Anti-inflammatory Limonoids From Cortex Dictamni

The root barks of perennial herb Dictamnus dasycarpus (Cortex Dictamni) were reported to be rich in anti-inflammation activity constituents, limonoids. Then, the investigation of anti-inflammation therapeutic limonoids from this plant was developed in the present study. Through the combination of various chromatographies isolation, six new limonoids, named dictamlimonol A (1), dictamlimonoside B (2), and dictamlimonols C–F (3–6), along with seven known ones (7–13), were obtained. Their structures were ascertained based on the extensive spectroscopic methods and ECD data analysis. Among them, compound 1 was the first 7,19-epoxy limonoid found in natural products. The anti-inflammatory effects of all limonoids were evaluated in lipopolysaccharide (LPS)-treated RAW 264.7 cell lines. Compounds 5, 7–11, and 13 were found to inhibit LPS-induced nitric oxide (NO) production. Moreover, dictamlimonol D (5), fraxinellone (11), and dasylactone A (13) were found to reduce the LPS-induced expressions of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and cyclooxygenase-2 (COX-2) at the protein levels in a dose-dependent manner. These findings support that the administration of Cortex Dictamni may be beneficial for inflammation.

The natural plants of Dictamnus genus (Rutaceae family) are main resources of limonoids. The limonoids obtained from them could be subdivided as limonoid aglycones, degraded limonoids, and limonoid glucosides (Lv et al., 2015). Dictamnus dasycarpus Turcz is a perennial herb belonging to Dictamnus genus, widely distributed in China. Its root barks (Cortex Dictamni) have been used to treat inflammation, scabies, rheumatic pain, jaundice, and other symptoms (Yang et al., 2017(Yang et al., , 2018. Though limonoids were reported as one of the major constituents in Cortex Dictamni, only fraxinellone obtained from it has been proved to possess in vitro anti-inflammatory effect (Kim et al., 2009;Lee et al., 2009;Wu et al., 2014) to date.
This study aims to clarify anti-inflammation limonoids in Cortex Dictamni by phytochemistry and bioactivity screening. Multiple chromatographies and spectral techniques were combined to isolate and identify limonoids. Then, the inhibitory activities of all obtained limonoids against nitric oxide (NO) production in RAW 264.7 cell lines induced by lipopolysaccharide (LPS) were evaluated. Furthermore, the antiinflammatory mechanism of activity compounds was studied by using Western blot assay.
NO is closely associated with inflammation. Agents that block NO production might be beneficial for the treatment of inflammatory responses. The LPS-stimulated RAW 264.7 cells were used as a potential in vitro antiinflammatory activity screening model to investigate the NO production inhibitory effect of limonoids 1-13 at a final concentration of 20 µM. Compared to the LPS group, compounds 5, 7-11, and 13 exhibited potential in vitro anti-inflammatory activities at 20 µM ( Table 7).
Within the active compounds, 5, 11, and 13 were selected for further research. It was found they inhibited NO release from RAW 264.7 cells in a concentration-dependent manner at 5, 10, and 20 µM (Figure 5).
The structure-activity relationships on NO production in LPS-stimulated RAW 264.7 cells were summarized as follows: In compounds 2 and 11-13, hydroxyl group or glucosyl group substitution at position 9 weakened the activity (11 and 13 > 2 and 12). Although the specimen was limited, carbonyl substitution at position 23 showed enhanced activity (13 > 11). In compounds 3-6, α type alkoxy group substitution showed stronger inhibitory effects on NO production than β type (5 > 3 and 4).
LPS can stimulate the acute inflammatory response by increasing expression of tumor necrosis factor (TNF-α) and interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and cyclooxygenase-2 (COX-2), successively (Ahujaa et al., 2019). In the process, the expressions of TNF-α, IL-6, iNOS/NO, NF-κB, as well as COX-2 will be upregulated, which will promote tissue damage and chronic disease. Therefore, the Western blot method was used to study the anti-inflammatory mechanism of compounds 5, 11, and 13 by determining the levels of these five proteins in LPS-stimulated RAW 264.7 cells.
Compared with the normal group, LPS treatment led to an obvious upregulation in the protein expressions of TNF-α, IL-6, NF-κB, iNOS, and COX-2. Compounds 5, 11, and 13 were found to inhibit the expression of the abovementioned proteins in a dose-dependent manner (Figures 6-8).
Among the anti-inflammatory active compounds, fraxinellone (11) had been found to inhibit LPS-induced NO production and reduce the LPS-induced expressions of iNOS and COX-2 at the mRNA and protein levels in a dose-dependent manner by regulating NF-κB in RAW 264.7 macrophage-like cells (Kim et al., 2009;Lee et al., 2009;Wu et al., 2014), which was identical to our experimental result, suggesting that our screening system was stable and suitable. Meanwhile, the effects and the mechanism of a new compound, dictamlimonol D (5), as well as the known compound, dasylactone A (13), in inflammation were characterized here firstly, which would provide new candidate drugs for inflammation-related diseases.
Since fraxinellone (11) is the main constituent in Cortex Dictamni, and the content of it is about 0.15% in Cortex Dictamni, we can predict that the anti-inflammatory activity of the plant is mainly derived from limonoids, especially fraxinellone. Further mechanism and clinical studies for it is necessary.

General Experimental Procedures
Optical rotations were measured on a Rudolph Autopol R IV automatic polarimeter (l = 50 mm) (Rudolph Research Analytical, Hackettstown NJ, USA). NMR spectra were determined on a Bruker 500-MHz NMR spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden, Switzerland) at 500 MHz for 1 H and 125 MHz for 13 C NMR (internal standard: TMS). IR spectra were recorded on a Varian 640-IR FT-IR spectrophotometer (Varian Australia Pty Ltd., Mulgrave, Australia). Negative-ion mode ESI-Q-Orbitrap-MS was obtained on a Thermo ESI-Q-Orbitrap MS mass spectrometer connected with the UltiMate 3000 UHPLC instrument via ESI interface (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Western Blot Analysis
Western blot method was used to study the anti-inflammatory mechanism of compounds 5, 11, and 13 by determining the levels of these five proteins (TNF-α, IL-6, COX-2, NF-κB, and iNOS) in LPS-stimulated RAW 264.7 cells as previously reported (He et al., 2018;Ruan et al., 2019). The raw quantification data were displayed in Figures S53-S55.

Statistical Analysis
Values were statistically analyzed by using SPSS 17.0 software. P < 0.05 was considered to indicate statistical significance. One-way analysis of variance (ANOVA) and Tukey's Studentized range test were used for the evaluation of the significant differences between means and post hoc, respectively.

CONCLUSION
In this study, 13 limonoids including 6 new ones, named dictamlimonol A (1), dictamlimonoside B (2), and dictamlimonols C-F (3-6), along with 7 known ones (7-13) were isolated from Cortex Dictamni by various chromatographies and identified by spectroscopies and chemical reactions. Among them, compound 1 was a first 7,19-epoxy limonoid found in natural products. Activity evaluation research showed several kinds of limonoids reduce expression of TNF-α, IL-6, iNOS, NF-κB, and COX-2 in LPS-stimulated RAW 264.7 cells. These findings support the idea that the administration of Cortex Dictamni may be beneficial for inflammation.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.