A comprehensive strategy based on ultra-high performance liquid chromatography with diode array detector fingerprinting and multi-component ultra-performance liquid chromatography with tandem mass spectrometry technology for quality control of Jiawei Huoxiang Zhengqi Pill.

Jiawei Huoxiang Zhengqi Pill (JHZP) is a commonly used Chinese patent medicine for the clinical treatment of headache, dizziness, chest tightness as well as abdominal distension, and pain caused by wind-cold flu. In this study, a comprehensive strategy combining ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD) fingerprinting and multi-component quantitative analysis was established and validated for quality evaluation of JHZP. A total of 49 characteristic common peaks were selected in a chromatographic fingerprinting study to assess the similarity of 15 batches of JHZP. Furthermore, 109 compounds were identified or preliminarily identified from JHZP by coupling with an advanced hybrid linear ion trap-Orbitrap mass spectrometer. For quantification, the optimized ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was employed for the simultaneous determination of 13 target compounds within 12 min. The sensitivity, precision, reproducibility, and accuracy of the method were satisfactory. This validated UPLC-MS/MS method was successfully applied to analyzing 15 batches of JHZP. The proposed comprehensive strategy combining UHPLC-DAD fingerprinting and multi-component UPLC-MS/MS analysis proved to be highly efficient, accurate, and reliable for the quality evaluation of JHZP, which can be considered as a reference for the overall quality evaluation of other Chinese herbal formulations.

Comprehensive polyphenolic profiling, antioxidant, antimicrobial, and cytotoxicity studies of Rhynchostylis retusa (L.) Blume: a medicinal orchid.

Rhynchostylis retusa (L.) Blume, commonly known as the Foxtail orchid, has garnered worldwide attention for its diverse medicinal properties. In this study, root extract and its fractions were evaluated for total polyphenols, flavonoids, targeted polyphenols, and antioxidant potential. The antimicrobial activity was assessed against Gram-positive and Gram-negative bacterial strains while cytotoxicity was assessed using the A549 and HCT-116 cell lines. The investigations showed that chloroform and ethyl acetate are the most effective solvents for fractionation of polyphenols from the parent extract. These fractions also exhibited strong antioxidant and cytotoxic potentials. The chloroform fraction showed maximum cell death of 87.35 and 92.36% in A549 and HCT- 116 cell lines respectively. All samples showed growth inhibition against bacterial strains except the n-hexane fraction, whereas the n-butanol fraction showed comparable antimicrobial activity with the tetracycline standard. The possible health benefits and thereby, application of R. retusa were thus revealed in this investigation.

Exploring the pharmacokinetics and tolerability of cyclooxygenase inhibitor ampiroxicam: a phase I study on single and multiple oral doses.

Introduction: Ampiroxicam is a long-acting, non-steroidal anti-inflammatory drug that selectively inhibits human cyclooxygenase, effectively mitigating fever, pain, and inflammation. This study evaluated the drug's tolerability and pharmacokinetics to support personalized dosing strategies. Methods: The study involved healthy participants and focused on the pharmacokinetics of ampiroxicam. Plasma levels of piroxicam, a key metabolite of ampiroxicam, were measured using ultra-performance liquid chromatography. Piroxicam was chosen due to its integral role in ampiroxicam's metabolic pathway. The analytical method underwent rigorous validation to ensure precision and accuracy, addressing potential interference from endogenous plasma substances. Results: Participants received ampiroxicam in single doses (low, medium, and high) and multiple doses. Pharmacokinetic parameters, including AUC0-216, AUC0-∞, and Cmax, exhibited a dose-dependent increase. No significant differences were noted across the dosage groups, and sex-specific differences were minimal, with the exception of mean residence time (MRT) in the multiple-dose group, which appeared influenced by body weight variations. Discussion: The findings affirm the safety and efficacy of ampiroxicam across different dosing regimens, validating its clinical utility and potential for personalized medicine in the treatment of pain and inflammation.

UPLC-MS/MS determination of 71 neuropsychotropic drugs in human serum.

In this study, a UPLC-MS/MS method was developed for the rapid detection of 71 neuropsychotropic drugs in human serum for drug concentration monitoring and toxicity screening. The analytes were separated from the biological matrix by protein precipitation using a methanol-acetonitrile solvent mixture. The chromatographic separation was performed on a Kromasil ClassicShell C18 column (2.1*50 mm, 2.5 µ m) with gradient elution using acetonitrile-0.2 % acetic acid and 10 mM ammonium acetate as the mobile phases (flow rate 0.4 mL/min, column temperature 40 °C, injection volume 5 µL). An electrospray ion source in both positive and negative ion modes with multiple ion monitoring was used. The total run time was 6 min. All compounds were quantified using the isotope internal standard method. Totally, 71 drugs were detected within their linear ranges with correlation coefficients greater than 0.990. The intra- and inter-batch precision relative standard deviations (RSDs) for the low, medium, and high concentration points were less than 15 %, with an accuracy of 90%-110 %. All compounds except Moclobemide N-oxindole are stabilised within 7 days. The relative matrix effect results for each analyte were within ±20 % of the requirements. The method is validated according to Clinical and Laboratory Standards Institute guidelines, easy to use, and has a low cost.

Exploring the active components and potential mechanisms of Alpiniae oxyphyllae Fructus in treating diabetes mellitus with depression by UPLC-Q-Exactive Orbitrap/MS, network pharmacology and molecular docking.

The growing incidence of diabetes mellitus (DM) and depression is a global public health issue. Alpiniae oxyphyllae Fructus (AOF) is a kind of medicinal and edible plant which be found with anti-diabetic property, and could improve depression-like symptoms. This study aimed to screen active targets and potential mechanisms of AOF in treating DM with depression. Injection of streptozotocin (STZ) and exposure to chronic unpredictable mild stress (CUMS) for 4 weeks were used to conduct the DM with depression mice model. Behavioral tests, indexes of glucose metabolism, monoamine neurotransmitters, inflammatory cytokine and oxidative stress were measured. Histopathological change of hippocampus tissue was observing by HE and Nissl staining. UPLC-Q-Exactive Orbitrap/MS, network pharmacology and molecular docking were used to explore the chemical components and mechanisms of AOF on the DM with depression. AOF showed a reversed effect on body weight in DM with depression mice. Glucose metabolism and insulin resistance could be improved by treatment of AOF. In addition, AOF could alleviate depression-like behaviors based on the results of behavior tests and monoamine neurotransmitters. AOF also attenuated STZ-CUMS induced neuron injury in hippocampus. Next, a total of 61 chemical components were identified in the UPLC-Q-Exactive Orbitrap/MS analysis of the extract of AOF. Network pharmacology analysis suggested that 12 active components and 227 targets were screened from AOF, and 1802 target genes were screened from DM with depression, finally 126 intersection target genes were obtained. Drug-disease targets network was constructed and implied that the top five components with a higher degree value includes quercetin, nootkatone, baicalein, (-)-epicatechin and nootkatol. Protein-protein interaction (PPI) network showed that MAPK1, FOS, AKT1, IL6 and TP53 may be the core intersection targets. The mechanism of the effect of AOF on DM with depression was analyzed through gene ontology (GO), and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, mainly involved in AGE/RAGE, PI3K/AKT, and MAPK signaling pathways. The results of molecular docking indicated that quercetin, nootkatone, baicalein, (-)-epicatechin and nootkatol all had good binding to the core intersection targets. Overall, our experimental researches have demonstrated that AOF could exert the dual effects of anti-diabetic and anti-depression on DM with depression mice, through multi-targets and multi-pathways.

Differences in the flavonoid composition of the leaves, fruits, and branches of mulberry are distinguished based on a plant metabolomics approach.

Mulberry is a common crop rich in flavonoids, and its leaves (ML), fruits (M), and branches (Ramulus Mori, RM) have medicinal value. In the present study, a total of 118 flavonoid metabolites (47 flavone, 23 flavonol, 16 flavonoid, 8 anthocyanins, 8 isoflavone, 14 flavanone, and 2 proanthocyanidins) and 12 polyphenols were identified by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The most abundant in ML were 8-C-hexosyl-hesperetin O-hexoside and astragalin, the most abundant in M were 8-C-hexosyl-hesperetin O-hexoside and naringenin, and the most abundant in RM were cyanidin 3-O-galactoside and gallocatechin-gallocatechin. The total flavonoid compositions of ML and RM were essentially the same, but the contents of flavonoid metabolite in more than half of them were higher than those in M. Compared with ML, the contents of flavone and flavonoid in RM and M were generally down-regulated. Each tissue part had a unique flavonoid, which could be used as a marker to distinguish different tissue parts. In this study, the differences between flavonoid metabolite among RM, ML, and M were studied, which provided a theoretical basis for making full use of mulberry resources.

Study on the molecular mechanisms of Liuwei Dihuang decoction against aging-related cognitive impairment based on network pharmacology and experimental verification.

Objective: Based on network pharmacology and experimental validation, this study aimed to screen the potential targets of Liuwei Dihuang decoction (LW) against mild cognitive impairment (MCI). Methods: Based on network pharmacology, this study preliminarily explored the targets and molecular mechanisms of LW in the treatment of MCI. The results showed that the mechanism of action of LW against MCI may be related to the cAMP pathway. Then, an aging cell and animal model was established to further verify its molecular mechanism. Results: A total of 23 active ingredients were identified in LW. In addition, through network pharmacological analysis, we found 22 anti-MCI active ingredients in LW, of which alisol B had the most significant effect, and predicted the potential mechanism pathway by which LW may improve MCI through the cAMP signaling pathway. Further in vivo and in vitro experiments confirmed that LW can alleviate cognitive dysfunction in aging mice and reduce D-galactose-induced senescent cells, which may be through activation of the cAMP/PKA/CREB signaling pathway. Conclusion: This study found that the traditional Chinese medicine formula LW may play a role in improving MCI by regulating the cAMP/PKA/CREB signaling pathway, which provides a reference for further clinical research on the anti-MCI effect of LW and its molecular mechanism.

Ultra-high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry-characterized extract of Aerides odorata Lour alleviates paracetamol-induced hepatotoxicity in animal model evidenced by biochemical, molecular, and computational studies.

BACKGROUND: Many kinds of orchids have significant health benefits although adequate research on their biological functions is yet to be carried out. This study investigated the paracetamol-induced liver damage-protecting effect of epiphytic Aerides odorata methanol extract (AODE). METHODS: The protective effects of AODE were studied by analyzing its effect on liver function parameters, messenger RNA (mRNA) expression, and tissue histopathological architecture. The results were confirmed by ligand-receptor interaction of molecular docking and multitarget interaction of network pharmacological analyses. RESULTS: AODE significantly (p < 0.05) minimized the dose-dependent increase in acid phosphatase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, lactate dehydrogenase, and total bilirubin compared to the reference drug silymarin. Malondialdehyde level decreased, and the antioxidant genes catalase (CAT), superoxide dismutase (SOD), ß-actin, paraoxonase-1 (PON1), and phosphofructokinase-1 (PFK-1) were upregulated in AODE-treated paracetamol-intoxicated rats. A total of 376 compounds comprising phenols and flavonoids were identified using ultra-high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UPLC-qTOF-MS). The online toxicity assessment using SwissADME and admetSAR exhibited drug-like, nontoxic, and potential pharmacological properties. Additionally, in silico analysis showed that isoacteoside, one of the identified compounds, exhibited the best docking score (-11.42) with the liver protein human pituitary adenylate cyclase-1 (Protein Data Bank ID: 3N94). Furthermore, network pharmacology analysis identified the top 10 hub genes, namely AKT1 (protein kinase B), CTNNB1 (catenin beta-1), SRC (proto-oncogene c-Src), TNF (tumor necrosis factor), EGFR (epidermal growth factor receptor), HSP90AA1 (heat shock protein 90α), MAPK3 (mitogen-activated protein kinase 3), STAT3 (signal transducer and activator of transcription 3), CASP3 (caspase protein), and ESR1 (estrogen receptor 1), which are responsible for hepatoprotective activity. CONCLUSION: The findings demonstrate that AODE could be a novel hepatoprotective target in drug-induced liver damage with a further single compound-based animal study.

Myoglobin as a molecular biomarker for meat authentication and traceability.

The global incidence of economically motivated meat adulteration represents a crucial issue for the food industry. Undeclared addition of cheaper or low-quality species to meat products of high commercial value has become a common practice that needs to be countered with specific measures. In this framework, myoglobin (Mb) is a sarcoplasmic haemoprotein, primarily responsible for meat colour and has been successfully used in meat fraud authentication. Mb is highly soluble in water, easily monitored at 409 nm and species-specific. Knowing that various analytical DNA-based and protein-based methods, as well as spectroscopic techniques have been developed over the years for the detection of meat fraud, the aim of the present review is to take stock of the situation regarding the possible use of Mb as a molecular biomarker for the easy and rapid detection of undeclared species in meat products, avoiding the need of sophisticated or expensive equipment and specialised operators.

A rapid quantitative UPLC-MS/MS method for analysis of key regulatory oxysterols in biological samples for liver cancer.

An UPLC-APCI-MS/MS method was developed for the simultaneous determination of cholesterol, 7-dehydrocholesterol(7DHC) and eight oxysterols including 27-hydroxycholesterol, 7α-hydroxycholesterol(7αOHC), 7ß-hydroxycholesterol(7ßOHC), 24S-hydroxycholesterol(24SOHC), 25-hydroxycholesterol(25OHC), 7α,24S-dihydroxycholesterol(7α,24SdiOHC), 7α,25-dihydroxycholesterol(7α,25diOHC), and 7α,27-dihydroxycholesterol(7α,27diOHC). It has been used for quantitative analysis of cholesterol, 7DHC and eight oxysterols in hepatocellular carcinoma (HCC) cells, plasma and tumor tissue samples. And the above compounds were extracted from the biological matrix (plasma and tissue) using liquid-liquid extraction with hexane/isopropanol after saponification to cleave the steroids from their esterified forms without further derivatization. Then cholesterol, 7DHC and oxysterols were separated on a reversed phase column (Agilent Zorbax Eclipse plus, C8) within 8min using a gradient elution with 0.1% formic acid in H2O and methanol and detected by an APCI triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) of the cholesterol, 7DHC and oxysterols ranged from 3.9ng/ml to 31.25ng/ml, and the recoveries ranged from 83.0% to 113.9%. Cholesterol, 7DHC and several oxysterols including 27OHC, 7αOHC and 7ßOHC were successfully quantified in HCC cells, plasma, tissues and urine of HCC mice. Results showed that 27OHC was at high levels in three kind of HCC cells and tumor tissues as well as plasma samples from both HepG2 and Huh7 bearing mice model，and the high levels of 27OHC in tumors were associated with HCC development. Moreover, the levels of cholesterol in HCC cells and tumor issues varied in different HCC cells and mice model. Oxysterols profiling in biological samples might provide complementary information in cancer diagnosis.

Bioactive compounds from dichloromethane extract of Artemisia rupestris L. alleviates CCl4/ConA-induced acute liver injury by inhibiting PI3K-AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia rupestris L. (AR) is a traditional medicinal herb commonly used in the Uyghurs and Kazakhs; it was first documented in the Supplement to Compendium of Materia Medica written by Zhao Xuemin in the Qing Dynasty of China and is used clinically to treat colds, hepatitis, and allergic diseases. AIM OF THE STUDY: The material basis and mechanisms of AR in acute liver injury (ALI) remain unclear. The purpose of this study was to reveal the possible active components involved in liver protection in AR and to preliminarily explore their pharmacological mechanisms. MATERIALS AND METHODS: The chemical composition of the ethanolic extract (ARA) was identified by UPLC-Q-Exactive-MS/MS and confirmed by 32 reference standards. The pharmacodynamic results were utilized to screen the active part within the ARA that contribute to the amelioration of CCl4/ConA-induced ALI. The main active components and core targets were predicted by network pharmacology and verified by molecular docking combined with qPCR and Western blotting. RESULTS: A total of 131 chemical components were identified in the ARA. The extraction parts of ARA had different therapeutic effects on ALI, among which the dichloromethane extract (ARA-D), which might constitute the main effective fraction of ARA, had significant anti-ALI effects. The network pharmacology results showed that targets including PIK3R1, AKT1, and EGFR, as well as 7 compounds, such as artemetin, vitexicarpin and rupestonic acid may play pivotal roles in treating CCl4/ConA-induced ALI. GO and KEGG pathway enrichment analyses revealed that the PI3K-AKT signaling pathway was the main pathway involved. In each model, ARA-D dose-dependently reduced the increase in ALT levels. High-dose ARA-D markedly decreased ALT activity from 196.79 ± 24.82 to 66.37 ± 16.19 U/L in the CCl4 model group and from 178.00 ± 28.39 to 50.67 ± 7.39 U/L in the ConA model group. Further studies revealed that ARA-D significantly inhibited TNF-α, IL-1ß, and IL-6 expression and inhibited the protein expression of PI3K, p-PI3K, and p-AKT in CCl4/ConA-induced ALI. CONCLUSION: ARA-D exhibits protective effects against ALI induced by CCl4/ConA, potentially through inhibition of the PI3K-AKT signaling pathway. These findings may help to determine the material basis and mechanisms of action of ARA-D for liver protection and provide ideas for future comprehensive studies.

Simultaneous measurement of COVID-19 treatment drugs (nirmatrelvir and ritonavir) in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

PAXLOVID™ (Co-packaging of Nirmatrelvir with Ritonavir) has been approved for the treatment of Coronavirus Disease 2019 (COVID-19). The goal of the experiment was to create an accurate and straightforward analytical method using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to simultaneously quantify nirmatrelvir and ritonavir in rat plasma, and to investigate the pharmacokinetic profiles of these drugs in rats. After protein precipitation using acetonitrile, nirmatrelvir, ritonavir, and the internal standard (IS) lopinavir were separated using ultra performance liquid chromatography (UPLC). This separation was achieved with a mobile phase composed of acetonitrile and an aqueous solution of 0.1% formic acid, using a reversed-phase column with a binary gradient elution. Using multiple reaction monitoring (MRM) technology, the analytes were detected in the positive electrospray ionization mode. Favorable linearity was observed in the calibration range of 2.0-10000 ng/mL for nirmatrelvir and 1.0-5000 ng/mL for ritonavir, respectively, within plasma samples. The lower limits of quantification (LLOQ) attained were 2.0 ng/mL for nirmatrelvir and 1.0 ng/mL for ritonavir, respectively. Both drugs demonstrated inter-day and intra-day precision below 15%, with accuracies ranging from -7.6% to 13.2%. Analytes were extracted with recoveries higher than 90.7% and without significant matrix effects. Likewise, the stability was found to meet the requirements of the analytical method under different conditions. This UPLC-MS/MS method, characterized by enabling accurate and precise quantification of nirmatrelvir and ritonavir in plasma, was effectively utilized for in vivo pharmacokinetic studies in rats.

UPLC-Q-TOF-MS/MS combined with machine learning methods for screening quality indicators of Hypericum perforatum L.

Hypericum perforatum L. (HPL), also known as St. John's wort, is one of the extensively researched domestically and internationally as a medicinal plant. In this study, non-targeted metabolomics combined with machine learning methods were used to identify reasonable quality indicators for the holistic quality control of HPL. First, the high-resolution MS data from different samples of HPL were collected, and visualized the chemical compounds through the MS molecular network. A total of 122 compounds were identified. Then, the orthogonal partial least squares-discriminant analysis (OPLS-DA) model was established for comparing the differences in metabolite expression between flower, leaf, and branches. A total of 46 differential metabolites were screened out. Subsequently, analyzing the pharmacological activities of these differential metabolites based on protein-protein interaction (PPI) network. A total of 25 compounds associated with 473 gene targets were retrieved. Among them, 13 highly active compounds were selected as potential quality markers, and five compounds were ultimately selected as quality control markers for HPL. Finally, three different classifiers (support vector machine (SVM), random forest (RF), and K-nearest neighbor (KNN)) were used to validate whether the selected quality control markers are qualified. When the feature count is set to 122 and 46, the RF model demonstrates optimal performance. As the number of variables decreases, the performance of the RF model degrades. The KNN model and the SVM model also exhibit a decrease in performance but still manage to satisfy the intended requirements. The strategy can be applied to the quality control of HPL and can provide a reference for the quality control of other herbal medicines.

Metabolomics analysis of the effects of chelerythrine on Ustilaginoidea virens.

Rice false smut (RFS) caused by Ustilaginoidea virens is widely distributed in major rice-producing regions. Previous studies have shown that treating RFS with chelerythrine can decrease the germination of fungus spores by 86.7% and induce fungal cell apoptosis. In the present study, the effects of chelerythrine on the metabolism of U. virens explored using metabolomics and analyses of differentially accumulated metabolites and altered metabolic pathways. The top 15 metabolites in random forest analysis were significantly different between groups. In positive ion mode, purine, phenylalanine metabolism, phenylalanine, tyrosine, tryptophan biosynthesis, pyrimidine metabolism, and nitrogen metabolism were dominant. Alanine, aspartate, glutamate metabolism, and phenylalanine metabolism were enriched in negative ion mode. Differentially expressed genes and altered metabolic pathways of U. virens were effected by chelerythrine. The findings support future research on the prevention and treatment of RFS by chelerythrine and provide a theoretical basis for targeted drug delivery.

Impact of Lactic Acid Bacteria Fermentation Based on Biotransformation of Phenolic Compounds and Antioxidant Capacity of Mushrooms.

Mushrooms contain phenolic compounds that possess health-promoting properties, including antioxidant effects. However, the low solubility and form of phenolic compounds affect their bioactivity and bioaccessibility. To overcome this limitation, our study investigates the fermentation of mushrooms to increase their free phenolic content and enhance their bioactivity. Our research focused on the impact of fermentation on both free and bound phenolic fractions (FPs and BPs, respectively) in Lentinula edodes and Lactarius deliciosus, which were successively fermented with Lactiplantibacillus plantarum LMG 17673 for 72 h. We examined the total phenolic content (TPC), phenolic profile, and antioxidant activity of both FPs and BPs. Our results showed that the TPC of BPs was higher than that of FPs in both mushrooms, with strong antioxidant capabilities. Fermentation significantly increased the TPC of FPs in both mushrooms, particularly after 24 h of fermentation. The TPC of BPs in mushrooms decreased during fermentation, indicating their release from the matrix. Additionally, we identified 30 bioactive compounds using UPLC-Q-TOF-MS/MS. Our study demonstrates for the first time that lactic acid bacteria fermentation of mushrooms with high phenolic content leads to the liberation of bound phenolics, enhancing their bioactivity and bioaccessibility.

Investigating the proliferative inhibition of HepG2 cells by exosome-like nanovesicles derived from Centella asiatica extract through metabolomics.

Nano-particles demonstrating excellent anticancer properties have gradually found application in cancer therapy. However, their widespread use is impeded by their potential toxicity, high cost, and the complexity of the preparation process. In this study, we achieved exosome-like Centella asiatica-derived nanovesicles (ADNVs) through a straightforward juicing and high-speed centrifugation process. We employed transmission electron microscopy and nanoparticle flow cytometry to characterize the morphology, diameter, and stability of the ADNVs. We evaluated the in vitro anticancer effects of ADNVs using Cell Counting Kit-8 and apoptosis assays. Through sequencing and bicinchoninic acid protein analysis, we discovered the abundant presence of proteins and microRNAs in ADNVs. These microRNAs can target various diseases such as cancer and infection. Furthermore, we demonstrated the effective internalization of ADNVs by HepG2 cells, resulting in an increase in reactive oxygen species levels, mitochondrial damage, cell cycle arrest at the G1 phase, and apoptosis. Finally, we analyzed changes in cellular metabolites post-treatment using cell metabolomics techniques. Our findings indicated that ADNVs primarily influence metabolic pathways such as amino acid metabolism and lipid biosynthesis, which are closely associated with HepG2 treatment. Our results demonstrate the potential utility of ADNVs as anticancer agents.

Network pharmacology study on fermented soybeans for the prevention of Alzheimer's disease in older individuals.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the disruption of synaptic communication among millions of neurons. Recent research has highlighted the potential therapeutic effectiveness of natural polyphenolic compounds in addressing AD. Soybeans are abundant in polyphenols, and their polyphenolic composition undergoes significant alteration through fermentation by Eurotium cristatum. Through comprehensive database searches, we identified active components within fermented soybean polyphenols and genes associated with AD. Subsequently, we utilized Venn diagrams to analyze the overlap between AD-related genes and these components. Furthermore, we visualized the network between intersecting targets and proteins using Cytoscape software. The anti-AD effects of soybeans were further explored through comprehensive analysis, including protein-protein interaction analysis, pathway enrichment analysis, and molecular docking studies. Our investigation unveiled 6-hydroxydaidzein as a major component of fermented soybean polyphenols, shedding light on its potential therapeutic significance in combating AD. The intersection between target proteins of fermented soybeans and disease-related targets in AD comprised 34 genes. Protein-protein interaction analysis highlighted key potential targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen synthase kinase 3 beta (GSK3B), amyloid precursor protein (APP), cyclin-dependent kinase 5 (CDK5), and beta-site APP cleaving enzyme 1 (BACE1). Molecular docking results demonstrated a robust binding effect between major components from fermented soybeans and the aforesaid key targets implicated in AD treatment. These findings suggest that fermented soybeans demonstrate a degree of efficacy and present promising prospects in the prevention of AD.

Metabolic profiling of Vitex Pubescens Vahl bark via UPLC-ESI-QTOF/MS/MS analysis and evaluation of its antioxidant and acetylcholinesterase inhibitory activities.

BACKGROUND: Alzheimer's disease is a neurodegenerative age-related disease that primarily affects the elderly population leading to progressive memory impairments and neural deficits. It is counted as a major cause of geriatric dependency and disability. The pathogenesis of Alzheimer's disease incidence is complex and involves various hypotheses, including the cholinergic hypothesis, deposition of ß-amyloid plaques, neuroinflammation, oxidative stress, and apoptosis. Conventional treatments such as donepezil aim to delay the symptoms but do not affect the progression of the disease and may cause serious side effects like hepatoxicity. The use of natural candidates for Alzheimer's disease treatment has drawn the attention of many researchers as it offers a multitargeted approach. METHODS: This current study investigates the metabolic profiles of total defatted methanolic extract of Vitex pubescens bark and its polar fractions, viz. ethyl acetate and n-butanol, using ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry(UPLC-ESI-QTOF/MS/MS) technique as well as evaluate the antioxidant using free radical scavenging assays, viz. DPPH and ABTS assays and in-vitro acetylcholinesterase inhibitory activities using Ellman's microplate assay. RESULTS: Metabolic profiling revealed a total of 71, 43, and 55 metabolites tentatively identified in the defatted methanolic extract, ethyl acetate, and n-butanol fractions, respectively. Phenolic acids were the most abundant class, viz. benzoic acids, and acyl quinic acid derivatives followed by flavonoids exemplified mainly by luteolin-C-glycosides and apigenin-C-glycosides. Quantification of the total phenolic and flavonoid contents in the total defatted methanolic extract confirmed its enrichment with phenolics and flavonoids equivalent to 138.61 ± 9.39 µg gallic acid/mg extract and 119.63 ± 4.62 µg rutin/mg extract, respectively. Moreover, the total defatted methanolic extract exhibited promising antioxidant activity confirmed through DPPH and ABTS assays with a 50% inhibitory concentration (IC50) value equivalent to 52.79 ± 2.16 µg/mL and 10.02 ± µg/mL, respectively. The inhibitory activity of acetylcholine esterase (AchE) was assessed using in-vitro Ellman's colorimetric assay, the total defatted methanolic extract, ethyl acetate, and n-butanol fractions exhibited IC50 values of 52.9, 15.1 and 108.8 µg/mL that they proved the significant inhibition of AchE activity. CONCLUSION: The results obtained herein unraveled the potential use of the total methanolic extract of Vitex pubescens bark and its polar fractions as natural candidates for controlling Alzheimer's disease progression.

Discovery and identification of natural alkaloids with potential to impact insulin resistance syndrome in Cyclocarya paliurus. (Batal) leaves by UPLC-QTOF-MS combined with HepG2 cells.

Cyclocarya paliurus (Batal.) leaves, which contain a range of bioactive compounds, have been used as a traditional Chinese medicine homologous food since ancient times. However, there is a paucity of literature on comprehensive studies of alkaloids in the leaves of Cyclocarya paliurus (Batal.). For the first time, this study aimed to discover and identify alkaloids extracted from Cyclocarya paliurus (Batal.) leaves by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-QTOF-MS). A total of ten alkaloids have been identified from Cyclocarya paliurus (Batal.) leaves based on accurate mass spectra (mass accuracy, isotopic spacing and distribution) and comparison to fragmentation spectra reported in the literature. In vitro, alkaloids alleviated insulin resistance by increasing glucose consumption and glycogen content in insulin resistance HepG2 cells. The RNA-seq and western blotting results showed that alkaloids could upregulate the expression of phosphatidylinositol 3-kinase (PI3K), and increase the phosphorylation of insulin receptor protein kinase B (AKT). This study not only clarified the chemical constituents and revealed that diverse alkaloids also presented from Cyclocarya paliurus (Batal.) leaves, also, it will provide chemical information on potential compounds for developing new drugs.

HPLC-QQQ-MS/MS-based authentication and determination of free and bound sialic acids content in human, bovine, sheep, goat milk, and infant formula.

An accurate method for qualitative and quantitative analysis of lipid-bound (LB), protein-bound (PB), oligosaccharides-bound, and free sialic acids in milk was developed by using high-performance liquid chromatography -triple quadrupole-tandem mass spectrometer. The profile of free and bound sialic acids in milk (human, bovine, goat, and sheep) and infant formula (IF) was examined in the present study. Human milk contains only N-acetylneuraminic acid (Neu5Ac) and was mainly present in the form of oligosaccharide-bound. The content of total Neu5Ac (T-Neu5Ac), free and bound Neu5Ac in human milk decreased with the prolongation of lactation. The most intriguing finding was the increase in the proportion of PB and LB sialic acids. The sialic acids in bovine and sheep milk were mainly PB and oligosaccharides-bound Neu5Ac. T-Neu5Ac in goat milk (GM) was 67.44-89.72 µg/mL and was mainly PB Neu5Ac, but total N-glycolylneuraminic acid (T-Neu5Gc) content of GM can be as high as 100.01 µg/mL. The concentration of T-Neu5Gc in sheep and GM was significantly higher than that of bovine milk (BM). T-Neu5Gc content of GM -based IF was 264.86 µg/g, whereas T-Neu5Gc content of BM -based IF was less (2.26-17.01 µg/g). Additionally, our results found that there were also sialic acids in IF ingredients, which were mainly bound with protein and oligosaccharides, primarily derived from desalted whey powder and whey protein concentrate.