Bioactive Diarylheptanoids from Alpinia coriandriodora

Eight new diarylheptanoids, coriandralpinins A–H (1–8), were isolated from the rhizomes of Alpinia coriandriodora, an edible plant of the ginger family. Their structures, including the absolute configurations, were established by extensive spectroscopic analysis and ECD calculations. Compounds 1–8 have a 1,5-O-bridged diarylheptanoid structure featuring polyoxygenated aryl units. When evaluated for intracellular antioxidant activity using t-BHP stressed RAW264.7 macrophages, all these compounds scavenged reactive oxygen species (ROS) in a concentration-dependent manner. Compounds 3 and 5 also showed inhibitory activity against NO release in LPS-induced RAW 264.7 cells. Six known flavonols, 7,4′-di-O-methylkaempferol, 7-O-methylquercetin, 7,4′-di-O-methylquercetin, 7,3′,4′-tri-O- methylquercetin, kaempferol 3-O-β-d-(6-O-α-l-rhamnopyranosyl)glucopyranoside, and 3-O-β-d-glucopyranuronosylquercetin were also isolated and characterized from the rhizomes.


Introduction
Metabolism of oxygen is crucial to life for the production of energy to support biological process. As a consequence of the aerobic metabolism, reactive oxygen species (ROS) are continuously generated in all living organisms, and controlled by several antioxidant mechanisms [1,2]. However, overproduction and/or mismanagement of ROS may evade cellular antioxidant defense systems, resulting in the general phenomenon of oxidative stress. The redox imbalance leads to cellular damage, which is implicated with inflammatory process and various chronic degeneration diseases such as cancer, cardiovascular disease and diabetes [3][4][5][6]. In recent years there has been increasing interest in antioxidants of plant origin [7][8][9][10], particularly those from edible plants, and many of these naturally occurring compounds have been shown to possess protective efficacies against oxidative stress and inflammation-related diseases [11][12][13][14].
As part of our going effort to isolate and identify antioxidant and anti-inflammatory compounds from edible and medicinal plants, our attention was drawn to Alpinia coriandriodora D. Fang (Zigiberaceae), a perennial herb distributed in Guangxi, China. Plants of the genus Alpinia are rich in diarylheptanoids, sesquiterpenoids, and monoterpenes, many of which possess antioxidant [15,16], anti-inflammatory [17], hepatoprotective [18], and anticancer activities [19,20]. Although A. coriandriodora has long been used by local inhabitants as food spices, few chemical investigations on the species have been reported so far. We now present herein the isolation, characterization, and biological activities of eight new diarylheptanoids, coriandralpinins A-G (1-8) (Fig. 1), from the rhizomes of this plant. The isolation of six known flavonoids is also described.

HO
Coriandralpinin F (6) had a molecular formula of C 23 H 28 O 9 according to HRESIMS data. Its UV spectrum was largely similar to those of 1-5 but differed in that the absorption maximum at the longest wavelength was significantly red-shifted (301 nm vs 271-280 nm), suggesting the presence of a chromophore with extended conjugated system. This finding was consistent with the 1 H and 13 C NMR data (Tables 1 and 2), which were comparable to those of 1 except the carbon signal indicating the presence of a conjugated ketone carbonyl (δ C 199.5) and the absence of proton and carbon resonances for C-7 methylene. Detailed analysis of the 1D and 2D NMR data readily derived a structure of 7-keto derivative of 1, including the relative stereochemistry. As the experimental ECD spectrum of 6 was significantly different from the spectrum of 1, theoretical computations of ECD spectrum were carried out in order to solve the absolute stereochemistry. As a result (Fig. 5), chiral carbons in 6 were shown to be inverted relative to those in 1-5, designating this compound as 1R,3S,5S. Coriandralpinin G (7) was a 3′'-O-demethyl derivative of 6 as elucidated by analysis of the 1D NMR (Tables 1 and 2), 2D NMR, and HRESIMS data. Its absolute stereochemistry was assigned to be the same as that of 6 based on the nearly parallel experimental ECD curves of the two compounds (Fig. 5). Thus, the structures of compounds 6 and 7 were established to be (1R,3S,5S)-1,5-epoxy-3-hydroxy-1,7-bis(4-hydroxy-3,5-dimethoxyphenyl)heptan-7-one and (1R,3S,5S)-1,5epoxy-3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(3,4-dihydroxy-5-methoxyphenyl)heptan-7-one, respectively.
The isolated known flavonoids were identified to be 7,4′-di-O-methylkaempferol (9)  Compounds 1-8 were evaluated for antioxidant and anti-inflammatory activities in respect that the two activities are of the most common and interesting biological properties of diarylheptanoids [28][29][30]. The antioxidant activity evaluation was conducted using a more physiologically relevant method, the cellular antioxidant activity (CAA) assay [29,30], with RAW 264.7 macrophages as the test cells. The concentrations of compounds used in the assay were 50 μM and lowers according to cell viability assay by MTT method, which showed that 1-8 had no observable toxicity to RAW 264.7 cells at the concentration up to 100 μM. As indicated in Fig. 8 and Table 3, all these compounds were capable of significantly decreasing t-BHP-induced ROS production in RAW 264.7 cells in a concentration-dependent manner (Fig. 8), although none of them was more potent than the positive control curcumin. Among them, 3′-or 3′'-OH bearing 3, 4, and 7 exhibited better activity (IC 50 : 12.0-18.0 μM) than their corresponding 3′-or 3′'-O-methyl derivatives 2, 5, and 6 ( Table 3). Besides, the change of 7-keto carbonyl to the methoxylated methine was shown to increase the activity (6 vs 8).
The potential anti-inflammatory activity of compounds 1-8 was evaluated by measuring their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells (Table 3). Among these diarylheptanoids, compounds 3 and 5 exhibited inhibitory activity against NO release with IC 50 values of 36.9 and 34.1 μM, respectively, which were comparable to that of the positive drug control dexamethasone (IC 50 : 33.6 μM). However, other isolated analogues were inactive in the assay (IC 50 > 50 μM).
In summary, phytochemical analysis of A. Coriandriodora rhizomes led to the characterization of a group of

General Experimental Procedures
Optical rotations were obtained on a Perkin-Elmer 341 polarimeter with MeOH as solvent. UV and ECD spectra were recorded in MeOH with a Chirascan CD spectrometer (Applied Photophysics LTD., England). 1

Plant Material
The rhizomes of A. Coriandriodora were purchased from Nanning, Guangxi, China, in January 2018. A voucher specimen (specimen number SC-42-3-7) was deposited at the Herbarium of South China Botanical Garden, Chinese Academy of Sciences.

Cell Viability Assay
The murine macrophage RAW 264.7 cell line was obtained from Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). The cells were cultured in DMEM medium supplemented with 10% heated-inactivated fetal bovine serum in a 37 °C, 5% CO 2 incubator. Compounds 1-8, curcumin, and t-BHP were assessed for cytotoxicity against RAW 264.7 cells at various concentrations (100, 50, 10, and 2 μM) by MTT method as previously described [31].

Cellular Antioxidant Activity Assay
In the MTT cell viability assay, 50 µM of t-BHP (Sigma-Aldrich, USA, TBH70X) treatment for 3 h provoked about 60% of cell death and this condition was selected for the subsequent experiments as reported [29,30]. Briefly, RAW 264.7 cells (4 × 10 4 cells/well) were seeded into a black 96-well plate and allowed to growth for 24 h, then treated with either compounds (100, 50, 25, 12.5, and 6.25 µM) or the positive control curcumin (20, 10, 5, 2.5, and 1.25 µM) for 24 h. After stained with 20 μM DCFH-DA (Sigma, USA) for 1 h in darkness, the cells were exposed to 50 μM t-BHP for 1 h to induce ROS generation. The supernatants of cell cultures were measured for intracellular ROS levels using the fluorescence intensity at excitation wavelength of 485 nm and the emission wavelength of 530 nm by microplate reader (Tecan Group Ltd., Swizerland).

Anti-Inflammatory Activity Evaluation
The in vitro anti-inflammatory activity was evaluated by measuring the inhibitory effects of compounds on NO production in LPS-stimulated RAW 264.7 macrophages as previously described [32]. Briefly, the supernatants of cells were obtained as described above and RAW 264.7 cells were pretreated with different concentrations of compounds 1-8 (50, 25, 12.5, 6.25, and 3.125 μM) at 37 °C for 1 h, followed by stimulated with LPS (0.1 µg/ml) for 24 h. DMSO (0.1%) and dexamethasone (50, 25, 12.5, 6.25 and 3.125 μM) were used as vehicle and positive controls, respectively. The levels of NO were determined using commercial NO assay kit (Beyotime Institute of Biotechnology, China). 50 μL of Griess reagent I and 50 μL of Griess reagent II were added to 100 μL of the supernatants of cells (9 × 10 5 /mL).
For calculations of the 13 C NMR shifts of 8, low-energy conformers of the stereoisomers, (1S,3R,5R,7R)-8 and (1S,3R,5R,7S)-8, obtained in above ECD simulations, were subjected to NMR calculations using the gauge including atomic orbitals (GIAO) method [40,41,42] at the mPW1PW91/6-311 + G(d,p)/PCM level [43]. The unscaled chemical shifts (δ u ) were computed using TMS as reference standard according to δ u = σ 0 − σ x (where σ x is the Boltzmann averaged shielding tensor and σ 0 is the shielding tensor of TMS computed at the same level employed for σ x ). The Boltzmann averaging was done at 298.15 K using the relative energies obtained from the single-point NMR calculations [44,45]. The goodness of fit between the predicted 13 C NMR data of the two stereoisomers and the experimental shifts of compound 8 were evaluated by the improved DP4 probability (DP4 +) [44,45].