Qualitative and quantitative analysis of chemical components in Qianggan capsule by UHPLC-Q-TOF-MS/MS and LC-sMRM.

Qianggan capsule (QGC) is a complex preparation composed of 16 traditional Chinese medicines (TCM) that can clear heat and dampness, fortify the spleen and blood, typify qi and relieve depression. However, the chemical composition of QGC remains incompletely understood, despite its clinical use in treating chronic hepatitis and liver injury. The objective of this study was to explore the quality markers of QGC through qualitative and quantitative analysis of its chemical components. First, the chemical composition of QGC was qualitatively analyzed using UHPLC-Q-TOF-MS/MS. Subsequently, the LC-sMRM method was developed and optimized to accurately quantify various chemical components of 10 batches of QGC. Finally, the variations in chemical components between batches were analyzed via multivariate statistical analysis. UHPLC-Q-TOF-MS/MS analysis revealed 167 chemical constituents in QGC, comprised of 48 flavonoids, 32 terpenoids, 18 phenolic acids, 9 coumarins, 9 phenylpropanoids, and 51 nucleosides, sugars, amino acids, anthraquinones, and other compounds. The LC-sMRM method was established for the quantitative analysis of 42 chemical components in 10 batches of QGC. The ultrasonic-assisted extraction parameters were optimized using RSM. Compared with conventional MRM, sMRM demonstrated superior sensitivity and precision. PCA and OPLS-DA identified eight chemical components with content differences among batches. This study established the chemical composition of QGC, offering useful guidance for assessing its quality.

Metabolic activation and cytochrome P450 inhibition of piperlonguminine mediated by CYP3A4.

Piperlonguminine (PLG) is a major alkaloid found in Piper longum fruits. It has been shown to possess a variety of biological activities, including anti-tumor, anti-hyperlipidemic, anti-renal fibrosis and anti-inflammatory properties. Previous studies have reported that PLG inhibits various CYP450 enzymes. The main objective of this study was to identify reactive metabolites of PLG in vitro and assess its ability to inhibit CYP450. In rat and human liver microsomal incubation systems exposed to PLG, two oxidized metabolites (M1 and M2) were detected. Additionally, in microsomes where N-acetylcysteine was used as a trapping agent, N-acetylcysteine conjugates (M3, M4, M5 and M6) of four isomeric O-quinone-derived reactive metabolites were found. The formation of metabolites was dependent on NADPH. Inhibition and recombinant CYP450 enzyme incubation experiments showed that CYP3A4 was the primary enzyme responsible for the metabolic activation of PLG. This study characterized the O-dealkylated metabolite (M1) through chemical synthesis. The IC50 shift assay showed time-dependent inhibition of CYP3A4, 2C9, 2E1, 2C8 and 2D6 by PLG. This research contributes to the understanding of PLG-induced enzyme inhibition and bioactivation.

Exposure to BDE-47 and BDE-209 impaired antioxidative defense mechanisms in Brachionus plicatilis.

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs) that pose serious challenges to aquatic animals and environments. Compared with BDE-47 which was one of the most toxic congeners known to date, BDE-209 is less toxic with higher abundance in biotic and abiotic samples. In this study, we have explored the effects of BDE-47 and BDE-209 at different concentrations on the radical oxygen species (ROS) levels and the antioxidant defense system of Brachionus plicatilis. Antioxidant indexes were measured, including total protein content (TSP), the activities of antioxidant enzymes, lipid peroxidation and DNA damage. The results indicated that while low concentrations of PBDEs could activate the antioxidant defense mechanisms, prolonged exposure to higher concentrations of PBDEs could impair the antioxidative capacity of B.plicatilis (P < 0.05). The overwhelming of the B.plicatilis antioxidant defense mechanism led to an accumulation of free radicals, resulting in the overactivation of lipid peroxidation and the increased frequency of DNA damage (P < 0.05). By studying the toxicity of PBDEs and the detoxification mechanism of B.plicatilis, our research has revealed useful indexes for detecting and monitoring the level of BDE-47 and BDE-209 in the future. Altogether, this study holds immense value in the field of ecotoxicology and environmental safety and will aid in the proper management of PBDEs pollution.

Feeding behavior toxicity in the marine rotifer Brachionus plicatilis caused by 2,2',4,4'-tetrabromodiphenyl ether (BDE-47): Characteristics and mechanisms.

Polybrominated diphenyl ether contamination in marine environments has received special attention due to its accumulation and magnification in the marine food web and toxicity to organisms. In the present study, a series of short-term toxicological tests were conducted with the marine rotifer Brachionus plicatilis to assess the effects on ingestion and digestive performance after treatment with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) at nonlethal concentrations under controlled laboratory conditions and to analyze the possible mechanism. The results showed that with accumulation in rotifers, BDE-47 caused a significant decline in the filtration and feeding rates in a concentration-dependent manner. Moreover, the activities of amylase (AMS) and protease were affected, indicating that BDE-47 impaired ingestion and digestion efficiency. BDE-47 exposure did not lead to abnormal microstructures in the main digestive tract (e.g., cilia around the corona, mastax, stomach, digestive gland and esophagus), but the gastric parietal cells shrank, suggesting nutritional deficiency. BDE-47 prominently induced the occurrence of irregular mitochondria at the cilia root, and mitochondrial and isocitrate dehydrogenase activity declined, indicating mitochondrial dysfunction. Furthermore, the activity of ATPase, which catalyzes ATP hydrolysis, decreased as the BDE-47 concentration rose, implying that BDE-47 retarded rotifer ATP dynamics, inevitably interfering with cilia movement to ingest food. Additionally, a significant decline in acetylcholine esterase activity was observed, which led to a hindrance in neurotransmission involved in food intake and digestion. Altogether, our results demonstrated that nonlethal concentrations of BDE-47 could induce feeding depression in rotifers, which is mainly attributed to stymied energy metabolism and nerve conduction.

Changes in the immune function of rainbow trout (Oncorhynchus mykiss) provide insights into strategies against BDE-47 stress.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous in marine ecosystems and have been suggested to bioaccumulate in aquatic food webs, with potentially negative impacts on marine organism. In this study, a 21-day experiment was performed under controlled laboratory conditions, in which 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), the most biotoxic PBDE in the marine environment, was fed to rainbow trout (Oncorhynchus mykiss) at concentrations of 50 and 500 ng g-1 in the diet. BDE-47 significantly decreased the specific growth rate of O. mykiss and was highly concentrated in the liver and head kidney, as evidenced by increased bioaccumulation factor (BAF) values. Tissue observation revealed impairment of the microstructure of the head kidney. Important immune factors in the skin, blood and head kidney were significantly inhibited by BDE-47 treatment (p < 0.05), whereas the respiratory burst activity of macrophages was enhanced. Additionally, immune-related genes were strongly downregulated following BDE-47 exposure (p < 0.05). In a bacterial challenge, the treatment groups had much higher mortality than did the control group (p < 0.05). BDE-47 accumulated and impaired immune organs, and the hierarchy of immune responses was impaired, consequently reducing O. mykiss resistance to pathogen invasion.

Toxic effect and mechanism of tris (1,3-dichloro-2-propyl)phosphate (TDCPP) on the marine alga Phaeodactylum tricornutum.

Tris (1,3-dichloro-2-propyl)phosphate (TDCPP) is an organophosphate-based plasticizer and flame retardant with a high production volume. The ubiquitous distribution and persistence of TDCPP in aquatic environment have led to concerns over its possible toxic effects on aquatic organism. However, data regarding the toxicity of TDCPP on algae are limited, and the molecular mechanism remains largely unknown. Therefore, we determined the growth characteristics, physiological changes and transcriptome profiles of Phaeodactylum tricornutum in response to 4 mg L-1 TDCPP for 24 h. TDCPP caused morphological damage and growth inhibition with an EC50 value of 3.71 mg L-1 at 96 h. A decline in pigments and photosynthetic activity was observed, indicating the occurrence of photosynthesis inhibition. Although the activities of both glutathione peroxidase and glutathione reductase were stimulated, oxidative stress was not relieved in the algal cells, as evidenced by the elevated levels of reactive oxygen species and lipid peroxidation. Transcriptomic analyses revealed 3312 differentially expressed genes (DEGs), and photosynthesis was a key target, as genes related to this process were greatly altered under TDCPP stress. Moreover, some DEGs were also enriched in amino acid metabolism, nitrogen metabolism, nucleotide metabolism and lipid metabolism, implying that TDCPP-induced damage towards algae by various pathways. Additionally, several TFs related to stress signaling were differentially expressed, suggesting roles in the TDCPP stress response. The results will provide critical data to understand the ecological risks and toxic mechanism of OPFRs entering into marine habitat.

Evaluation of the toxic response induced by BDE-47 in a marine alga, Phaeodactylum tricornutum, based on photosynthesis-related parameters.

The pollution of polybrominated diphenyl ethers (PBDEs) is becoming a pressing environmental problem in aquatic environments, and its threat to aquatic organism has received much attention. In this study, Phaeodactylum tricornutum was treated with 0.8 and 4 mg L-1 2,2',4,4'-tetrabrominated biphenyl ether (BDE-47), the most toxic PBDEs, for 96 h. BDE-47 inhibited cell growth in a time- and concentration-dependent manner. Observation of cell ultrastructure suggested the damage of the chloroplasts morphology. BDE-47 also decreased the chlorophyll content and the oxygen evolution rate, and altered the performance of photosystems. Transcriptomic analysis revealed differential expression of 62 genes related to photosynthesis in BDE-47 treatments (4 mg L-1) and transcription suppression of 58 genes involved in chlorophyll synthesis, antenna proteins, oxygen evolution, electron transport and downstream carbon fixation, implying potential toxicity targets in cells. Additionally, the levels of reactive oxygen species (ROS) and lipid peroxidation increased under BDE-47 stress and were positively correlated with photosynthesis inhibition. Pretreatment with the ROS scavenger N-acetyl-l-cysteine reduced the extent of inhibition, suggesting that ROS was responsible for these effects. Another experiment with the electron transport chain inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea showed that the generation of ROS was partially blocked, primarily indicating that photosynthetic inhibition induced by BDE-47 contributed to ROS overproduction. Thus, BDE-47 inhibited the photosynthesis by down-regulating the gene expression. This change stimulated ROS production, further leading to chloroplast membrane damage to aggravate this inhibition via a feedback loop. These effects of BDE-47 had adverse outcomes on the entire physiological state and the population growth of the microalgae.

The responses of Oncorhynchus mykiss coping with BDE-47 stress via PXR-mediated detoxification and Nrf2-mediated antioxidation system.

The low brominated polybrominated diphenyl ether (PBDE) 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is ubiquitous in the marine environment. To elucidate the stress response and possible mechanisms underlying BDE-47, the rainbow trout fish Oncorhynchus mykiss were selected and orally fed bait with BDE-47 concentrations of 50 ng/g and 500 ng/g. BDE-47 was found to be mainly accumulated in head kidney and caused lipid peroxidation after prolonged exposure. We studied the detoxification system genes pregnane X receptor (PXR) and downstream genes (cytochrome 3 A, CYP3 A; glutathione S-transferase, GST) and their corresponding enzyme activity and found that the above indicators in the treatment groups increased first and then decreased with time, while the 500 ng/g group showed more significant changes. Further, the antioxidant system gene expression levels of the NF-E2-related factor 2 (Nrf2) and downstream genes (superoxide dismutase, SOD; catalase, CAT) were found significantly up-regulated with concentration and time. The change in the enzyme activity of SOD and CAT showed the same tendency as that of indicators of detoxifying system. The results showed that BDE-47 can accumulated in head kidney and caused activate and fast increase of genes and enzymes of detoxification and antioxidant system in the short-term and then damage the response systems in longer times. After Pearson correlation analysis, the Integrated Biomarker Response (IBR) Index was established with malondialdehyde (MDA) content; PXR, Nrf2, SOD, and CAT gene expression; and CYP3 A, GST, and CAT enzymatic activity, which were significantly related to BDE-47 bioaccumulation (P < 0.5). The IBR value can indicate the ecotoxicological responses of the head kidney to different BDE-47 concentrations exposure, but the high activity of the antioxidant system might obscure the damage of the detoxification system.

Responses of the rotifer Brachionus plicatilis to flame retardant (BDE-47) stress.

A series of short-term toxicological tests were conducted on the rotifer Brachionus plicatilis to assess the toxicity of the flame retardant 2,2',4,4'-tetrabrominated biphenyl ether (BDE-47). BDE-47 increased mortality, morphological damage, and altered population dynamics and fecundity of rotifer. Antioxidant enzymes were differentially changed to maintain the balance between antioxidant and pro-oxidant activity. However, with increases in the concentration of BDE-47, the metabolic and antioxidant activity decreased. Moreover, the reactive oxygen species (ROS) and malondialdehyde contents increased and the ratio between glutathione and glutathione-SH decreased, indicating oxidative stress. The addition of the ROS-inhibitor N-acetylcysteine alleviated the degree of damage and stimulated the activity of xenobiotic-metabolizing and antioxidant system, which suggested that ROS were the most important loop in the stress response.