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Patients with bladder cancer (BLCA) still show high recurrence after surgery and chemotherapy. Hesperetin (HE), as a natural compound, has attracted researchers' attention due to its low toxicity and easy access. However, the inhibitory effect of HE on BLCA remains unknown. The hub genes and enrichment pathways regulated by HE in the treatment of BLCA were predicted by network pharmacology. The molecular docking of HE and hub proteins was visualized. Colony and CCK8 assays were used to test cell proliferation, and BLCA migration was confirmed by transwell and wound healing assays. In addition, the occurrence of apoptosis and ferroptosis was demonstrated by Hoechst staining, transmission electron microscopy (TEM) and ROS (reactive oxygen species) assay. Western Blotting was performed to validate the hub proteins, target functions and pathways. SRC, PIK3R1 and MAPK1 were identified as hub targets for HE in BLCA, involving the PI3k/AKT pathway. Furthermore, HE inhibited the proliferation and migration of BLCA cells. The MMP2/MMP9 proteins were significantly inhibited by HE. The increased expression of Bax and cleaved caspase-3 indicated that HE could promote BLCA cell apoptosis. In addition, Hoechst staining revealed concentrated and illuminated apoptotic nuclei. The activation of ROS and the decline of GPX4 expression suggested that HE might induce ferroptosis as an anti-BLCA process. Shrunk mitochondria and apoptotic bodies were observed in BLCA cells treated with HE, with reduced or absent mitochondrial cristae. We propose for the first time that HE could inhibit the proliferation and migration of BLCA cells and promote apoptosis and ferroptosis. HE may act by targeting proteins such as SRC, PIK3R1 and MAPK1 and the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Traditional Tibetan medicine has extensively documented the health benefits of Dracocephalum heterophyllum. However, there are few reports on the chemical composition of furanocoumarins, probably because of their complicated isolation and purification procedures. In this study, four antioxidative furanocoumarins were isolated from Dracocephalum heterophyllum by medium‐ and high‐pressure liquid chromatography in combination with on‐line high‐performance liquid chromatography−1,1‐diphenyl‐2‐picrylhydrazyl recognition. Crude samples were sequentially pretreated by medium‐pressure liquid chromatography using silica gel, MCI GEL CHP20P, and diol as stationary phases, whereas on‐line high‐performance liquid chromatography−1,1‐diphenyl‐2‐picrylhydrazyl system was used to recognize antioxidant peaks in target fractions. Thereafter, the antioxidative peaks were separated and purified through high‐pressure liquid chromatography to obtain four furanocoumarins with purities greater than 95%; namely isodemethylfuropinarine, demethylfuropinarine, alloimperatorin, and alloisoimperatorin. Finally, the antioxidant capacity of the isolated furanocoumarins was determined using in vitro experiments (1,1‐diphenyl‐2‐picrylhydrazyl assays, molecular docking, and cellular validation) and it was concluded that nuclear factor erythroid 2‐related factor 2 protein is a potential target of these compounds for their antioxidation effects. Thus, the proposed methodology exhibits excellent efficacy for the preparative isolation of high‐purity antioxidative furanocoumarins from extracts of Dracocephalum heterophyllum and it can be efficiently utilized for isolating antioxidants from other natural products. [ABSTRACT FROM AUTHOR]

Dracocephalum heterophyllum Benth is well-known for its ability to alleviate liver heat. In this study, Dracocephalum heterophyllum Benth ethyl acetate extracts were evaluated on mouse models of nonalcoholic steatohepatitis and liver fibrosis. After 6 and 8 weeks of treatment, serum parameters and gene expressions in tissue samples, as well as stained tissue sections, demonstrated that the ethyl acetate extracts were effective in treating these liver diseases. The principal bioactive constituent (rosmarinic acid) was identified and screened by high pressure liquid chromatography-1,1-diphenyl-2-picrylhydrazyl and affinity ultrafiltration-HPLC. The rosmarinic acid was separated from extracts with high purity by medium- and high-pressure liquid chromatography. Finally, the interactions between rosmarinic acid and the key targets of lipid metabolism, oxidative stress and inflammation were verified by molecular docking. Thereby, an indirect regulation of lipid and cholesterol metabolism and inhibition of liver inflammation and liver fibrosis by the studied extract has been observed. This study demonstrated that Dracocephalum heterophyllum Benth ethyl acetate extracts have the potential to treat nonalcoholic steatohepatitis and liver fibrosis, revealing their multi-target and multi-pathway therapeutic characteristics. [ABSTRACT FROM AUTHOR]

The health benefits of Dracocephalum heterophyllum are widely reported in traditional Tibetan medicines, but the reported chemical composition is limited, probably due to difficulties in separating and purifying compounds. In this study, antioxidative phenylethanoids were isolated from an extract of Dracocephalum heterophyllum using medium- and high-pressure liquid chromatography, coupled with on-line HPLC–1,1-diphenyl-2-picrylhydrazyl recognition. Firstly, crude samples (1.3 kg) of Dracocephalum heterophyllum were pretreated via silica gel medium-pressure liquid chromatography to yield 994.0 g of Fr2, of which 10.8 g was then pretreated via MCI GEL®CHP20P medium-pressure liquid chromatography. The resulting Fr23 and Fr25 were further separated and purified using high-pressure liquid chromatography, and yielded 8.08 mg of Fr2391, 9.76 mg of Fr2551, 16.09 mg of Fr2581, and 8.75 mg of Fr2582. Furthermore, analysis of the purity and structures of the phenylethanoids suggested that Fr2391, Fr2551, Fr2581, and Fr2582 corresponded to decaffeoylverbascoside, rosmarinic acid, acteoside, and 2′-O-acetylplantamajoside, respectively, with all being over 95% pure. Finally, the antioxidant potential of the compounds was explored based on their ability to scavenge 1,1-diphenyl-2-picrylhydrazine, as well as through molecular docking of proteins related to antioxidant pathways. Altogether, our findings revealed that the proposed method is promising for separating pure antioxidative phenylethanoids from other natural compounds. [ABSTRACT FROM AUTHOR]