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Background: Activation of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in gout. Selaginella moellendorffii has been confirmed effective for the treatment of gout in hospital preparations. Flavonoids, such as amentoflavone (AM), are the main active components of this medicine.Purpose: We aimed to investigate the flavonoid extract (TF) and AM's effects on NLRP3 inflammasome in vitro and their preventive effects on gout in vivo.Methods: LC-MS method was employed to investigate the chemical profile of TF. The cellular inflammation model was established by lipopolysaccharide (LPS) or monosodium urate (MSU) stimulation. The cell membrane integrality and morphological characteristics were determined by using Lactate dehydrogenase (LDH) assay kits, propidium iodide (PI) stain, and scanning electron microscopy (SEM). The inflammatory cytokines and NLRP3 inflammasome activation were determined using enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (RT-PCR), immunofluorescence staining, and western blotting. The acute gout mouse model was induced by MSU injection into footpads, and then the paw edema, inflammatory mediators, and histological examination (HE) were analyzed.Results: The main constituents in TF are AM and robustaflavone. In the cellular inflammation model, TF down-regulated the levels of nitric oxide (NO), TNF-α, and LDH, suppressed NLRP3 inflammasome-derived interleukin-1β (IL-1β) secretion, decreased caspase-1 activation, repressed mature IL-1β expression, inhibited ASC speck formation and NLRP3 protein expression. In an acute gout mouse model, oral administration of TF to mice effectively alleviated paw edema, reduced inflammatory features, and decreased the levels of IL-1β in mouse foot tissue. Similarly, the characteristic constituent AM was also able to down-regulated the levels of NO, TNF-α, and LDH, down-regulate the mRNA expression of IL-1β, TNF-α, caspase-1, and NLRP3. Besides, the foot thickness, lymphocyte infiltration, and IL-1β level were also prevented by AM.Conclusion: The results indicated that TF and its main constituent AM alleviate gout arthritis via NLRP3/ASC/Caspase-1 axis suppression.

Seed is not only the main reproductive organ of most of herbal plants but also an important part of Traditional Chinese Medicine (TCM). Seed TCMs possess important medicinal properties and have been widely used as components of pharmaceutical products. In parallel with the increasing popularity and accessibility of seeds as medicinal products in recent years, numerous substitutes and adulterants have also appeared on the market. Due to the small volume and similar appearances of many seed TCMs, they are very difficult to accurately identify the constituent plant species through organoleptic methods. Usage of the wrong herb may be ineffective or may worsen the condition and even cause death. Correct identification of seed herbal medicines is therefore essential for their safe use. Here, we acquired 177 ITS2 sequences and 15 psbA-trnH sequences from 51 kinds of seed TCMs belonging to 64 species that have been described in the Chinese Pharmacopoeia. Tree-building analysis showed that the ITS2 sequences of 48 seed TCMs can be differentiated from each other, and they formed distinct, non-overlapping groups in the maximum-likelihood tree. Furthermore, all of the sequences acquired in this study have been submitted to the public DNA barcoding system for herbal medicine, and this integrated database was used to identify 400 seed TCM samples purchased from medicinal markets, drug stores, and the Internet, enabling the identification of 7.5% of the samples as containing non-declared species. This study provides a brief operating procedure for the identification of seed TCMs found in herbal medicine. In the future, researchers and traditional herbal medicine enterprises can use this system to test their herbal materials.