Acylphloroglucinol Derivatives from Garcinia multiflora with Anti-Inflammatory Effect in LPS-Induced RAW264.7 Macrophages

Two new acylphloroglucinol derivatives, 13,14-didehydroxygarcicowin C (1) and 13,14-didehydroxyisoxanthochymol (2), have been isolated from the stems of Garcinia multiflora, together with seven known compounds (3–9). The structures of new compounds 1 and 2 were elucidated by MS and extensive 1D/2D NMR spectroscopic analyses. Among the isolates, 13,14-didehydroxy-isoxanthochymol (2) and sampsonione B (3) exhibited inhibition against lipopolysaccharide (LPS)-induced NF-κB activation in macrophages at 30 μM with relative luciferase activity values (inhibitory %) of 0.75 ± 0.03 (24 ± 4%) and 0.12 ± 0.03 (88 ± 4%), respectively. Additionally, sampsonione B (3) reduced LPS-induced nitric oxide (NO) production in murine RAW264.7 macrophages and did not induce cytotoxicity against RAW 264.7 cells after 24 h treatment. Compound 3 is worth further investigation and may be expectantly developed as an anti-inflammatory drug candidate.

Molecules 2018, 23, 2587 2 of 12 responsive to lipopolysaccharide (LPS) and activated macrophages produce multiple pro-inflammatory molecules (such as nitric oxide (NO)). Nuclear factor κB (NF-κB) [12,13] is a transcription factor mediating inflammatory responses and known as a drug target for anti-inflammatory strategy. In our research on the anti-inflammatory constituents of Formosan plants, numerous species had been screened for inhibitory activity on LPS-induced NF-κB activation, and G. multiflora was found to be an active species. Phytochemical investigations on the stems of G. multiflora has resulted in the isolation of two new acylphloroglucinol derivatives, 13,14-didehydroxygarcicowin C (1) and 13,14-didehydroxy-isoxanthochymol (2), along with seven known compounds. We evaluated the anti-inflammatory effect of the isolated compounds in LPS-stimulated RAW264.7 macrophages and found that 13,14-didehydroxyisoxanthochymol (2) and sampsonione B (3) decreased NF-κB activity. Moreover, sampsonione B (3) inhibited the production of nitric oxide (NO) in LPS-activated macrophages. In this article, the structural elucidation of 1 and 2 and the inhibitory activity of the isolates on LPS-induced NF-κB activation are described.
LPS-mediated NF-κB activation results in upregulation of pro-inflammatory molecules, such as NO, in macrophages [12,26]. Thus, NO generation is a hallmark of inflammatory responses. Our study further evaluated the potential anti-inflammatory compounds, 13,14-didehydroxyisoxanthochymol (2) and sampsonione B (3) on NO production. The result showed that 13,14didehydroxyisoxanthochymol (2) did not obviously affect LPS-induced NO generation in RAW264.7 macrophages and did not display cytotoxicity against RAW 264.7 cells after 24 h treatment ( Figure  9A,C). In contrast, sampsonione B (3) could suppress LPS-induced NO generation in a concentrationdependent manner ( Figure 9B) without causing significant cytotoxicity ( Figure 9D).
It is observed that the inhibition on NO production is in a close correlation with NF-κB activation (e.g. compound 3 in Figures 8B and 9B). Thus, compound 3 may be involved in NF-κB-dependent NO regulation.
LPS-mediated NF-κB activation results in upregulation of pro-inflammatory molecules, such as NO, in macrophages [12,26]. Thus, NO generation is a hallmark of inflammatory responses. Our study further evaluated the potential anti-inflammatory compounds, 13,14-didehydroxy-isoxanthochymol (2) and sampsonione B (3) on NO production. The result showed that 13,14-didehydroxyisoxanthochymol (2) did not obviously affect LPS-induced NO generation in RAW264.7 macrophages and did not display cytotoxicity against RAW 264.7 cells after 24 h treatment ( Figure 9A,C). In contrast, sampsonione B (3) could suppress LPS-induced NO generation in a concentration-dependent manner ( Figure 9B) without causing significant cytotoxicity ( Figure 9D).

Biological Assay
The effect of the isolates on LPS-induced NF-κB activation in RAW 264.7/Luc-P1 macrophage was assessed by determining the luminescence resulted from luciferase activity in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by MS and NMR.

Cells and Culture Medium
The RAW 264.7/Luc-P1 cell is an LPS-responsive cell line with an integrated reporter gene (pELAM1-Luc) [23]. The murine RAW 264.7 macrophage and RAW 264.7/Luc-P1 cells were cultured and originated conditions as described previously [23,24].

Nitric Oxide (NO) Production
The RAW 264.7 cells (4 × 10 4 cells in 96-well plates) were treated with 13,14-didehydroxyisoxanthochymol (2), sampsonione B (3) and 0.1% DMSO for 1 h and then incubated with LPS (1µg/mL) for 23 h. The 100 µL of cell culture medium with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 2.5% phosphoric acid) in a 96-well plate was incubated for 10 min. The absorbance at 550 nm was measured by using a Model 680 Microplate Reader (Bio-rad, Hercules, CA, USA). The level of NO production was calculated from sodium nitrite (NaNO 2 ) standard curve [27].

Statistical Analysis
The data are displayed as mean ± SD from three independent experiments. Statistical analysis was performed using Student's t test. Differences were considered as statistically significant when p < 0.05.

Conclusions
Our research on the phytochemical investigation of G. multiflora has led to the isolation of two new (1, 2) and seven known (3)(4)(5)(6)(7)(8)(9) compounds. The structures of these isolates were established by spectroscopic data. Based on the results of our bioactivity assays, among the isolates, 13,14-didehydroxyisoxanthochymol (2) and sampsonione B (3) exhibited inhibition against lipopolysaccharide (LPS)-induced NF-κB activation in macrophages at 30 µM with relative luciferase activity values of 0.75 ± 0.03 and 0.12 ± 0.03, respectively. Furthermore, samposonione B (3) showed LPS-induced NO generation in concentration dependent manner. Thus, our research suggests G. multiflora and its isolated compound (especially 3) are worth further study and may be expectantly developed as the candidates for the prevention or treatment of diverse inflammatory diseases.