Different behavior of food-related benzoic acids toward iron and copper.

Benzoic acids, which are commonly found in food, are also produced by human microbiota from other dietary phenolics. The aim was to investigate the interactions of 8 food-related benzoic acids with the physiological metals iron and copper under different (patho)physiologically relevant pH conditions in terms of chelation, reduction, impact on the metal-based Fenton chemistry, and copper-based hemolysis. Only 3,4-dihydroxybenzoic acid behaved as a protective substance under all conditions. It chelated iron, reduced both iron and copper, and protected against the iron and copper-based Fenton reaction. Conversely, 2,4,6-trihydroxybenzoic acid did not chelate iron and copper, reduced both metals, potentiated the Fenton reaction, and worsened copper-based hemolysis of rat red blood cells. The other tested compounds showed variable effects on the Fenton reaction. Interestingly, prooxidative benzoic acids mildly protected human erythrocytes against Cu-induced lysis. In conclusion, 3,4-dihydroxybenzoic acid seems to have a protective effect against copper and iron-based toxicity under different conditions.

Developing an off-on fluorescence sensor based on red copper nanoclusters wrapped by sulfhydryl and polymer double ligands for sensitive detection of N-acetyl-L-cysteine.

N-acetyl-L-cysteine (NAC) as a class of thiols is commonly used in the treatment of lung diseases, detoxification and prevention of liver damage. In this paper, 4-mercaptobenzoic acid (4-MBA) coated and polyvinylpyrrolidone (PVP) attached copper nanoclusters (4-MBA@PVP-CuNCs) were successfully synthesized using a simple one-pot method with an absolute quantum yield of 10.98 %, and its synthetic conditions (like effects of single/double ligands and temperature) were studied intensively. Then Hg2+ could quench the fluorescence of the 4-MBA@PVP-CuNCs and its fluorescence was restored with the addition of NAC. Based on the above principles, an off-on switching system was established to detect NAC. That is, the 4-MBA@PVP-CuNCs-Hg probe was prepared by adding Hg2+ to switch off the fluorescence of the CuNCs by static quenching, and then NAC was added to switch on the fluorescence of the probe based on the chelation of NAC and Hg2+. Moreover, the effects of metal ion types and mercury ion doses for the probe construction were also further discussed. The method showed excellent linearity in the range of 0.05-1.25 µM and low detection limit of 16 nM. Meanwhile, good recoveries in real urine, tablets and pellets were observed, which proved the reliability of the method and provided a convenient, fast and sensitive method for NAC detection.

Enhancing the sensitivity of immunomagnetic assay for 3-phenoxybenzonic acid: a novel approach utilizing magnetosome-nanobody complexes and gold nanoparticles probes.

The 3-phenoxybenzoic acid (3-PBA) residues in environment are posing a significant challenge to our daily lives. To establish a more sensitive and rapid detection method, anti-3-PBA nanobodies (Nbs) were immobilized onto magnetosomes (bacterial magnetic nanoparticles, termed as BMPs), forming a robust BMP-Nb complex. The 3-PBA derivative was labeled with horseradish peroxidase (HRP) and further associated with gold nanoparticles (AuNPs) to create a highly sensitive probe (3-PBA-HRP-AuNP). An innovative immunoassay that combined BMP-Nb complex with 3-PBA-HRP-AuNP was developed for determinaton of 3-PBA. This method enabled the determination of 3-PBA with a half-maximum signal inhibition concentration (IC50) of 1.03 ng/mL, which was more sensitive than that of using 3-PBA-HRP as tracer with an IC50 of 2.18 ng/mL. The reliability of the assay was evidenced by the quantitative recovery of 3-PBA from water and soil samples ranging from 76.85 to 95.64%. The 3-PBA residues determined by this assay in actual water samples were between < LOD and 2.54 ng/mL and were between < LOD and 11.25 ng/g (dw) in real soils, respectively, which agreed well with those of liquid chromatography mass spectrometry (LC-MS). Collectively, the BMP-Nb and 3-PBA-HRP-AuNP-based immunoassay provides a powerful tool for the precise detection of 3-PBA residues in environment matrices, reinforcing our capacity to monitor and mitigate potential ecological and health impacts associated with this prevalent pollutant.

Development of Nitric Oxide-Donating Netarsudil Derivatives as a Synergistic Therapy for Glaucoma with Reduced Ocular Irritation.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a series of NO-donating Netarsudil derivatives were designed, synthesized, and their activities in vitro and in vivo were evaluated. Among them, (S)-10e released an appropriate amount of NO in aqueous humor in vitro and displayed potent ROCK inhibition. Topical administration of (S)-10e significantly lowered intraocular pressure in an acute ocular hypertension rabbit model and protected retinal ganglion cells in a magnetic microbead occlusion mouse model. A metabolism investigation revealed that (S)-10e released 7a, a metabolite after NO releasing, and 13, an active metabolite of (S)-Netarsudil, in rabbit eyes. Notably, introducing an NO donor moiety attenuated ROCK inhibition-induced ocular irritation in an sGC-independent manner, suggesting that the attenuated conjunctival hyperemia effect of (S)-10e is related to the NO-induced protein S-nitrosation of phosphodiesterase 3A (PDE3A). Overall, (S)-10e is a promising candidate for glaucoma treatment.

A Luminescent MOF-Based Sensor for Monitoring of an Anticancer Drug and a Pyrethroid Fungicide Biomarker in Wastewater and Biological Fluids.

The extensive use of insecticides, such as pyrethroids, and pharmaceutical drugs, such as doxorubicin (DOX) has significantly increased to meet the growing demand for food production and disease treatment. Among them, 3-phenoxybenzoic acid (3-PBA), a metabolite of pyrethroid insecticides, poses various health and environmental risks. Similarly, DOX is a well-known anticancer drug and has been continuously used for many years. The high demand and unregulated disposal of these substances raise concerns for both humans and the environment. To address this issue, there is a pressing need to monitor the presence of these analytes in wastewater to protect our ecosystems. This challenge has inspired us to develop an MOF-based fluorometric dual sensor capable of rapid and selective detection of these analytes in aqueous solutions. This work represents the first MOF-based dual probe for detecting these targeted analytes. There was a 98% fluorescence quenching upon the introduction of DOX whereas about a 11-fold increment of the probe's fluorescence intensity took place in the presence of 3-PBA. The sensitivity of the probe is notably high as limits of detection (LOD) are 8.7 nM for DOX and 1.2 nM for 3-PBA. Our designed probe has the highest KSV value for DOX which is 3.37 × 106 M-1. The MOF demonstrated remarkable rapid response time of just 5 and 10 s for DOX and 3-PBA, respectively. The MOF exhibited outstanding selectivity in detecting DOX and 3-PBA, even when other interfering substances were present. We tested the probe's sensing abilities in various environments, such as serum, urine, wastewater, and different pH levels. These findings underscore the sensor's practicality and usefulness in real-world applications. The underlying mechanisms driving the sensing processes were thoroughly investigated by using various modern analytical methods.

Prediction of Degradation Profiles for Various Sartans under Solvent-Free Mechanochemical Conditions.

For the approval of a drug, the stability data must be submitted to regulatory authorities. Such analyses are often time-consuming and cost-intensive. Forced degradation studies are mainly carried out under harsh conditions in the dissolved state, often leading to extraneous degradation profiles for a solid drug. Oxidative mechanochemical degradation offers the possibility of generating realistic degradation profiles. In this study, a sustainable mechanochemical procedure is presented for the degradation of five active pharmaceutical ingredients (APIs) from the sartan family: losartan potassium, irbesartan, valsartan, olmesartan medoxomil, and telmisartan. High-resolution mass spectrometry enabled the detection of impurities already present in untreated APIs and allowed the elucidation of degradation products. Significant degradation profiles could already be obtained after 15-60 min of ball milling time. Many of the identified degradation products are described in the literature and pharmacopoeias, emphasizing the significance of our results and the applicability of this approach to predict degradation profiles for drugs in the solid state.

The Nicotiana tabacum UGT89A2 enzyme catalyzes the glycosylation of di- and trihydroxylated benzoic acid derivatives.

Glycosyltransferases catalyze the transfer of a glycoside group to a wide range of acceptor compounds to produce glycoconjugates with diverse biological and pharmacological activities. The present work reports the identification and biochemical characterization of Nicotiana tabacum UGT89A2 glycosyltransferase (NtUGT89A2). The enzyme is a monomer in solution that catalyzes the O-ß-glucosylation of di- and tri-hydroxylated and chlorinated derivatives of benzoic acid. NtUGT89A2 has a preference for 2,5-dihydroxybenzoic acid (2,5-DHBA) over 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,4-dihydroxybenzoic acid (2,4-DHBA). Other substrates that can be used by NtUGT89A2 include 3,4,5-trihydroxybenzoic acid and chlorinated derivatives such as 2-chloro-5-hydroxybenzoic acid (2-Cl-5-HBA). The substrates of NtUGT89A2 were identified by thermal stability experiments, where we observed a maximum increase of the thermal denaturation midpoint (Tm) of 10 °C in the presence of 2,5-DHBA and UDP-glucose. On the other hand, the highest specific activity was obtained with 2,5-DHBA (225 ± 1.7 nkat/mg). Further characterization revealed that the enzyme has a micromolar affinity for its substrates. Notably, the enzyme retains full activity after incubation at 70 °C for 1 h. These results provide a basis for future functional and structural studies of NtUGT89A2.

Benzoate glycosides from Gentiana scabra Bge. and their lipid-lowering activity.

Seven undescribed benzoate glycosides (1-7) and five known ones (8-12) were isolated from the rhizomes of Gentiana scabra Bge. Their structures were characterized by comprehensive NMR and MS spectroscopic data analysis. The lipid-lowering effects of these compounds were evaluated by measuring the triglyceride (TG) contents and intracellular lipid droplets (LDs) in oleic acid (OA)-treated HepG2 cells. The results showed that compounds 1, 5, 7, and 11 significantly reduced the TG content at 20 µM, and the Bodipy staining displayed that OA enhanced the levels of LDs in the cell, while these compounds reversed the lipid accumulation caused by OA. These findings provide a basis for further development and utilization of G. scabra as a natural source of potential lipid-lowering agents.

Rapid, Simple and Low-Cost Analytical Method Development for Quantification of Eltrombopag Olamine in Tablet Dosage by UV Spectroscopy Method.

BACKGROUND: Eltrombopag Olamine is a drug used to treat thrombocytopenia, a disorder where blood platelet counts get lower and severe aplastic anemia. It serves as a thrombopoietin receptor agonist, which give rise to platelet production in the bone marrow. OBJECTIVES: The objective of this study is to develop a simple, specific, accurate, precise and economical Ultraviolet spectroscopy method to estimate the amount of Eltrombopag Olamine in bulk and tablet dosage form. METHODS: The developed method was performed using methanol for identification and physicochemical characterization of the drug. The validation parameters like linearity, precision, accuracy, robustness limits of detection and quantitation, and specificity were assessed as per ICH Q2 (R2). RESULTS: The maximum absorbance wavelength (λmax) of the drug was found at 247 nm in methanol. The linearity was found in the concentration range of 2-14 µg/ml with regression equation y = 0.0619x - 0.0123 and r² = 0.999. The standard addition method was used to determine the accuracy of the developed method. The result was found in the % recovery range of 98-99%. The precision was done on λmax with respect to the parameters such as repeatability, intraday, and interday. The method was found to be precise as the % RSD value was found to be <2%. The detection limit value (LOD) and quantitation limit value (LOQ) were 0.0524 µg/ml and 0.1588 µg/ml, respectively. CONCLUSION: The developed method is simple, economical, accurate and selective. The developed method was adaptable for the estimation of Eltrombopag Olamine analysis in pharmaceutical dosage form and routine quality control laboratory.

Novel Brassinosteroid Analogues with 3,6 Dioxo Function, 24-Nor-22(S)-Hydroxy Side Chain and p-Substituted Benzoate Function at C-23-Synthesis and Evaluation of Plant Growth Effects.

Brassinosteroids (BRs) are an important group of polyhydroxylated naturally occurring steroidal phytohormones found in the plant kingdom in extremely low amounts. Due to the low concentrations in which these compounds are found, much effort has been dedicated to synthesizing these compounds or their structural analogs using natural and abundant sterols. In this work, we report the synthesis of new brassinosteroid analogs obtained from hyodeoxycholic acid, with a 3,6 dioxo function, 24-Nor-22(S)-hydroxy side chain and p-substituted benzoate function at C-23. The plant growth activities of these compounds were evaluated by two different bioassays: rice lamina inclination test (RLIT) and BSI. The results show that BRs' analog with p-Br (compound 41f) in the aromatic ring was the most active at 1 × 10-8 M in the RLIT and BSI assays. These results are discussed in terms of the chemical structure and nature of benzoate substituents at the para position. Electron-withdrawing and size effects seems to be the most important factor in determining activities in the RLIT assay. These results could be useful to propose a new structural requirement for bioactivity in brassinosteroid analogs.

Rapid intracellular pH measurement based on electroporation- surface-enhanced Raman scattering.

In this study, electroporation-surface-enhanced Raman scattering (SERS) was applied to rapidly measure intracellular pH. The generation of a sensitive SERS probe for measuring pH in the range of 6.0-8.0 was accomplished through the conjugation of the pH-sensitive molecule 4-mercaptobenzoic acid (4-MBA) to the surface of gold nanoparticles (Au NPs) through its thiol functional group. This bioprobe was then rapidly introduced into nasopharyngeal carcinoma CNE-1 cells by electroporation, followed by SERS scanning and the fitting of intensity ratios of each detection point's Raman peaks at 1423 cm-1 and 1072 cm-1, to create the pH distribution map of CNE-1 cells. The electroporation-SERS assay introduces pH bioprobes into a living cell in a very short time and disperses the nanoprobe throughout the cytoplasm, ultimately enabling rapid and comprehensive pH analysis of the entire cell. Our work demonstrates the potential of electroporation-SERS for the biochemical analysis of live cells.

Effects of Raman Labeling Compounds on the Stability and Surface-Enhanced Raman Spectroscopy Performance of Ag Nanoparticle-Embedded Silica Nanoparticles as Tagging Materials.

Surface-enhanced Raman spectroscopy (SERS) tagging using silica(SiO2)@Ag nanoparticles (NPs) is easy to handle and is being studied in various fields, including SERS imaging and immunoassays. This is primarily due to its structural advantages, characterized by high SERS activity. However, the Ag NPs introduced onto the SiO2 surface may undergo structural transformation owing to the Ostwald ripening phenomenon under various conditions. As a result, the consistency of the SERS signal decreases, reducing their usability as SERS substrates. Until recently, research has been actively conducted to improve the stability of single Ag NPs. However, research on SiO2@Ag NPs used as a SERS-tagging material is still lacking. In this study, we utilized a Raman labeling compound (RLC) to prevent the structural deformation of SiO2@Ag NPs under various conditions and proposed excellent SiO2@Ag@RLC-Pre NPs as a SERS-tagging material. Using various RLCs, we confirmed that 4-mercaptobenzoic acid (4-MBA) is the RLC that maintains the highest stability for 2 months. These results were also observed for the SiO2@Ag NPs, which were unstable under various pH and temperature conditions. We believe that SERS tags using SiO2@Ag NPs and 4-MBA can be utilized in various applications on based SERS because of the high stability and consistency of the resulting SERS signal.

Effect of substituents at the C3´, C3´N, C10 and C2-meta-benzoate positions of taxane derivatives on their activity against resistant cancer cells.

We tested the effect of substituents at the (1) C3´, C3´N, (2) C10, and (3) C2-meta-benzoate positions of taxane derivatives on their activity against sensitive versus counterpart paclitaxel-resistant breast (MCF-7) and ovarian (SK-OV-3) cancer cells. We found that (1) non-aromatic groups at both C3´ and C3´N positions, when compared with phenyl groups at the same positions of a taxane derivative, significantly reduced the resistance of ABCB1 expressing MCF-7/PacR and SK-OV-3/PacR cancer cells. This is, at least in the case of the SB-T-1216 series, accompanied by an ineffective decrease of intracellular levels in MCF-7/PacR cells. The low binding affinity of SB-T-1216 in the ABCB1 binding cavity can elucidate these effects. (2) Cyclopropanecarbonyl group at the C10 position, when compared with the H atom, seems to increase the potency and capability of the derivative in overcoming paclitaxel resistance in both models. (3) Derivatives with fluorine and methyl substituents at the C2-meta-benzoate position were variously potent against sensitive and resistant cancer cells. All C2 derivatives were less capable of overcoming acquired resistance to paclitaxel in vitro than non-substituted analogs. Notably, fluorine derivatives SB-T-121205 and 121,206 were more potent against sensitive and resistant SK-OV-3 cells, and derivatives SB-T-121405 and 121,406 were more potent against sensitive and resistant MCF-7 cells. (4) The various structure-activity relationships of SB-T derivatives observed in two cell line models known to express ABCB1 favor their complex interaction not based solely on ABCB1.

Ergosterol and its metabolites as agonists of Liver X receptor and their anticancer potential in colorectal cancer.

Aberrant cholesterol homeostasis is a well-recognized hallmark of cancer and is implicated in metastasis as well as chemotherapeutic resistance, the two major causes of cancer associated mortality. Liver X receptors (LXRs) are the key transcription factors that induce cholesterol efflux via enhancing the expression of ABCA1 and ABCG1. Therefore, a comprehensive analysis of several novel sterols namely ergosta-7,22,24(28)-trien-3ß-ol (Erg1), ergosta-5,22,25-trien-3-ol (Erg2), ergosta-5,7,22,24(28)-tetraen-3ß-ol (Erg3), and ergosta-7,22-dien-3ß-ol (Erg4) as LXR agonists has been performed. Molecular docking studies have shown that these sterols possess higher binding affinities for LXRs as compared to the reference ligands (GW3965 and TO901317) and also formed critical activating interactions. Molecular dynamic (MD) simulations further confirmed that docking complexes made of these sterols possess significant stability. To assess the extent of LXR activation, ABCA1 promoter was cloned into luciferase reporter plasmid and transfected into HCT116 cells. It was observed that treatment with Erg, Erg2 and Erg4 led to a significant LXR activation with an EC50 of 5.64 µM, 4.83 and 3.03 µM respectively. Furthermore, a significant increase in mRNA expression of NR1H2 and LXR target genes i.e. ABCA1, ABCG1 and ApoE was observed upon Erg treatment. Flow cytometric analysis have revealed a significant increase in the accumulation of ABCA1 upon Erg treatment. Cytotoxicity studies conducted on colorectal cancer cell and normal epithelial cell line showed that these sterols are selectively toxic towards cancer cells. Taken together, our findings suggests that ergosterol activates LXRs, have significant anticancer activity and could be a likely candidate to manage aberrant cholesterol homeostasis.

An in silico investigation of the toxicological effects and biological activities of 3-phenoxybenzoic acid and its metabolite products.

We aimed to elucidate the toxic effects and biological activities of 3-phenoxybenzoic acid (3PBA) and its metabolite products.Numerous in silico methods were used to identify the toxic effects and biological activities of 3PBA, including PASS online, molecular docking, ADMETlab 2.0, ADMESWISS, MetaTox, and molecular dynamic simulation.Ten metabolite products were identified via Phase II reactions (O-glucuronidation, O-sulfation, and methylation).All of the investigated compounds were followed by Lipinski's rule, indicating that they were stimulants or inducers of hazardous processes.Because of their high gastrointestinal absorption and ability to reach the blood-brain barrier, the studied compounds' physicochemical and pharmacokinetic properties matched existing evidence of harmful effects, including haematemesis, reproductive dysfunction, allergic dermatitis, toxic respiration, and neurotoxicity.The studied compounds have been linked to the apoptotic pathway, the reproductivity system, neuroendocrine disruptors, phospholipid-translocating ATPase inhibitors, and JAK2 expression.An O-glucuronidation metabolite product demonstrated higher binding affinity and interaction with CYP2C9, CYP3A4, caspase 3, and caspase 8 than 3PBA and other metabolite products, whereas metabolite products from methylation were predominant and more toxic.Our in silico findings partly meet the 3Rs principle by minimizing animal testing before more study is needed to identify the detrimental effects of 3PBA on other organs (liver, kidneys).Future research directions may involve experimental validation of in silico predictions, elucidation of molecular mechanisms, and exploration of therapeutic interventions.These findings contribute to our understanding of the toxicological profile of 3PBA and its metabolites, which has implications for risk assessment and regulatory decisions.

Key properties & pharmacokinetics of 3PBA & its metabolites were reportedMetabolite products from methylation were predominant and more toxicMain toxics: haematemesis, reproductive dysfunction, toxic respiration, dermatitis.

Sensitive SERS assay for L-cysteine based on functionalized silver nanoparticles.

L-cysteine, an indispensable amino acid present in natural proteins, plays pivotal roles in various biological processes. Consequently, precise and selective monitoring of its concentrations is imperative. Herein, we propose a Surface-enhanced Raman Scattering (SERS) sensor for detecting L-cysteine based on the anti-aggregation of 4-mercaptobenzoic acid (4-MBA) and histidine (His) functionalized silver nanoparticles (Ag NPs). The presence of Hg2+ ions can induce the aggregation of Ag NPs@His@4-MBA due to the unique nanostructures of Ag NPs@His@4-MBA, resulting in a robust SERS intensity of 4-MBA. However, in the presence of L-cysteine, the stronger affinity between L-cysteine and Hg2+ reduces the concentration of free Hg2+, causing the dispersion of the aggregated functionalized Ag NPs and the reduction of the SERS signal intensity of 4-MBA. The developed SERS platform demonstrates excellent performance with a low detection limit of 5 nM (S/N = 3) and linear detection capabilities within the range of 0.01-100 µM for L-cysteine. Additionally, the method was successfully employed for the determination of L-cysteine in spiked serum samples, yielding recoveries ranging from 95.0 % to 108.1 % with relative standard deviations of less than 3.3 %. This study not only presents a novel approach for fabricating highly sensitive and specific SERS biosensors for biomolecule detection but also offers a significant strategy for the development and construction of SERS substrates using anti-aggregation design.

Quality by design-aided acid-free synthesis of self P, N, S-doped black seed-derived carbon quantum dots for application as a nanosensor for eltrombopag environmental and bioanalysis and pharmacokinetic assay.

Herein, we developed a rapid, one-step, and cost-effective methodology based on the fabrication of water-soluble self-nitrogen, sulfur, and phosphorus co-doped black seed carbon quantum dots (BSQDs) via microwaveirradiation in six minutes. Our synthesis approach is superior to those in the literature as they involved long-time heating (12 h) with sulfuric acid and sodium hydroxide and/or high temperatures (200 °C). A full factorial design was applied to obtain the most efficient synthesis conditions.BSQDs displayed excitation-independent emissions, demonstrating the purity of the synthesized BSQDs, with a maximum fluorescence at 425 nm after excitation at 310 nm. Eltrombopag olamine is an anti-thrombocytopenia drug that is also reported to cause toxicity in river water based on its Persistence, Bioaccumulation, and Toxicity (PBT). The synthesized BSQDs were employed as the first fluorometric sensor for environmental and bioanalysis of eltrombopag. The fluorescence of BSQDs decreased with increasing concentrations of eltrombopag, with excellent selectivity and sensitivity down to 30 ppb. BSQDs were successfully applied as sensing probes for the detection of eltrombopag in medical tablets, spiked and real human plasma samples, and river water samples, with an overall recovery of at least 97 %. The good tolerance to high levels of foreign components and co-administered drugs indicates good selectivity and versatility of the proposed methodology. Plasma pharmacokinetic parameters such as t1/2, Cmax, and t max of eltrombopag were evaluated to be 9.91 h, 16.0 µg mL-1, and 5 h, respectively. Moreover, the green character of the BSQDs as a sensor was proved by various analytical greenness scales.

UPLC-ESI-Q-TOF-MS/MS based metabolomics investigation on chemical constituent consistency of Zhenwu Decoction before and after compatibility.

Zhenwu Decoction (ZWD), a classic formula from Zhang Zhongjing's "Treatise on Typhoid Fever" in the Han Dynasty, consists of five traditional Chinese medicines: Aconiti Lateralis Radix Praeparata (ALRP), Paeoniae Radix Alba, Poria Cocos, Ginger, and Rhizoma Atractylodis Macrocephalae. To evaluate the chemical constituent consistency of ZWD before and after compatibility, an ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry was established to comprehensively study the constituents of ZWD. By normalizing the peak area, the pairwise compatibility of ALRP and the other four medicinal herbs, as well as the compatibility of the entire formula were studied, respectively. Multivariate statistical analysis was used to identify the differences. The processed data were analyzed by principal component analysis and supervised orthogonal partial least squared discriminant analysis, and an S-plot was generated to compare the differences in the chemical composition of the two types of decoction samples. The results showed that during the decoction process of ZWD, a total of seven components were recognized as differential compounds before and after compatibility of ZWD, namely 6-gingerol, zingerone, benzoylhypaconine, hypaconitine, benzoylaconine, paeoniflorin and fuziline. The results of this study provide basic data reference for understanding the law of ZWD compatibility and are valuable for the compatibility study of other herbal medicines.

Toxic effect and mechanism of ß-cypermethrin and its chiral isomers on HTR-8/SVneo cells.

Beta-cypermethrin (ß-CYP) consists of four chiral isomers, acting as an environmental estrogen and causing reproductive toxicity, neurotoxicity, and dysfunctions in multiple organ systems. This study investigated the toxic effects of ß-CYP, its isomers, metabolite 3-phenoxybenzoic acid (3-PBA), and 17ß-estradiol (E2) on HTR-8/SVneo cells. We focused on the toxic mechanisms of ß-CYP and its specific isomers. Our results showed that ß-CYP and its isomers inhibit HTR-8/SVneo cell proliferation similarly to E2, with 100 µM 1S-trans-αR displaying significant toxicity after 48 h. Notably, 1S-trans-αR, 1R-trans-αS, and ß-CYP were more potent in inducing apoptosis and cell cycle arrest than 1R-cis-αS and 1S-cis-αR at 48 h. AO/EB staining and flow cytometry indicated dose-dependent apoptosis in HTR-8/SVneo cells, particularly at 100 µM 1R-trans-αS. Scratch assays revealed that ß-CYP and its isomers variably reduced cell migration. Receptor inhibition assays demonstrated that post-ICI 182780 treatment, which inhibits estrogen receptor α (ERα) or estrogen receptor ß (ERß), ß-CYP, its isomers, and E2 reduced HTR-8/SVneo cell viability, whereas milrinone, a phosphodiesterase 3 A (PDE3A) inhibitor, increased viability. Molecular docking studies indicated a higher affinity of ß-CYP, its isomers, and E2 for PDE3A than for ERα or ERß. Consequently, ß-CYP, its isomers, and E2 consistently led to decreased cell viability. Transcriptomics and RT-qPCR analyses showed differential expression in treated cells: up-regulation of Il24 and Ptgs2, and down-regulation of Myo7a and Pdgfrb, suggesting the PI3K-AKT signaling pathway as a potential route for toxicity. This study aims to provide a comprehensive evaluation of the cytotoxicity of chiral pesticides and their mechanisms.