[Difference in liver injury induced by dictamnine between males and females: based on untargeted metabolomics].

In recent years, reports of adverse reactions related to traditional Chinese medicine(TCM) have been on the rise, especially some traditionally considered "non-toxic" TCM(such as Dictamni Cortex). This has aroused the concern of scholars. This study aims to explore the metabolomic mechanism underlying the difference in liver injury induced by dictamnine between males and females through the experiment on 4-week-old mice. The results showed that the serum biochemical indexes of liver function and organ coefficients were significantly increased by dictamnine(P<0.05), and hepatic alveolar steatosis was mainly observed in female mice. However, no histopathological changes were observed in the male mice. Furthermore, a total of 48 differential metabolites(such as tryptophan, corticosterone, and indole) related to the difference in liver injury between males and females were screened out by untargeted metabolomics and multivariate statistical analysis. According to the receiver operating characteristic(ROC) curve, 14 metabolites were highly correlated with the difference. Finally, pathway enrichment analysis indicated that disorders of metabolic pathways, such as tryptophan metabolism, steroid hormone biosynthesis, and ferroptosis(linoleic acid metabolism and arachidonic acid metabolism), may be the potential mechanism of the difference. Liver injury induced by dictamnine is significantly different between males and females, which may be caused by the disorders of tryptophan metabolism, steroid hormone biosynthesis, and ferroptosis pathways.

Psoraleae Fructus Ethanol Extract Induced Hepatotoxicity via Impaired Lipid Metabolism Caused by Disruption of Fatty Acid ß-Oxidation.

Herb-induced liver injury (HILI) is gradually increasing, and Psoraleae Fructus (PF) has been reported to induce hepatotoxicity. However, its underlying toxicity mechanism has been only poorly revealed. In this paper, we attempted to explore the liver injury and mechanism caused by Psoraleae Fructus ethanol extract (PFE). First, we administered PFE to mice for 4 weeks and evaluated their serum liver function indices. H&E staining was performed to observe the pathological changes of the livers. Oil red O staining was used to visualize hepatic lipids. Serum-untargeted metabolomics and liver proteomics were used to explore the mechanism of PF hepatotoxicity, and transmission electron microscopy was determined to assess mitochondria and western blot to determine potential target proteins expression. The results showed that PFE caused abnormal liver biochemical indicators and liver tissue injury in mice, and there was substantial fat accumulation in liver tissue in this group. Furthermore, metabolomic analysis showed that PFE changed bile acid synthesis, lipid metabolism, etc., and eight metabolites, including linoleic acid, which could be used as potential biomarkers of PFE hepatotoxicity. Proteomic analysis revealed that differential proteins were clustered in the mitochondrial transmembrane transport, the long-chain fatty acid metabolic process and purine ribonucleotide metabolic process. Multiomics analysis showed that eight pathways were enriched in both metabolomics and proteomics, such as bile secretion, unsaturated fatty acid biosynthesis, and linoleic acid metabolism. The downregulation of SLC27A5, CPT1A, NDUFB5, and COX6A1 and upregulation of cytochrome C and ABCC3 expressions also confirmed the impaired fatty acid oxidative catabolism. Altogether, this study revealed that PFE induced hepatotoxicity by damaging mitochondria, reducing fatty acid ß-oxidation levels, and inhibiting fatty acids ingested by bile acids.

Generation of Monoclonal Antibodies Against Natural Products.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

[Comparative study on preparation of Polygoni Multiflori Radix based on hepatotoxic bioassay].

Toxicity of different processed was evaluated Polygoni Multiflori Radix by determining the hepatotoxic potency for selecting processing technology. Process Polygoni Multiflori Radix using high pressure steamed, Black Bean high pressure steamed, atmospheric steamed for different time. Using normal human hepatocytes (L02) as evaluation model, hepatotoxic potency as index to evaluate hepatotoxic potency of different processed Polygoni Multiflori Radix. Analysis chemical composition of some processed products by UPLC-MS. Hepatotoxic bioassay method cloud evaluate the toxicity of different Polygoni Multiflori Radix samples. Different processing methods can reduce the toxicity of Polygoni Multiflori Radix, high pressure steamed three hours attenuated was better. Different processing methods have different effects on chemical constituents of Polygoni Multiflori Radix. Comparing with crude sample, the contents of gallic acid, 2,3,5,4-tetrahydroxyl diphenylethylene-2-O-glucoside, emodin-8-O-beta glucoside and emodin were decreased in processed products with 3 kinds of different methods. The change trend of 2,3,5,4-tetrahydroxyl diphenylethylene-2-O-glucoside content was similar with hepatotoxic potency. Different processing methods can reduce the toxicity of Polygoni Multiflori Radix. Processing methods and time attenuated obvious impact on toxicity. Recommended further research on the attehuated standard control of Polygoni Multiflori Radix concocted.

[Comparison of processed and crude Polygoni Multiflori Radix induced rat liver injury and screening for sensitive indicators].

To investigate the difference of liver injury in rats gavaged with crude and processed Polygoni Multiflori Radix. The 75% ethanol extract of crude and processed Polygoni Multiflori Radix (50 g · kg(-1) crude medicine weight/body weight) were continuous oral administered to rats for 6 weeks. Serum biochemical indicators were dynamically detected, the change of liver histopathology was assessed 6 weeks later. Principal component analysis (PCA) was adopted to screen sensitive indicator of the liver damage induced by polygoni multiflori radix. Biochemical tests showed that the crude Polygoni Multiflori Radix group had significant increase of serum ALT, AST, ALP, DBIL and TBIL (P < 0.01 or P < 0.05) and significant decreases of serum IBIL and TBA (P < 0.01 or P < 0.05), while the processed Polygoni Multiflori Radix group showed no obvious changes, compared to the untreated normal group. Histopathologic analysis revealed that crude Polygoni Multiflori Radix group exhibited significant inflammatory cells infiltration in portal area around the blood vessels, tissue destruction and local necrosis of liver cells. There were not obvious pathological changes in processed Polygoni Multiflori Radix group. The results demonstrated that the injury effect of processed Polygoni Multiflori Radix on liver injury of rats was significantly lower than that of unprocessed, and that processing can effectively reduce the hepatotoxicity of Polygoni Multiflori Radix. Traditional transaminase liver function indicators were not sensitive for crude Polygoni Multiflori Radix induced liver damage. The serum content of DBIL and TBIL can reflect the liver damage induced by crude Polygoni Multiflori Radix early and can be sensitive indicators for clinical monitoring the usage of it.

The protective and toxic effects of rhubarb tannins and anthraquinones in treating hexavalent chromium-injured rats: the Yin/Yang actions of rhubarb.

Chromium nephrotoxicity (CrNT) is thought to occur through the oxidant lesion mechanism. There is still a lack of specific remedies against CrNT. We primarily screened Chinese herbal medicines with a potential protective effect against CrNT, e.g., rhubarb (Rheum palmatum L.). However, the active constituents in rhubarb and its mechanisms remain unclear. In this study, the total rhubarb extract (TR) was successively separated into three parts: total anthraquinone extract (TA), total tannin extract (TT) and remaining component extract (RC). The effects of each extract on the potassium dichromate (K(2)Cr(2)O(7))-induced nephrotoxicity in rats were comparatively assessed. The results showed that only the administration of TT protected the kidney function in K(2)Cr(2)O(7)-injured rats. Besides, TT showed significant activity to scavenge hydroxyl radicals, which is considered to be the dominant lesion product generated by hexavalent chromium. TT also showed a reduced ability to transform toxic high valence chromium ions into non-toxic low valence ions. And TT was able to further precipitate chromium ions. These results suggested that rhubarb tannins treat CrNT as a free radical scavenger, reductant, and metal precipitant. The multiple protective routes of the plant tannins reveal a superior option for development into a promising natural remedy against CrNT. In addition, the opposite effects of rhubarb anthraquinones in treating CrNT were observed compared to rhubarb tannins, which suggested the duo-directional effects (Yin and Yang) of herbal medicines should be addressed.