The ethanolic extract of Artemisia anomala exerts anti-inflammatory effects via inhibition of NLRP3 inflammasome.

BACKGROUND: Artemisia anomala S. Moore (Compositae), known as "Nan-Liu-Ji-Nu" in traditional Chinese medicine (TCM), has been used to treat many inflammatory diseases, including enteritis, acute icteric hepatitis, rheumatism, toothache, tonsillitis, and chronic bronchitis, for centuries. Our preliminary studies have demonstrated that the ethanolic extract of A. anomala (EAA) might be with the potential of inhibiting the activation of the NLRP3 inflammasome. However, the anti-inflammatory activity of EAA based on NLRP3 inflammasome inhibition is still unclear. PURPOSE: This work aimed to elucidate the anti-inflammatory mechanism of EAA by inhibiting NLRP3 inflammasome activation. METHODS: Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) were used to evaluate the inhibitory effects on NLRP3 inflammasome activation. The level of IL-1ß was determined by ELISA. The expression levels of IL-1ß, caspase-1, NLRP3, and ASC were assayed using western blot analysis. ASC oligomerization and speck formation were detected by immunofluorescence microscopy. The measurements of intracellular chloride and potassium were conducted using N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) probe assay and inductively coupled plasma-optical emission spectrometry (ICP-OES), respectively. Mitochondrial reactive oxygen species (mtROS) were examined using the MitoSOX method. Acridine orange (AO) staining was used to detect the permeability of the lysosomal membrane. A DSS-induced ulcerative colitis model was established to evaluate the anti-inflammatory effects of EAA in vivo. Finally, high-performance liquid chromatography (HPLC) was employed to identify and quantify the major constituents of EAA. RESULTS: In BMDMs, EAA significantly inhibited the release of IL-1ß induced by LPS. The mechanistic study revealed that EAA inhibited NLRP3 inflammasome activation by blocking the oligomerization of ASC and suppressed the LPS-induced priming step. Furthermore, EAA protected lysosomes by inhibiting the TAK1-JNK pathway, thereby inhibiting the assembly of downstream NLRP3 inflammasome and the production of IL-1ß. In addition, EAA exerted potent protective effects in an ulcerative colitis model by decreasing the content of colonic IL-1ß and alleviating the process of ulcerative colitis. HPLC analysis identified eight main components of EAA, including isofraxidin (1), quercetin-7-O-ß-D-glucopyranoside (2), apigenin-7-O-ß-D-glucopyranoside (3), 7-methoxycoumarin (4), quercetin (5), luteolin (6), kaempferol (7), and eupatorin (8), Of these compounds, quercetin and kaempferol were found to be the most potent ingredients. CONCLUSION: These findings collectively reveal that EAA exerts anti-inflammatory effects by both suppressing the NLRP3 priming step and protecting lysosomes to inhibit NLRP3 inflammasome activation, suggesting that this traditional herbal medicine might be used to treat NLRP3-driven inflammatory diseases.

Synthesis and evaluation of new compounds bearing 3-(4-aminopiperidin-1-yl)methyl magnolol scaffold as anticancer agents for the treatment of non-small cell lung cancer via targeting autophagy.

Magnolol and honokiol are the two major active ingredients with similar structure and anticancer activity from traditional Chinese medicine Magnolia officinalis, and honokiol is now in a phase I clinical trial (CTR20170822) for advanced non-small cell lung cancer (NSCLC). In search of potent lead compounds with better activity, our previous study has demonstrated that magnolol derivative C2, 3-(4-aminopiperidin-1-yl)methyl magnolol, has better activity than honokiol. Here, based on the core of 3-(4-aminopiperidin-1-yl)methyl magnolol, we synthesized fifty-one magnolol derivatives. Among them, compound 30 exhibited the most potent antiproliferative activities on H460, HCC827, H1975 cell lines with the IC50 values of 0.63-0.93 µM, which were approximately 10- and 100-fold more potent than those of C2 and magnolol, respectively. Besides, oral administration of 30 and C2 on an H460 xenograft model also demonstrated that 30 has better activity than C2. Mechanism study revealed that 30 induced G0/G1 phase cell cycle arrest, apoptosis and autophagy in cancer cells. Moreover, blocking autophagy by the autophagic inhibitor enhanced the anticancer activity of 30in vitro and in vivo, suggesting autophagy played a cytoprotective role on 30-induced cancer cell death. Taken together, our study implied that compound 30 combined with autophagic inhibitor could be another choice for NSCLC treatment in further investigation.

Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra.

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1ß while pulvones C showed better suppression effect on IL-1ß production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKß kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKß kinase.

Identification, characterization and HPLC quantification of formulation-related impurities of honokiol, an antitumor natural drug candidate in clinical trials.

Natural products and their derivatives have historically been invaluable as a source of therapeutic agents. Honokiol, as a well-known natural product in Chinese herbal medicine Houpu, is finally being studied in a Phase I clinical trial (CTR20170822) in patients with Advanced Non-Small Cell Lung Cancer (NSCLS) in China this year. During the honokiol liposome formulation process, five major impurities were present in the range of 0.05-0.1% based on the HPLC analysis. These five major impurities were obtained from the forced degradation product of honokiol through countercurrent chromatography and prep-HPLC. The structure were elucidated with 1H NMR, 13C NMR, 2D NMR and MS spectral data. The proposed HPLC method was validated for specificity, linearity (concentration range 0.01-1.62, 0.003-0.96, 0.05-7.98, 0.04-6.52, 0.03-5.18 µg/ml for impurities I-V respectively, R2 > 0.9988), accuracy (99.11-100.67%), precision (CV < 1.6%), and sensitivity (LOD 3.3, 0.1, 16.7, 13.3, 10.0 ng/ml for impurities I-V respectively). The validated method was employed in the further study of the honokiol drug substance.