Two New Stilbenoids from the Aerial Parts of Arundina graminifolia (Orchidaceae)

Two new phenanthrene derivatives, a phenanthrenequinone named arundiquinone (1) and a 9,10-dihydrophenanthrene named arundigramin (2) together with a known lignin dimer (3) and seven known stilbenoids (4–10) were isolated from the aerial parts of the Asian orchid Arundina graminifolia. The structures of the isolated compounds were elucidated by spectroscopic methods, including extensive 1D, 2D NMR (heteronuclear single quantum coherence (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and HR-ESI-MS techniques, as well as comparison with respective literature reports. The cytoprotective activity of the isolated compounds were evaluated for their ability to reduce beta amyloid induced toxicity on undifferentiated PC12 cells. Compound 8 showed moderate cytoprotective activity at 0.5 µmol/L (71% of cell viability) while the other compounds showed no significant activity at the highest concentration tested.

Molecules 2016, 21, 1430; doi:10.3390/molecules21111430 www.mdpi.com/journal/molecules As part of our continuing efforts in contributing to the phytochemical and biological evaluation of tropical orchids [33][34][35], the ethyl acetate extract of the aerial parts of A. graminifolia collected from Chiang Mai Province (Thailand) was screened for its neuroprotective activity against beta amyloid (βA) induced cytotoxicity on PC12 cells and showed promising results. Based on the aforementioned preliminary screening, the extensive investigation on the chemical entities in the plant was pursued. Two new constituents, 1-4 phenanthrenequinone (1) and 9,10-dihydrophenanthrene (2) along with a known lignan dimer (3) and seven known stilbenoids (4-10) were isolated and characterized. The cytoprotective activity of the compounds was then assessed to establish whether or not it is responsible for the cytoprotective activity of the tested extract.

Results and Discussion
The ethyl acetate (EtOAc) extract of the aerial parts of A. graminifolia was subjected to a series of chromatographic techniques (silica gel, Sephadex LH-20, Sigma Aldrich, Saint-Louis, MO, USA), and semi-preparative RP-HPLC) to afford two new stilbenoids: arundiquinone and arundigramin (1 and 2), as well as eight known compounds that were identified by comparison of their spectroscopic data to previously published reports as rac-syringaresinol (3) [36,37], orchinol (4) [38], ephemeranthoquinone (5) [39], densiflorol B (6) [40], coelonin (7) [41], lusianthridin (8) [42], batatasin III (9) [43], and flavanthrin (10) [44], (Figure 1). It is noteworthy to add that, besides the two new isolated stilbenoids (1 and 2), compound 3 syringaresinol is herein reported as its first occurrence in A. graminifolia. As part of our continuing efforts in contributing to the phytochemical and biological evaluation of tropical orchids [33][34][35], the ethyl acetate extract of the aerial parts of A. graminifolia collected from Chiang Mai Province (Thailand) was screened for its neuroprotective activity against beta amyloid (βA) induced cytotoxicity on PC12 cells and showed promising results. Based on the aforementioned preliminary screening, the extensive investigation on the chemical entities in the plant was pursued. Two new constituents, 1-4 phenanthrenequinone (1) and 9,10-dihydrophenanthrene (2) along with a known lignan dimer (3) and seven known stilbenoids (4-10) were isolated and characterized. The cytoprotective activity of the compounds was then assessed to establish whether or not it is responsible for the cytoprotective activity of the tested extract.
Molecules 2016, 21, 1430 4 of 9 located on C-5 and C-7, respectively. This was also supported by NOESY correlations from 5-OCH3 to H-4 and H-6 as well as from 7-OCH3 with H-6 and H-8. The remaining two hydroxyl groups were thus assigned to C-1 and C-2 with the help of the HMBC cross peaks correlations between H-3 and H-4, respectively, to the last two oxygenated quaternary carbons C-1 and C-2. Thus, the structure of compound 2 was defined as 5,7-dimethoxy-9,10-dihydrophenanthrene-1,2-diol, named arundigramin. Cell viability, expressed as a percentage relative to the untreated control cells, decreased by more than 60% after exposure to βA25-35 alone. The EtOAc extract of A. graminifolia aerial parts showed promising cytoprotective activity against βA induced cytotoxicity on undifferentiated PC12 cells (86% cell viability at 75 mg/L) (Figure 3). Despite this effect, the isolated compounds (1-10) were tested for their potential cytoprotective effect. Only compound 8 displayed moderate cytoprotective activity at 0.5 µmol/L, and the other compounds did not exhibit any significant effect at the tested concentrations ( Figure 4). Furthermore, compounds 1, 2, 3, 5 and 7 were cytotoxic to the cells at the highest tested concentration (50 µmol/L).  Cell viability, expressed as a percentage relative to the untreated control cells, decreased by more than 60% after exposure to βA 25-35 alone. The EtOAc extract of A. graminifolia aerial parts showed promising cytoprotective activity against βA induced cytotoxicity on undifferentiated PC12 cells (86% cell viability at 75 mg/L) (Figure 3). Despite this effect, the isolated compounds (1-10) were tested for their potential cytoprotective effect. Only compound 8 displayed moderate cytoprotective activity at 0.5 µmol/L, and the other compounds did not exhibit any significant effect at the tested concentrations ( Figure 4). Furthermore, compounds 1, 2, 3, 5 and 7 were cytotoxic to the cells at the highest tested concentration (50 µmol/L).

General Experimental Procedures
Optical rotations were measured with a Perkin Elmer 341 polarimeter (Perkin-Elmer Inc., Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV-2401 PC spectrometer (Shimadzu, Kyoto, Japan). IR spectra were obtained on a 380 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The 1D and 2D NMR spectra were performed on a 500 MHz proton operating system on a Bruker Avance III spectrometer (Bruker BioSpin, Rheinstetten, Germany) Acetone-d 6 (Euriso-Top, Saint-Aubin, France) was used as deuterated solvent and its protonated residual signal was used as internal standard at 2.05 ppm relative to TMS. The HR-ESI-MS analyses were performed on an HPLC-DAD/UV-MS Agilent 1200 Series coupled to a 6520 Q-ToF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The acquisition of mass spectra was conducted in ESI positive ion mode. A Varian LC-920 HPLC-DAD/UV system (Varian Inc., Palo-Alto, CA, USA) equipped with a Kinetex XB-C18 column (100 mm× 3.0 mm i.d, 2.6 µm) (Phenomenex, Torrance, CA, USA) was used for HPLC-DAD/UV analysis. The prepacked Solid Phase Extraction (SPE) Chromabond cartridge (SiOH, 50 g/150 mL) was purchased from Macherey-Nagel (Macherey-Nagel, Düren, Germany), and SPE fractions were monitored by TLC. The spots were visualized either under UV light (254 nm) and under visible light after heating the plates sprayed with 2% sulfuric vanillin reagent. Sephadex LH-20 (Sigma Aldrich) was used for gel chromatography eluting with methanol. Semi-preparative RP-HPLC experiments were conducted on a Gilson LC system (Gilson Inc., Limburg an der Lahn, Germany) equipped with a semi preparative Kinetex Axia C-18 Column (100 mm × 21.2 mm i.d, 5 µm) (Phenomenex, Torrance, CA, USA). Analytical TLC plates were carried out on pre-coated alumina silica gel 60F 254 plates (0.25 mm thickness) (Merck, Darmstadt, Germany). Analytical grade solvents of HPLC quality were purchased from Sigma Aldrich.

Extraction and Isolation
The air-dried powder of the aerial parts of A. graminifolia (100 g) was subjected to successive extraction using cyclohexane, EtOAc and CH 3 OH. Each extraction was performed by maceration for 30 min followed by sonicating in an ultrasonic bath for 10 min at room temperature (1 g raw material per 15 mL of organic solvent) and filtered. Extractions were repeated three times, and the filtrates were combined and evaporated under reduced pressure to afford cyclohexane extract (0.61 g), EtOAc extract (2.49 g) and CH 3 OH extract (11.01 g).

Cytoprotective Protocol
Undifferentiated PC12 cells were grown in DMEM-Glutamax supplemented with 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 15% fetal horse serum, and 2.5% fetal bovine serum at 37 • C in a humidified atmosphere of 5% CO 2 . Cells were seeded at a density of 2 × 10 4 cells/well in 96-well culture plates. After 24 h, cells were incubated with the EtOAc extract (10, 25, 50, 75 and 100 mg/L) and compounds 1-10 were screened at a concentration of 0.5, 5 and 50 µmol/L in the presence or absence of βA 25-35 (5 µM). The extract and compounds were dissolved in DMSO at a final concentration of 0.1%.
The cell viability was determined by the colorimetric MTT reduction assay. After treatment (24 h), PC12 cells were incubated with 0.5 mg/mL (DMEM) of MTT for 3 h at 37 • C. The resulting dark blue formazan crystals were dissolved with 100 µL of DMSO. Absorbance values were read at 540 nm on a microplate reader (MRX Dynex, Dynex Technologies, Denkendorf, Germany) [49]. Cell viability was expressed as a percentage of control cells at 100% viability.
Statistical analysis was performed using GraphPad Prism (version 7.0, GraphPad Software Inc. San Diego, CA, USA). All data are expressed as mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc analysis using Dunnett's multiple test. Differences were considered significant at p < 0.05.

Conclusions
Two new phenanthrene derivatives, arundiquinone (1) and arundigramin (2) together with eight known compounds (4-10) were isolated from the EtOAc extract of A. graminifolia aerial parts, a well-studied Asian orchid. The structures of the new phenanthrenes were elucidated by means of NMR and HR-ESI-MS, as well as comparisons with previous literature reports. Compound 3 was isolated here from A. graminifolia for the first time, whereas the presence of compounds 4-10 was already signaled in this species. Cytoprotective activity of the isolated compounds was evaluated on their ability to reduce beta amyloid induced toxicity on undifferentiated PC12 cells; however, only compound 8 showed moderate activity while the other isolated compounds did not display any significant activity at the tested concentrations. The EtOAc extract is particularly rich and contains more than 40 compounds based on the HPLC-DAD/UV profiling of this extract and only ten compounds have been isolated and tested. We thus envisage that some minor components that we have yet to isolate may have contributed to the potent bioactivity of the EtOAc extract. Further experiments geared towards identifying these minor components that may be responsible for the displayed cytoprotective activity in the EtOAc extract is in progress.