Dihydrostilbene glycosides and lignan from Camellia sasanqua

Cite this paper: Vietnam J. Chem., 2020, 58(5), 661-665 Article DOI: 10.1002/vjch.202000062 661 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Dihydrostilbene glycosides and lignan from Camellia sasanqua Nguyen Thi Cuc1,2, Nguyen Xuan Nhiem1,2, Bui Huu Tai1,2, Phan Van Kiem1,2*, Vu Kim Thu3* 1Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam 2Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam 3Hanoi University of Mining and Geology, Pho Vien, Duc Thang, Bac Tu Liem district, Hanoi 10000, Viet Nam Submitted April 27, 2020; Accepted May 17, 2020 Abstract Three dihydrostilbene glycosides, 3,5-dihydroxydihydrostilbene 4′-O-β-D-glucopyranoside (1), 3,5- dimethoxydihydrostilbene 4′-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (2), 5,4′-dihydroxydihydrostilbene 3-O-β-D-glucopyranoside (3), and one lignan, nudiposide (4) were isolated from the methanol extract of leaves of Camellia sasanqua Thunb. Their chemical structures were determined by using ESI-MS and NMR spectra as well as in comparison with the reported data. Compounds 3 and 4 were reported from Camellia genus for the first time. Keywords. Camellia sasanqua, dihydrostilbene, lignan. 1. INTRODUCTION Camellia sasanqua (Theaceae) is an evergreen shrub growing to 10m. The leaves are broad elliptic, 3-7 cm long and 1.2-3 cm broad, with a finely serrated margin. The flowers are 5-7 cm in diameter, with 5- 8 white to dark pink petals.[1] Phytochemical studies revealed that this plant contained terpenoids and phenolics.[2-4] These compounds have shown the potential significant biological effects as anti- inflammatory and anticancer activities.[2,3] This paper reported the isolation and structural elucidation of three known dihydrostilbene glycosides and one known lignan from the methanol extract of C. sasanqua leaves. 2. MATERIALS AND METHODS 2.1. Plant materials The leaves of Camellia sasanqua Thunb. were collected in Nguyen Binh, Cao Bang province, Viet Nam in April 2019, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources. A voucher specimen (NCCT-P85) was deposited at the Institute of Marine Biochemistry, VAST. 2.2. General experimental procedures See reference: [8] 2.3. Extraction and isolation The dried powder leaves of C. sasanqua (6.0 kg) were sonicated with hot MeOH (3 times × 15 L) to obtain MeOH extract (650 g) under reduced pressure. The MeOH extract was suspended in water and successively partitioned with n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc) to yield corresponding n-hexane (CSA1A, 9.2 g), dichloromethane (CS1B, 95.0 g), ethyl acetate (CS1C, 54.0 g) residues, and water layer (CS1D). CS1D was chromatographed on a Diaion HP-20 column, first eluting with water to remove sugar components, then increasing concentration of MeOH in water (25, 50, 75, and 100 %) to obtain four fractions, CS1D1-CS1D4. CS1D2 was chromatographed on a silica gel CC eluting with gradient solvent of CH2Cl2/MeOH (20/1, 10/1, and 5/1, v/v) to give three fractions, CS1D2A-CS1D2C. CS1D2A was subjected on a RP-18 column eluting with acetone/water (1/3, v/v) to give three smaller fractions, CS1D2A1-CS1D2A3. CS1D2A1 was subjected to HPLC (J’sphere H-80 column, length 250 mm × 20 mm ID, eluting with 18 % acetonitrile in water, a flow rate of 3 mL/min) to yield compound 4 (13.0 mg). CS1D2B was chromatographed on a RP-18 column eluting with MeOH/water (1/1.5, v/v) to give three fractions, CS1D2B1-CS1D2B3. CS1D2B1 was subjected to HPLC (J’sphere H-80 column, length 250 mm × 20 Vietnam Journal of Chemistry Phan Van Kiem et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 662 mm ID, eluting with 22 % acetonitrile in water, a flow rate of 3 mL/min) to yield compounds 1 (60.0 mg) and 3 (30.0 mg). The CS1D4 fraction was applied to a silica gel column, eluting with a gradient solvents of CH2Cl2/MeOH (20/1, 10/1, and 5/1, v/v) to give three fractions, CS1D4A-CS1D4C. CS1D4B was chromatographed on a RP-18 column, eluting with MeOH/water (1/1, v/v) to give three smaller sub-fractions, CS1D4B1-CS1D4B3. Compound 2 (12.0 g) was obtained from CS1D4B3 on a sephadex LH-20 column, eluting with MeOH/water (1/1, v/v). Figure 1: The chemical structures of compounds 1-4 3,5-Dihydroxydihydrostilbene 4′-O-β-D- glucopyranoside (1): white amorphous powder; [α]D25: -39.0 (c 0.1, MeOH); ESI-MS m/z 391 [M-H]-; 1H- and 13C-NMR (CD3OD) data, see table 1. 3,5-Dimethoxydihydrostilbene 4′-O-α-L- rhamnopyranosyl-(1→6)-β-D-glucopyranoside (2): white amorphous powder; [α]D25: -63.0 (c 0.1, MeOH); ESI-MS m/z 565 [M-H]-; 1H- and 13C-NMR (CD3OD) data, see table 1. 5,4′-Dihydroxydihydrostilbene 3-O-β-D- glucopyranoside (3): white amorphous powder; [α]D25: -36.0 (c 0.1, MeOH); ESI-MS m/z 391 [M-H]-; 1H- and 13C-NMR (CD3OD) data, see table 1. Nudiposide (4): white amorphous powder; [α]D25: -68.0 (c 0.1, MeOH); ESI-MS m/z 551 [M-H]-; 1H-NMR (CD3OD) δH 2.70 (d, J = 6.0 Hz, H-1)/2.71 (d, J = 5.5 Hz, H-1), 1.73 (m, H-2), 2.06 (m, H-3), 4.25 (d, J = 7.0 Hz, H-4), 6.43 (s, H-2′/H- 6′), 3.64 (m, H-2α), 3.62 (m, H-3α)/3.82 (m, H-3α), 3.34 (s, 5-OMe), 3.87 (s, 7-OMe), 3.77 (s, 3′/5′- OMe), Xyl: 4.12 (d, J = 7.5 Hz, H-1′′), 3.22 (dd, J = 7.5, 9.0 Hz, H-2′′), 3.29 (t, J = 9.0 Hz, H-3′′), 3.52 (m, H-4′′), 3.15 (dd, J = 10.5, 11.5 Hz, H-5′′)/3.87 (m, H-5′′); 13C-NMR (CD3OD) δC 34.0 (C-1), 40.7 (C-2), 46.9 (C-3), 43.3 (C-4), 147.6 (C-5), 138.9 (C- 6), 148.7 (C-7), 107.8 (C-8), 130.1 (C-9), 126.3 (C- 10), 139.6 (C-1′), 107.0 (C-2′/C-6′), 149.0 (C-3′/C- 5′), 134.6 (C-4′), 66.1 (C-2α), 71.2 (C-3α), 60.0 (5- OMe), 56.6 (7-OMe), 56.8 (3′/5′-OMe), Xyl: 105.0 (C-1′′), 75.0 (C-2′′), 78.0 (C-3′′), 71.3 (C-4′′), 67.1 (C-5′′). 3. RESULTS AND DISCUSSION Compound 1 was obtained as a white amorphous powder. The 1H-NMR spectrum of 1 showed the signals of four protons of a p-substituted aromatic ring at δH 7.01 (2H, d, J = 8.5 Hz) and 7.08 (2H, d, J = 8.5 Hz); three protons of an 1,3,5-trisubstituted aromatic ring at δH 6.13 (1H, d, J = 2.0 Hz) and 6.16 (2H, d, J = 2.0 Hz); two methylene groups at δH 2.71 (2H, t, J = 7.0 Hz) and 2.81 (2H, t, J = 7.0 Hz); and one anomeric proton at δH 4.88 (1H, d, J = 7.5 Hz). The 13C-NMR and HSQC spectra of 1 showed the signals of 20 carbons, including 5 non-protonateds at δC 137.1, 145.3, 157.2, and 159.2×2; 12 methines at δC 71.3, 74.9, 77.9×2, 101.2, 102.4, 108.1×2, 117.6×2 and 130.3×2; and 3 methylenes at δC 37.8, 39.1, and 62.5. The analysis of 1H- and 13C-NMR data suggested that structure of 1 was similar to 3,5- dihydroxydihydrostilbene 4′-O-β-D- glucopyranoside.[5] The position of hydroxy groups at C-3 and C-5 were confirmed by HMBC correlation from H-4 (δH 6.13) to C-2/C-6 (δC 108.1)/C-3/C-5 (δC 159.2). The HMBC correlation between H-2′/H-6′ (δH 7.08) and C-1′ (δC 137.1)/C-4′ (δC 157.2)/C-α′ (δC 37.8), between glc H-1′′ (δH 4.88) and C-4′ (δC 157.2) confirmed the position of β-D-glucopyranosyl at C-4′. Based on the above data Vietnam Journal of Chemistry Dihydrostilbene glycosides and lignin © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 663 and ESI-MS result (m/z 391 [M-H]-, corresponding to the molecular formula of C20H24O8) the structure of compound 1 was determined as 3,5- dihydroxydihydrostilbene 4′-O-β-D- glucopyranoside. This compound was isolated from C. oleifera. [5] Table 1: The 1H- and 13C-NMR data for compounds 1-3 C 1 2 3 δCa) δHa) (mult., J in Hz) δCa) δHa) (mult., J in Hz) δCa) δHa) (mult., J in Hz) 1 145.3 - 145.3 - 145.6 - 2 108.1 6.16 (d, 2.0) 107.6 6.32 (d, 2.0) 109.3 6.42 (dd, 1.5, 2.0) 3 159.2 - 162.1 - 160.0 - 4 101.2 6.13 (d, 2.0) 98.9 6.30 (d, 2.0) 102.7 6.40 (dd, 2.0, 2.0) 5 159.2 - 162.1 - 159.2 - 6 108.1 6.16 (d, 2.0) 107.6 6.32 (d, 2.0) 110.8 6.32 (dd, 1.5, 2.0) α 39.1 2.71 (t, 7.0) 39.4 2.82 (m) 39.4 2.76 (m) α′ 37.8 2.81 (t, 7.0) 37.9 2.83 (m) 37.9 2.79 (m) 1′ 137.1 - 137.1 - 134.0 - 2′, 6′ 130.3 7.08 (d, 8.5) 130.4 7.11 (d, 8.5) 130.4 6.98 (d, 8.5) 3′, 5′ 117.6 7.01 (d, 8.5) 117.8 7.01 (d, 8.5) 116.0 6.69 (d, 8.5) 4′ 157.2 - 157.3 - 156.3 - 3,5-OMe 55.6 3.72 (s) Glc 1′′ 102.4 4.88 (d, 7.5) 102.6 4.83 (d, 7.5) 102.2 4.82 (d, 7.5) 2′′ 74.9 3.43 (dd, 9.0, 7.5) 74.9 3.47 (dd, 9.0, 7.5) 74.9 3.44 (dd, 8.5, 7.5) 3′′ 77.9 3.48 (t, 9.0) 78.0 3.48 (t, 9.0) 78.0 3.47 (t, 9.0) 4′′ 71.3 3.43 (t, 9.0) 71.5 3.37 (t, 9.0) 71.4 3.41 (t, 9.0) 5′′ 77.9 3.49 (m) 76.8 3.55 (m) 78.0 3.41 (m) 6′′ 62.5 3.73 (dd, 12.0, 5.0) 3.91 (dd, 12.0, 1.5) 67.9 3.63 (dd, 11.0, 6.0) 4.03 (dd, 11.0, 2.0) 62.5 3.73 (dd, 12.0, 5.0) 3.91 (dd, 12.0, 1.0) Rha 1′′′ 102.1 4.74 (d, 1.5) 2′′′ 72.1 3.88 (dd, 1.5, 3.0) 3′′′ 72.4 3.74 (dd, 3.0, 9.0) 4′′′ 74.0 3.39 (t, 9.0) 5′′′ 69.8 3.68 (m) 6′′′ 17.9 1.24 (d, 6.5) a)recorded in CD3OD; Glc, glucopyranosyl; Rha, rhamnopyranosyl. Compound 2 was isolated as a white amorphous powder. Similar to 1, the 1H-NMR spectra of 2 exhibited the signals of one dihydrostilbene, and two sugar moieties. The 13C-NMR and HSQC spectra of 2 showed the signals of 28 carbons, including 5 non- protonateds, 17 methines, 3 methylenes, 1 methyl, and 2 methoxy carbons. The analysis of 1H- and 13C- NMR data of 2 were found to be similar to those of 3,5-dimethoxydihydrostilbene 4′-O-α-L-rhamnopy- ranosyl-(1→6)-β-D-glucopyranoside.[5] The HMBC correlations between H-4 (δH 6.30) and C-2/C-6 (δC 107.6)/C-3/C-5 (δC 162.1), between methoxy group (δH 3.72) and C-3/C-5 (δC 162.1) confirmed the position of methoxy groups at C-3 and C-5. The HMBC correlations from H-2′/H-6′ (δH 7.11) to C-1′ (δC 137.1)/C-4′ (δC 157.3)/C-α′ (δC 37.9), from rha H-1′′′ (δH 4.74) to glc C-6″ (δC 67.9), and from glc H-1′′ (δH 4.83) to C-4′ (δC 157.3) indicated the sugar linkage as α-L-rhamnopyranosyl-(1→6)-β-D- glucopyranosyl and at C-4′. Furthermore, the ESI- MS of 2 exhibited an ion peak at m/z 565 [M-H]-, corresponding to the molecular formula of C28H38O12. Consequently, the structure of 2 was elucidated as 3,5-dimethoxydihydrostilbene 4′-O-α- L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside. This compound was isolated from C. oleifera.[5] Compound 3 also was isolated as a white amorphous powder. The 1H-NMR spectrum of 3 showed the signals of one dihydrostilbene and one sugar unit. The 13C-NMR and HSQC spectra of 3 showed the signals of 20 carbons, of which, 14 carbons assigned to one dihydrostilbene and 6 Vietnam Journal of Chemistry Phan Van Kiem et al. © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 664 carbons to one β-D-glucopyranosyl unit. The analysis of NMR and ESI-MS data of 3 suggested that structure of 3 was a dihydrostilbene glucoside, similar to 5,4′-dihydroxydihydrostilbene 3-O-β-D- glucopyranoside.[6] The position of hydroxy groups at C-5 and C-4′ were confirmed by HMBC correlation from H-6 (δH 6.32) to C-2 (δC 109.3)/C-4 (δC 102.7)/C-5 (δC 159.2), from H-2′/H-6′ (δH 6.98) to C-1′ (δC 134.0)/C-4′ (δC 156.3)/C-α′ (δC 37.9). 13C-NMR data of the sugar from C-1′′ to C-6′′ were at δC 102.2, 74.9, 78.0, 71.4, 78.0, and 62.5, respectively, together with the coupling constant between H-1′′ and H-2′′, J = 7.5 Hz suggesting a β- D-glucopyranoside. The HMBC correlation from glc H-1′′ (δH 4.82) to C-3 (δC 160.0) confirmed the position of β-D-glucopyranosyl at C-3. Thus, the structure of 3 was determined as 5,4′- dihydroxydihydrostilbene 3-O-β-D-glucopyranoside. Figure 2: The key HMBC correlations of compounds 1-4 The 1H-NMR spectrum of 4 showed the signals of two protons of a 1,3,4,5-tetrasubstituted aromatic ring at δH 6.43 (2H, s); one proton of a penta- substituted aromatic ring at δH 6.59 (1H, s); four methoxy groups at δH 3.34 (3H, s), 3.77 (6H, s), and 3.87 (3H, s); and one anomeric proton at δH 4.12 (1H, d, J = 7.5 Hz). The 13C-NMR and HSQC spectra of 4 showed the signals of 27 carbons, including 9 non-protonateds, 10 methines, 4 methylenes, and 4 methoxy carbons. The analysis of 1H- and 13C-NMR data of 4 were found to be similar to those of with those of nudiposide.[7] The location of glucose unit at C-3α was determined by the downfield chemical shift of C-3α (δC 71.2) as well as by HMBC correlation between xyl H-1′′ (δH 4.12) to C-3α (δC 71.2). From the above evidence, compound 4 was identified as nudiposide. 4. CONCLUSION Three dihydrostilbene glycosides, 3,5- dihydroxydihydrostilbene 4′-O-β-D-glucopyranoside (1), 3,5-dimethoxydihydrostilbene 4′-O-α-L- rhamnopyranosyl-(1→6)-β-D-glucopyranoside (2), 5,4′-dihydroxydihydrostilbene 3-O-β-D- glucopyranoside (3), and one lignan, nudiposide (4) were isolated from the methanol extract of leaves of Camellia sasanqua Thunb. Their chemical structures were determined by using ESI-MS and NMR spectra as well as by comparison with the reported data. Compounds 3 and 4 were reported from Camellia genus for the first time. Acknowledgment. This research is funded by Graduate University of Science and Technology under grant number GUST.STS.ĐT2018-HH01. REFERENCES 1. D. H. Bich, D. Q. Trung, B. X. Chuong, N. T. Dong, D. T. Dam, V. N. Lo, P. D. Mai, P. K. Man, D. T. Nhu, N. Tap, T. Toan, Medicinal plants and medicinal animals in Vietnam. Vol. 1. 2004, Hanoi: Science and Technics Publishing House, 419-422. 2. H. Matsuda, S. Nakamura, K. Fujimoto, R. Moriuchi, Y. 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Vu Kim Thu Hanoi University of Mining and Geology Pho Vien, Duc Thang, Bac Tu Liem district, Hanoi 10000, Viet Nam E-mail: vukimthu@humg.edu.vn.