Aculebiphenyl A–B, new biphenyl derivatives from Ruscus aculeatus

Abstract The investigation of chemical constituents from the rhizomes of Ruscus aculeatus resulted in the isolation of two new biphenyl derivatives, aculebiphenyls A and B (1-2), together with two known analogs (3-4). Their chemical structures were elucidated based on extensive spectroscopic interpretation and HR-ESI-MS analysis. Compounds 3-4 were isolated from the Ruscus genus for the first time. The isolated compounds were tested for anti-inflammatory activities and antibacterial activities. Compound 1 exhibited significant inhibitory effects on LPS-induced NO production and COX-2 with IC50 values of 10.8 µM and 0.4 µM. Compound 1 also significantly down-regulated the levels of inflammatory cytokines IL-1β, IL-6, and TNF-α. Compound 1 showed moderate antibacterial activities.


Introduction
Ruscus genus, belonging to the Liliaceae family, is native to the Mediterranean, Southern, and Western Europe, and is represented by perennial, rhizomatous, and evergreen shrubs [1].Among approximately seven species spread throughout Europe up to Iran, Ruscus aculeatus Linn, locally referred to as "Jia-Ye-Shu" in Chinese is the most widely distributed and appreciated.As an ancient phlebo therapeutic agent, R. aculeatus is the most studied species.During the middle ages, the young shoots of R. aculeatus were used not only as food but also as medicinal agents for the treatment of urinary disorders and abdominal pain [2].The hydroalcoholic extract of R. aculeatus rhizomes is traditionally used as a vascular preventive and tonic for venous system disorders, including venous fragility and varicose veins [3].The underground parts of R. aculeatus have been also used as diuretic and anti-inflammatory agents for the treatment of hemorrhoids and atherosclerosis [4].The most studied phytochemical investigations of this plant are steroidal saponins [5-7], while other constituents have been also isolated, including triterpenes, flavonoids, coumarins, sparteine, tyramine, and glycolic acid [8].In order to exploit and utilize the compound resources of R. aculeatus, to provide a theoretical basis for exploring the chemical composition and biosynthetic pathways of the Ruscus genus, and to search for inflammatory inhibitors, we launched a systematic study to investigate the chemical constituents of R. aculeatus.Thus, we reported the isolation, structure elucidation, anti-inflammatory activities, and antibacterial activities of the biphenyl derivatives isolated from R. aculeatus.The structures of compounds 1-4 are shown in Figure 1.
Aculebiphenyl B (2) was obtained as a colorless crystal (MeOH), and this structure exhibited a protonated molecular ion peak at m/z 315.1579 [M þ H] þ in HR-ESI-MS, corresponding to the molecular formula C 19 H 22 O 4 , which indicated 9 degrees of unsaturation.The UV spectrum of compound 2 was similar to that of compound 1.
Moreover, the isoprenyl moiety was located at C-3 of benzene ring A of biphenyl by HMBC correlations (Figure 2) of H-7 (d 3.37) with C-2 (d 128.0),C-3 (d 130.4), and C-4 (d 156.7), and H-8 (d 5.35) with C-3 (d 130.4).Furthermore, the HMBC correlations of methoxy group protons at d 3.87 with C-4 (d156.7)indicated that the methoxy group was located at C-4 of benzene ring A, adjacent to isoprenyl moiety.Additionally, the HMBC correlations of another methoxy group protons at d 3.94 with C-3 0 (d147.0)suggested that the methoxy group was linked at C-3 0 of benzene ring B of biphenyl.Moreover, the NOESY correlations (Figure 2) of 4-OCH 3 with H-5 and 3 0 -OCH 3 with H-2 0 confirmed the positions of the methoxy groups.The structure of compound 2 was determined (Figure 1) and named as aculebiphenyl B.
By comparing the physico-chemical and spectroscopic properties with those reported in literature, two known compounds were elucidated mesembrine (3) [10] and mesembrenone (4) [11].Compounds 3 and 4 were isolated from the Ruscus genus for the first time.
Considering the traditional use of R. aculeatus and a goal to obtain NO and COX-2 inhibitors for inflammatory disorders, all isolated compounds (1-4) were tested individually for NO production inhibition in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and COX-2 inhibitory effects.The isolated compounds 1-4 showed remarkable NO inhibitory effects with IC 50 values of 10.8 ± 0.1 mM, 41.2 ± 0.6 mM, 28.9 ± 0.4 mM, and 30.5 ± 1.3 mM.Compared with the positive control of aminoguanidine hydrochloride (IC 50 ¼ 25.8 mM), compound 1 exhibited more potent inhibitory activity against NO production.Compound 1 inhibited COX-2 activity in a dose-dependent manner in 0.031-0.5 mM concentration (Figure S1).The IC 50 value of the inhibitory effect of compound 1 against COX-2 was 0.4 mM (Table S1, Figure S1).Using dexamethasone (DEX) as the positive control (1.0 mM), we tested the effects of compound 1 on the concentration of inflammatory cytokines in RAW 264.7 cells.The results showed that compound 1 remarkably downregulated the levels of the inflammatory cytokines IL-1b, IL-6, and TNF-a at 2.0 mM (Table S2, Figure 3).
The antibacterial activities of four compounds isolated from R. aculeatus were evaluated by determining the minimum inhibitory concentration (MIC).The results showed that compound 1 had moderate antibacterial activity against the four standard strains, and MIC values against Staphylococcus aureus, Streptococcus pneumoniae, Salmonella typhimurium, and Escherichia coli were 0.2, 0.4, 0.4, and 0.4 mg/ml, respectively.Compared with S. typhimurium and E. coli, the isolated compounds showed better antibacterial activity against S. aureus and S. pneumoniae, indicating that these compounds may have a better antibacterial effect against Gram-positive bacteria (Table S3).
In this study, two new biphenyl derivatives were isolated, which were rarely reported from R. aculeatus.Additionally, these derivatives enriched the structural resources of the Ruscus genus.The biological evaluation of the isolated biphenyl derivatives indicated that compound 1 has shown great application potential as a lead compound from natural sources in the research and development of anti-inflammatory drugs.

Plant material
Dried rhizomes of Ruscus aculeatus were purchased in June 2016 from Anguo Market of Hebei Province, China.The plant material was authenticated by Prof. Wei Ning (College of Horticulture, Shenyang Agricultural University).The voucher specimen (RA-2016061502) was deposited at the Department of Animal Pharmacy of Shenyang Agricultural University.

NO production inhibition assays
The nitrite concentration in the medium was measured as an indicator of NO production in accordance with the Griess reaction [12].The NO inhibitory effects of 1-4 were tested in LPS-activated RAW 264.7 cells.Then, RAW 264.7 cells were cultured in LPS and phenol red-free RPMI 1640 medium with 10% FBS at 37 � C in a humidified atmosphere with 5% CO 2 .Cells were seeded in 96-well plates at 1 � 10 5 cells/well and incubated for 24 h.After stimulation with LPS at 1 mg/ml, the compounds were added at different concentrations; then, cells were incubated for 24 h at 37 � C in a humidified atmosphere with 5% CO 2 .The level of nitrite formation in supernatants was measured using a Griess reagent by measuring the absorbance at 540 nm.The percent inhibition for nitrite production was determined by comparing it with the vehicle control.IC 50 values were calculated from dose curves, and aminoguanidine hydrochloride was used as the positive control.Nitrite concentrations and inhibitory rates were calculated by the calibration curve prepared with sodium nitrite standards.Experiments were conducted in triplicate, and data were presented as mean ± SD of three independent experiments.

COX-2 inhibition assays
COX-2 assay buffer, Milli-Q grade water, DMSO, and other appropriate solvents were used to prepare appropriate concentrations of compound 1.The effect of compound 1 on COX-2 enzyme activity was determined by fluorescence assay.Groups included the blank control group, 100% enzyme active control group, compound 1 with different concentrations (31.25, 62.5, 125, 250, and 500 nM) groups, and positive control group (50 nM Celecoxib).Corresponding reagents were added to the 96-well plate successively.Referring to the instruction manual of the COX-2 inhibitor screening kit for the other steps, the relative fluorescence unit was measured by using a microplate reader.The inhibition of COX-2 enzyme activity by compound 1 was analyzed by GraphPad Prism 8, and the IC 50 value was obtained.

Inflammatory cytokine assays
RAW 264.7 cells (5 � 10 5 cell/ml) were cultured in a 96-well plate under 5% CO 2 at 37 � C for 48 h and were incubated with LPS (1 mg/ml) for 1 h followed by the addition of compound 1 (2 mM) and DEX (1 mM) for 1 h.The cell supernatant was collected, and the concentration of the cytokines IL-1b, IL-6, and TNF-a was determined by ELISA.Experimental data were presented as mean ± SD of five independent experiments.

Antibacterial activity assays
Broth microdilution [13] was used to determine the antibacterial activities of the four isolated compounds.S. aureus, S. pneumoniae, S. typhimurium, and E. coli were cultured in a nutrient broth medium at 37 � C and 150 rpm until OD 600 ¼ 0.4-0.6.Then, the bacterial solution was diluted with an MHB medium at 1 � 10 5 CFU/ml as the using concentration.Using the continuous twofold dilution method, gentamicin sulfate (control antibiotic) was diluted to 256-0.5 mg/ml concentration gradient with MHB medium.Each compound was prepared as a concentration from 6400 to 12.5 mg/ml in MHB medium.The diluted compounds 1-4 and 100 ml of gentamicin sulfate were added to wells 1-10 of the 96-well plate in descending order of concentration.Then, 100 ml of diluted bacterial solution (1 � 10 5 CFU/ml) was added to wells 1-10 of each row.At this point, the concentration of compounds in wells 1-10 was 3200, 1600, 800, 400, 200, 100, 50, 25, 12.5, and 6.25 mg/ml, respectively.Next, 100 ml of MHB medium was added in the 11 th well for blank control, and 100 ml of diluted bacterial solution was added in the 12 th well for control.Two parallel tests were conducted for each compound.The bacterial solution and medicinal solution in each well were mixed, covered, and then incubated at 37 � C for 18-24 h to observe the phenomenon.The MIC of the four compounds against S. aureus, S. pneumoniae, S. typhimurium, and E. coli were determined by using this method.