Fifteen unreported compounds in Anemarrhena asphodeloides, iriflophene (3), hostaplantagineoside C (7), tuberoside G (8), spicatoside B (9), platycodin D (14), platycoside A (15), platycodin D2 (16), polygalacin D2 (17), platycodin D3 (18), isovitexin (20), vitexin (21), 3,4-dihydroxyallylbenzene-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (22), iryptophan (24), adenosine (25), α-d-Glucose monoallyl ether (26), together with eleven known compounds (1, 2, 4–6, 10–13, 19 and 23), were isolated from the rhizomes of Anemarrhena asphodeloides. The chemical structures of these compounds were characterized using HRMS and NMR. The anti-inflammatory activities of the compounds were evaluated by investigating their ability to inhibit LPS-induced NO production in N9 microglial cells. Timosaponin BIII (TBIII) and trans-hinokiresinol (t-HL) exhibited significant inhibitory effects on the NO production in a dose-dependent manner with IC₅₀ values of 11.91 and 39.08 μM, respectively. Immunoblotting demonstrated that TBIII and t-HL suppressed NO production by inhibiting the expressions of iNOS in LPS-stimulated N9 microglial cells. Further results revealed that pretreatment of N9 microglial cells with TBIII and t-HL attenuated the LPS-induced expression tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) at mRNAs and protein levels. Moreover, the activation of nuclear factor-κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were inhibited by TBIII and t-HL, respectively. Our findings indicate that the therapeutic implication of TBIII and t-HL for neurogenerative disease associated with neuroinflammation.

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D₁ receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D₁ receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca²⁺ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

The objective of our present work was to explore the possible enhanced anti-neuroinflammatory ability of Aspergillus oryzae fermented hemp seed in lipopolysaccharide (LPS)-stimulated N9 microglial cells and elucidate its underlying mechanism. The water extract of hemp seed was fermented by Aspergillus oryzae. LPS-stimulated N9 microglial cells were employed for the inflammatory cell model. The release of nitric oxide (NO) was determined by Griess assay. The cytokines and inflammatory mediator expression were measured by qPCR and ELISA. The phosphorylated key signaling proteins, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K/Akt), were quantified by western blot analysis. The production of intracellular reactive oxygen species (ROS) was measured by DCFH oxidation. Fermented hemp seed (FHS) reduced NO production by downregulating inducible nitric oxide synthase (iNOS) expression in LPS-stimulated N9 microglial cells. FHS treatment decreased LPS-stimulated expression of inflammatory cytokines either on mRNA or protein levels. Moreover, FHS inhibited LPS-stimulated phosphorylation of NF-κB, MAPKs, and PI3K/Akt signaling pathways. Furthermore, FHS significantly reduced the ROS production in the cells. It was concluded that FHS exerted its anti-neuroinflammatory activities by suppressing ROS production, thus inhibiting NF-κB, MAPKs, and PI3K/Akt activation, consequently decreasing the expression levels of inflammatory mediators and cytokines.

The identification of exogenous ligands from natural products is an alternative strategy to explore the unrevealed physiological functions of orphan G-protein-coupled receptors (GPCRs). In this study, we have successfully identified and pharmacologically characterized licoisoflavone A (LIA) as a novel selective antagonist of BRS-3, an orphan GPCR. Functional studies showed that pretreatment with LIA ameliorated hydrogen peroxide (H₂O₂)-induced cardiomyocyte injury. Furthermore, LIA pretreatment significantly restored the activities of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), as well as lactate dehydrogenase (LDH) levels, in H9c2 cells following H₂O₂ exposure. The protective effect of LIA was also evident in primary cardiomyocytes from rats and mice against H₂O₂-induced cell injury but was absent in primary cardiomyocytes derived from bombesin receptor subtype-3 knockout (Brs3^−/y) mice, strongly confirming the mechanism of LIA’s action through BRS-3 antagonism. Proteomics studies further revealed that LIA exerted its protective effects via activating the integrin/ILK/AKT and ERK/MAPK signaling pathways. Complementary findings from Bantag-1, a well-recognized antagonist of BRS-3, in human embryonic kidney 293 mBRS-3 (HEK293-mBRS-3) stable cells and B16 cell lines, which demonstrated resistance to H₂O₂-induced damage, further supported the pivotal role of BRS-3 in oxidative stress-induced cell injury. Our study contributes to expanding our understanding of the potential pharmacological functions of BRS-3, unveiling previously unknown pharmacological functionality of this orphan receptor.

• BRS3 and EP3 form dimers on HEK-293T cell membranes upon their co-expression. • BRS3 and EP3 crosstalk switches to couple Gα_s protein and induce P38 phosphorylation. • BRS3 suppresses PGE2/EP3-mediated F-actin rearrangement and migration of B16 melanoma cells.