Isolation and structure elucidation of three flavonoids from the seed of ziziphus mauritiana lam

Journal of Science and Technology 54 (2C) (2016) 409-416 ISOLATION AND STRUCTURE ELUCIDATION OF THREE FLAVONOIDS FROM THE SEED OF ZIZIPHUS MAURITIANA LAM Vu Thi Thanh Loan1, Le Huyen Tram2, Tran Thuong Quang2, * 1Phu Tho Medical College, Truong Thinh, Phu Tho, Vietnam 2Hanoi University of Science and Technology, 01 Đại Cồ Việt, Hanoi, Vietnam *Email: quang.tranthuong@hust.edu.vn Received: 15 June 2016; Accepted for publication: 28 October 2016 ABSTRACT Three flavonoids, spinosin (1), 6′′′-feruloyl spinosin (2) and 6′′′-sinapoyl spinosin (3) were isolated from EtOH extract of seed of Ziziphus mauritiana. Their structures were elucidated by the analysis of their spectroscopic data (ESI-MS,1H-NMR,13C-NMR, DEPT, HSQC và HMBC) and compared with literatures. These compounds were isolated for the first time in Vietnam from the seed of Ziziphus mauritiana Keywords: Ziziphus mauritiana, spinosin, 6′′′- feruloyl spinosin and 6′′′-sinapoyl spinosin. 1. INTRODUCTION The genus Ziziphus (Rhamnaceae), comprises of approximately 170 species and 12 variants, and is distributed in the warm-temperate and subtropical regions throughout the world. Ziziphus mauritiana Lam., an evergreen shrub up to 15 m height, is distributed in Vietnam. It is also widely growing throughout low-latitudes of Asia, Africa and Australia. In the Vietnamese Pharmacopoeia, the dry seeds of Z. mauritiana Lam have been used traditionally to tranquilize and relax the mind, soothing nerves (anxiolytic), and reducing sweating (anti-hydronic) effect [1]. Alkaloids [2, 6], flavonoids [2 - 6], triterpenoids [6] have been identified from the seeds of Z. mauritiana on the world. However, no chemical study was reported on the seeds of Z. mauritiana in Vietnam. The phytochemical investigation on the seeds of Z. mauritiana was carried on. This led to the isolation of Spinosin (1), 6′′′- feruloyl spinosin (2) và 6′′′-sinapoyl spinosin (3). 2. MATERIALS AND METHODS 2.1. General Experimental Procedures 1D and 2D NMR spectra were performed on Bruker AM 500 FT-NMR spectrometers with TMS as an internal standard. ESI-MS spectra were recorded on AGILENT 1200 series LC-MSD Ion Trap. TLC was performed on precoated TLC plates (DC-Alufolien 60 F254 and RP18 F254 Vu Thi Thanh Loan, Le Huyen Tram, Tran Thuong Quang 410 Merck) with compounds visualized by spraying the dried plates with 10 % aqueous H2SO4 followed by heating until the plate was dry or UV lamp (245 nm). Silica gel (240-430 mesh, Merck), reversed silica gel (ODS-60-14/63, Fujisilisa – Japan), diaion HP20SS (Supelco-USA) and Sephadex LH20 (Sigma) were used for column chromatography (CC). 2.2. Plant Material The seeds of Z. mauritiana were collected from Phu Tho province and identified by Prof. Tran Huy Thai (The Institute of Ecology and Biological Resources). A voucher sample has been deposited in Hanoi University of Science and Technology. 2.3. Extraction and Isolation The air-dried seeds (2 kg) of Z. mauritiana were powdered, and extracted three times per 3 hours with EtOH at 40 °C. The extracts (312 g) was suspended in H2O and degreased by n- hexan and ethyl acetate. The H2O-soluble layer was applied to a Diaion HP20SS column, eluting with H2O/MeOH (1:0→0:1), to give fractions W1–W5. Compound 1 (53 mg) was obtained by crystallization from fraction W2. Fraction W3 was subjected to Sephadex LH-20 chromatography column, eluting with MeOH to derive the subfractions W3a, W3b và W3c. Subfraction W3b was subjected to silica gel (EtOAc–MeOH, 1:0 → 0:1) to give W3b1 and W3b2. Subfraction W3b1 was further separated by reversed silica gel ODS-60 (MeOH) to provide compounds 2 (17 mg). Subfraction W3b2 was further separated by reversed silica gel ODS-60 (MeOH) to provide compounds 3 (12 mg). Spinosin (1): A yellowish crystal with a molecular formula C28H32O15. ESI-MS m/z 609,01 [M+H]+. For 1H NMR (500 MHz DMSO-d6, room temperature) and 13C NMR (125 MHz, DMSO-d6) spectral data see Table 1. 6′′′-feruloyl spinosin (2): A yellowish amorphous powder with a molecular formula C38H40O18. ESI-MS m/z 785,01 [M+H]+. For 1H NMR (500 MHz DMSO-d6, room temperature) and 13C NMR (125 MHz, DMSO-d6) spectral data see Table 2. 6′′′-sinapoyl spinosin (3): A yellowish amorphous powder with a molecular formula C39H42O19. ESI-MS m/z 837.10 [M+Na]+. For 1H NMR (500 MHz DMSO-d6, room temperature) and 13C NMR (125 MHz, DMSO-d6) spectral data see Table 2. 3. RESULT AND DISCUSSION Compound 1 was isolated as a yellowish crystal, Its molecular formula was deduced to be C28H32O15 on the basis of ESI-MS (m/z 609.01 [M+H]+). Its 1H and 13C-NMR spectra showed signals for a flavonoid glycoside [2]. In 1H-NMR spectrum, compound 1 exhibited a singlet at δH 13.62, 13.5 (1H, s, 5-OH) which was ascribed to 5-OH, substituted at carbon C-5 at δC 160.55/159.68. The 1H NMR spectrum of 1 indicated six aromatic proton signals due to aglycone moiety, i.e., a characteristic singlet signal at δ 6.83/ 6.85 due to the H-3 proton, a singlet at δ 6.77/6.80 (1H, s, H-8) indicating A-ring with three substituents, an AA′BB′ aromatic proton system appearing at δ 7.98/7.96 (1H, d, J = 8.5 Hz, H-2’, 6′) and 6.95/6.93 (1H, d , J = 8.5 Hz, H-3′, 5′) indicating a C-4′ substituted B-ring, and a singlet at δ 3,89 (3H, s, 7-OCH3) due to methoxyl protons. In 13C- NMR spectrum, the carbon signal at δC 182.30/181.99 was assigned to the carbonyl carbon C-4. Isolation and structure eluciddation of tree flavonoids from the seed of ziziphus mauritiana Lam 411 The signals of two anomeric protons at δ 4.70/4.68 (1H, d, J = 9.5 Hz, H-1′′) and 4.16 (1H, d, J = 7.8 Hz, H-1′′′) and 10 protons at δ 2.55-4.70 (4.70/4.68 (1H, d, J = 9.5, H-1′′), tại δ 4.47/4.29 (1H, t, J = 9.0, H-2′′) và tại các δ 3.44/4.43 (H-3′′), 3.16 (H-4′′), 3.18 (H-5′′) 3.70/3.68 (2H, H-6′′), 4.16 (1H, t, J = 9.0, H-1′′′), 2.86 (1H, m, H-3′′′), 2.98 (1H, H-4′′′), 2.55/2.75 (1H, m, H-5′′′), δ 3.16/2.98 (2H, b, H-6′′′)) together with the 13C NMR spectral data indicated the presence of two β-D-glucosyl moieties. The resonances for the carbons and protons of the aglycone and the sugar moiety had a close resemblance to those of spinosin, and they were assigned according to the 1H and 13C NMR spectral data for spinosin as well as its own HSQC spectrum. The linkage between the anomeric C-1′′ and C-6 and the interglucosidic linkage between C-2′′ and C-1′′′ were determined by the HMBC correlations of H-1′′ (δ 4.47/4.29)/ C-7 (δ 165.08/163.87) and C-5 (δ 160.55/159.68) and H-1′′′(δ 4.16)/C-2′′(δ 81.23/80.76) (figure 1 (1)). The chemical shift value of the anomeric carbon C-1′′ and HMBC correlation between H-1′′ (δ 4.47/4.29) and C-6 (δ 108.53/108.58) indicated that the anomeric carbon (C-1′′, δ 71.05/70.72) of β-glucose was connected to C-6 through a C-linkage. Table 1. 1H-NMR and 13C-NMR spectra of spinosin. Pos. δH (J, Hz) δH (J, Hz)* δC δC* 2 163.81/163.79, C 163,66/163,66 3 6.83/6.85(s) 6.83/6.84 103.10/103.00, CH 102.90/102.99 4 182.30/181.99, C 181.82/182.15 5 160.55/159.68, C 159.57/160.42 6 108.53/108.58, C 108.58/108.58 7 165.08/163.87, C 163.72/164.94 8 6.77/6.80(s) 6.67/ 6.80 90.78/90.31, CH 90.20/ 90.67 9 157.09/156.97, C 156.85/156.96 10 104.44/104.19, C 104.09/ 04.36 1′ 121.08/121.04, C 120.92/120.92 2′ 6′ 7.98/7.96 (d, 8.5) 7.97 128.55, C 128.34/128.34 3′ 5′ 6.95/6.93 (d, 8,5) 6.95 116.00, CH 115.59/115.89 4′ 161.28, C 160.78/161.22 7-OMe 3.89 (s) 3.92 56.54/56.12, CH3 55.99/ 56.42 1′′ 4.70/4.68 4.67/4.69 71.05/70.72, CH 70.64/ 70.99 2′′ 4.47/4.29 81.23/80.76, CH 80.58 /81.06 3′′ 3.44/3.43 78.68/78.29, CH 78.19/ 78.55 4′′ 3.16 70.46, CH 70.41 /70.41 Vu Thi Thanh Loan, Le Huyen Tram, Tran Thuong Quang 412 * Reference spectral data Thus, the spectral data of compound 1 were similar to Spinosin, previously reported in the literature [4]. The similar NMR phenomenon to that of spinosin, i.e. the appearance of serial separate signals, was observed. It is proposed that rotational isomers produced by the rotational barriers 7-OCH3 in flavones-6-C-glycoside must exist in compound 1 [4]. Figure 1. Structure and key HMBC correlations of compounds 1-3. Compound 2 was isolated as a yellowish amorphous powder. The molecular formula was determined to be C38H40O18 from the [M+H]+ ion peak at m/z 785,01 in the ESI-MS. The resonances for the carbons and protons located on the flavonoid framework had a close resemblance to those of the spinosin based on the 1H and 13C NMR spectral data as well as its own HSQC spectrum. The downfield shift of C-6′′′ (δ 62.18 + 2.1 ppm) and the upfield shift of C-5′′′ (δ73.45, - 2.8 ppm) relative to the corresponding signals of spinosin revealed the acylation of C-6′′′, which is also supported by the corresponding signals of 6′′′- feruloyl spinosin. The spinosin moiety accounted for a partial molecular formula of C28H31O15. Thus, the remaining molecular formula should be C10H9O3 as a substituent attached to the spinosin group at C-6′′′. The resonances for the carbons and protons of the substituent in the 1H and 13C NMR as well as HSQC spectra included an ABX aromatic proton system appearing at δ 6.78 (1H, d, J = 8.5, H-5′′′′), δ 6.93/6.79 (1H, dd, J = 8,0, 2,0 Hz, H-6′′′′), δ 7.17/7.04 (1H, d, J = 2,0, H-2′′′′), a 5′′ 3.18 81.91/81.62, CH 81.42 /81.67 6′′ 3.70/3.68(m) 61.48, CH2 61.40 1′′′ 4.16 4.15/4.17 105.43/105.25, CH 105.08/105.22 2′′′ 2.83/2.85 74.73/74.56, CH 74.48 /74.62 3′′′ 2.86 (m) 76.38/76.34, CH 76.25/76.25 4′′′ 2.98 (m) 69.47/69.18, CH 69.23/69.52 5′′′ 2.55/2.75 (m) 76.66/76.44, CH 76.25 /76.25 6′′′ 3.16/2.98 60.08/60.62, CH2 60.10 /60.59 5-OH 13,62/13,5 Isolation and structure eluciddation of tree flavonoids from the seed of ziziphus mauritiana Lam 413 methoxyl (δ 3.80 (3H, s, 3-OMe; δC 55.71/55.66), and a carbonyl (δC 166.42/166.32 (C-9′′′′)). The geometry of the trans C-7′′′′– C-8′′′′ double bond was confirmed on the basis of large coupling constant 15.5 Hz (7.22/7.80, d, J = 15.5, H-7′′′′ and 6.24/6.17, d, J = 15.5, H-8′′′′). The HMBC correlation between H-6′′′′/C-7′′′, H-2′′′′/C-7′′′′ as well as the proton of methoxyl and C- 3′′′′ indicated the presence of a feruloyl moiety (figure. 1 (2)). Thus, the spectral data of compound 2 were similar to 6′′′-feruloylspinosin, previously reported in the literature [3, 4]. Table 2. 1H-NMR and 13C-NMR spectra of compounds 2 and 3. Pos. 2 3 δH (J, Hz) δC δH (J, Hz) δC 2 163.96/163.68 6.69/6.54(s) 164.08/163.6 3 6.85/6.71(s) 103.24 103.91 4 182.33/181.88 181.75 5 160.84/159.53 159.433 6 108.77 108.67 7 165.27/164.21 165.10 8 90.68/89.98 90.45/89.86 9 157.10/156.93 156.97 10 104.51/104.03 103.91 1′ 121.24 7.84/7.82(d,8.5) 121.16 2′ 6′ 7.82/7.80(d,7.5) 128.64/128.53 7.84/7.82(d, 8.5) 128.51/128/ 41 3′ 5′ 6.90/6.88(d,9) 116.09/115.88 6.91/6.86(d,8.5) 115.75 4′ 161.23 161.42 7-OMe 3.90/3.87(s) 56.49/56.03 3.91/3.86(s) 56.36/56.07 1′′ 4.69/4.68(d, 9.5) 71.06 4.70/4.69(d, 9.5) 70.59 2′′ 4.48/4.26(m) 82.00/81.69 4.48/4.25(m) 81.937/81.63 3′′ 78.85/78.64 3.45(m) 78.78/78.53 4′′ 3.16(m) 70.31 3.17(m) 70.27 5′′ 3.16(m) 82.00 3.17(m) 81.63 6′′ 3.74(m) 61.54 3.82(m) 61.43 1′′′ 4.28(d, 8) 105.81 4.29(d,8) 105.03 Vu Thi Thanh Loan, Le Huyen Tram, Tran Thuong Quang 414 2′′′ 2.93(m) 74.48 2.89(m) 74.42/74.36 3′′′ 3.12(m) 76.33 3.11(m) 76.42 4′′′ 3.08(m) 68.99 3.09(m) 68.57 5′′′ 2.95(m) 73.45 2.95(m) 73.31 6′′′ 3.83/3.65(m) 62.18 4.05(m) 62.01 1′′′′ 125.59/125.42 128.21 2′′′′ 7.17/7.04(d,2) 110.89/110.78 6.86/6.69(s) 110.79 3′′′′ 147.97/147.92 149.19 4′′′′ 149.29 140.45 5′′′′ 6.78(d, 8.5) 115.83/115.42 149.19 6′′′′ 6.93/6.79(dd, 8, 2) 123.19/123.02 6.86/6.69(s) 110.79 7′′′′ 7.22/7.80(d, 15.5) 144.85/144.77 7.24/7.10(d,16) 147.85 8′′′′ 6.24/6.17(d,15.5) 114.10/113.66 6.27/6.19(d,15.5) 113.59 9′′′′ 166.42/166.31 166.27 3′′′′-Ome 3.80(s) 55.71/55.66 3.81/3.79(s) 55.59/55.54 5′′′′-Ome 3.81/3.79(s) 55.59/55.54 5- OH 13.62/13.50 Compound 3 was isolated as a yellowish amorphous powder. The molecular formula was determined to be C39H42O18 from the [M+Na]+ ion peak at m/z 837,10 in the ESI-MS. All the protons and carbons of 3 appeared as pair signals in the 1H and 13C NMR spectra, which is characteristic of signals arising from a spinosin skeleton. In addition, a set of signals arising from one carbonyl (δC 166.27, C-9''''), two methoxyl [δC 55.59/55.54 (OCH3 x 2)), four olefinic methines [δC 110,79/110,68 (CHx2), 147,85(CH), 113,59(CH)], and four aromatic quaternary carbons [δC 128,21(C), 149.19(CHx2), 140,45(C)] were observed in the 13C NMR (DEPT) spectra. Figure 2. Key HMBC correlations of sinapoyl moiety. O O H3CO HO OCH3 OHO HO OH 6'''9 '''' 7''''2'''' 6'''' 3'''' 5'''' Isolation and structure eluciddation of tree flavonoids from the seed of ziziphus mauritiana Lam 415 The 1H NMR spectrum displayed the existence of a symmetric 1,3,4,5-tetra-substituted aromatic ring [δH 6.86/6.69 (2H, s)], one trans double bond [δH 7.24/7.10, 6.27/6.19 (each 1H, d, J = 15.5 Hz, H-7'''', H-8'''')] and two methoxyl groups [δH 3.81/3.79 (6H, s, 3'''', 5''''-OMe) and didn’t displayed the proton of 4′′′′. The aforementioned data suggested that 3 was a spinosin derivative acylated with a 3,5-dimethoxyl,4-hydroxyl-(E) cinamoyl (sinapoyl moiety). In the HMBC spectrum of 3, correlations between the H-6''' (δH 4.05/3.57) and C-9'''' (δC 166.27) indicated the linkage of the sinapoyl moiety with C-6''' (δC 62.01) of spinosin unit (figure 2, figure 1 (3)). Thus, the spectral data of compound 3 were similar to 6′′′-sinapoyl spinosin, compared with literature [6]. 4. CONCLUSION Three flavonoids, spinosin (1), 6’’’- feruloyl spinosin (2) and 6’’’-sinapoyl spinosin (3) were isolated from EtOH extract of seed of Ziziphus mauritiana. Their structures were elucidated by the analysis of their spectroscopic data (ESI-MS,1H-NMR,13C-NMR, DEPT, HSQC and HMBC) and compared with literatures. The 1H and 13C NMR spectra of 1, 2 and 3 showed the same phenomenon of separate signals because of rotational isomers which are produced by the rotational barriers 7-OCH3 in flavone-6-C-glycoside. It was proved that the multiplicity collapsed to a first-order spectrum when measuring at high temperature. Thus, structural elucidation of this type of compounds will be easy when measuring all spectra at high temperature. These compounds were isolated for the first time in Vietnam from the seed of Ziziphus mauritiana Acknowledgment. This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2014.47. REFERENCES 1. Đỗ Tất Lợi - The medicinal plants and herbs in Vietnam, Medical Publishing, Hanoi, 2004, p. 446-447. 2. Lin Z., Zhi L. X, Chun F. W., Jing Y. Y., Yoshihiro K. & Dan Y. - Two new flavonoid glycosides from Semen Ziziphi Spinosae, Journal of Asian Natural Products Research 14 (2012) 121–128. 3. Yuan Y. X., Zhi L. X., Hui W., Yoshihiro K. & Dan Y. - A novel spinosin derivative from Semen Ziziphi Spinosae, Journal of Asian Natural Products Research 13 (2011) 1151– 1157. 4. Gong C., Yanjing B., Yuying Z., Jing T., Yi L., Guangzhong T., Libin M., Ning L., Xiaojie X. - Flavonoids from Ziziphus jujuba Mill var. spinose, Tetrahedron 56 (2000) 8915-8920. 5. Yi W., Feng H., Qin P., Yao S., Zhida M., and Jingyu L. - C-glucosyl flavones from the seeds of Ziziphus jujuba var. spinosae, Chemistry of Natural Compounds 47 (3) (2011) 369-372. 6. Bin W., Hong T. Zhu, Dong W., Chong R. Y., Min X., and Ying J. Z. - New spinosin derivatives from the seeds of Ziziphus mauritiana, Nat. Prod. Bioprospect. 3 (3) (2013) 93–98. Vu Thi Thanh Loan, Le Huyen Tram, Tran Thuong Quang 416 TÓM TẮT PHÂN LẬP VÀ XÁC ĐỊNH CẤU TRÚC BA HỢP CHẤT FLAVONOID TỪ HẠT TÁO TA Vũ Thị Thanh Loan1, Lê Huyền Trâm2, Trần Thượng Quảng2, * 1Trường Cao đẳng Y tế Phú Thọ, Khu 5 Trường Thịnh TX. Phú Thọ - Phú Thọ 2Viện Kỹ thuật Hoá học, Trường ĐH Bách Khoa Hà Nội, 01 Đại Cồ Việt Hà Nội *Email: quang.tranthuong@hust.edu.vn Ba hợp chất spinosin (1), 6′′′- feruloyl spinosin (2) và 6′′′- sinapoyl spinosin (3) đã được phân lập từ cao chiết phân đoạn nước của nhân hạt táo ta Ziziphus mauritiana Lam. Cấu trúc của chúng được xác định dựa trên số liệu phổ khối phun mù điện tử ESI-MS và phổ cộng hưởng từ hạt nhân NMR (1H, 13C, DEPT, HSQC và HMBC) và so sánh với các tài liệu tham khảo. Các hợp chất này được phân lập lần đầu tiên ở Việt Nam từ hạt táo ta Ziziphus mauritiana Lam. Từ khóa: táo ta, Ziziphus mauritiana, spinosin, 6′′′- feruloyl spinosin và 6′′′-sinapoyl spinosin.