Lignans from the stems of Clerodendrum inerme Gaertn

Cite this paper: Vietnam J. Chem., 2021, 59(2), 187-191 Article DOI: 10.1002/vjch.202000164 187 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Lignans from the stems of Clerodendrum inerme Gaertn. Tran Thi Minh * , Nguyen Thi Minh Thuong School of Chemical Engineering, Hanoi University of Science and Technology 1 Dai Co Viet, Hai Ba Trung, Hanoi 10000, Viet Nam Submitted September 19, 2020; Accepted November 11, 2020 Abstract The polar constituents of Clerodendrum inerme collected from coastal area of Thai Binh province were investigated. From the water soluble fraction of the C. inerme stems, four lignan and lignan glucosides (1-4) along with a phenolic glucoside (5) were isolated. Their chemical structures were established as icariol A2 (1), syringaresinol-4-O- -glucopyranoside (2), lariciresinol-4-O--D-glucopyranoside (3), dehydrodiconiferyl alcohol-4-O--D- glucopyranoside (4) and leonuriside A (5) by NMR spectroscopic and mass spectrometric analysis as well as comparison with those reported in the literature. Compounds 1, 2, 3 and 5 were found for the first time from this plant. Keywords. Clerodendrum inerme, stems, lignan, lignan glucosides. 1. INTRODUCTION Clerodendrum inerme Gaertn. (Verbenaceae) is widely distributed near the seashore from the north to the south of Vietnam. [1] This plant has been used as a folk medicine in Thailan, China, and Vietnam for treatment of various diseases such as skin diseases, topical burns, malaria, rheumatism and hepatitis. [2] This species has been reported to contain flavonoids, [3] diterpenes, [4,5] phenylethanoid glycosides, [6,7] megastigmane and iridoid glycosides. [8] Previously, we have reported the isolation and structural determination of andrographolide and lupeol hexacosanoate ester from the ethyl acetate fraction of methanolic extract obtained from the leaves of C. inerme. [9] As a continuation of our investigation on this plant, we wish to describe the isolation and structural determination of five polar compounds comprising four lignan and lignan glucosides (1-4) along with a phenolic glucoside (5) from the water layer of methanolic extract of C. inerme stems collected in the beach of Thai Binh province. 2. MATERIALS AND METHODS 2.1. General experimental procedures ESI-MS were recorded on an ESI-LC/MS/MS-Xevo TQMS spectrometer. NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Silica gel 60 (0.04-0.063 mm, Merck), RP-18 resins (150 m, YMC), Diaion HP 20 (Mitsubishi chemical Co.), and Sephadex LH-20 (25-100 m, Sigma-Aldrich) were used for column chromatography (CC). The thin layer chromatography (TLC) was performed on Merck precoated TLC DC-Alufolien silica gel 60F254 and RP-18F254S. The plates were visualized under UV fluorescence or by dipping in 1 % vanillin-H2SO4 and heating at 100 o C for 1-2 min. 2.2. Plant materials The stems of C. inerme (Verbenaceae) were collected from the coastal area of Thai Binh province, Vietnam, in 2018. The plant was identified by Prof. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST). A voucher specimen (HUST.N02) was deposited in the laboratory of the Organic Department, Hanoi University of Science and Technology (HUST), Vietnam. 2.3. Extraction and isolation The dry and powdered stems of C. inerme (5.0 kg) were extracted three times with 80 % aqueous methanol at 50 o C using a sonicator. After evaporation of the solvent under reduced pressure, the methanol extract (185.0 g) was suspended in water (3 L) and partitioned with n-hexane followed by ethyl acetate (EtOAc) to give n-hexane (27.1 g), Vietnam Journal of Chemistry Tran Thi Minh et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 188 EtOAc (17.0 g), and water (95.5 g) residues. The water soluble fraction (95.5 g) was subjected to column chromatography on a Diaion HP 20 eluting with the solvent systems of methanol/water, increasing concentration of methanol in water (0, 20, 40, 60, 100 %). The fraction eluted with 40-60 % methanol (14.6 g) was chromatographed on a silica gel column eluting with a gradient solvent system of dichloromethane/methanol (10/1, 5/1, 2.5/1, 1/1, v/v) to give eight sub-fractions (CI.1-CI.8). Sub- fractions CI.2, CI.3, CI.6, and CI.7 were further separated on sephadex LH 20 column eluting with methanol, then followed by RP-18 column eluting with methanol/water (1/2, v/v) to afford compounds 1 (55.9 mg), 2 (17.8 mg), 4 (12.5 mg), and 5 (62.1 mg), respectively. CI.5 (1.27 g) was similarly separated on a Sephadex LH 20 column eluting with methanol, then purified on a RP-18 column eluting with methanol/water (1/2.5, v/v) to give compound 3 (16.7 mg). Icariol A2 (1): White amorphous powder, ESI- MS (positive): m/z 419 [M+H-H2O] + , 401 [M+H- 2H2O] + ; 1 H-NMR (500 MHz, DMSO) H: 6.64 (4H, s, H-2, H-6, H-2’, H-6’), 4.83 (2H, d, J = 8.0 Hz, H- 7, H-7’), 3.76 (12H, s, OCH3), 3.49 (4H, m, H2-9, H2-9’), 2.14 (2H, m, H-8, H-8’); 13 C-NMR (125 MHz, DMSO) C: 147.8 (C-3, C-5, C-3’, C-5’), 134.6 (C-1, C-1’), 133.2 (C-4, C-4’), 103.7 (C-2, C- 6, C-2’, C-6’), 82.0 (C-7, C-7’), 60.4 (C-9, C-9’), 55.9 (3, 5, 3’, 5’-OCH3), 53.4 (C-8, C-8’). Syringaresinol-4-O--glucopyranoside (2): White amorphous powder, ESI-MS (positive): m/z 419 [M+H-Glucose] + ; 1 H-NMR (500 MHz, CD3OD) and 13 C-NMR (125 MHz, CD3OD)  (ppm): given in table 1. Lariciresinol-4-O--D-glucopyranoside (3): White amorphous powder, ESI-MS (positive): m/z 343 [M+H-Glucose] + , 523 [M+H] + ; 1 H-NMR (500 MHz, CD3OD) and 13 C-NMR (125 MHz, CD3OD)  (ppm): given in table 1. Dehydrodiconiferyl alcohol-4-O--D-glucopyranoside (4): White amorphous powder, ESI-MS (positive): m/z 341 [M+H-Glucose] + , 521 [M+H] + ; 1 H-NMR (500 MHz, CD3OD) and 13 C-NMR (125 MHz, CD3OD)  (ppm): given in table 1. Leonuriside A (5): White amorphous powder, ESI-MS (positive): m/z 355 [M+Na] + ; 1 H-NMR (500MHz, DMSO)  (ppm): 6.07 (2H, s, H-3, H-5), 4.65 (1H, d, J = 7.5 Hz, H-1’), 3.69 (6H, s, 2,6- OCH3), 3.61 (1H, m, H-6’a), 3.43 (1H, m, H-6’b), 3.19 (2H, m, H-2’, H-5’), 3.14 (1H, m, H-4’), 3.02 (1H, m, H-3’); 13C-NMR (500MHz, DMSO)  (ppm): 153.9 (C-4), 153.1 (C-2, C-6), 127.5 (C-1), 103.5 (C-1’), 93.8 (C-3, C-5), 77.0 (C-3’), 76.4 (C- 5’), 74.2 (C-2’), 70.0 (C-4’), 61.0 (C-6’), 56.1 (2,6- OCH3). 3. RESULTS AND DISCUSSION The separation of water residue of C. inerme stems using chromatography methods yielded five compounds 1-5 (figure 1). The chemical structures of all five compounds were identified based on comparison of spectral data with those published. Compound 1 was obtained as a white amorphous powder. The molecular formula C22H28O9 was determined by ESI-MS and NMR spectroscopic data. The 13 C-NMR spectrum of 1 showed eight carbon signals indicating the presence of two symmetrical halves to the molecule, which was further confirmed by the symmetrical proton signals on 1 H-NMR. The 1 H-NMR spectrum of 1 indicated proton signals of two 1,3,4,5-tetrasubstituted benzene rings [H 6.64 (4H, s, H-2, H-6, H-2’, H- 6’)], two oxymethine groups [H 4.83 (2H, d, J = 8.0 Hz, H-7, H-7’)], four methoxy groups [H 3.76 (12H, s, OCH3)], two oxymethylene groups [H 3.49 (4H, m, H2-9, H2-9’)], and two methine groups [H 2.14 (2H, m, H-8, H-8’)]. Analysis of ESI-MS, 1H and 13 C-NMR data indicated the structure of 1 was identical to icariol A2. [10] The HMBC spectrum was examined to confirm this structure. The HMBC correlations from H-7/H-7’ to C-8/C-8’, C-9/C-9’, C-2,6/C-2’,6’, and from H-8/H-8’ to C-1/C-1’ confirmed the presence of 7,7’-monoepoxy type lignan skeleton. The positions of methoxy groups at C-3,5 and C-3’,5’ were determined by the HMBC correlation between proton signal of methoxy groups (H 3.76) to C-3,5/C-3’5’. The large coupling constant of H-7/H-7’ (J = 8.0 Hz) suggested trans orientation between H-7/H-8 and H-7’/H-8’. Thus, the chemical structure of compound 1 was determined as icariol A2. [10] This compound was found in the Neoalsomitra integrifoliola genus and showed weak anti-inflammatory activity. [ 11] Compound 2 was obtained as a white amorphous powder. The molecular formula of 2 was clarified as C28H36O13 based on the ion peak at m/z 419 [M+H- Glucose] + in ESI-MS and NMR spectral data. The Vietnam Journal of Chemistry Lignans from the stems of © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 189 1 H NMR spectrum of 2 showed the proton signals of two symmetric 1,3,4,5-tetrasubstituted benzene rings [δH 6.73 (2H, s, H-2, H-6) and 6.67 (2H, s, H-2’, H- 6’)], two oxymethylene groups [δH 4.30 (2H, dd, J = 8.5, 5.0 Hz, H-9a, H-9a’) and 3.93 (2H, dd, J = 9.0, 3.0 Hz, H-9b, H-9b’)], two oxymethine groups [δH 4.87 (1H, d, J = 3.6 Hz, H-7) and 4.73 (1H, d, J = 4.3 Hz, H-7’)], two methine groups [δH 3.14 (2H, m, H-8, H-8’)], four methoxy groups [δH 3.87 and 3.86 (each, 6H, s)], an anomeric proton signal [δH 4.88 (1H, d, J = 7.5 Hz, H-1”)], and other proton signals of sugar from 3.22 to 3.55 ppm. The 13 C-NMR and HSQC spectra of 2 showed twenty eight carbon signals comprising six carbon signals of a hexose unit and 22 carbon signals of aglycone moiety. The sugar unit was identified as -D-glucose based on the chemical shift of carbons (δC 105.4, 78.3, 77.8, 75.7, 71.3, 62.6) and the coupling constant of anomeric proton (J = 7.5 Hz). [12] The above mentioned NMR data indicated 2 to be a furofuran lignan glucoside. Moreover, the presence of furofuran lignan skeleton was further confirmed by the significant 1 H- 1 H COSY cross peaks of H-7/H- 8/H-9 and H-7’/H-8’/H-9’ along with the key HMBC correlations of H-7/C-2,6, C-9; H-7’/C-2’,6’, C-9’; and H-8,8’/C-1, C-1’. The positions of methoxy groups at C-3,5 and C-3’,5’ were determined by HMBC correlations from the proton signals of methoxy groups (δH 3.87, 3.86) to C-3,5 and C-3’,5’. The linkage of glucose at C-4 was determined by the HMBC correlation from H-1” (H 4.88) to C-4 (C 135.6). The small coupling constants of H-7 (J = 3.6 Hz) and H-7’(J = 4.3 Hz) indicated cis orientation between H-7/H-8 and H-7’/H-8’. Thus, the structure of compound 2 was identified as syringaresinol-4-O--D- glucopyranoside by comparison of its NMR spectral data with those published. [12] Compound 3 was obtained as a white amorphous powder. The molecular formula of C26H34O11 was derived from its pseudomolecular ion peak at m/z 523 [M+H] + in ESI-MS and NMR spectral data. The 1 H and 13 C NMR spectra (table 1) of 3 showed the presence of two 1,3,4-trisubstituted benzene rings, two methoxy groups, two oxymethylene groups, one oxymethine group, three methine groups, and a hexose moiety. The sugar unit was identified as β-D- glucopyranose by comparing the 1 H and 13 C NMR data with those published. [12,13] The analysis of 1 H and 13 C NMR data indicated 3 to be a lariciresinol lignan glucoside. [13] The linkage of glucose at C-4 was confirmed by the HMBC correlation between the anomeric proton H-1” (H 4.90) and C-4 (C 147.3). The relative configuration of 3 was indicated based on NOESY spectroscopic analysis. The NOESY correlation of H-8 (H 2.37)/H-8’(H 2.73) and the absence of the NOESY correlation of H- 7/H-8 indicated cis orientation between H-8/H-8’ and trans orientation between H-7/H-8, respectively. The anti-periplanar orientation of H-7 and H-8 was further confirmed by the large coupling constants of H-7 (J = 6.5 Hz). Thus, compound 3 was identified as lariciresinol-4-O--D-glucopyranoside by comparison of its NMR spectral data with those published. [13] Compound 4 had a molecular formula of C26H32O11, which was suggested by its ESI-MS, NMR and DEPT data. The 13 C NMR and DEPT spectra of 4 indicated the presence of a hexose unit and 20 carbon signals for the aglycone moiety. The 1 H NMR spectrum of 4 showed signals of five aromatic protons [δH 7.17 (d, J = 8.0 Hz), 7.05 (d, J = 2.0 Hz), 6.95 (dd, J = 8.0, 2.0 Hz), 6.96 and 6.97 (each, 1H, s)], two methoxy groups [δH 3.85 and 3.90 (each, 3H, s)], two trans-olefinic protons which appeared as AB part of an ABX2 spin system [δH 6.57 (d, J = 16.0 Hz) and 6.24 (dt, J = 15.5, 6.0 Hz)], a dihydrobenzofuran ring, and an anomeric proton [δH 4.91 (d, J = 7.5 Hz)]. These data suggested that the aglycone of compound 4 was a dehydrodiconiferyl alcohol type lignan. [14] The 13 C NMR data of the sugar moiety were consistent with those of β-D-glucose.[12-14] The long-range correlation from H-1” (H 4.91) to C-4 (C 147.7) confirmed that the -D-glucopyranosyl was attached to C-4 of the aglycone. The trans orientation of H-7 and H-8 was indicated by the large coupling constants of H-7 (J = 6.5 Hz). Thus, compound 4 was identified as dehydrodiconiferyl alcohol-4-O-- D-glucoside by comparison of its NMR spectral data with those published. [14] Compound 5 was identified as leonuriside A, via the comparison of its NMR spectral data with those published. [15] To our best knowledge, this is the first isolation of compounds 1, 2, 3 and 5 from C. inerme, whereas the compound 4 was previously isolated from this genus growing in Thailand. [8] Acknowledgements. This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2018.36. Vietnam Journal of Chemistry Tran Thi Minh et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 190 Table 1: 1 H and 13 C-NMR spectral data of compounds 2-4 (in CD3OD) C position 2 3 4 C H (mult., J in Hz) C H (mult., J in Hz) C H (mult., J in Hz) 1 139.5 - 139.5 - 138.1 - 2 104.9 6.73 (s) 111.4 7.01 (s) 111.2 7.05 (d, 2.0) 3 154.4 - 150.9 - 151.0 - 4 135.6 - 147.3 - 147.7 - 5 154.4 - 118.0 7.16 (d, 8.0) 118.1 7.17 (d, 8.0) 6 104.9 6.73 (s) 119.6 6.90 (d, 8.0) 119.4 6.95 (dd, 8.0, 2.0) 7 87.1 4.78 (d, 3.6) 83.8 4.86 (d, 6.5) 88.8 5.60 (d, 6.5) 8 55.5 3.14 (m) 54.1 2.37 (m) 55.3 3.48 (m) 9 72.8 3.93 (dd, 9.0, 3.0) 4.30 (dd, 8.5, 5.0) 60.5 3.89 (m), 3.67 (m) 64.9 3.79 (dd, 11.0, 7.5) 3.86 (m) 3-OCH3 56.9 3.87 (s) 56.7 3.88 (s) 56.7 3.85 (s) 5-OCH3 56.9 3.87 (s) - - - - 1’ 133.1 - 133.5 - 132.7 - 2’ 104.6 6.67 (s) 113.4 6.81 (s) 112.2 6.97 (s) 3’ 149.3 - 149.0 - 145.3 - 4’ 136.3 - 145.8 - 149.2 - 5’ 149.3 - 116.2 6.74 (d, 8.0) 130.1 - 6’ 104.6 6.67 (s) 122.1 6.66 (d, 8.0) 116.5 6.96 (s) 7’ 87.5 4.73 (d, 4.3) 33.6 2.94 (m), 2.52 (m) 131.9 6.57 (d, 16.0) 8’ 55.7 3.14 (m) 43.8 2.73 (m) 127.6 6.24 (dt, 15.5, 6.0) 9’ 72.9 3.93 (dd, 9.0, 3.0) 4.30 (dd, 8.5, 5.0) 73.7 4.02 (m) 3.75 (m) 63.8 4.22 (dd, 5.5, 1.0) 3’-OCH3 57.1 3.86 (s) 56.4 3.85 (s) 56.8 3.90 (s) 5’-OCH3 57.1 3.86 (s) - - - - 1” 105.4 4.88 (d, 7.5) 102.9 4.90 (d, 7.0) 102.8 4.91 (d, 7.5) 2” 75.7 3.50 (m) 74.9 3.49 (m) 74.9 3.51 (m) 3” 77.8 3.44 (m) 77.9 3.49 (m) 77.8 3.49 (m) 4” 71.3 3.44 (m) 71.4 3.41 (m) 71.3 3.41 (m) 5” 78.3 3.22 (m) 78.2 3.41 (m) 78.2 3.41 (m) 6” 62.6 3.69 (dd, 12.0, 5.0) 3.80 (dd, 12.0, 2.5) 62.5 3.69 (m), 3.89 (m) 62.5 3.70 (m), 3.89 (m) Figure 1: Structure of compounds 1-5 from C. inerme stems Vietnam Journal of Chemistry Lignans from the stems of © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 191 REFERENCES 1. 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Characterization of lariciresinol glucosides from Osmanthus asisticus, Heterocycles, 1993, 36, 117-121. 14. O. Salama, R. K. Chaudhuri, O. Sticher. A lignan glucoside from Euphrasia rostkoviana, Phytochemistry, 1981, 20, 2603-2604. 15. H. Otsuka, M. Takeuchi, S. Inoshiri, T. Sato, K. Yamasaki. Phenolic compounds from Coix lachryma- jobi var. ma-yuen, Phytochemistry, 1989, 28, 883-886. Corresponding author: Tran Thi Minh School of Chemical Engineering Hanoi University of Science and Technology 1 Dai Co Viet, Hai Ba Trung, Hanoi 10000, Viet Nam E-mail: minh.tranthi@hust.edu.vn Tel.: +84- 988557877. Figure 2: The key HMBC correlations of compounds 1-3