Five 2-(2-Phenylethyl)chromones from Sodium Chloride-Elicited Aquilaria sinensis Cell Suspension Cultures

Five 2-(2-phenylethyl)chromones including a new one, (5S,6R,7S,8R)-5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (1), and four known ones (2–5), were isolated from 150 mM NaCl-elicited Aquilaria sinensis cell suspension cultures. In addition, three feruloyl amides (6–8), six nucleosides (9–14), (+)-syringaresinol (15), indole-3-carboxaldehyde (16), and two glycosides (17–18) were also obtained. The structures were unambiguously identified by analysis of their UV, IR, NMR, and HRESIMS data. The absolute configuration of the new 2-(2-phenylethyl)chromone (1) was established by a dimolybdenum tetraacetate-induced circular dichroism experiment. Compared to un-elicited cell lines, the appearance of 2-(2-phenylethyl)chromones in NaCl-treated cells occurred on the 3rd and 5th days of their treatment. 2-(2-Phenylethyl)chromones, feruloyl amides, nucleosides, and lignins have been reported to be closely related to plant defense; therefore, the identification of these compounds from NaCl-elicited A. sinensis cell suspension cultures would be useful for further exploring the mechanism of agarwood formation.


Introduction
2-(2-Phenylethyl)chromones are a subgroup of plant polyphenols specifically produced by Aquilaria, Gonystulus, and Gyrinops species (Thymelaeaceae) in response to biotic or abiotic stress. Hitherto, more than 100 congeners of 2-(2-phenylethyl)chromones have been reported, and many of them have potentially anti-inflammatory [1], neuroprotective [2], antitumor activities, etc. [3][4][5]. Under stress conditions, such as wounding or fungal infections, some resin-impregnated heartwood containing a variety of 2-(2-phenylethyl)chromones are slowly forming in the trunk and branches of some species of Thymelaeaceae. Those resinous heartwoods are commercially called agarwood, which have long been used pharmaceutically as a digestive, sedative, and antiemetic in oriental medical treatments [6] and as incense in many cultures because of its unique fragrance. The finest agarwood pieces are considerably valued in some Asian incense ceremonies. An extremely long growing cycle and an increased level of consumption are probably the main reasons of the over-exploitation and serious depletion of agarwood, leading to the relative rarity and high cost of its wild resource. Since 2005, accordingly, Aquilaria and Gyrinops have been listed in Appendix II (potentially threatened species) of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [7]. Hence, it should be of great interest to unveil the mechanisms of agarwood formation and thereby to artificially manipulate the process of agarwood formation [8].
Because it takes timber species a considerably long time for their resinous portions to form inside of the wood of agarwood-producing plants, researchers tend to use cell suspension cultures to study the mechanisms of agarwood formation. Therefore, establishing cell suspension cultures with a high rate of cell growth and a high production of 2-(2-phenylethyl)chromones, the characteristic components of agarwood, would be undoubtedly meaningful. Here, cell suspension cultures of Aquilaria sinensis, the only origin species of agarwood in China, were established, and the chemical constituents of NaCl-elicited A. sinensis cell suspension cultures were investigated, resulting in the isolation of five 2-(2-phenylethyl)chromones (1)(2)(3)(4)(5) together with 13 other known compounds (6-18) ( Figure S1). The structural elucidation of the new 2-(2-phenylethyl)chromone (1) is comprehensively discussed herein.  [9] revealed that these two compounds shared a very similar skeleton. The only difference was the presence of two methines at a relatively higher field (δ C 52.0, 58.3) in the 13 C-NMR spectrum of 1, suggesting these two carbons were both chlorinated. In the HMBC spectrum, the long range correlations between the proton at δ H 4.94 (1H, br.s), which was attached at C-5 (δ C 52.0), and the carbonyl carbon at δ C 179.4 definitively established that one of the two chlorine atoms were attached at C-5. The other chlorine atom at C-8 was deduced from the HMBC correlations of H-8/C-10 and H-7/C-8 ( Figure 1). Accordingly, the planar structure of 1 was elucidated as 5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (see Figures S2-S9).  [7]. Hence, it should be of great interest to unveil the mechanisms of agarwood formation and thereby to artificially manipulate the process of agarwood formation [8].

Results and Discussion
Because it takes timber species a considerably long time for their resinous portions to form inside of the wood of agarwood-producing plants, researchers tend to use cell suspension cultures to study the mechanisms of agarwood formation. Therefore, establishing cell suspension cultures with a high rate of cell growth and a high production of 2-(2-phenylethyl)chromones, the characteristic components of agarwood, would be undoubtedly meaningful. Here, cell suspension cultures of Aquilaria sinensis, the only origin species of agarwood in China, were established, and the chemical constituents of NaCl-elicited A. sinensis cell suspension cultures were investigated, resulting in the isolation of five 2-(2-phenylethyl)chromones (1-5) together with 13 other known compounds (6-18) ( Figure S1). The structural elucidation of the new 2-(2-phenylethyl)chromone (1) is comprehensively discussed herein. Comparison of the NMR data with those of the known Compound 2 [9] revealed that these two compounds shared a very similar skeleton. The only difference was the presence of two methines at a relatively higher field (δC 52.0, 58.3) in the 13 C-NMR spectrum of 1, suggesting these two carbons were both chlorinated. In the HMBC spectrum, the long range correlations between the proton at δH 4.94 (1H, br.s), which was attached at C-5 (δC 52.0), and the carbonyl carbon at δC 179.4 definitively established that one of the two chlorine atoms were attached at C-5. The other chlorine atom at C-8 was deduced from the HMBC correlations of H-8/C-10 and H-7/C-8 ( Figure 1). Accordingly, the planar structure of 1 was elucidated as 5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (see Figures S2-S9). The relative configuration of 1 was established by analysis of the coupling constants between the involved protons and further confirmed by a NOESY experiment. In the 1 H-NMR spectrum, the relatively large coupling constant between H-7 and H-8 (J = 8.0 Hz) revealed the trans pseudoaxialaxial relationships between H-7 and H-8. In contrast, the relatively small coupling constants between H-5, H-6, and H-7 revealed the cis relationships between these protons, which were confirmed by the NOE correlations of H-5/H-6, H-5/H-7 in the NOESY spectrum of 1.

Results and Discussion
The absolute configurations of C-6 and C-7 were solved according to the method reported by Snatzke and Frelek [10]. DMSO solutions of 1 and dimolybdenum tetraacetate [Mo2(AcO)4] were homogeneously mixed, and the induced circular dichroism (ICD) spectrum was recorded. In the ICD spectrum of 1 (Figure 2), the positive Cotton effect at 310 nm suggested a positive dihedral angle of The relative configuration of 1 was established by analysis of the coupling constants between the involved protons and further confirmed by a NOESY experiment. In the 1 H-NMR spectrum, the relatively large coupling constant between H-7 and H-8 (J = 8.0 Hz) revealed the trans pseudoaxial-axial relationships between H-7 and H-8. In contrast, the relatively small coupling constants between H-5, H-6, and H-7 revealed the cis relationships between these protons, which were confirmed by the NOE correlations of H-5/H-6, H-5/H-7 in the NOESY spectrum of 1.

General Experimental Procedures
UV spectra were obtained using a Shimadzu UV-2450 spectrophotometer (Shimadzu Corporation, Tokyo, Japan). NMR spectra were recorded on a Varian INOVA-500 spectrometer (Varian Corporation, Palo Alto, CA, USA) operating at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR. HRESIMS was recorded on an LCMS-IT-TOF system, fitted with a Prominence UFLC system and an ESI interface (Shimadzu Corporation). Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter (Rudolph Corporation, Flanders, NJ, USA). IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrophotometer (Thermo Corporation, Waltham, MA, USA) with KBr pellets. CD spectra were recorded using a Jasco J810 spectropolarimeter (YMC Corporation, Kyoto, Japan). HPLC was performed on a Shimadzu LC-20AT pump system (Shimadzu Corporation), equipped with a SPD-M20A photodiode array detector. A semi-preparative HPLC column (YMC-Pack C 18 , 250ˆ10 mm, 5 µm, YMC Corporation) was employed for the isolation. TLC was performed using GF 254 plates.

Cell Culture and Viability
Fresh young leaves of A. sinensis were cut into pieces, surface-sterilized using sodium hypochlorite (2.5% v/v) for 10 min, and rinsed with sterile distilled water four times. These processed leaves were then inoculated onto solid Murashige-Skoog (MS) basal media. Calli were initiated from the plant tissues after incubation in dark conditions for one month at 25˝C, subcultured in a MS liquid medium with 2 mg/L naphthal acetic acid, 1 mg/L 6-benzyladenin, 1 mg/L kinetin, 1 mg/L vitamin B5, and 1 mg/L 2,4-dichlorophenoxyacetic acid, and supplemented with sucrose (4% w/v) as a carbon source. The cells were cultivated via shaking at 250 rpm at 25˝C in the dark. The cell suspension cultures were subcultured into fresh medium every two weeks.

Elicitation and Harvesting Cells
Cell suspensions cultured for two weeks (5.28 L) were treated with NaCl (Beijing Chemicals, Beijing, China) at a final concentration of 150 mM. Control cell suspensions received no treatment. After different times of NaCl treatment, cells were harvested by filtration.

Absolute Configuration of Compound 1
According to the published procedure [10], Compound 1 (0.27 mg) was added to a stock solution of 0.6-0.7 mg/mL Mo 2 (AcO) 4 (Strem Chemicals, Newburyport, MA, USA) in absolute anhydrous dimethylsulfoxide (DMSO) to achieve the ligand-to-metal ratio is approximately 0.6/0.8 up to 1.0/1.2. CD spectra of Compound 1 were recorded using a Jasco J810 spectropolarimeter, with a 0.1-cm cell in DMSO (Aladdin, Shanghai, China) and at room temperature, see Figure 2.

Conclusions
We have firstly reported five 2-(2-phenylethyl)chromones including a new one, together with 13 other known compounds from NaCl-elicited cell suspension cultures of A. sinensis. Most of these compounds have been previously demonstrated to be closely related to plant defense. Thus, it would be meaningful to further explore and understand the mechanism of pharmaceutically important and commercially valued agarwood formation.