bDepartment of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People's Armed Police Forces, Tianjin 300309, China;
cTaiJi Pharmaceutical Research Institute, Chongqing 400118, China;
dGuangdong Junyuan Pharmaceutical Co., Ltd., Guangzhou 525432, China
Aquilariaspecies (Thymelaeaceae),large evergreen trees native to Southeast Asia,are mainly cultivated in Guangdong and Guangxi provinces (China),and the Taiwan district .Aquilaria sinensis(Lour.) Gilg,is the major source of agarwood (Chen-Xiang in Chinese),one of the most highly valuable forest products currently traded internationally . Leaves ofA. sinensiscanbeconsumedonadailybasisasa healthy tea in southern China,and also be used traditionally for trauma-related illness such as fractures and bruising . Up to date, the chemical constituents of the aerial part ofA. sinensishave been identified as flavones [4, 5],xanthones ,benzophenones [5, 6], sesquiterpenes [7, 8, 9],and chromone derivatives [10, 11, 12].
In order to comprehensively utilize and develop the resources, we undertook a chemical investigation on the leaves of this tropical plant,leading to the discovery of five new benzophenone glycosides,namely aquilarinensides A-E (1-5) (Fig. 1). In the present study,we herein report the isolation and structure elucidation of those compounds.2. Experimental
Plant material: The leaves ofA. sinensiswere purchased from Dianbai Nanyao Medicine Co.,Ltd. (Guangdong province,China) on August 8,2011,and were identified by one of authors (P.-F. Tu). A voucher specimen (No. AS08232011) was deposited at the Herbarium of Peking University Modern Research Center for Traditional Chinese Medicines.
Extraction and isolation: The dried leaves (6.0 kg) ofA. sinensis were extracted with aqueous ethanol (70%,50 L×2h×2), followed by concentration in vacuum to obtain the ethanolic extract (700 g). The extracts (700 g) were suspended in water and extracted with chloroform and n-butanol,successively. The nBuOH extract (300 g) was chromatographed on silica gel (3000 g,19 mm×700 mm),stepwise eluting with CHCl3-MeOH-H2O (98:2:0.1→1:1:0.1,v/v/v) to yield five fractions (IAB1-IAB5). Fraction IAB3 (50 g) was subjected to Sephadex LH-20,eluting with MeOH to give four subfractions (IAB3D1-IAB3D4). The subfraction IAB3D1 (1.2 g) was then fractionated by ODS column (MeOH-H2O 10%→60%),semi-preparative HPLC (CH3CN-H2O 26:74),silica gel column (CHCl3-MeOH-H2O 85:15:1.5) successively to afford compounds 1 (2.0 mg). The subfraction IAB3D3 (2.3 g) was transferred to ODS column with gradient 10%-60% MeOH-H2O as the elution,then separated by semi-preparative HPLC with CH3CN-H2O (32:68),and further purified by silica gel column chromatography with CHCl3-MeOH-H2O 9:1:0.1 to give compounds 2(7.3 mg),and 3(5.2 mg). The subfraction IAB3D4 (2.1 g) was transferred to ODS column with gradient 10%-60% MeOH- H2O as the elution,then separated by semi-preparative HPLC with CH3CN-H2O (45:55) to give compounds 4(3.5 mg). Fraction IAB2 (10 g) was subjected to ODS column,eluting with aqueous MeOH (10%-80%) to give six subfractions (IAB2A-IAB2F). The subfraction IAB2F (0.9 g) was subjected to silica gel H column with CHCl3-MeOH-H2O 9:1:0.1 to give compounds5(2.6 mg).
Acid hydrolysis of 1-5: A solution of each compound (3.0 mg) in 1.0 mol/L CF3COOH (2.0 mL) was heated under reflux for 2 h. After cooling,the residue was centrifuged to obtain the supernatant. The supernatant was then partitioned with EtOAc (2 mL for three times) to yield the aqueous layer. The aqueous layers of the hydrolysates of 1-5 were evaporated to dryness to give the residues,which were dissolved in anhydrous pyridine (300mL). ThenL-cysteine methyl ester hydrochloride (3.0 mg) was added. The mixture was heated by thermostat oil bath at 60°C for 2 h. After reaction,0.5 mL HMDS-TMCS-pyridine (3:1:9) was added and heated by oil bath at 60°C for another 1.5 h. Afterwards, solvent was evaporated under reduced pressure. The product was dissolved byn-hexane and purified by extracting with water. The n-hexane layer was filtered and analyzed by GC. GC analysis was performed using an Agilent 6890N GC under the following conditions: HP-5 column (30 m×0.32 mm×0.25mm); column temperature: 230°C; carrier gas: He (1 mL/min); injector temperature: 260°C; ion source temperature: 280°C; detector: FID. The retention times of the persilylated monosaccharide derivatives were as follows:D-quinovose (11.8 min),D-fucose (11.3 min),D-xylose (9.2 min),and L-rhamnose (10.2 min), which were confirmed by comparison with those of authentic standards. 3. Results and discussion
Compounds 1-5 showed bands at 3541-3419,1650-1643,and 1608-1451 cm-1 in the FT-IR spectra (Fig. S3 in Supporting information),suggesting the presence of hydroxy group,carbonyl group,and aromatic group absorptions,respectively. Compound 1  had the molecular formula of C25H30O13 on the basis of the observed peak at m/z 537.1598 [M_H]-in HR-ESI-MS spectrum(Fig. S1 in Supporting information),suggesting the 11 degrees of unsaturation. In the 1H NMR spectrum (Fig. S4 in Supporting information),two groups of doublets at δH 7.62 (d,2H,J=8.7Hz)and 6.83 (d,2H,J= 8.7 Hz) indicated the existence of apara-substituted aromatic ring with two equivalent pairs ofortho-coupled protons. The signals at δH 6.27 (d,1H,J= 2.0 Hz) and 6.08 (d,1H,J=2.0Hz) displayed another tetrasubstituted benzene ring. Moreover,two anomeric protons at δH 5.24 (d,1H,J=1.6Hz) and 5.09 (d,1H, J= 1.5 Hz),together with two terminal methyl groups at δH 1.23 (d,3H,J= 6.5 Hz) and 1.22 (d,3H,J=5.5Hz) couldbeobserved, indicating the presence of two methylpentose moieties. The 13C NMR spectrum of 1 (Fig. S5 in Supporting information) presented 25 signals,including one carbony 1 carbon,two benzene rings and two rhamnopyranosyl units. All the above-mentioned information suggested that compound1was a derivative of iriflophenone containing two rhamnose moieties [5, 6],which was further confirmed by 1H-1HH COSY,HSQC,and HMBC spectra (Fig. 2). The Lconfiguration of the rhamnopyranosyl moieties was determined by comparison of the NMR data,and GC analysis after acidic hydrolysis and derivatization of 1.The α-configuration could be deduced based on the chemical shifts and coupling constants of their anomeric protons Rha H-1" (J=1.5Hz) and Rha H-1"' (J= 1.5 Hz). The linkage positions of the two sugar moieties were established as a-L-rhamnopyranosyl-(1→4)-a-L-rhamnopyranosyl based on the 13C NMR shift values and the HMBC correlation between Rha H-1"'and Rha C-4" . In addition,the key HMBC correlation of Rha H-1" with C-2 suggested the rhamnopyranosyl moiety was located at C-2 (Fig. 2). Therefore,the structure of1was determined to be iriflophenone-2-O-α-L-rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranoside and assigned the trivial name aquilarinenside A.
On the basis of HRESIMS spectra (Figs. S11 and S20 in Supporting information),aquilarinenside B (2)  and aquilarinenside C (3)  were determined to share the same molecular formula of C25H30O13 as that of compound1. Inspection of 1D NMR data of 2 and 3 revealed these two compounds closely resembled that of 1,with the only difference in the sugar residues. The outer rhamnopyranosyl in compound 1 was replaced by a fucopyranosyl in 2 and a quinovopyranosyl in 3, respectively,which was further confirmed by 2D NMR and acid analysis. By comparing with the known compound ,the J value of H-1"'(7.0 Hz) indicated the b-D-configuration of fucopyranosyl,while theJ value of H-1"'(7.8 Hz) indicated the b-D-configuration of quinovopyranosyl. The structures of aquilarinenside B (2) and aquilarinenside C (3) were thus determined to be iriflophenone 2-O-β-D-fucopyranosyl-(1→4)-O-α-L-rhamnopyranoside and iriflophenone 2-O-β-D-quinovopyranosyl-(1→4)-Oa-L-rhamnopyranoside,respectively.
Aquilarinenside D (4)  gave the molecular formula of C24H28O13 on the basis of the detected peak atm/z 523.1442 [M_H]-(calcd. for C24H27O13,523.1457) in HRESIMS spectrum (Fig. S30 in Supporting information). The 1H NMR and 13C NMR spectra of1and4displayed near-identical similarity for all signals, and the main difference observed was that outera-L-rhamnopyranoside unit was replaced by ab-D-xylopyranoside unit. It was confirmed by GC analysis of the hydrolyzed product and by the coupling constant of the anomeric proton Xyl H-1"'(d,J= 7.7 Hz). Therefore,aquilarinenside D (4) was determined as iriflophenone 2-O-β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranoside.
Aquilarinenside E (5)  showed the molecular formula of C21H22O10 by analysis of its HRESIMS spectrum (m/z 435.1247 [M+H]-,calcd. for C21H23O10: 435.1227) (Fig. S42 in Supporting information). Comparison of the NMR spectroscopic data (Table 1) revealed a similar structure to 1. Compound 5incorporated an acetyl group instead of the outer rhamnopyranosyl found in1, which was further confirmed by 2D NMR spectra. The key HMBC correlation between H-4" ((δH 4.79) and C-1"'(δC 172.7) indicated the acetyl group was located at C-4" position of the rhamnopyranosyl. Thus,aquilarinenside E (5) was determined as iriflophenone 2-O-α-L-(4"-acetyl)-rhamnopyranoside.
From the leaves ofA. sinensis,five new benzophenone glycosides(1-5) were isolated. Their structures were elucidated by a combination of 1D and 2D NMR,HRMS,and chemical analysis.Acknowledgment
This research work was supported by the Special Program for New Drug Innovation of the Ministry of Science and Technology, China (Nos. 2009ZX311-004,2009ZX0308-004).Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.07.013.
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|||Aquilarinenside A (1): flavescent crystalline powder (MeOH); [α]D25 -25.0 (c 0.08, MeOH); UV (MeOH) λmax (log ε): 291 nm (0.17) and 205 nm (0.53); IR nmax 3514, 2982, 2932, 1643, 1606, 1510, 1449, 1388, 1320, 1277, 1168, 1138, 1051, 919, 881, 839, 801, 702, 627, 580 cm-1; HR-ESI-MS m/z, 537.1598 [M-H]-(calcd. for C25H30O13, 537.1614).|
|||P.K. Agrawal, NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides, Phytochemistry 31 (1992) 3307-3330.|
|||Aquilarinenside B (2): flavescent crystalline powder (MeOH); [α]D25 -70.0 (c 0.08, MeOH); UV (MeOH) λmax (log ε): 291 nm (0.27) and 205 nm (0.81); IR nmax 3420, 2926, 1648, 1608, 1510, 1452, 1378, 1320, 1167, 1073, 784, 468 cm-1; HRESI-MS m/z, 537.1617 [M-H]-(calcd. for C25H30O13, 537.1614).|
|||Aquilarinenside C (3): flavescent crystalline powder (MeOH); [α]D25 -26.0 (c 0.08, MeOH); UV (MeOH) λmax (log ε): 290 nm (0.22) and 205 nm (0.61); IR νmax 3419, 2978, 2931, 1650, 1604, 1512, 1451, 1317, 1274, 1168, 1064, 1026, 926, 828, 766, 695, 581, 533cm-1; HR-ESI-MS m/z, 537.1632 [M-H]-(calcd. for C25H30O13, 537.1614).|
|||Aquilarinenside D (4): flavescent crystalline powder (MeOH); [α]D25 -30.0 (c 0.14, MeOH); UV (MeOH) λmax (log ε): 289 nm(0.44) and 205 nm (1.29); IR νmax 3419, 2978, 2933, 1605, 1511, 1453, 1319, 1275, 1167, 1072, 1044, 972, 925, 893, 819, 696, 652, 623, 584, 532 cm-1; HR-ESI-MS m/z, 523.1442 [M-H]-(calcd. for C24H27O13, 523.1457).|
|||Aquilarinenside E (5): flavescent crystalline powder (MeOH); [α]D25 -3.33 (c 0.30, MeOH); UV (MeOH) λmax (log ε): 289 nm (0.32) and 205 nm (0.96); IR νmax 3582, 3519, 3380, 2931, 1712, 1627, 1591,1555, 1514, 1450,1380, 1314, 1291, 1212, 1171, 1100, 1071, 1047, 922, 836, 805, 712, 596, 532 cm-1; HR-ESI-MS m/z, 435.1247 [M+H]+ (calcd. for C21H23O10, 435.1227).|