Nghiên cứu thành phần hóa học và khảo sát hoạt tính gây độc tế bào của các hợp chất phân lập từ vỏ cây dà quánh (ceriops decandra)

Received: 16 April 2020 Revised: 20 August 2020 Accepted: 25 August 2020 DOI: 10.1002/mrc.5091 LETTER - SPECTRAL ASSIGNMENT Structure elucidation of two new diterpenes from Vietnamese mangrove Ceriops decandra Nguyen Van Thanh1 | Kieu Thi Phuong Linh1 | Pham Thanh Binh1 Nguyen Phuong Thao1 | Nguyen The Cuong2 | Tran Thi Bich Ha6 | | Nguyen Van Chien3 | Nguyen Quoc Trung4 | Vu Huy Thong5 | Nguyen Xuan Cuong1 | Nguyen Hoai Nam1 | Chau Van Minh1 Department of Bioactive Natural Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam Melinh Station for Biodiversity, Institute of Ecology and Biological Resourses, Vietnam Academy of Science and Technology, Hanoi, Vietnam Department of Corrosion and Protection of Metals, Institute for Tropical Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam Department of Chemistry and Catalytic Materials, Institute of Materials Science, Vietnam Academy of Science and Technology, Hanoi, Vietnam Department of Basic Science, University of Fire Fighting and Prevention, Hanoi, Vietnam Faculty of Quality Standards and Reference Substances, Institute of Drug Quality Control Ho Chi Minh city, Ho Chi Minh, Vietnam Correspondence Nguyen Van Thanh, Department of Bioactive Natural Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam Email: thanhcmgu@yahoo.com, nvthanh1977@imbc.vast.vn Funding information Vietnam Academy of Science and Technology, Grant/Award Number: TĐPCCC.04/18-20 | INTRODUCTION Ceriops decandra (Griff.) W.Theob (Rhizophoraceae), a true mangrove plant, occur in Africa, Australia, South Asia, and many countries of Southest Asia [1] The bark of C decandra is an Indian folk medicine used for the treatment of diarrhea, amoebiasis, hemorrhage, and malignant ulcers [2] The leaf extract has been reported to exhibite antinociceptive activity [3] Previous phytochemical investigations on this plant resulted in the isolation of lupane- and ursane-type triterpenoids from the leaf [4], beyerane-, pimarane-, kaurane-, and abietanetype diterpenoids from the roots [1, 5–7], and abietaneand podocarpane-type diterpenoids from the barks [2, 8] In an ongoing search for bioactive natural products from mangroves [9, 10], we report here the isolation, structure elucidation, and cytotoxicity assay of two new diterpenes, ceridecandrin A (1) and B (2), from C decandra stem barks Their structures were elucidated by analysis of HR-QTOF-MS and 1D and 2D NMR spectroscopic data It should be noted that compound is the Magn Reson Chem 2020;1–6 first example of a 8,15-epoxypimarane-type diterpenoid possessing a 14,16-ether bridge, and compound is the second member of a rare class of 10,19-epoxyrosane-type diterpenoid (Figure 1) | R E S U L T S AN D D I S C U S S I O N Ceridecandrin A (1) was obtained as white, amorphous powder Its molecular formula was determined to be C20H32O3 on the basis of 13C NMR data (Table 1) and HR-QTOF-MS ion peaks at m/z 321.2416 [M + H]+ (calcd for C20H33O3+, 321.2424), m/z 343.2244 [M + Na]+ (calcd for C20H32O3Na+, 343.2244), and m/z 338.2687 [M + NH4]+ (calcd for C20H36O3N+, 338.2690), indicating five index of hydrogen deficiency The 13C NMR and HSQC spectra disclosed 20 carbon signals, corresponding to four methyls, seven sp3 methylenes (including one oxymethylene at δC 74.2), five sp3 methines (consisting three bearing oxygen at δC 70.7, 84.1, and 85.1), and four sp3 quartenary carbons (one oxygenated at δC 89.0) The wileyonlinelibrary.com/journal/mrc © 2020 John Wiley & Sons, Ltd VAN THANH ET AL FIGURE Structures of Compounds and TABLE 1 H (500 MHz) and 13C NMR (125 MHz) data for (in CDCl3) and (in CD3OD) Position δC δ H (J in Hz) δC δ H (J in Hz) 40.5 β 1.63 (1H, overlapped) α 0.85 (1H, overlapped) 31.2 β 1.77 (1H, overlapped) α 1.69 (1H, overlapped) 18.5 β 1.48 (1H, overlapped) α 1.37 (1H, overlapped) 29.8 β 1.95 (1H, dddd, 4.5, 7.0, 14.0, 16.5) α 1.71 (1H, overlapped) 41.8 β 1.41 (1H, overlapped) α 1.17 (1H, ddd, 3.5, 13.5, 13.5) 75.4 3.56 (1H, br d, 4.0) 32.6 - 49.0 - 45.2 1.28 (1H, overlapped) 45.6 2.20 (1H, dd, 5.0, 14.0) 26.6 1.69 (2H, overlapped) 18.4 β 1.47 (1H, overlapped) α 1.42 (1H, overlapped) 70.7 4.11 (1H, br s) 27.7 α 1.62 (1H, overlapped) β 1.26 (1H, overlapped) 89.0 - 32.9 1.67 (1H, overlapped) 49.8 1.25 (1H, overlapped) 40.0 - 10 36.4 - 91.2 - 11 19.3 β 1.75 (1H, dddd, 7.0, 14.0, 14.0, 14.0) α 1.44 (1H, overlapped) 32.4 α 1.78 (1H, overlapped) β 1.25 (1H, overlapped) 12 31.2 β 2.00 (1H, dddd, 1.5, 7.0, 14.0) α 1.59 (1H, overlapped) 33.6 β 1.55 (1H, ddd, 3.5, 13.5, 14.0) α 1.24 (1H, overlapped) 13 46.3 - 37.1 - 14 85.1 3.61 (1H, br s) 42.0 α 1.27 (1H, overlapped) β 1.23 (1H, overlapped) 15 84.1 4.01 (1H, br s) 152.3 5.82 (1H, dd, 10.5, 17.5) 16 74.2 α 3.93 (1H, dd, 1.0, 8.0) β 3.78 (1H, br d, 8.0) 109.3 α 4.93 (1H, dd, 1.0, 17.5) β 4.87 (1H, overlapped) 17 18.4 1.06 (3H, s) 22.7 1.02 (3H, s) 18 33.5 0.91 (3H, s) 16.1 0.97 (3H, s) 19 22.2 0.86 (3H, s) 77.4 β 3.68 (1H, d, 8.5) α 3.64 (1H, d, 8.5) 20 15.7 0.90 (3H, s) 15.5 0.96 (3H, s) presence of five oxygenated carbons and the absence of any unsaturated carbon, in combination with the molecular formula C20H32O3, suggested that compound was a pentacyclic diterpenoid with two ether linkages The 1H NMR spectrum exhibited four singlet methyl signals at δH 0.86 (s, H-19), δH 0.90 (s, H-20), δH 0.91 (s, H-18), and δH 1.06 (s, H-17), three oxygenated methine protons at δH 3.61 (br s, H-14), δH 4.01 (br s, H-15), and δH 4.11 (br s, VAN THANH ET AL H-7), and a pair of oxymethylene protons at δH 3.78 (br d, J = 8.0 Hz, H-16β)/3.93 (dd, J = 1.0, 8.0 Hz, H-16α) Careful interpretation of correlations observed in the COSY and HMBC spectra revealed that the planar structure of (Figure 2) was similar to that of 8,15Repoxypimaran-16-ol [7] and ent-8,15R-epoxypimaran-16-ol [11], except for the presence of an additional hydroxyl group at C-7, and an ether bridge between C-14 and C-16 in Indeed, the HMBC correlation from H-15 (δH 4.01) to C-8 (δC 89.0) established the 8,15-epoxy linkage The hydroxyl group was located at C-7 due to COSY crosspeaks of H-5/H-6/H-7, as well as the HMBC correlations from H-5 (δH 1.28) to C-7 (δC 70.7) The connection of C-14 and C-16 through an oxygen atom was confirmed by HMBC correlation from H-14 (δH 3.61) to C-16 (δC 74.2) The relative configuration of was determined by the analysis of coupling constant and NOESY spectrum The small vicinal coupling constant of H-7 (δH 4.11, br s) and the large vicinal coupling constant of H-3α (δH 1.17, J = 3.5, 13.5, 13.5 Hz) and H-11β (δH 1.75, J = 7.0, 14.0, 14.0, 14.0 Hz) suggested the equatorial orientation of H-7 and the axial orientations of both H-3α and H-11β The NOESY correlations of H-3α/H3-18, H3-18/H-5, H-5/H- FIGURE ( Key COSY ( ) of and ) and HMBC correlations 1α, H-1α/H-2α, H-1α/H-9, H-9/H-11α, H-9/H-12α, H-9/ H-14, H-14/H3-17, H3-20/H-1β, H3-20/H-2β, H3-20/H11β, and H-2β/H319 confirmed the structure of pimarane diterpenoid skeleton of [5] and α-configuration of H-14 (Figure 3) The trans-fusion between the B and C rings of pimarane scaffold, together with NOESY cross-peaks of H-16β/H-7 and H-16α/H3-17, indicated that H-15 was β-orientation Thus, structure of was determined as 8,15:14,16-diepoxy-7α-hydroxy-pimarane Ceridecandrin B (2) was isolated as white, amorphous powder The molecular formula of 2, C20H32O2, was deduced from the 13C NMR data, and [M + Cl]− ion peaks at m/z 339.2071 and 341.2059 with a ratio of 3:1 (calcd for C20H32O2Cl−, 339.2096, and 341.2067) in the HR-QTOF-MS, corresponding to five index of hydrogen deficiency The 13C NMR and HSQC spectra revealed the presence of 20 carbons, including four nonprotonated carbons (one bearing oxygen at δC 91.2), nine methylenes (one sp2 carbon at δC 109.3 and one oxygenated at δC 77.4), four methine (one sp2 carbon at δC 152.3 and one oxymethine at δC 75.4), and three methyls The presence of two sp2 carbons and three oxygenated carbons, along with the HR-MS data analysis, indicated that was a tetracyclic diterpenoid with an ether bridge The 1H NMR spectrum showed signals for one monosubstituted double bond at δH 5.82 (dd, J = 10.5, 17.5 Hz, H-15), δH 4.87 (H16β)/4.93 (dd, J = 1.0, 17.5 Hz, H-16α), one oxymethine group at δH 3.56 (br d, J = 4.0 Hz, H-3), one oxymethylene group at δH 3.64 (d, J = 8.5 Hz, H19α)/3.68 (d, J = 8.5 Hz, H-19β), and three singlet methyls at δH 0.96 (s, H-20), δH 0.97 (s, H-18), and δH 1.02 (s, H-17) Detailed analysis of COSY and HMBC correlations (Figure 2) revealed that the planar structure of was closely related to that of euphomianol A [12], a rosane-type diterpenoid with a 10,19-oxygen bridge, except for the absence of a hydroxy group at C-5 in This was confirmed by the COSY cross-peaks of H-5/H2-6/ H2-7/H-8/H2-14 and the HMBC correlations from H2-19 (δH 3.64/3.68) to C-3 (δC 75.4), C-4 (δC 49.0), C-5 (δC 45.6), C-10 (δC 91.2), and C-18 (δC 16.1) According to the coupling constant values between H-3 and H-2β (J = 4.5 Hz), between H-2β and H-1α (J = 14.0 Hz), and between H-12β and H-11α (J = 13.5 Hz), H-3 was assigned as equatorial orientation, and both H-2β and H-12β were in axial orientation The NOESY correlations of H-3/H-2β, H-2β/H-19α, H-2β/H1β, H-19β/H-6β, H-6β/H3-20, and H3-20/H-12β revealed that these protons were cofacial and they were in β-configuration (Figure 3) By contrast, NOE cross-peaks of H-1α/H-5, H-5/H-6α, H-5/H-8, H-8/H3-17, and H3-17/ H-12α demonstrated that these protons were α-oriented 4 VAN THANH ET AL FIGURE ( TABLE | M A T E R I A L S A ND M E T HO D Cytotoxicity of Compounds and IC50 (μg/mL) 3.1 | General Compound SK-LU-1 HepG2 MCF7 58.36 ± 5.83 60.28 ± 2.77 44.17 ± 3.23 22.10 ± 2.65 27.53 ± 1.53 20.02 ± 1.55 0.41 ± 0.05 0.47 ± 0.03 0.35 ± 0.04 Ellipticine a Key NOESY correlations ) of and a Positive control substance Therefore, the structure of was identified as 10,19-epoxy-3α-hydroxy-rosane Compounds and were evaluated for cytotoxicity against three cancer cell lines: SK-LU-1, HepG2, and MCF7 Ellipticine was used as a positive control The results showed that both compounds exhibited weak cytotoxicity against three cell lines with IC50 values in the range of 20.02 to 60.28 μg/mL (Table 2) Optical rotations were measured using a JASCO P2000 polarimeter (JASCO, Oklahoma, OK, US) The HR-QTOF-MS were recorded on an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (CA, USA) Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany) and YMC*GEL resins (ODS-A, 12 nm S-150 μm, YMC Co., Ltd.) Analytical thin layer chromatography (TLC) systems were performed on precoated silica gel 60 F254 (1.05554.0001, Merck) and RP18 F254S plates (1.15685.0001, Merck), and the isolated compounds were visualized by spraying with 10% H2SO4 in water and then heating for 1.5–2 All procedures were carried out with solvents purchased from commercial sources that were used without further purification VAN THANH ET AL 3.2 | NMR spectra NMR spectra were recorded on a Bruker Ascend 500/Avance III HD spectrometer at temperature of 303 K Compounds and were dissolved in CDCl3 and CD3OD, respectively, and transferred into 5-mm NMR tubes 1H and 13C chemical shifts (δ) were referenced to tetramethylsilane (TMS) at 0.00 ppm Coupling constants (J) were expressed in Hertz (Hz) The 1H NMR experiments were carried out with spectrometer frequency (SF) = 500.20 MHz, spectral width in Hz (SWH) = 10,000 Hz, acquisition time (AQ) = 3.2768 s, number of scans (NS) = 16, relaxation delay (D1) = 1.0 s, 90 pulse width (P1) = 10.00 μs, Fourier transform size (SI) = 65,536, and line broadening (LB) = 0.3 Hz The 13 C NMR spectrum was acquired with SF = 125.77 MHz, SWH = 31,250 Hz, AQ = 1.048 s, NS = 1,536, D1 = 2.0 s, P1 = 10.00 μs, SI = 32,768, and LB = 1.0 Hz The 2D NMR spectra were recorded using Bruker library pulse sequence condition as follows: for HSQC, NS = 4, D1 = 2.0 s, SWH = 6009.615 Hz, time domain data points (TD) = 2048, AQ = 0.1704 s; for HMBC, NS = 16, SWH = 3012.048 Hz, AQ = 0.3399 s, D1 = 1.3 s, TD = 2048; for COSY, SWH = 3067.485 Hz, TD = 2048, NS = 2, AQ = 0.3338 s, D1 = 1.8 s; and for NOESY, SWH = 2994.012 Hz, TD = 2048, NS = 8, AQ = 0.3420 s, D1 = 1.8 s 3.3 | Plant material The stem barks of C decandra were collected from Ca Mau province, Vietnam, in July 2018 and identified by Dr Nguyen The Cuong A voucher specimen (PCCC01-CD) was deposited in the Department of Bioactive Natural Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST) 3.4 | Extraction and isolation The air-dried, powdered stem barks of C decandra (9 kg) were extracted with EtOAc three times at room temperature in ultrasonic bath The crude residue (100 g) was separated on a silica gel CC and eluted with n-hexane/EtOAc mixtures of increasing polarity (100:0 ! 0/100) to obtained 12 fractions, E1–E12 Fraction E5 was divided into eight fractions, E5A– E5H, by a silica gel CC (n-hexane/EtOAc, 15/1) Fraction E5H was subjected to a silica gel CC (nhexane/EtOAc, 2.5/1) to give three fractions, E5H1– E5H3 Fraction E5H1 was further chromatographed on a silica gel CC (n-hexane/acetone, 2/1) to yield two fractions, E5H1A and E5H1B Fraction E5H1B was separated on a silica gel CC (CH2Cl2/EtOAc, 4/1), followed by a silica gel CC (n-hexane/acetone, 10/1) to obtained three fractions, E5H1B1–E5H1B3 Fraction E5H1B1 was purified by a silica gel CC (nhexane/acetone, 10/1) to yield Compound (1.7 mg) Compound (1.3 mg) was purified by YMC CC (MeOH/H2O, 6/1) from fraction E5H1B2 3.5 | Physical and spectroscopic data Ceridecandrin A (1): white, amorphous powder, α25 D +52.8 (c 0.8, MeOH); 1H NMR (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz) spectral data, see Table 1; HR-QTOF-MS: m/z 321.2416 [M + H]+ (calcd for C20H33O3+, 321.2424), m/z 343.2244 [M + Na]+ (calcd for C20H32O3Na+, 343.2244), and m/z 338.2687 [M + NH4]+ (calcd for C20H36O3N+, 338.2690) Ceridecandrin B (2): white, amorphous powder, α25 D +60.7 (c 0.4, MeOH); 1H NMR (CD3OD, 500 MHz) and 13 C NMR (CD3OD, 125 MHz) spectral data, see Table 1; HR-QTOF-MS: m/z 339.2071, and 341.2059 [M + Cl]− (calcd for C20H32O2Cl−, 339.2096, and 341.2067) 3.6 | Cytotoxicity assays The cytotoxicity assays for ceridecandrin A (1) and B (2) were performed using the SRB method [13] as a reviously reported protocol [14] The raw NMR data files of the spectra including the relevant fid(s) are given in the Supporting Information ACKNOWLEDGEMENTS This research was supported by the Vietnam Academy of Science and Technology (project code: TĐPCCC.04/18-20) We thank Dr Dang Vu Luong, Institute of Chemistry, VAST, for NMR measurement and Prof Do Thi Thao, Institute of Biotechnology, VAST, for cytotoxicity assays PE ER RE VI EW The peer review history for this article is available at https://publons.com/publon/10.1002/mrc.5091 ORCID Nguyen Van Thanh 5999 https://orcid.org/0000-0002-5473- RE FER EN CES [1] Z.-P Jiang, L.-W Tian, L Shen, J Wu, Fitoterapia 2018, 130, 272 6 [2] H Wang, M Y Li, T Satyanandamurty, J Wu, Planta Med 2013, 79, 666 [3] S J Uddin, J A Shilpi, J Barua, R Rouf, Fitoterapia 2005, 76, 261 [4] C Ponglimanont, P Thongdeeying, Aust J Chem 2005, 58, 615 [5] A S R Anjaneyulu, V Lakshmana Rao, Phytochemistry 2002, 60, 777 [6] A S R Anjaneyulu, V L Rao, E Lobkovsky, J Clardy, J Nat Prod 2002, 65, 592 [7] A S R Anjaneyulu, V Lakshmana Rao, Phytochemistry 2003, 62, 1207 [8] H Wang, M Y Li, F Z Katele, T Satyanandamurty, J Wu, G Bringmann, Beilstein J Org Chem 2014, 10, 276 [9] N V Thanh, L H Hieu, P T T Huong, L T Vien, T M Linh, N T Cuong, N X Cuong, N H Nam, N D Quang, C Van Minh, Phytochem Lett 2018, 25, 52 [10] N Van Thanh, H J Jang, L B Vinh, K T P Linh, P T T Huong, N X Cuong, N H Nam, C Van Minh, Y H Kim, S Y Yang, Bioorg Chem 2019, 88, 102921 [11] W Herz, P Kulanthaivel, Phytochemistry 1983, 22, 715 [12] S.-N Liu, D Huang, S L Morris-Natschke, H Ma, Z.-h Liu, N P Seeram, J Xu, K.-H Lee, Q Gu, Org Lett 2016, 18, 6132 VAN THANH ET AL [13] A Monks, D Scudiero, P Skehan, R Shoemaker, K Paull, D Vistica, C Hose, J Langley, P Cronise, A Vaigro-Wolff, M Gray-Goodrich, H Campbell, J Mayo, M Boyd, J Natl Cancer Inst 1991, 83, 757 [14] N H Nam, N T Ngoc, T T H Hanh, N X Cuong, N V Thanh, D T Thao, D C Thung, P V Kiem, C V Minh, Steroids 2018, 138, 57 SU PP O R TI N G I N F O RMA TI O N Additional supporting information may be found online in the Supporting Information section at the end of this article How to cite this article: Van Thanh N, Linh KTP, Binh PT, et al Structure elucidation of two new diterpenes from Vietnamese mangrove Ceriops decandra Magn Reson Chem 2020;1–6 https://doi.org/10.1002/mrc.5091