Benzophenone glycosides from the pericarps of Aquilaria yunnanensis S. C. Huang

Abstract Two new benzophenone glycosides, aquilarisides A (1) and B (2), together with six known analogues (3-8) were isolated from the pericarps of Aquilaria yunnanensis S. C. Huang. Their structures were elucidated on the basis of 1D and 2D NMR and mass spectroscopic analyses, and the absolute configuration of compound 1 was determined by experimental and calculated electronic circular dichroism (ECD) spectra. Anti-inflammatory activities of all compounds 1–8 were evaluated for their inhibitory activities against lipopolysaccharide (LPS)-stimulated induced nitric oxide (NO) production in RAW 264.7 cells using the Griess assay. Compound 2 indicated a weak inhibition of NO production. Graphical Abstract


Introduction
The genus Aquilaria (Thymelaeaceae) comprises 21 species all over the world (Hashim et al. 2016), mainly distributed in East and Southeast Asia (Pripdeevech et al. 2011). Two species Aquilaria sinensis (Lour.) Gilg and Aquilaria yunnanensis S. C. Huang are native to China. The resinous woods of Aquilaria species, namely agarwood, is known in folk medicine to possess various functions such as sedative, analgesic, digestive, carminative and antiemetic effects (He et al. 2012;Liu et al. 2017;Zhao et al. 2016). The wild Aquilaria species are in peril of extinction because of indiscriminate cutting in search of the resin, and they have been listed in Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITIES) since 2004 (Hashim et al. 2016). In addition to the resinous wood, the pericarps of Aquilaria species have been used in traditional Chinese medicine as antitumor, and antibacterial activities (Zhang et al. 2013). In a continuing search for anti-inflammatory agents from Aquilaria plants (Huo et al. 2017), the EtOAc-soluble fraction from a 95% EtOH extract of the pericarps of A. yunnanensis shows inhibition of NO production in LPS-stimulated RAW 264.7 cells. Subsequent isolation of the bioactive fractions afforded two new benzophenone glycosides, aquilariside A (1) and B (2), together with six known analogues (3-8) ( Figure 1). Herein, we describe the isolation and structural elucidation of the new compounds (1-2) as well as an evaluation of their anti-inflammatory activities based on the inhibition of NO production in LPS-stimulated RAW 264.7 cells.  Figure S1), and 13 C NMR spectroscopic data, indicated 12 degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3377 cm À1 ), carbonyl (1667 cm À1 ), phenyl (1617 cm À1 ) groups ( Figure S2  methine carbons (d C 107.9, 103.2, 90.1), one sp 3 oxygenated tertiary carbon (d C 122.8), three sp 3 oxygenated methine carbons (d C 86.2, 82.7, 78.7), one sp 3 oxygenated methylene carbon (d C 62.8) and one sp 3 methylene carbon (d C 32.3). Analysis of 1 H and 13 C NMR spectroscopic data of 1 (Table S1) showed a close structural resemblance to aquilarinoside A (Qi et al. 2009). The major differences in the structure of compound 1 and aquilarinoside A is the presence of an additional hydroxy group at C-7, as well as the replacement of the hydroxyl and sp 2 methine at C-4a and C-5a with an sp 2 oxygenated tertiary carbon in 1 to form a xanthone. The assignment was supported by the deshielded C-7 resonance (d C 154.0; D d C þ 39.3) and the shielded C-6 (d C 145.9; D d C -15.7), C-8 (d C 107.9; D d C -24.0) resonances. Taking the indices of hydrogen deficiency into account, the structure of 1 required the presence of an additional ring. The HMBC correlations from H-5 to C-6/C-7/C-8a, and H-8 to C-5a/C-7/C-9 ( Figure S7) indicated the linkage of two benzene rings via (4a,O,5a)-ether bond forming a xanthone skeleton. The relative configuration of 1 was established on the basis for the NOESY spectrum ( Figure S8), which showed NOE correlations of H-5 0 /H-3 0 , H-4 0 /H 2 -6 0 , and H-3 0 / H-1 0 a. The absolute configuration of 1 was established by the experimental and quantum chemical calculated electronic circular dichroism (ECD) spectra. The ECD spectra of (2 0 R,3 0 R,4 0 R,5 0 S)-1 ( Figure S9) was calculated using time-dependent density functional theory (TDDFT) at the B3LYP/6-31G level with the CPCM model in MeOH. The experimental and calculated ECD spectra showed good agreement. Thus, the structure and the absolute configuration of aquilariside A (1) was defined as shown.

Results and discussion
The molecular formula of compound 2 was determined as C 26 H 30 O 13 by HRESIMS (m/z 551.1786 [M þ H] þ , calcd for C 26 H 31 O 13 , 551.1759) (Figure S10), and 13 C NMR spectroscopic data, indicated 12 indices of hydrogen deficiency. The IR spectrum showed for the presence of hydroxy (3394 cm À1 ), ester carbonyl (1724 cm À1 ), and benzyl (1604, 1513 cm À1 ) groups ( Figure S11). The 1 H NMR spectrum ( Figure S12) Figure S13). These signals are due to one keto carbonyl carbon (d C 197.6), two ester carbonyl carbons (d C 173.2, 172.2), four sp 2 oxygenated tertiary carbons (d C 163.4, 163.0, 160.5, 157.9), two sp 2 quaternary carbons (d C 132.9, 109.6), six sp 2 methine carbons (d C 132.7 Â 2, 116.2 Â 2, 98.1, 95.1), one sp 3 oxygenated tertiary carbon (d C 70.9), two sp 3 methylene carbons (d C 46.3, 45.9), one sp 3 methyl carbon (d C 28.0) and one methoxy carbon (d C 52.0). The remaining six signals in the 13 C NMR spectrum were assigned to a rhamnopyranosyl moiety. The NMR data of 2 (Table S1) were similar to those of compound 3 which was previously isolated from A. sinensis (Yuan et al. 2017), except for the presence of resonances for a methoxy group (d H 3.67 and d C 52.0). On acid hydrolysis, it afforded L-rhamnose, which was detected by TLC and HPLC analyses. The linkage of methoxy group in 2 was established at C-5 000 by an HMBC experiment ( Figure S14), which showed crosspeaks between the signals at d H 3.67 (methoxy group) and d C 73.2 (C-5 000 ). Therefore, the structure of compound 2 was established as shown, and named aquilariside B.  (Tables S2-S7).
Compounds 1-8 were evaluated for their inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages using the Griess assay (Li et al. 2014;Huang et al. 2015). Indomethacin served as a positive control (IC 50 = 35.7 lM). Compound 2 showed weak inhibition of NO production in LPS-stimulated RAW 264.7 cells, with IC 50 value of 95.4 ± 1.4 lM. Compounds 1, and 3-8 were inactive (<50% inhibition at 100 lM, the highest concentration tested).

General experimental procedures
Optical rotations were determined by a Rudolph Autopol IV automatic polarimeter. UV spectra were measured with a Shimadzu UV-2450 spectrophotometer. IR spectra were obtained on a Thermo Nicolet Nexus 470 FT-IR spectrophotometer with KBr pellets. 1D and 2D spectra were recorded on a Varian INOVA-500 spectrometer (CA, USA) operating at 500 MHz for 1 H and 125 MHz for 13 C, respectively. Rotary evaporator (BUCHI, Switzerland) was used for concentrating solvent. HRESIMS was recorded on an LCMS-IT-TOF system, fitted with a Prominence UFLC system and an ESI interface (Shimadzu, Kyoto, Japan). Silica gel (200 -300 mesh, Qingdao Marine Chemical Inc., Qingdao, People' Republic of China), and Sephadex LH-20 (Pharmacia) were used for separation and purification. A semipreparative RP-HPLC column (YMC-Pack C 18 , 250 Â 10 mm, 5 lm) was employed for isolation. Analytical HPLC was performed on a LC-20A chromatography system composed of a diode array detector (DAD) (Shimadzu, Tokyo, Japan), and an Agilent XDB C 18 column (4.6 Â 250 mm, 5 lm).

Plant material
The dried pericarps of A. yunnanensis were collected in August 2015 in Dehong, Yunnan Province, People's Republic of China. The plant material was authenticated by one of the authors (P.-F. Tu). A voucher specimen (JLI-AY-201508) has been deposited in the Modern Research center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine.

Acid hydrolysis
Compound 2 was dissolved in 1.0 N HCl (2.0 mL) at 80 C in water bath for 5.0 h. The reaction mixture was dissolved in H 2 O after evaporation and extracted exhaustively with CH 2 Cl 2. The aqueous layers containing sugar was evaporated to dryness under reduced pressure, then analyzed using silica gel TLC by comparison with standard sugars. The residue was dissolved in pyridine (2.0 mL) containing L-cysteine methyl ester hydrochloride (2.0 mg) and heated at 60 C for 2.0 h. O-tolyl isothiocyanate (10.0 mL) was added to the mixture and then heated at 60 C for another 2.0 h. The standard Lrhamnose was subjected to the same procedures. The retention time of L-rhamnose (t R = 28.8 min) was confirmed by comparison with standards (Tanaka et al. 2007;Wu et al. 2017;Wang et al. 2015).

Cell culture and measurement of NO production
The murine macrophage RAW 264.7 cells were purchased from Peking Union Medical College (PUMC) Cell Bank (Beijing, People's Republic of China). Cell maintenance, experimental procedures, and data presentation for the inhibition of NO production and viability assay were the same as previously described (Li et al. 2014;Huang et al. 2015). Indomethacin (IC 50 value of 35.7 lM) was used as a positive control. All the compounds were prepared as stock solution in DMSO (final solvent concentration less than 0.2% in all assays).

Conclusion
Two new benzophenone glycosides, aquilarisides A (1) and B (2), together with six known analogues were obtained from the pericarps of A. yunnanensis. Compound 2 showed mild inhibition of NO production in LPS-stimulated RAW 264.7 cells.

Disclosure statement
No potential conflict of interest was reported by the authors.