-NMR spectrum confirmed the presence of four doublet proton signals of eight aromatic protons at δH 7.07 (2H, d, 8.5 Hz, H-2’’, H-6’’), 7.03 (2H, d, 9.0 Hz, H-2’, H-6’), 6.65 (2H, d, 9.0 Hz, H-3’, H-5’) and 6.62 (2H, d, 8.5 Hz, H-3’’, H-5’’). These observations suggested the presence of two para-disubstituted benzene groups in the structure of the compound. At the lower frequency range from 1.70 to 4.30 ppm, the 1H-NMR spectrum displayed two oxygenated methine protons, two methine protons and two methylene protons corresponding to five carbon signals from 44.0 to 79.6 ppm in the 13C-NMR spectrum. These proton and carbon signals belonged to a cyclopentane ring which was confirmed through the COSY correlations of H-1/H-2/H-3/H-4/H-5/H-1 (See Fig. 2). The HMBC correlations of H-2 with C-1’, C-2’, C-6’, of H-2’ with C-2, of H-3 with C-2’’, C-6’’ and of H-2’’ with C-3 suggested the attachment of two 4-hydroxyphenyl groups to the cyclopentane ring at its C-2 and C-3. On the basis of the above analysis, the gross structure of 1 was 1,4-dihydroxy-2,3-di-(4hydroxyphenyl)cyclopentane. The relative configuration of 1 was further proven by the NOESY spectrum. The NOESY spectrum exhibited cross peaks between H-3 and H-4, between H-4 and H-5b, between H-5b and H1, between H-1 and H-3 (See Fig. 3), therefore these four hydrogen atoms were on the same side of the ring. The NOESY correlation of H-5a with H-2 proved that these two hydrogen atoms were on the same side of the plane, and on the opposite side comparing to the four mentioned above atoms. Therefore, 1 was thus determined to be (1β,2α,3β,4β)-1,4-dihydroxy-2,3-di-(4-hydroxyphenyl) cyclopentane and was named sonnerphenolic A. Compound 2 was obtained from an ethyl acetate fraction as a yellowish oil, ( [α ]D = -282.8, c 25
0.18, MeOH) with a molecular formula of C18H18O3 determined by the pseudo-molecular ion peak in the HR-ESI-MS at m/z 305.1160 [M+Na]+ (Calcd. for C18H18O3Na, 305.1154). The 1H-NMR spectrum displayed the presence of seven aromatic protons including three signals at δH 6.90 (1H, brs, H-2’), 6.81 (1H, d, 8.0 Hz, H-5’) and 6.80 (1H, brd, 8.0 Hz, H-6’) of an ABX system of an Abenzene ring and two signals at δH 7.09 (2H, d, 8.4 Hz, H-2’’ and H-6’’) and 6.78 (2H, d, 8.4 Hz, H-3’’ and H-5’’) of an AA'BB' system of a B-benzene ring. The 1 H-NMR and HSQC spectra also showed the presence of two olefinic methylene protons, three olefinic methine protons of two double bonds in the zone from 5.15 to 6.50 ppm, a down field shifted saturated methine proton at δH 4.54 (1H, dd, 9.6, 6.4 Hz, H-3) and a signal at δH 3.89 (3H, s) of a methoxy group. These corresponded to the observation of sixteen carbon signals including two signals appearing as double intensity at δC 129.0 and 115.6 belonging to an AA'BB' system in the 13C-NMR spectrum. Moreover, the COSY spectrum revealed the correlations of the proton H-1 at δH 6.49 and proton H2 at δH 5.67, of proton H-2 and proton H-3 at δH 4.54, of proton H-3 and proton H-4 at δH 6.01, and of H-4 and protons H-5 at δH 5.20 and 5.17 (See Fig. 2). These findings confirmed the presence of a 3
1,3-diarylpenta-1,4-diene moiety in the structure. The coupling constant of 11.6 Hz assigned the 1,2-double bond as cis. HMBC correlations of H-1 at δH 6.49 to the three aromatic carbons at δC 130.9 (C-1’), 115.0 (C-2’), 120.8 (C-6’) suggested the linkage of an aryl group to this penta-1,4-diene moiety at its C-1. The 1 H-NMR and HSQC spectra showed that H-2’ at δH 6.90 was a broad singlet and H-6’ at δH 6.80 was a broad doublet with the coupling constant of 8.0 Hz. These signals suggested that these two protons were meta to each other. Additionally, H-6’ had a COSY cross peak with a doublet proton signal at δH 6.81 (1H, d, 8.0 Hz, H-5’). This implied that the A-benzene ring possessing the ABX system with two other substituents at C-3’ and C-4’ was attached to the penta-1,4-diene moiety at its C-1. The HMBC correlations of the hydroxyl proton at δH 5.59 with carbons C-2’ and C-3’, and of methoxy protons at δH 3.89 with C-4’ indicated that the hydroxyl group and the methoxy group linked to the A-benzene ring at its C-3’ and C-4’, respectively. The hydroxyl group at C-4’’ of the B-benzene ring was determined by the HMBC correlations of the hydroxyl proton at δH 4.80 with carbons C-3’’ (C-5’’) and C-4’’. The attachment of this 4’’-hydroxyphenyl group to C3 of the penta-1,4-diene moiety was confirmed via the HMBC correlations of H-2, H-3, and H-4 with C-1’’, and vice versa the correlations of the aromatic proton signals of the B-ring at δH 7.09 (2H, d, 8.4 Hz, H-2’’ and H-6’’) with C-3. The absolute configuration of C-3 was not determined. However, 2 was levorotatory, therefore it was proposed to be (–)-1-(3-hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)penta-1,4diene and was named sonnerphenolic B. Compound 23 was obtained from an ethyl acetate fraction as a colorless oil, ( [α]D = +33.4, c 25
1.8, MeOH), with a molecular formula of C16H24 O8 determined by the pseudo-molecular ion peak in the HR-ESI-MS at m/z 367.1344 [M+Na]+ (Calcd. for C16H24O8Na, 367.1369). The 1H-NMR spectrum of 23 displayed three typical signals of an ABX spin system at δH 6.88 (1H, d, 1.5 Hz, H2’), 6.91 (1H, d, 8.0 Hz, H-5’), and 6.78 (1H, dd, 8.0, 2.0 Hz, H-6’). It corresponded to the presence of six aromatic carbon signals from 116.0 to 144.0 ppm in the 13C-NMR spectrum. The 13C-NMR spectrum also showed the presence of a glucopyranosyl moiety with carbon signals at δC 101.5 (C1’’), 73.3 (C-2’’), 75.9 (C-3’’), 69.6 (C-4’’), 75.8 (C-5’’), and 60.7 (C-6’’) which, in turn, had the HSQC correlations with proton signals at δH 4.50 (H-1’’), 3.30 (H-2’’), 3.53 (H-3’’), 3.43 (H-4’’), 3.42 (H-5’’), 3.75 (H-6’’a) and 3.91 (H-6’’b). The large coupling constant of the anomeric proton at δH 4.50 (d, 8.0 Hz) indicated the β-configuration of the glucose unit. At the lower frequency range, the 1 H-NMR spectrum displayed a three-proton doublet signal at δH 1.32 (H-1) of a methyl group which showed the COSY cross peak with an oxygenated proton at δH 3.95 (H-2) (See Fig. 2). This proton H-2 revealed the COSY cross peak with both proton signals at δH 1.91 (H-3a) and 1.81 (H-3b) of a methylene group. The COSY correlations of protons H-3a and H-3b with a two-proton signal at δH 2.65 (H-4) were observed. It corresponded to the presence of the four remaining carbon signals at δC 20.5 (C-1), 76.7 (C-2), 37.5 (C-3), and 29.9 (C4) in the 13C-NMR spectrum. These findings suggested the presence of a 3-oxygenated butyl moiety. The down field shifted of methylene protons H-4 appeared as a triplet signal at δH 2.65 indicating that this methylene group was adjacent to a benzene ring. This was confirmed via the HMBC correlations of H-4 with both carbons C-2’ and C-6’, vice versa the HMBC correlations of H-2’ (δH 6.88) and H-6’ (δH 6.78) with carbon C-4 at δC 29.9. Furthermore, the correlations of the 4
proton signal H-6’ at δH 6.78 with 13C signal at δC 141.8 confirmed that this signal belonged to C-4’ and therefore, carbon C-3’ resonated at δC 143.8. The attachment of the β-D-glucose at C-2 was assigned by the HMBC correlations of H-2 and C-1’’, as well as of H-1’’ and C-2. From the above NMR analysis, the gross chemical structure of 23 was determined as 4-(3,4dihydroxyphenyl)butane-2-ol 2-O-β-D-glucopyranoside. Compound (2R)-4-(3,4-dihydroxyphenyl)butane-2-ol 2-O-β-D-glucopyranoside or 20 tripodantoside was isolated from Tripodanthus acutifolius. It possessed a negative specific rotation with the value of –47.1 (c 0.232, CH3OH), and the aglycone of tripodantoside was also levorotatory ( [α ]D = –18.1, c 0.312, CH3OH). Compound 23 possessed a positive specific rotation 25
with the value of +33.4 (c 1.8, CH3OH), and its aglycone was dextrorotatory ( [α ]D = +13.1, c 0.13, 25
CH3OH). Therefore, 23 was an epimer at C-2 of tripodantoside. This was supported by the different chemical shift values in the same deuterated solvent of carbons of 23 with the corresponding ones of tripodantoside (Table 2). 23 was identified as (2S)-4-(3,4-dihydroxyphenyl)butane-2-ol 2-O-β-Dglucopyranoside and was named sonnerphenolic C. Compound 3 was obtained from a petroleum ether fraction as a white amorphous powder, ( [α ] = +8.8, c 0.5, CH3OH), with a molecular formula of C40H75O9N determined by the pseudo25 D
molecular ion peak in the HR-ESI-MS at m/z 736.5346 (Calcd. for C40H75O9NNa, 736.5340). The 1 H-, and HSQC-NMR spectra showed that 3 possessed four olefinic protons at δH 5.72 (H-5), 5.48 (H-4), 5.43 (2H, H-13, H-14), seven protons of a sugar unit, a methine bearing a nitrogen atom, two oxygenated methines and an oxygenated methylene group at δH 4.20−3.10, some methylene groups in the high field zone at δH 2.20−1.20 and a six-proton triplet signal at δH 0.90 of two terminal methyl groups. In the 13C-NMR spectrum, an amide carbon signal at δC 177.2, four olefinic carbon signals at δC 134.4, 132.0, 131.2 and 130.7, one anomeric carbon at δC 104.7, some oxygenated carbon signals at δC 78.0–62.7 and one nitrogenated methine carbon at δC 54.7, some aliphatic methylene carbon signals at δC 36.0−23.0 and one signal for two terminal methyl groups at δC 14.4. The presence of a β-D-glucopyranosyl moiety was demonstrated by the doublet signal of an anomeric proton at δH 4.27 (d, J=8.0 Hz, H-1”) which had COSY correlations with H-2”/H-3”/H4”/H-5”/H-6” in the range from 3.18 ppm to 3.86 ppm. The linkage of this sugar to the aglycone at C-1 was confirmed by HMBC correlations of the anomeric proton H-1’’ with a carbon signal at δC 69.8 (C-1) and of the methylene protons H-1 at δH 4.12 and 3.72 with carbon C-1’’ at δC 104.7. Up to this point, 3 was a cerebroside composing of a D-glucose, a fatty acid and a fatty amino-alcohol. The acid hydrolysis in methanol of 3 yielded a mixture of a fatty acid methyl ester (3A), sphingosine (3B), and a methyl D-glucopyranoside. The fragment 3A was determined as methyl 2-hydroxyhexadecanoate by GC-MS analysis. The HR-ESI-MS spectrum of 3B proved that this fragment was an aliphatic 18-carbon chain containing two hydroxyl groups, one amino group and two double bonds. The positions of the two hydroxyl groups, one amino group and the first double bond could be determined on the analysis of the 2D-NMR data of 3. The COSY spectrum showed the correlations of two oxygenated methylene protons at δH 4.12 (H-1a) and 3.72 (H-1b) with the methine proton bearing nitrogen at δH 3.99 (H-2). This methine proton had COSY cross peak with the methine proton at δH 4.14 (H-3) which correlated with the olefinic proton at δH 5.48 (H-4). All these COSY correlations suggested the presence of two hydroxyl groups at C-1 and C-3, 5
an amino group at C-2 and a double bond at C-4 in 3B. HMBC experiments well supported these assignments. The position of the second double bond of 3B could be proposed at C-13 based on the result of MS/MS on 3 with a fragment at m/z = 667 [M−C5H10 +Na]+ (Fig. 4). The 2D-NMR data of 3 also supported these assignments. The double bond at C-4 with the E-configuration was evidenced by the large vicinal coupling constant (J4,5 =15.5 Hz) and by the chemical shift of the adjacent carbon at δC 33.6 (C-6).21 The protons of olefinic carbons C-13 and C-14 resonated at the same value at δH 5.43 but as evidenced by the chemical shifts of their adjacent carbons at δC 33.3 (C-12) and 33.7 (C-15), the E-configuration was assigned for the double bond at C-13. All data suggested that 3 was a cerebroside with two straight long chains as shown in Fig. 4. By considering biogenetic relationships steric factors,22 the chemical shift values of H-2, C-1, C-2, C-3 and C-2’ could be utilized to determine the absolute stereochemistry of glucosphingolipids and sphingolipids.23,24 The chemical shifts values of proton H-2 at δH 3.99, and of carbons at δC 69.8 (C-1), 54.7 (C-2), 72.9 (C-3), 73.1 (C-2’) of 3 were in good agreement with those reported for glycosphingosine (as model structure) with 2S, 3R, 2’R configurations. With these considerations, the structure of 3 was proposed as 1-O-β-D-glucopyranosyl-(2S,3R,2’R,4E,13E)-2-N-(2’hydroxyhexadecanoylamino)octadeca-4,13-dien-3-ol and was named Sonnercerebroside. In the in vitro cytotoxic assay using the Sulforhodamine B method (SRB), described by Skehan25 with camptothecin as the positive control, compounds (5, 6, 23) exhibited cytotoxicities against only the MCF-7 cancer cell line with IC50 values of 146.9±9.0, 114.5±7.2, and 112.8±9.4 µM, respectively (see Table 3), while they showed non toxic with the normal cell (PHF) with IC50s > 277 µM. Compound (22) showed inhibitory activity against MCF-7 and NCI-H460 cancer cell lines with IC50 values of 222.2±10.8, and 286.6±8.5 µM, respectively. However this compound also showed toxic with the normal cell (PHF) with IC50 of 185.7±4.7 µM. The AChE inhibitory activity of 4 extracts and 15 isolated compounds from the S. ovata leaves was tested in vitro by Ellman’s method26 with galanthamine as the positive control. Among the tested samples, only (S)-rhodolatouchol (22) showed inhibition against AChE with an IC50 value of 96.1±14.5 µM (Table 3). The results showed that the above compounds are less active than positive controls (Table S1 and S2 in Supplementary material), and also less active than extracts of other Sonneratia species as well as some other compounds isolated from Sonneratia species in previous reports. However, in our studies, some of the isolated compounds have the potential cytotoxicity and AChE inhibition and represent a rich bio-resource which could be easily collected in mangrove forests, so S. ovata deserves to be more thoroughly studied to obtain valuable pharmaceutical products. Acknowledgements This research was supported by the Department of Science and Technology – Ho Chi Minh city, grant #1058/QĐ-SKHCN. N.T.H.T would like to thank Vietnamese government for a scholarship at Roskilde University under the 322 projects, to thank Chulalongkorn University for a scholarship at Chulalongkorn University under the One Semester Scholarships Program for ASEAN Countries. Supplementary data Supplementary data associated with this article can be found in the online version (The preliminary screening results of bioassay, HR-ESI-MS, 1D, 2D-NMR spectra of 1-3 and 23, 6
detailed experimental procedures including general experimental procedures, plant material, extraction and isolation procedure for compounds 1–23, cytotoxicity assay and AChE inhibition assay) References 1. Bandaranayake, W. M. Wetl. Ecol. Manag. 2002, 10, 421. 2. Wetwitayaklung, P.; Limmatvapirat, C.; Phaechamud, T. Indian J. Pharm. Sci. 2013, 75, 649. 3. Conforti, F.; Rigano, D.; Menichini, F.; Loizzo, M.R.; Senatore, F. Fitoterapia 2009, 80, 62. 4. Tian, M.; Dai, H.; Li, X.; Wang, B. Chinese J. Oceanol Limnol 2009, 27, 288. 5. Wu, S. B.; Wen, Y.; Li, X. W.; Zhao, Y.; Zhao, Z.; Hu, J. F. Biochem. Syst. Ecol. 2009, 37, 1. 6. Zheng, Z.; Pei, Y. H. Journal of Shenyang Pharmaceutical University 2008, 25, 35. 7. Iida, Y.; Oh, K. B.; Saito, M.; Matsuoka, H.; Kurata, H.; Natsume, M.; Abe, H. J. Agric. Food Chem. 1999, 47, 584. 8. Li, L.; Seeram, N. P. J. Agric. Food Chem. 2010, 58, 11673. 9. Kuang, H. X.; Xia, Y. G.; Yang, B. Y.; Wang, Q. H.; Lü, S. W. Arch. Pharm. Res. 2009, 32, 329. 10. Wu, Q. L.; Wang, M.; Simon, J. E.; Yu, S. C.; Xiao, P. G.; Ho, C. T. Acs. Sym. Ser. 2003, 859, 292. 11. Jutiviboonsuk, A.; Zhang, H.; Tan, G. T.; Nguyen, V. H.; Nguyen, M. C.; Ma, C.; Bunyapraphatsara, N.; Soejarto, D. D.; Fong, H. H. Phytochemistry 2005, 66, 2745. 12. Wen, Q.; Lin, X.; Liu, Y.; Xu, X.; Liang, T.; Zheng, N.; Kintoko, K.; Huang, R. Molecules 2012, 17, 12330. 13. Yan, L. H.; Xu, L. Z.; Lin, J.; Zou, Z. M.; Zhao, B. H.; Yang, S. L. J. Chin. Mater. Med. 2008, 33, 1839. 14. Lemes, G. F.; Ferri, P. H.; Lopes, M. N. Quim. Nova. 2011, 34, 39. 15. Gnoatto, S. C.; Dassonville-Klimpt, A.; Nascimento, D. S.; Galera, P.; Boumediene, K.; Gosmann, G.; Sonnet, P.; Moslemi, S. Eur. J. Med. Chem. 2008, 43, 1865. 16. Hsu, F. L.; Lee, Y. Y.; Cheng, J. T. J. Nat. Prod. 1994, 57, 308. 17. Lu, Y.; Foo, L. Y. Food Chem. 1999, 65, 1. 18. Isaza, J. H.; Ito, H.; Yoshida, T. Phytochemistry 2001, 58, 321. 19. Li, H. Z.; Song, H. J.; Li, H. M.; Pan, Y. Y.; Li, R. T. Arch. Pharm. Res. 2012, 35, 1887. 20. Soberon, J. R.; Sgariglia, M. A.; Sampietro, D. A.; Quiroga, E. N.; Sierra, M. G.; Vattuone, M. A. J. Appl. Microbiol. 2010, 108, 1757. 21. Yang, N. Y.; Ren, D. C.; Duan, J. A.; Xu, X. H.; Xie, N. Helv. Chim. Acta. 2009, 92, 291. 22. Thomas, K.; Konrad, S. Angew. Chem. Int. Ed. 1999, 38, 1532. 23. Kang, S. S.; Kim, J. S.; Xu, Y. N.; Kim, Y. H. J. Nat. Prod. 1999, 62, 1059. 24. Tang, J.; Meng, X.; Liu, H.; Zhao, J.; Zhou, L.; Qiu, M.; Zhang, X.; Yu, Z.; Yang, F. Molecules 2010, 15, 9288. 25. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107. 26. Ellman, G. L.; Courtney, D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
7
Table 1. NMR spectral data of compounds of 1 and 2 Table 2. NMR spectral data of compounds 3 and 23 Table 3. The IC50 values of cell growth inhibitory effects and AChE inhibitory activity of some isolated compounds
Figure 1. Structures of compounds isolated from the leaves of S. ovata Figure 2. Selected COSY and HMBC correlations of 1, 2, and 23 Figure 3. Selected NOESY correlations of 1 Figure 4. Selected COSY and HMBC correlations and ESI-MS/MS fragment analysis of 3
8
Figure 1. Structures of compounds isolated from the leaves of S. ovata
9
Figure 2. Selected COSY and HMBC correlations of 1, 2, and 23
Figure 3. Selected NOESY correlations of 1
Figure 4. Selected COSY and HMBC correlations and ESI-MS/MS fragment analysis of 3
10
Table 1. NMR spectral data of compounds of 1 and 2 Position
Compound 1a δH (J in Hz) 4.10 (ddd, 8.5, 8.5, 6.0) 3.34 (dd, 13.0, 8.5) 3.14 (dd, 13.0, 4.5) 4.21 (ddd, 6.5, 4.5, 2.0) 1.74 (ddd, 14.5, 6.0, 2.0) 2.63 (ddd, 14.5, 8.5, 6.0)
Compound 2b δH (J in Hz) 6.49 (d, 11.6) 5.67 (dd, 10.8, 10.4) 4.54 (dd, 9.6, 6.4) 6.01 (ddd, 16.8, 10.4, 6.0) 5.20 (brd, 18.4) 5.17 (brd, 9.2)
δC 1 79.6 2 55.8 3 56.7 4 74.5 5a 44.0 5b 1’ 134.0 2’ 7.03 (d, 9.0) 129.9 6.90 (brs) 3’ 6.65 (d, 9.0) 116.1* 4’ 156.6 5’ 6.65 (d, 9.0) 116.1* 6.81 (d, 8.0) 6’ 7.03 (d, 9.0) 129.9 6.80 (brd, 8.0) 1’’ 130.8 2’’, 6’’ 7.07 (d, 8.5) 131.5 7.09 (d, 8.4) 3’’, 5’’ 6.62 (d, 8.5) 115.8* 6.78 (d, 8.4) 4’’ 156.6 3’-OH 5.59 (s) 4’-OMe 3.89 (s) 4’’-OH 4.80 (s) 1 1 Assignments were done by H- H COSY, HSQC, HMBC experiments a Measured at 500 MHz for 1H and 125 MHz for 13C using methanol-d4 as solvent b Measured at 400 MHz for 1H and 100 MHz for 13C using chloroform-d as solvent * The assignments interchangeable in the vertical column
11
δC 128.8 132.3 47.0 140.8 115.2 130.9 115.0 145.5 145.8 110.6 120.8 135.8 129.0 115.6 154.2 56.1
Table 2. NMR spectral data of compounds 3 and 23
2
Compound 3a δH (J in Hz) δC 4.12 (m) 69.8 3.72 (dd, 3.5, 10.5) 3.99 (m) 54.7
3
4.14 (m)
72.9
4 5 6 7−11 12 13 14 15 16 17 18 1’ 2’
5.48 (dd, 15.5, 7.5) 5.72 (dt, 15.5, 6.5) 2.07 (m) 1.29−1.33 (m) 1.98−2.07 (m) 5.43 (m) 5.43 (m) 2.07 (m) 1.29−1.33 (m) 1.29−1.33 (m) 0.90 (t, 7.0)
131.2 134.4 33.6 30.2−30.8 33.3* 130.7 132.0 33.7* 33.1 23.7 14.4 177.2 73.1
Position 1
Compound 23b δH (J in Hz)
δC
Tri ** δC
1.32 (d, 6.5)
20.5
18.2
3.95 (m) 1.91 (m) 1.81 (m) 2.65 (t, 7.5)
76.7
75.0
37.5
37.2
29.9
29.2
135.4 143.0 3.99 (m) 6.88 (d, 1.5) 116.2 115.3 1.71 (m) 3’ 35.9 143.8 141.0 1.55 (m) 4’ 1.42 (m) 26.2 141.8*** 134.5 5’ 6.91 (d, 8.0) 116.2 115.5 1.29−1.33 (m) 30.2−30.8 6’ 6.78 (dd, 8.0, 2.0) 120.6 119.9 1.29−1.33 (m) 30.2−30.8 7’−13’ 1.29−1.33 (m) 30.2−30.8 14’ 33.1 1.29−1.33 (m) 15’ 23.7 1.29−1.33 (m) 16’ 0.90 (t, 7.0) 14.4 1’’ 4.27 (d, 8.0) 104.7 4.50 (d, 8.0) 101.5 101.8 2’’ 3.20 (t, 9.0, 8.0) 75.0 3.30 (dd, 9.0, 8.0) 73.3 72.3 3’’ 3.36 (dd, 9.0, 9.0) 78.0 3.53 (dd, 9.0, 9.0) 75.9 75.1 4’’ 3.28 (m) 71.6 3.43 (m) 69.6 74.9 5’’ 3.28 (m) 77.9 3.42 (m) 75.8 68.8 3.86 (dd, 11.5, 1.5) 3.75 (dd, 12.0, 5.0) 6’’ 62.7 60.7 61.8 3.68 (dd, 11.5, 5.0) 3.91 (brd, 12.0) 1 1 Assignments were done by H- H COSY, HSQC, HMBC experiments a Measured at 500 MHz for 1H and 125 MHz for 13C using methanol-d4 as solvent b Measured at 500 MHz for 1H and 125 MHz for 13C using deuterium oxide-d2 as solvent * The assignments interchangeable in the vertical column ** Tri: Tripodantoside (using deuterium oxide-d2 as solvent)20 *** In the HMBC spectrum, the proton signal of H-6’ at δH 6.78 correlated with 13C signal at δC 141.8 (C-4’) and did not with 13C signal at δC 143.8 (C3’)
12
Table 3. The IC50 values (µM) of cell growth inhibitory effects and AChE inhibitory activity of some isolated compounds IC50 values (µM)
Compounds MCF-7
NCI-H460
PHF
AChE
5
146.9±9.0
-
>277.8
-
6
114.5±7.2
-
297.7±17.8
-
22
222.2±10.8
286.6±8.5
185.7±4.7
96.1±14.5
23
112.8±9.4
-
>290.7
-
Camptothecin
0.108±0.039
0.022±0.002
4.772±2.414
-
Galanthamine
-
-
-
0.141±0.024
13
Graphical abstract
14
