A novel electrochemical immunosensor for sensitive detection of depression marker Apo-A4 based on bipyridine-functionalized covalent organic frameworks.

A novel electrochemical immunosensor for detecting potential depression biomarker Apolipoprotein A4 (Apo-A4) was developed using a multi-signal amplification approach. Firstly, the sensor utilized a modified electrode material, NG-PEI-COF, combining bipyridine-functionalized covalent organic framework (COF) and polyethyleneimine-functionalized nitrogen-doped graphene (NG-PEI), providing high surface area and excellent electron transfer capability for the first-stage amplification in electrical signal conduction. Subsequently, gold nanoparticles (AuNPs) were further electrodeposited onto the electrode, providing good biocompatibility and abundant binding sites for immobilizing the target antigen, thus achieving the second-stage amplification in target recognition and binding. To address the lack of redox properties of the antigen, a tracer probe was formed by loading AuNPs, anti-Apo-A4, and toluidine blue (TB) successively onto COF, leading to the third-stage amplification in signal conversion. The constructed electrochemical immunosensor TB/Ab/AuNPs/COF-Apo-A4/AuNPs/NG-PEI-COF/GCE exhibited excellent detection performance against Apo-A4 with a linear range of 0.01 to 300 ng mL-1 and had a low detection limit of 2.16 pg mL-1 (S/N = 3). In addition, the biosensor had good reproducibility (RSD = 2.31%), stability, and significant anti-interference performance toward other depression biomarkers. The sensor has been successfully used for the quantitative detection of Apo-A4 in serum, providing potential applications for detecting Apo-A4 in the clinic and serving as a reference for constructing sensing methods based on COF.

Development of a dual-template molecularly imprinted electrochemical sensor for the simultaneous detection of depression markers 5-HT and Glu.

A dual-template molecularly imprinted electrochemical sensor was developed for the simultaneous detection of serotonin (5-HT) and glutamate (Glu). First, amino-functionalized reduced graphene oxide (NRGO) was used as the modification material of a GCE to increase its electrical conductivity and specific surface area, using Glu and 5-HT as dual-template molecules and o-phenylenediamine (OPD) with self-polymerization ability as functional monomers. Through self-assembly and electropolymerization, dual-template molecularly imprinted polymers were formed on the electrode. After removing the templates, the specific recognition binding sites were exposed. The amount of NRGO, polymerization parameters, and elution parameters were further optimized to construct a dual-template molecularly imprinted electrochemical sensor, which can specifically recognize double-target molecules Glu and 5-HT. The differential pulse voltammetry (DPV) technique was used to achieve simultaneous detection of Glu and 5-HT based on their distinct electrochemical activities under specific conditions. The sensor showed a good linear relationship for Glu and 5-HT in the range 1 ~ 100 µM, and the detection limits were 0.067 µM and 0.047 µM (S/N = 3), respectively. The sensor has good reproducibility, repeatability, and selectivity. It was successfully utilized to simultaneously detect Glu and 5-HT in mouse serum, offering a more dependable foundation for objectively diagnosing and early warning of depression. Additionally, the double signal sensing strategy also provides a new approach for the simultaneous detection of both electroactive and non-electroactive substances.

Integrated metabolomic and transcriptomic analysis of triterpenoid accumulation in the roots of Codonopsis pilosula var. modesta (Nannf.) L.T.Shen at different altitudes.

INTRODUCTION: Codonopsis Radix is a beneficial traditional Chinese medicine, and triterpenoid are the major bioactive constituents. Codonopsis pilosula var. modesta (Nannf.) L.T.Shen (CPM) is a precious variety of Codonopsis Radix, which is distributed at high mountain areas. The environment plays an important role in the synthesis and metabolism of active ingredients in medicinal plants, but there is no report elaborating on the effect of altitude on terpenoid metabolites accumulation in CPM. OBJECTIVES: This study aims to analyse the effects of altitude on triterpenoid biosynthetic pathways and secondary metabolite accumulation in CPM. MATERIAL AND METHODS: The untargeted metabolomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 10 triterpenoids based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method were analysed at the low-altitude (1480 m) and high-altitude (2300 m) CPM fresh roots. The transcriptome based on high-throughput sequencing technology were combined to analyse the different altitude CPM triterpenoid biosynthetic pathways. RESULTS: A total of 17,351 differentially expressed genes (DEGs) and 55 differentially accumulated metabolites (DAMs) were detected from the different altitude CPM, and there are significant differences in the content of the 10 triterpenoids. The results of transcriptome study showed that CPM could significantly up-regulate the gene expression levels of seven key enzymes in the triterpenoid biosynthetic pathway. CONCLUSIONS: The CPM at high altitude is more likely to accumulate triterpenes than those at low altitude, which was related to the up-regulation of the gene expression levels of seven key enzymes. These results expand our understanding of how altitude affects plant metabolite biosynthesis.

Research on the brewing technology of Dangshen Huangjiu with low biogenic amines and high functional factors.

BACKGROUND: Biogenic amines (BA) are hazardous components in Huangjiu (HJ). To ensure the quality of Dangshen Huangjiu (DSHJ), an orthogonal experiment L9 (33) was proposed to optimize the process by the main brewing factors (pre-fermentation temperature, pre- and post-fermentation time) that may affect BA and functional factors in DSHJ. DSHJ was produced with low BA content and high functional factors. Gas chromatography-ion mobility spectrometry combined with a multivariate statistical method (GC-IMS-MSM) was used to analyze the volatile components in the brewing process of DSHJ. RESULTS: The optimum brewing process parameters of DSHJ were as follows: pre-fermentation temperature, 28 °C; pre-fermentation time, 9 days; post-fermentation time, 18 days. The average content of BA in DSHJ was 33.12 mg L-1, and the sensory score, total phenol content and DPPH free radical scavenging rate of DSHJ were significantly higher than those of HJ. A total of 14 esters, 7 acids, 7 alcohols, 1 ketone, 5 aldehydes and 1 pyrazine in DSHJ and HJ were identified by GC-IMS. There were no significant differences (P > 0.05) in DSHJ and HJ in the soaking rice and saccharification stage. 11 components, such as ethyl acetate, and 12 components, such as acetic acid, were the different components of HJ and DSHJ in pre-fermentation and post-fermentation stages, respectively. In the post-fermentation stage, the contents of 8 components in DSHJ such as ethyl acetate were higher than in HJ. CONCLUSION: The preparation process parameters of DSHJ optimized by orthogonal experiments can ensure that DSHJ has the advantages of low BA content, high total phenol content and good antioxidant activity. Sensory score and GC-IMS-MSM analysis found that DSHJ prepared using the optimal process had the characteristics of good taste and rich aroma. © 2024 Society of Chemical Industry.

Cooperative effects of three preservatives on physiological quality, endophytic bacterial community and volatile organic compounds of postharvest Codonopsis pilosula var. modesta roots.

BACKGROUND: Codonopsis pilosula var. modesta (CPVM) is a famous medicinal and edible plant of Campanulaceae. However, fresh CPVM roots (FCPVR) are prone to softening, browning and spoilage after concentrated harvesting in the main production area of Gansu Province, China in autumn, which poses great challenges to their large-scale storage and modern processing. In this study, effects of chitosan (CS), natamycin (NA) and modified atmosphere agent (MA) on the postharvest quality of FCPVR were first investigated. The roots after different treatments were stored at 4 °C and relative humidity of 75 ± 5% for 100 days. Their overall quality changes were evaluated from three perspectives: physiological quality, endophytic bacterial community and volatile organic compounds. RESULTS: The clustering heatmap and principal component analysis results indicated that CS (2 g kg-1), NA (0.5 g kg-1) and MA (5 g) had a synergistic effect on physiological quality. The roots in the CS + NA + MA group maintained better physiological state, effective components and antioxidant capacity throughout the storage process. On this basis, compared with room temperature storage, the relative abundance of the main spoilage bacterium Pseudomonas in the CS + NA + MA group roots decreased by 44% on the 100th day of storage. Furthermore, after CS + NA + MA composite treatment, the roots produced richer esters with fruit aroma during low-temperature storage. CONCLUSIONS: The CS + NA + MA composite treatment could maintain the physiological quality and flavor of FCPVR, inhibit spoilage by microbial contamination and maintain the optimal quality during low-temperature storage for up to 100 days. © 2024 Society of Chemical Industry.

Analysis of 14 terpenoids and sterols and variety discrimination of Codonopsis Radix using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

Recently, we confirmed that the 95% ethanol-extracted fraction of Codonopsis Radix, which contains several triterpenoids and sterols, possesses pharmacological activities. However, due to the low content and diverse types of triterpenoids and sterols, their similar structures, lack of ultraviolet absorption, and difficulty in obtaining controls, few studies have so far assessed their contents in Codonopsis Radix. We accordingly constructed an ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry technique for the simultaneous quantitative determination of 14 terpenoids and sterols. Separation was performed on the Waters Acquity UPLC HSS T3 C18 column (100 × 2.1 mm, 1.8 µm) with 0.1% formic acid (A) and 0.1% formic acid in methanol (B) as mobile phase under gradient elution. The determination coefficients for each of the matrix calibration curves were ≥0.9925. The average recovery ranged from 81.25% to 118.05%, with relative standard deviations of <4%. The contents of 14 components in 23 batches were quantified and further analyzed through chemometrics. Linear discriminant analysis can distinguish sample varieties. The quantitative analysis method can accurately determine the contents of 14 components and thereby provide the chemical basis for the quality control of Codonopsis Radix. It also could be a valuable approach for the classification of different Codonopsis Radix varieties.

Evaluation of the Quality of Codonopsis Radix in Different Growth Years by the AHP-CRITIC Method.

The quality of traditional Chinese medicines (TCM) has a significant correlation with the source and growth years. However, there is no research on the relationship between the growth period and the quality of Codonopsis Radix (CR). This work aims to evaluate the quality of CR in different growth years (2-5 years). First, the content of 6 efficacy-related and 28 nutrient-related components in different growth years of CR was analyzed. The results showed that with the increase in growth years of CR, the content of some components increased, while some decreased. Then, the AHP-CRITIC method was performed to score the CR in different growth years, the results showed that the comprehensive score of CR increased with the increase of growth years, and the 5-year-CR had the highest score. Finally, in vitro activity assays were designed to verify the evaluation results. The results demonstrated that with the increase of growth years, the spleen lymphocyte proliferation activity and DPPH free radical scavenging activity of CR were enhanced, which proved that the AHP-CRITIC method is reasonable to evaluate the quality of CR in different growth years. The aforementioned findings demonstrated that CR quality improved with longer planting years.

Electrochemical immunosensor AuNPs/NG-PANI/ITO-PET for the determination of BDNF in depressed mice serum.

A novel electrochemical immunosensor was developed for highly sensitive detection of brain-derived neurotrophic factor (BDNF), a well-known depression marker. The immunosensor was fabricated by modifying indium tin oxide-coated polyethylene terephthalate (ITO-PET) with N-doped graphene-polyaniline (NG-PANI) and gold nanoparticles (AuNPs) to enhance the conductivity and protein loading capacity. Subsequently, BDNF was immobilized onto the electrode surface via gold-sulfur bonds, followed by the attachment of biotinylated antibody (Biotin-Ab) and horseradish peroxidase-avidin (HRP-Avidin) to create the final immunosensor (HRP-Avidin-Biotin-Ab-BDNF-AuNPs/NG-PANI/ITO-PET). The proposed immunosensor exhibited a linear range of determination (0.781-400 pg/mL) with a low limit of detection (LOD) of 0.261 pg/mL (S/N = 3) and excellent reproducibility (RSD = 1.4%) and stability (92.7%, RSD = 3.1%). Additionally, the immunosensor demonstrated good anti-interference performance and good recovery (98.1-107%). To evaluate the practical utility of the immunosensor, BDNF levels were quantified in the serum of mice with depression induced by chronic unpredictable mild stress (CUMS). The results indicated that the serum BDNF levels were significantly decreased in the depression model group compared with the control group, highlighting the potential of this immunosensor for clinical detection of BDNF in depression diagnosis and treatment.

Physiological Analysis and Genetic Mapping of Short Hypocotyl Trait in Brassica napus L.

Hypocotyl length is a botanical trait that affects the cold tolerance of Brassica napus L. (B. napus). In this study, we constructed an F2 segregating population using the cold-resistant short hypocotyl variety '16VHNTS158' and the cold-sensitive long hypocotyl variety 'Tianyou 2288' as the parents, and BSA-seq was employed to identify candidate genes for hypocotyl length in B. napus. The results of parental differences showed that the average hypocotyl lengths of '16VHNTS158' and 'Tianyou 2288' were 0.41 cm and 0.77 cm at the 5~6 leaf stage, respectively, after different low-temperature treatments, and '16VHNTS158' exhibited lower relative ion leakage rates compared to 'Tianyou 2288'. The contents of indole acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) in hypocotyls of '16VHNTS158' and 'Tianyou 2288' increased with decreasing temperatures, but the IAA and GA contents were significantly higher than those of 'Tianyou 2288', and the BR content was lower than that of 'Tianyou 2288'. The genetic analysis results indicate that the genetic model for hypocotyl length follows the 2MG-A model. By using SSR molecular markers, a QTL locus associated with hypocotyl length was identified on chromosome C04. The additive effect value of this locus was 0.025, and it accounted for 2.5% of the phenotypic variation. BSA-Seq further localized the major effect QTL locus on chromosome C04, associating it with 41 genomic regions. The total length of this region was 1.06 Mb. Within this region, a total of 20 non-synonymous mutation genes were identified between the parents, and 26 non-synonymous mutation genes were found within the pooled samples. In the reference genome of B. napus, this region was annotated with 24 candidate genes. These annotated genes are predominantly enriched in four pathways: DNA replication, nucleotide excision repair, plant hormone signal transduction, and mismatch repair. The findings of this study provide a theoretical basis for cloning genes related to hypocotyl length in winter rapeseed and their utilization in breeding.

Characterization of chemical composition variations in raw and processed Codonopsis Radix by integrating metabolomics and glycomics based on multiple chromatography-mass spectrometry technology.

Codonopsis Radix, a popular food homology medicine, is widely used in clinical traditional Chinese medicine and food supplement, raw products and three types of processed products are the main forms of decoction pieces in China. However, there is no scientific basis for comprehensive chemical characterization of raw and three types of processed products. Herein, we investigated qualitatively and quantificationally secondary and primary metabolites in raw Codonopsis Radix and three types of processed products by metabolomics and glycomics employing multiple chromatography-mass spectrometry technology combined with chemometric analysis further to look for differential compounds and propose the processing-induced chemical mechanisms. The results indicated that Codonopsis Radix became dark-colored and the smell of burnt incense odor was observed after processing. The principal component analysis demonstrated that secondary metabolome and glycome were significantly altered between raw and processed products, and 36 differential secondary metabolites and 11 differential primary metabolites were finally screened through orthogonal partial least-squares-discriminant analysis. The main types of compounds are alkaloids, terpenoids, glycosides, amino acids, monosaccharides, oligosaccharides, and furfural derivatives. Meanwhile, Chemical mechanisms could be involved, including oxidation, glycosidic hydrolysis, esterification, dehydration, and Maillard reaction. This work supplies a chemical basis for the application of various types of Codonopsis Radix decoction pieces.

Electrochemical immuno determination of connective tissue growth factor levels on nitrogen-doped graphene.

Connective tissue growth factor (CTGF) is a disease marker of rheumatoid arthritis (RA), and its rapid and sensitive detection is essential for the diagnosis of RA. In this work, a three-dimensional pore structure of alkali-activated nitrogen-doped graphene (aN-G) was used as an electrode modification material, and a label-free electrochemical immunosensor for the sensitive detection of CTGF was successfully constructed by the formation of an amide bond between amino groups in protein and carboxyl groups on the carbon surface. Under optimized conditions, the sensor achieved accurate detection of CTGF in the wide range of 0.0625 ~ 2000 pg mL-1. It had good accuracy (95.0 ~ 100.1%), repeatability (1.2 ~ 2.2%), stability, selectivity, and a low limit of detection (0.0424 pg mL-1, S/N = 3). The sensor was used in serum samples of patients with RA, and CTGF was also successfully detected. Based on this, the electrochemical sensor is expected to become an effective method for RA diagnosis and treatment effect evaluation.

Quality evaluation of Codonopsis Radix and processed products based on the analysis of monosaccharides and oligosaccharides by liquid chromatography coupled with charged aerosol detector.

INTRODUCTION: Codonopsis Radix (CR) is an edible food and traditional Chinese herb medicine that is widely used in China and Southeast Asia. Saccharides, including fructo-oligosaccharides (FOS) and polysaccharides, are among the most important active substances in CR. However, a quality evaluation of CR based on oligosaccharides has not been conducted. OBJECTIVE: This study aimed to establish a high-performance liquid chromatography coupled with charged aerosol detector method (HPLC-CAD) for the quality evaluation of CR and processed products based on analysis of monosaccharides and oligosaccharides. METHOD: A sensitive and rapid HPLC-CAD method for the simultaneous determination of two monosaccharides (D-fructose and D-glucose), sucrose, and FOS (GF2-GF6) was established to evaluate the quality of CR for the first time. In the present study, 65 batches of CR from three species of the genus Codonopsis were analysed using multivariate statistical techniques. Furthermore, the effects of cultivation management measures (plant growth retardants supply, harvesting time, and growth period) and primary process (drying methods) in the production areas on the target compounds were studied by analysing 34 batches of processed samples. RESULTS: Different varieties of CR resulted in considerably different saccharide contents. Cultivation management measures and processing method remarkably affected the quality of CR. Low concentration of plant growth retardants was recommended. The best harvest time is in October after 4 years of growth. Dryer-drying was suggested to meet the requirement for large-scale processing. CONCLUSION: This method would provide an efficient analytical tool for monosaccharides and oligosaccharides of CR and contribute to the improvement of CR quality.

[Influence of processing factors on honey-fried Codonopsis Radix].

The present study investigated the influence of heating and honey addition on the appearance, chemical component content, and pharmacological activity of Codonopsis Radix decoction pieces in the honey-frying process, and explored the processing mechanism of honey-fried Codonopsis Radix. The color, sweetness, and content of macromolecular components(e.g., oligosaccharides and polysaccharides) and small molecular components(e.g., lobetyolin and atractylenolide Ⅲ) of raw Codonopsis Radix, fried Codonopsis Radix, honey-mixed Codonopsis Radix, and honey-fried Codonopsis Radix were determined, and the antioxidant activities in vitro of their water extract, polysaccharide extract, and oligosaccharide extract were compared. The results showed that in terms of color and sweetness, compared with the raw Codonopsis Radix, the fried Codonopsis Radix slightly changed, the honey-mixed Codonopsis Radix changed significantly, and the honey-fried Codonopsis Radix changed with high significance. In terms of the content of lobetyolin, atractylenolide Ⅲ, and polysaccharides, the samples were ranked as raw Codonopsis Radix > fried Codonopsis Radix > honey-mixed Codonopsis Radix > honey-fried Codonopsis Radix, which indicated that heating and honey addition could reduce the content of these three components. In terms of the content of oligosaccharides, the samples were ranked as honey-fried Codonopsis Radix ≈ honey-mixed Codonopsis Radix > fried Codonopsis Radix ≈ raw Codonopsis Radix, indicating that honey addition could increase the content of oligosaccharides. In terms of antioxidant activity in vitro, ABTS radical scavenging ability of water extract, polysaccharides, and oligosaccharides of honey-fried Codonopsis Radix was most potent, while the change of antioxidant activity in vitro of each extract in the other three processed products was different. In short, both heating and honey addition can affect the appearance, chemical component content, and antioxidant activity in vitro of Codonopsis Radix decoction pieces, but the effect of the combination of the two factors is the best. The comprehensive analysis of the effects of heating and honey addition on Codonopsis Radix decoction pieces indicates that honey addition followed by heating at high temperature is the necessary condition for honey-fried Codonopsis Radix to enhance its activity.

Echinacoside exerts anti-tumor activity via the miR-503-3p/TGF-ß1/Smad aixs in liver cancer.

BACKGROUND: Echinacoside (ECH) is the main active ingredient of Cistanches Herba, which is known to have therapeutic effects on metastatic tumors. However, the effects of ECH on liver cancer are still unclear. This study was to investigate the effects of ECH on the aggression of liver cancer cells. METHODS: Two types of liver cancer cells Huh7 and HepG2 were treated with different doses of ECH at different times and gradients. MTT and colony formation assays were used to determine the effects of ECH on the viability of Huh7 and HepG2 cells. Transwell assays and flow cytometry assays were used to detect the effects of ECH treatment on the invasion, migration, apoptosis and cell cycle of Huh7 and HepG2 cells. Western blot analysis was used to detect the effects of ECH on the expression levels of TGF-ß1, smad3, smad7, apoptosis-related proteins (Caspase-3, Caspase-8), and Cyto C in liver cancer cells. The relationship between miR-503-3p and TGF-ß1 was detected using bioinformatics analysis and Luciferase reporter assay. RESULTS: The results showed that ECH inhibited the proliferation, invasion and migration of Huh7 and HepG2 cells in a dose- and time-dependent manner. Moreover, we found that ECH caused Huh7 and HepG2 cell apoptosis by blocking cells in S phase. Furthermore, the expression of miR-503-3p was found to be reduced in liver tumor tissues, but ECH treatment increased the expression of miR-503-3p in Huh7 and HepG2 cells. In addition, we found that TGF-ß1 was identified as a potential target of miR-503-3p. ECH promoted the activation of the TGF-ß1/Smad signaling pathway and increased the expression levels of Bax/Bcl-2. Moreover, ECH could trigger the release of mitochondrial Cyto C, and cause the reaction Caspases grade. CONCLUSIONS: This study demonstrates that ECH exerts anti-tumor activity via the miR-503-3p/TGF-ß1/Smad aixs in liver cancer, and provides a safe and effective anti-tumor agent for liver cancer.

The metabolic effect of gut microbiota on drugs.

There are more than 1000 species of microbes reside in the human gut, umbering∼1014 microbes. As the invisible organ of human beings, gut microbiota can usually participate in drug metabolism by producing specific enzymes, such as reductase and hydrolytic enzyme, thus affecting the efficacy, toxicity, and bioavailability of drugs. At least 30 commercially available drugs have been shown to be substrates of gut microbes-derived enzymes, and an increasing number of drugs may have the potential to contact with the distal gut with the help of improved release systems or poor solubility/permeability, more drugs are expected to be found to be metabolized through the gut flora. By collecting examples of intestinal flora participating in the metabolism of synthetic drugs and traditional Chinese medicine components, this article provides a comprehensive reference for future researchers to study drug metabolism by intestinal flora. Noticeably, the composition and quantity of intestinal flora varies among individuals, and can be affected by some drug administration (such as antibiotics) or environmental changes (acute plateau hypoxia). This seems to suggest that intestinal flora could have the potential to be a new drug target to affect the efficacy of drugs which can be metabolized by Intestinal flora. Accordingly, understanding the impact of intestinal flora on drug metabolism and clarifying the drug transformation process is of great significance for guiding rational clinical use, individualized use, toxicological evaluation, and promoting drug discovery and development.

Identification of anti-inflammatory active ingredients from Tumuxiang by ultra-performance liquid chromatography/quadrupole time-of-flight-MSE.

The dried roots of Inula helenium L. (IH) and Inula racemosa Hook f. (IR) are used commonly as folk medicine as 'tumuxiang' (TMX). The mixing and sharing of IH and IR in clinical use is a universal phenomenon. Modern pharmacological studies confirmed that IH and IR display anti-inflammatory activities. However, the difference in anti-inflammatory pharmacodynamic substances between these two herbs is still unknown. In the present study, the fingerprints of 18 IH and nine IR samples were established using UPLC/QTOF-MSE . A dimethylbenzene-induced mouse ear vasodilation model was applied in evaluating the anti-inflammatory properties of all 27 samples. Then, the spectrum-efficacy model between chemical characteristic peaks and anti-inflammatory activities was investigated using principal component regression and partial least squares. Finally, the combination of UNIFI Scientific Information System with a library search of traditional Chinese medicines was employed to automatically characterize the peaks. UNIFI identified a total of 80 chemical components. Among the components, the 53 characteristic peaks showed correlation with anti-inflammatory activities, pointing to phenolic and organic acids as primary anti-inflammatory ingredients of TMX. This approach can efficiently and intelligently facilitate the identification of bioactive components from traditional Chinese medicine.

Pharmacokinetics and tissue distribution of five active ingredients of Eucommiae cortex in normal and ovariectomized mice by UHPLC-MS/MS.

1. Pinoresinol di-O-ß-d-glucopyranoside (PDG), geniposide (GE), geniposidic acid (GA), aucubin (AN) and chlorogenic acid (CA) are the representative active ingredients in Eucommiae cortex (EC), which may be estrogenic. 2. The ultra high-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the five ingredients showed good linearity, low limits of quantification and high extraction recoveries, as well as acceptable precision, accuracy and stability in mice plasma and tissue samples (liver, spleen, kidney and uterus). It was successfully applied to the comparative study on pharmacokinetics and tissue distribution of PDG, GE, GA, AN and CA between normal and ovariectomized (OVX) mice. 3. The results indicated that except CA, the plasma and tissue concentrations of PDG, GE, GA in OVX mice were all greater than those in normal mice. AN could only be detected in the plasma and liver homogenate of normal mice, which was poorly absorbed in OVX mice and low in other measured tissues. PDG, GE and GA seem to be better absorbed in OVX mice than in normal mice proved by the remarkable increased value of AUC0-∞ and Cmax. It is beneficial that PDG, GE, GA have better plasma absorption and tissue distribution in pathological state.

Ultrasensitive cortisol electrochemical immunosensor amplifying by Au single-atom nanozymes and HRP enzymes.

Cortisol, a corticosteroid hormone as a primary stress hormone response to internal and external stress, has been regarded as a gold standard reliable biomarker to evaluate human mental stress. The double enzymes strategy, using nanozyme and enzyme amplifying the electrochemical signal, has been widely used to improve the performance of electrochemical biosensors. An ultra-sensitive electrochemical cortisol sensor based on Au single-atom nanozymes had been fabricated through HRP labeled anti-cortisol antibody binding with Au by Au-S bond. Based on the high catalytic activity of Au single-atom nanozymes and the high selectivity of HRP-labeled anti-cortisol antibodies, the cortisol electrochemical sensor-based Au single-atom nanozymes had an excellent response to cortisol, such as high electrochemical activity, high sensitivity, high selectivity, and wide linear range (0.15-300 ng mL-1) and low detection (0.48 pg mL-1) through the four-parameter logistic model with 95% confidence. The electrochemical cortisol sensor was used to determine the cortisol concentration of human saliva at different times.

Neuroprotective effects of total phenolics from Hemerocallis citrina Baroni leaves through the PI3K/AKT pathway.

Neurological injury, as a major pathogenic mechanism in depression, holds significant importance in the research and development of antidepressant drugs. Hemerocallis citrina Baroni (H. citrina), referred to as "Forgetting Sadness Grass," has been confirmed to possess remarkable neuroprotective effects. Studies have identified that the total phenolics in H. citrina Baroni leaves (HLTP) consist of flavonoids and phenolic acids and numerous studies have substantiated the neuroprotective effects of them. Based on this, we propose that HLTP may possess neuroprotective properties. To confirm this hypothesis, we initially employed network pharmacology techniques to predict potential targets for the neuroprotective effects of HLTP based on the Swiss Target Prediction database. GO and KEGG analyses were conducted to predict potential pathways, and a component-target-pathway network was constructed. Molecular docking experiments were then performed to analyze the binding abilities of the selected active components with the main targets. Furthermore, we validated the neuroprotective effects of HLTP and key targets selected through network pharmacology using a corticosterone-induced PC12 neuronal cell damage model. Network pharmacology research has identified that in the HLTP, Quercetin, Rutin, Apigenin, and Isoquercitrin are potential active components that may exert neuroprotective effects by modulating key targets such as AKT1, TNF, TP53, and CASP3 through crucial pathways including PI3K/AKT and apoptosis. Molecular docking revealed that 4-O-Caffeoylquinic acid, 5-O-Caffeoylshikimic acid, 4-p-Coumaroylquinic acid, and 5-O-Feruloylquinic acid exhibit low binding energies with key targets. Particularly, 4-O-Caffeoylquinic acid forms stable binding through hydrogen bonding with residues such as LYS389, GLU49, GLN47, LYS30, ASP44, and GLU40 in AKT1. PC12 cells were stimulated with 200 µmol/L Corticosterone (Cort) for 24 h, and then treated with 50, 100 and 200 µg/mL of HLTP for 24 h. The cell viability of damaged cells were significantly increased in a dose-dependent manner by 9.50%, 10.42% and 21.25%, respectively (P < 0.01). Western blot analysis confirmed that HLTP significantly (P < 0.01) increased the protein expression of PI3K and AKT by 15.24%, 30.44%, 41.03%, and 21.78%, 43.63%, 12.86%, respectively. In addition, through biochemical method, flow cytometry and WB analysis, we found that different concentrations of HLTP can all improve cell damage by reducing ROS, MDA, Ca2+, Cyt-C, Caspase-3, TNF-α and IL-1ß, and increasing SOD, CAT, MMP, Bcl-2/Bax and IL-10. In particular, the HLTP at 200 µg/mL, compared with the Model group, decreased by 140.2%, 54.66%, 51.34%, 65.26%, 40.32%, 63.87%, and 55.38%, and increased by 39.65%, 35.45%, 38.38%, 28.54%, and 39.98%, respectively. Through the above experiments, we verified that HLTP may exert neuroprotective effects by mediating the PI3K/AKT signaling pathway to counteract oxidative stress damage, improve mitochondrial dysfunction, and alleviate inflammatory injury.

Protective effect of Cistanche deserticola on gentamicin-induced nephrotoxicity in rats.

Objective: Gentamicin (GM) is a commonly used aminoglycoside antibiotic, however, renal toxicity has limited its usage. The present study was designed to evaluate the ameliorative effect of Cistanche deserticola on GM-induced nephrotoxicity in rats. Methods: The nephrotoxicity in rats was induced by intraperitoneal administration of GM (100 mg/kg) for 10 consecutive days. Glomerular filtration rate, blood urea nitrogen, creatinine and kidney histopathology were detected to assess the GM-induced nephrotoxicity. The oxidative stress (catalase, superoxide dismutase, glutathione and malondialdehyde) was assessed. The inflammatory response (tumor necrosis factor-α, interleukin-6, myeloperoxidase and nuclear factor-kappa B) and apoptotic marker (Bax and Bcl-2) were also evaluated. Results: The results showed that water and 75% ethanol extracts of C. deserticola (named CDW and CDE, respectively) (100, 200 and 400 mg/kg) in combination with GM could recover the reduction of glomerular filtration rate and enhance the renal endogenous antioxidant capability induced by GM. The increase in the expression of renal inflammatory cytokines (tumor necrosis factor-α and interleukin-6), nuclear protein of nuclear factor-kappa B (p65) and the activity of myeloperoxidase induced by GM was significantly decreased upon CDW or CDE treatment. In addition, CDW or CDE treatment could decrease the Bax protein expression and increase the Bcl-2 protein expression in GM-induced nephrotoxicity in rats significantly. Conclusion: The study demonstrated that C. deserticola treatment could attenuate kidney dysfunction and structural damage in rats induced by GM through the reduction of inflammation, oxidative stress and apoptosis.