Flavonoids from the leaves of aralia hiepiana - Nguyen Thi Dieu Thuan

Vietnam Journal of Science and Technology 56 (4A) (2018)259-265 FLAVONOIDS FROM THE LEAVES OF ARALIA HIEPIANA Nguyen Thi Dieu Thuan 1,* , Nguyen Thi Thu Hien 1,2 , Tran Minh Hao 3 , Pham Van Huyen 1 , Nguyen Huu Toan Phan 1,2 1 Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology (VAST), 116 Xo Viet Nghe Tinh, DaLat 2 Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, HaNoi 3 Dalat University, 1 Phu Dong Thien Vuong, Da Lat * Email: ngtdthuan@gmail.com Received:23 July 2018; Accepted for publication: 30 September 2018 ABSTRACT Aralia hiepiana J.Wen & Lowry (Araliaceae), a new species from southern Vietnam, is described and illustrated by J. Wen & Lowry in 2002. Up to now, there are not any chemical data from this endemic species. By various chromatography methods, fiveflavonoids namely kaempferitrin (1), kaempferol 3-O-β-D-glucopyranosyl-7-O--L-rhamnopyranoside(2), kaempferol (3), quercetin (4),andapigetrin (5) were isolated from the methanol extract of leaves of Aralia hiepianacollected in Da Lat, Lam Dong province. Their structures were elucidated using 1-D and 2-D NMR techniques and by comparison with the literature data. This is the first time to isolate these compounds from Aralia hiepiana. Keywords:Aralia hiepiana,Araliaceae, kaempferitrin, kaempferol3-O-β-D-glucopyranosyl-7-O- -L-rhamnopyranoside, kaempferol, quercetin, apigetrin. 1. INTRODUCTION Aralia genus belonging to Araliaceae family, comprises of 74 species over the world [1] and15 species in Vietnam [2]. Several species have been used as in traditional herbal medicine for the treatment of gastric ulcer, hepatitis, rheumatic, arthritis, cancer and other diseases [2]. There have been many publications on the chemical compositions and biological activity of the Aralia species such as Aralia elata [3, 4], A. taibaiensis [5, 6], and A. armata [7]. Compounds mainly found in this genus were triterpenoid saponin and flavonoid glycosides [3-7]. Aralia hiepiana J.Wen & Lowry (Araliaceae) is widely distributed in the west highland of Vietnam. No phytochemical work, however, has been performed on this plant to date.This paper mainly describes the isolation andstructural identification of five flavonoids compounds, namely kaempferitrin (1), kaempferol 3-O-β-D-glucopyranosyl-7-O--L-rhamnopyranoside (2), kaempferol (3), quercetin (4), and apigetrin (5) from the methanol extract of the leaves of A.hiepiana. Nguyen Thi DieuThuan et al. 260 2. EXPERIMENT 2.1. General experimental procedures 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectrawere measured on a Bruker AM500 FT-NMR spectrometer. The Electrospray Ionization – Mass Spectroscopy (ESI-MS) spectra were obtained from an Agilent 1260 series single quadrupole LC/MS system. Column chromatography (CC) was carried out on silica gel (Si 60, 230-400 mesh, Merck) and RP-C18 column. All solvents were redistilled before use. Thin-layer chromatography was performed on silica gel 60 F254 sheets (Merck) and RP-18 F254S plates (Merck).Compounds were visualized under UV radiations (254, 365 nm) and by spraying plates with 10% H2SO4 followed by heating. 2.2. Plant material The leaves of Aralia hiepiana J.Wen & Lowry were collected in April 2017 at DaLat, Lam Dong province and were identified by Dr. Nong Van Duy from Tay Nguyen Institute for Scientific Research, VAST. A voucher specimen (No.TN3/129) is deposited at Tay Nguyen Institute for Scientific Research, VAST. 2.3. Extraction and isolation The powdered leaves of A.hiepiana (4.5 kg) were extracted with methanol at 40 o C three times at room temperature. The solvent was evaporated and concentrated under low pressure to give the crude extract (619.0g). The crude extract was dissolved in methanol:water (50:50, v/v) and then partitioned with hexane, chloroform, ethyl acetateto provide the corresponding extracts of: hexane (187.6g), CHCl3(17.6g), EtOAc (52.3g) and a water layer. The chloroform extract was fractionated by silica gel column chromatography using a mixture of chloroform-methanol (100:0 - 0:100, v/v)to yield twelve fractions (C1 - C12). Fraction C10 (177mg) was applied to a silica gel CC and eluted with ethylacetate-methanol (6:1, v/v) to give three sub-fractions (C10.1 - C10.3). The sub-fraction C10.2 (132mg) was chromatographed on RP-C18, eluted with methanol-water (1:4, v/v) to obtain compound 1. Fraction C11 was separated on an RP-18CC using the mobile phase of methanol-water (1:3 - 1:0, v/v) to obtain six sub-fractions (C11.1 - C11.6). Compound 2 was purified from sub-fraction C11.5 (39 mg) by silica gel CC eluting with chloroform-methanol (5:1, v/v). The ethyl acetate extract was separated on a silica gel CC, eluted with chloroform-methanol (100:0 - 0:100, v/v) to yield five fractions. Fraction E4 (23.6g) was chromatographed on a silica gel CC using a mixture of dichloromethane-acetone-methanol (10:1:1 - 5:1:1, v/v/v) to give twenty sub-fractions (E4.1 – E4.20). The sub-fraction E4.7 (1.7g) was subjected on a Sephadex LH-20 column using a gradient of methanol-water (1:3 – 1:0, v/v) to obtain sixsub-fractions (E4.7.1 – E4.7.6). The sub-fraction E4.7.6 (30 mg) was further purified by solid-phase extraction (SPE) to obtain compound 3. Fraction E5 (30.3g) was chromatographed over a silica gel CC, eluted with chloroform-methanol (100:0 - 0:100, v/v) to afford fourteenth sub-fractions (E5.1- E5.14). Compounds 4 and 5 were purified from sub-fraction E5.8 (596 mg) by SPE eluting with methanol-water (0:1-1:0, v/v). Kaempferitrin (1): yellow crystals; 1 H-NMR (500 MHz, CD3OD-d4) and 13 C-NMR (125 MHz, CD3OD-d4) see Table 1. ESI-MS (m/z): 579 [M+H] + (C27H30O14, M = 578). Flavonoids from the leaves of Aralia hiepiana 261 O R2 O OH R3 R1 OH Figure 1.Structures of compounds 1-5. Kaempferol 3-O-β-D-glucopyranosyl-7-O--L-rhamnopyranoside (2): yellow amorphous powder; 1 H-NMR (500 MHz, DMSO-d6) and 13 C-NMR (125 MHz, DMSO-d6), see Table 1; ESI-MS: m/z 617 [M+Na] + , 593 [M-H] - (C27H30O15, M = 594). Kaempferol (3): yellow powder; 1 H-NMR (500MHz, CD3OD-d4) H (ppm): 8.10 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.93 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.20 (1H, d, J = 2.0 Hz, H-6), 6.42 (1H, d, J = 2.0 Hz, H-8); 13 C-NMR (125 MHz, CD3OD-d4) see Table 2; ESI-MS m/z: 285 [M- H] - , 287 [M+H] + (C15H10O6, M = 286). Quercetin (4): yellow amorphous powder; 1 H-NMR (500 MHz, CD3OD-d4) H (ppm): 7.76 (1H, s, H-2'), 7.66 ( 2H, d, J = 8.5 Hz, H-6'), 6.91 (2H, d, J = 8.5 Hz, H-5'), 6.41 (1H, s, H- 8), 6.21 (1H, s, H-6); 13 C-NMR (125 MHz, CD3OD-d4) see table 2; ESI-MS m/z: 301 [M-H] - (C15H10O7, M = 302). Apigetrin (5): yellow amorphous powder; 1 H-NMR (500 MHz, DMSO-d6)7.96 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.96 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.45 (1H, d, J = 2.0 Hz, H-6), 6.84 (1H, d, J = 2.0 Hz, H-8), 6.86 (1H, s, H-3), 5.07 (1H, d, J = 7.5 Hz, H-1"), 3.49-3.17 (4H, H-2", H-3", H-4", H-5"), 3.72 (1H, m, H-6"), 3.48 (1H, m, H-6"); 13 C-NMR (125 MHz, DMSO-d6) see Table 2; ESI-MS m/z: 431 [M-H] - (C21H20O10, M = 432). 3. RESULTS AND DISCUSSION Compound 1 was obtained as yellow crystals. The 1 H- and 13 C-NMR spectra (Table 1)showed the aromatic proton signals characteristic for the 1,4-disubstituted ring B of a flavonoid glycoside, at H 7.81 (2H, d, J = 8.5 Hz, H-2', H-6') and 6.96 (2H, d, J = 8.5 Hz, H-3', H-5'). Two doublet signals observed at H 6.46 (1H, d, J = 2.0 Hz) and 6.75 (1H, d, J = 2.0 Hz) were assigned for protons H-6 and H-8, respectively. Compound 1 displayed two anomeric proton signals at H 5.42 (d, J = 2.0 Hz, rha-H-1") and 5.58 (d, J = 2.0 Hz, rha-H-1"') in the 1 H NMR spectrum, and also exhibited two anomeric carbon signals at C 103.53 (rha-C-1") and 99.88 (rha-C-1''') in the 13 C-NMR spectrum. The sugar linkages were determined based on HMBC spectrum. A long-range correlation was observed between a proton signal at H 5.42 (rha-H-1") and a carbon signal at C 136.50 (C-3) of the aglycone moiety, while the other anomeric proton signal at H 5.58 (rha-H-1''') showed a correlation with a carbon signal at (C 163.57 (C-7). These data and other NMR data thus allowed us to identify compound 1 as kaempferitrin (Figure 1)[8]. Kaempferitrin exerts immunostimulatory effects on immune responses mediated by splenocytes, macrophages, PBMC and NK cells [9]. (1) R1 = R2 = O-Rha, R3 = H (2) R1= O-Rha, R2 = O-Glu, R3 = H (3) R1 = R2 = OH, R3 = H (4) R1 = R2 = R3 = OH (5) R1 = O-Glc, R2 = R3 = H Nguyen Thi DieuThuan et al. 262 Compound 2 was isolated as a yellow amorphous powder. The 1 H- and 13 C-NMR spectra of 2 were very similar to those of 1 (Table 1), except for one sugar unit signals. The addition of a carbon signal of the sugar residue at δC 60.87 (C-6'')and the chemical shift value of the carbon signals at δC100.78 (C-1''), 74.22 (C-2''), 76.44 (C-3''), 69.93 (C-4''), and 77.55 (C-5'') showed the presence of a glucose at 2 instead of rhamnose at 1. This β-D-glucopyranosyl unit was determined to attach on carbon C-3 of the flavonoid ring system by the HMBC correlations of anomeric proton signal [δH5.47 (d, J = 7.0 Hz, H-1'')] to carbon C-3 (δC 133.48). From the spectroscopic data, the structure of compound 2 was determined as kaempferol 3-O-β-D- glucopyranosyl-7-O--L-rhamnopyranoside, isolated also from Brugmansiasuaveolens[10] (Figure 1). Table 1.The 1 H and 13 C-NMR spectroscopic data of compounds 1 and 2. 1 2 Position aC C H (J in Hz) bC C H (J in Hz) 2 159.90 159.82 156.84 156.79 3 136.60 136.50 133.52 133.48 4 179.90 179.80 177.68 177.64 5 163.10 163.04 160.91 160.88 6 100.56 100.56 6.46, d, 2.0 99.42 99.39 6.44, d, 2.0 7 163.60 163.57 161.63 161.59 8 95.70 95.61 6.75, d, 2.0 94.54 94.5 6.82, d, 2.0 9 158.20 158.11 156.03 155.99 10 107.60 107.58 105.71 105.68 1 122.50 122.42 120.75 120.70 2 132.00 132.00 7.81, d, 8.5 131.03 131 8.07, d, 9.0 3 116.60 116.59 6.96, d, 8.5 115.21 115.18 6.89, d, 9.0 4 161.80 161.78 160.23 160.24 5 116.60 116.59 6.96, d, 8.5 115.21 115.18 6.89, d, 9.0 6 132.00 132.00 7.81, d, 8.5 131.03 131 8.07, d, 9.0 1 103.53 103.53 5.42, d, 2.0 100.82 100.78 5.47, d, 7.0 2 71.92 71.92 4.24, dd, 2.0, 3.5 74.24 74.22 3.20, m 3 72.20 72.13 3.74, dd, 3.5, 9.0 76.46 76.44 3.22, m 4 73.20 73.19 3.38, m 69.94 69.93 3.08, m 5 72.00 72.00 3.38, m 77.57 77.55 3.08, m 6 17.70 17.66 1.28, d, 6.0 60.87 60.87 3.56, m; 3.32, m 1 99.90 99.88 5.58, d, 2.0 98.42 98.4 5.55, s 2 71.70 71.70 4.04, m 70.09 70.07 3.43, m 3 72.14 72.09 3.85, m 70.30 70.26 3.63, m 4 73.60 73.61 3.50, t, 9.5 71.66 71.62 3.30, m 5 71.30 71.30 3.63, m 69.84 69.82 3.84, m 6 18.10 18.07 0.96, d, 6.0 17.94 17.91 1.11, d, 6.0 aC of kaempferitrin (125 MHz. CD3OD-d4) [8] bC of kaempferol 3-O-β-D-glucopyranosyl-7-O--L-rhamnopyranoside (125 MHz.CD3OD-d4) [10]. Compound 3 was isolated as the yellow powder. The 1 H-NMR spectrum of compound 3 displayed pattern of flavonol, with characteristic signals of two doublet signals in ring A at δH 6.20 (1H, d, J = 2.0 Hz, H-6) and 6.42 (1H, d, J = 2.0 Hz, H-8), and signals of an AA'BB' spin system (ring B) at δH 8.10 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.93 (2H, d, J = 8.5 Hz, H-3', H-5'). The 13 C-NMR spectrum of compound 3 contained 15 carbon signals, including 8 quaternary Flavonoids from the leaves of Aralia hiepiana 263 aromatic carbons, a carbonyl group, and 6 methine aromatic carbons (Table 2). By comparison of the spectroscopic data with those in published literature [11], compound 3 was identified as kaempferol (Figure 1), which is commonly found in many plant species. Table 2.The 13 C-NMR data of compounds (3-5). 3 4 5 Position cC C dC C eC C 2 148.1 148.08 147.9 148.03 164.9 164.31 3 137.1 137.13 137.2 137.20 103.8 103.09 4 177.3 177.39 177.3 177.35 182.7 182.00 5 162.4 162.52 162.5 162.51 162.1 161.10 6 99.3 99.29 99.3 99.25 100.2 99.54 7 165.5 165.59 165.7 165.57 163.6 162.97 8 94.5 94.48 94.4 94.42 95.5 94.87 9 158.2 158.28 158.2 158.25 157.6 156.95 10 104.5 104.56 104.4 104.53 106.0 105.34 1 123.7 123.75 124.1 124.17 121.7 120.99 2 130.7 130.69 116.0 116.02 129.3 128.61 3 116.3 116.32 146.2 146.23 116.7 116.03 4 160.5 160.56 148.7 148.03 161.7 161.42 5 116.3 116.32 116.2 116.24 116.7 116.03 6 130.7 130.69 121.6 121.69 129.3 128.61 1 100.6 99.93 2 73.8 73.11 3 77.8 77.17 4 70.2 69.58 5 77.1 76.44 6 61.3 60.61 cC of kaempferol (125 MHz, CD3OD-d4) [11], dC of quercetin (75 MHz, CD3OD-d4) [12], eC of apigetrin (100 MHz, DMSO-d6) [13]. Nguyen Thi DieuThuan et al. 264 Compound 4 was obtained as a yellow amorphous powder. The 1 H and 13 C NMR spectral data of 4 were similar to those of 3 (Table 2), except for the appearance of an ABX system at δH 7.76 (1H, br s, H-2'), 7.66 (1H, d, J = 8.5 Hz, H-6'), and 6.91 (1H, d, J = 8.5 Hz, H-5') in 4 (Figure 1). Based on the NMR data and comparison of the data given in theliterature, compound 4 was determined as quercetin (Figure 1) [12]. Compound 5 was isolated as a yellow amorphous powder. The 1 H NMR spectra displayed pattern of flavone glycoside, with characteristic signals of two doublet signals in ring A at δH 6.45 (1H, d, J = 2.0 Hz, H-6) and 6.84 (1H, d, J = 2.0 Hz, H-8), signals of 1,4-disubstituted ring B of a flavonoid at δH 7.96 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.96 (2H, d, J = 8.5 Hz, H-3', H-5'), and a singlet signal at δH 6.86 (H-3). Compound 5 also displayed an anomeric proton signal at δH 5.07 (d, J = 7.5 Hz, glc-H-1") in the 1 H-NMR spectrum, and exhibited an anomeric carbon signal at δC 99.93 (glc-C-1") in the 13 C-NMR spectrum. This β-D-glucopyranosyl residue was determined to attach on carbon C-7 of the flavonoid ring system by the HMBC correlations of anomeric proton signal [δH 5.47 (H-1'')] to carbon C-7 (δC 162.97). Based on the NMR data and comparison of the data given in the literature, the structure of compound 5 was identifed as apigetrin (Figure 1) [13]. This compound has antioxidant properties as well as immunomodulating effects upon splenocytes, NK and CTL cells, and macrophages [14]. 4. CONCLUSION From the methanol extract of leaves of Aralia hiepina, five flavonoids namely kaempferitrin (1), kaempferol 3-O-β-D-glucopyranosyl-7-O--L-rhamnopyranoside (2), kaempferol (3), quercetin (4), andapigetrin (5) were isolated. Their structures were elucidated by 1D- and 2D-NMR spectral data as well as comparison with those reports. This is the first report on the chemical constituents of this plant. REFERENCES 1. on 1st July 2018). 2. Moi L. D., Minh C. V., Sung T. V., Long P. Q., Kiem P. V., Thai T. 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