Proteomic and Metabolomic Characterization of COVID-19 Patient Sera.

Early detection and effective treatment of severe COVID-19 patients remain major challenges. Here, we performed proteomic and metabolomic profiling of sera from 46 COVID-19 and 53 control individuals. We then trained a machine learning model using proteomic and metabolomic measurements from a training cohort of 18 non-severe and 13 severe patients. The model was validated using 10 independent patients, 7 of which were correctly classified. Targeted proteomics and metabolomics assays were employed to further validate this molecular classifier in a second test cohort of 19 COVID-19 patients, leading to 16 correct assignments. We identified molecular changes in the sera of COVID-19 patients compared to other groups implicating dysregulation of macrophage, platelet degranulation, complement system pathways, and massive metabolic suppression. This study revealed characteristic protein and metabolite changes in the sera of severe COVID-19 patients, which might be used in selection of potential blood biomarkers for severity evaluation.

Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Multitissue Circadian Proteome Atlas of WT and Per1-/-/Per2-/- Mice.

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46 h with an interval of 2 h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

Proteomics Investigation of Diverse Serological Patterns in COVID-19.

Serum antibodies IgM and IgG are elevated during Coronavirus Disease 2019 (COVID-19) to defend against viral attacks. Atypical results such as negative and abnormally high antibody expression were frequently observed whereas the underlying molecular mechanisms are elusive. In our cohort of 144 COVID-19 patients, 3.5% were both IgM and IgG negative, whereas 29.2% remained only IgM negative. The remaining patients exhibited positive IgM and IgG expression, with 9.3% of them exhibiting over 20-fold higher titers of IgM than the others at their plateau. IgG titers in all of them were significantly boosted after vaccination in the second year. To investigate the underlying molecular mechanisms, we classed the patients into four groups with diverse serological patterns and analyzed their 2-year clinical indicators. Additionally, we collected 111 serum samples for TMTpro-based longitudinal proteomic profiling and characterized 1494 proteins in total. We found that the continuously negative IgM and IgG expression during COVID-19 were associated with mild inflammatory reactions and high T cell responses. Low levels of serum IgD, inferior complement 1 activation of complement cascades, and insufficient cellular immune responses might collectively lead to compensatory serological responses, causing overexpression of IgM. Serum CD163 was positively correlated with antibody titers during seroconversion. This study suggests that patients with negative serology still developed cellular immunity for viral defense and that high titers of IgM might not be favorable to COVID-19 recovery.

Proteomic profiling of ovarian clear cell carcinomas identifies prognostic biomarkers for chemotherapy.

Clear cell ovarian carcinoma (CCOC) is a relatively rare subtype of ovarian cancer (OC) with high degree of resistance to standard chemotherapy. Little is known about the underlying molecular mechanisms, and it remains a challenge to predict its prognosis after chemotherapy. Here, we first analyzed the proteome of 35 formalin-fixed paraffin-embedded (FFPE) CCOC tissue specimens from a cohort of 32 patients with CCOC (H1 cohort) and characterized 8697 proteins using data-independent acquisition mass spectrometry (DIA-MS). We then performed proteomic analysis of 28 fresh frozen (FF) CCOC tissue specimens from an independent cohort of 24 patients with CCOC (H2 cohort), leading to the identification of 9409 proteins with DIA-MS. After bioinformatics analysis, we narrowed our focus to 15 proteins significantly correlated with the recurrence free survival (RFS) in both cohorts. These proteins are mainly involved in DNA damage response, extracellular matrix (ECM), and mitochondrial metabolism. Parallel reaction monitoring (PRM)-MS was adopted to validate the prognostic potential of the 15 proteins in the H1 cohort and an independent confirmation cohort (H3 cohort). Interferon-inducible transmembrane protein 1 (IFITM1) was observed as a robust prognostic marker for CCOC in both PRM data and immunohistochemistry (IHC) data. Taken together, this study presents a CCOC proteomic data resource and a single promising protein, IFITM1, which could potentially predict the recurrence and survival of CCOC.

Exploration of the cysteine reactivity of human inducible Hsp70 and cognate Hsc70.

Hsp70s are multifunctional proteins and serve as the central hub of the protein quality control network. Hsp70s are also related to a number of diseases and have been established as drug targets. Human HspA1A (hHsp70) and HspA8 (hHsc70) are the major cytosolic Hsp70s, and they have both overlapping and distinct functions. hHsp70 contains five cysteine residues, and hHsc70 contains four cysteine residues. Previous studies have shown these cysteine residues can undergo different cysteine modifications such as oxidation or reaction with electrophiles to regulate their function, and hHsp70 and hHsc70 have different cysteine reactivity. To address the mechanism of the differences in cysteine reactivity between hHsp70 and hHsc70, we studied the factors that determine this reactivity by Ellman assay for the quantification of accessible free thiols and NMR analysis for the assessment of structural dynamics. We found the lower cysteine reactivity of hHsc70 is probably due to its lower structural dynamics and the stronger inhibition effect of interaction between the α-helical lid subdomain of the substrate-binding domain (SBDα) and the ß-sheet substrate-binding subdomain (SBDß) on cysteine reactivity of hHsc70. We determined that Gly557 in hHsp70 contributes significantly to the higher structural dynamics and cysteine reactivity of hHsp70 SBDα. Exploring the cysteine reactivity of hHsp70 and hHsc70 facilitates an understanding of the effects of redox reactions and electrophiles on their chaperone activity and regulation mechanisms, and how these differences allow them to undertake distinct cellular roles.

Assembly and comparative analysis of the complete multichromosomal mitochondrial genome of Cymbidium ensifolium, an orchid of high economic and ornamental value.

BACKGROUND: Orchidaceae is one of the largest groups of angiosperms, and most species have high economic value and scientific research value due to their ornamental and medicinal properties. In China, Chinese Cymbidium is a popular ornamental orchid with high economic value and a long history. However, to date, no detailed information on the mitochondrial genome of any species of Chinese Cymbidium has been published. RESULTS: Here, we present the complete assembly and annotation of the mitochondrial genome of Cymbidium ensifolium (L.) Sw. The mitogenome of C. ensifolium was 560,647 bp in length and consisted of 19 circular subgenomes ranging in size from 21,995 bp to 48,212 bp. The genome encoded 35 protein-coding genes, 36 tRNAs, 3 rRNAs, and 3405 ORFs. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 915 dispersed repeats, 162 simple repeats, 45 tandem repeats, and 530 RNA editing sites. Analysis of codon usage showed a preference for codons ending in A/T. Interorganellar DNA transfer was identified in 13 of the 19 chromosomes, with plastid-derived DNA fragments representing 6.81% of the C. ensifolium mitochondrial genome. The homologous fragments of the mitochondrial genome and nuclear genome were also analysed. Comparative analysis showed that the GC content was conserved, but the size, structure, and gene content of the mitogenomes varied greatly among plants with multichromosomal mitogenome structure. Phylogenetic analysis based on the mitogenomes reflected the evolutionary and taxonomic statuses of C. ensifolium. Interestingly, compared with the mitogenomes of Cymbidium lancifolium Hook. and Cymbidium macrorhizon Lindl., the mitogenome of C. ensifolium lost 8 ribosomal protein-coding genes. CONCLUSION: In this study, we assembled and annotated the mitogenome of C. ensifolium and compared it with the mitogenomes of other Liliidae and plants with multichromosomal mitogenome structures. Our findings enrich the mitochondrial genome database of orchid plants and reveal the rapid structural evolution of Cymbidium mitochondrial genomes, highlighting the potential for mitochondrial genes to help decipher plant evolutionary history.

Rapid screening and isolation of 5-lipoxygenase inhibitors in Inonotus obliquus and mechanism of action in the treatment of asthma.

Accurate screening and targeted preparative isolation of active substances in natural medicines have long been two technical challenges in natural medicine research. This study outlines a new approach to improve the efficiency of natural product preparation, focusing on rapidly and accurately screening potential active ingredients in Inonotus obliquus as well as efficiently preparing 5-lipoxidase (5-LOX) inhibitors, to provide new ideas for the treatment of asthma with Inonotus obliquus. First, we used ultrafiltration (UF) mass spectrometry to screen for three potential inhibitors of 5-LOX in Inonotus obliquus. Subsequently, the inhibitory effect of the active ingredients screened in the UF assay on 5-LOX was verified using the molecular docking technique, and the potential role of the active compounds in Inonotus obliquus for the treatment of asthma was analyzed by network pharmacology. Finally, based on the above activity screening guidelines, we used semi-preparative liquid chromatography and consecutive high-speed countercurrent chromatography to isolate three high-purity 5-LOX inhibitors such as betulin, lanosterol, and quercetin. Obviously, through the above approach, we have seamlessly combined rapid discovery, screening, and centralized preparation of the active ingredient with molecular-level interactions between the active ingredient and the protease.

Screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Poria Cum Radix Pini and mechanism of action in the treatment of gout disease.

Poria Cum Radix Pini is a rare medicinal fungus that contains several potential therapeutic ingredients. On this basis, a particle swarm mathematical model was used to optimize the extraction process of total triterpenes from P. Cum Radix Pini, and xanthine oxidase inhibitors were screened using affinity ultrafiltration mass spectrometry. Meanwhile, the accuracy of the ultrafiltration assay was verified by molecular docking experiments and molecular dynamics analysis, and the mechanism of action of the active compounds for the treatment of gout was analyzed by enzymatic reaction kinetics and network pharmacology. A high-speed countercurrent chromatography method combined with the consecutive injection and the economical two-phase solvent system preparation using functional activity coefficient of universal quasichemical model (UNIFAC) mathematical model was developed for increasing the yield of target compound. In addition, dehydropachymic acid and pachymic acid were used as competitive inhibitors, and 3-O-acetyl-16alpha-hydroxydehydrotrametenolic acid and dehydrotrametenolic acid were used as mixed inhibitors. Then, activity-oriented separation and purification were performed by high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography and the purity of the four compounds isolated was higher than 90%. It will help to provide more opportunities to discover and develop new potential therapeutic remedies from health care food resources.

High-Speed Countercurrent Chromatography Isolation of Active Components from Evodia Rutaecarpa and Affinity Ultrafiltration Screening for Their Acetylcholinesterase Inhibitor Activity.

Acetylcholinesterase inhibitors from Evodia rutaecarpa were screened, prepared, and evaluated. To screen the lipophilic alkaloid active constituents in E. rutaecarpa, we improved and optimized an ultrafiltration system. Three acetylcholinesterase (AChE) inhibitors, dehydroevodiamine, evodiamine, and rutecarpine, were screened. Addressing the limitations of the traditional response surface methodology (RSM) for multiobjective screening, we integrated RSM with the Non-dominated Sorting Genetic Algorithm III to achieve the optimal extraction of these active ingredients. High-speed countercurrent chromatography was used to isolate the active components using a two-phase solvent system: n-hexane/ethyl acetate/methanol/water (3.0:2.5:3.5:2.0, v/v/v/v) and ethyl acetate/methanol/water (3.0:1.0:4.0, v/v/v). The nuclear magnetic resonance spectroscopy confirmed the structures of the compounds, and molecular docking and dynamics simulations assessed the inhibitory effects of the chemical components on AChE, which were consistent with the findings of the ultrafiltration experiments. We also confirmed the neuroprotective properties of these compounds against glutamate-induced apoptosis in PC12 cells. Overall, we achieved the systematic optimization of multitarget compound extraction and lipophilic alkaloid ultrafiltration screening, as well as preparation and activity validation, laying the groundwork for the development of AChE inhibitors from lipophilic alkaloids.

Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Quaternity method for integrated screening, separation, extraction optimization, and bioactivity evaluation of acetylcholinesterase inhibitors from Sophora flavescens Aiton.

INTRODUCTION: Sophora flavescens Aiton (Fabaceae), a ubiquitous plant species in Asia, contains a wide range of pharmacologically active compounds, such as flavonoids, with potential anti-Alzheimer's disease (anti-AD) effects. OBJECTIVES: The objective of the study is to develop a quaternity method for the screening, isolation, extraction optimization, and activity evaluation of acetylcholinesterase (AChE)-inhibiting compounds from S. flavescens to realize high-throughput screening of active substances in traditional Chinese medicine and to provide experimental data for the development of anti-AD drugs. METHODS: With AChE as the target molecule, affinity ultrafiltration and liquid chromatography-mass spectrometry were applied to screen for potential inhibitors of the enzyme in S. flavescens. Orthogonal array experiments combined with the multi-objective Non-Dominated Sorting Genetic Algorithm III was used for the first time to optimize the process for extracting the active substances. Enzyme inhibition kinetics and molecular docking studies were performed to verify the potential anti-AD effects of the active compounds. RESULTS: Five AChE-inhibiting compounds were identified: kushenol I, kurarinone, sophoraflavanone G, isokurarinone, and kushenol E. These were successfully separated at purities of 72.88%, 98.55%, 96.86%, 96.74%, and 95.84%, respectively, using the n-hexane/ethyl acetate/methanol/water (4.0/5.0/4.0/5.0, v/v/v/v), n-hexane/ethyl acetate/methanol/water (5.0/5.0/6.0/4.0, v/v/v/v), and n-hexane/ethyl acetate/methanol/water (4.9/5.1/5.7/4.3, v/v/v/v) mobile phase systems. Enzyme inhibition kinetics revealed that kushenol E had the best inhibitory effect. CONCLUSION: This study elucidates the mechanism of action of five active AChE inhibitors in S. flavescens and provides a theoretical basis for the screening and development of anti-AD and other therapeutic drugs.

Efficient and fast screening and separation based on computer-aided screening and complex chromatography methods for lipoxygenase inhibitors from Ganoderma lucidum.

INTRODUCTION: Accurate screening and targeted preparative isolation of active substances from natural medicines have long been technical challenges in natural medicine research. OBJECTIVES: This study outlines a new approach for improving the efficiency of natural product preparation, focusing on the rapid and accurate screening of potential active ingredients in Ganoderma lucidum and efficient preparation of lipoxidase inhibitors, with the aim of providing new ideas for the treatment of Alzheimer's disease with G. lucidum. METHODS: The medicinal plant G. lucidum was selected through ultrafiltration coupled with liquid chromatography and mass spectrometry (UF-LC-MS) and computer-assisted screening for lipoxygenase (LOX) inhibitors. In addition, the inhibitory effect of the active compounds on LOX was studied using enzymatic reaction kinetics, and the underlying mechanism is discussed. Finally, based on the earlier activity screening guidelines, the identified ligands were isolated and purified through complex chromatography (high-speed countercurrent chromatography and semi-preparative high-performance liquid chromatography). RESULTS: Five active ingredients, ganoderic acids A, B, C2, D2, and F, were identified and isolated from G. lucidum. We improved the efficiency and purity of active compound preparation using virtual computer screening and enzyme inhibition assays combined with complex chromatography. CONCLUSION: The innovative methods of UF-LC-MS, computer-aided screening, and complex chromatography provide powerful tools for screening and separating LOX inhibitors from complex matrices and provide a favourable platform for the large-scale production of bioactive substances and nutrients.

Immersive Virtual Reality Versus Video Distraction for the Management of Emergence Delirium in Children: A Randomized Controlled Study.

PURPOSE: Emergence delirium (ED) presents challenges for both parents and health care providers in pediatric surgical settings. This study aims to evaluate the effectiveness of immersive virtual reality (VR) distraction and video distraction combined with parental presence in reducing ED in preschool-aged children undergoing elective surgery. DESIGN: A prospective, randomized, controlled clinical trial was conducted with 90 children ages 4 to 7. Participants were randomly assigned to three groups: VR distraction (group V), tablet video distraction with parental presence (group T), and standard care (group C). The primary endpoints were the incidence of ED and Pediatric Anesthesia Emergence Delirium Scale scores, with secondary measures encompassing scores from the Parental Separation Anxiety Scale and the Faces, Legs, Activity, Cry, Consolability (FLACC) scale. METHODS: Participants were assigned to one of the three intervention groups, and relevant scales were used to assess ED, parental separation anxiety, and postoperative pain. The immersive VR distraction and video distraction with parental presence interventions were compared against standard care. FINDINGS: Immersive VR distraction significantly reduced the incidence of ED (6.67% in group V vs 40% in group T and 60% in group C), and the incidence of ED in group V was notably lower than in the other groups (P = .023 vs group T and P = .004 vs group C). Children in group V displayed significantly lower FLACC compared with the other groups as well (P < .05). However, no significant differences between the 3 groups were observed in perioperative anxiety as assessed by the Parental Separation Anxiety Scale scores (P = .27). CONCLUSIONS: This study underscores the potential of immersive VR distraction as an effective intervention for mitigating ED in pediatric surgical patients. The findings suggest that incorporating VR technology during the perioperative period can positively impact postoperative outcomes. Further research in diverse surgical contexts is recommended to validate these findings and explore the broader applicability of VR distraction in pediatric health care settings.

An Analysis of Clinical and Pathologic Features, RecurIndex Genomic Profiles, and Survival Outcomes in HER2-Low Breast Cancer.

BACKGROUND: In recent years, breast cancer has become the most common cancer in the world, increasing women's health risks. Approximately 60% of breast cancers are categorized as human epidermal growth factor receptor 2 (HER2)-low tumors. Recently, antibody-drug conjugates have been found to have positive anticancer efficacy in patients with HER2-low breast cancer, but more studies are required to comprehend their clinical and molecular characteristics. METHODS: In this study, we retrospectively analyzed the data of 165 early breast cancer patients with pT1-2N1M0 who had undergone the RecurIndex testing. To better understand HER2-low tumors, we investigated the RecurIndex genomic profiles, clinicopathologic features, and survival outcomes of breast cancers according to HER2 status. RESULTS: First, there were significantly more hormone receptor (HR)-positive tumors, luminal-type tumors, and low Ki67 levels in the HER2-low than in the HER2-zero. Second, RI-LR (P = .0294) and RI-DR (P = .001) scores for HER2-low and HER2-zero were statistically significant. Third, within HER2-negative disease, HR-positive/HER2-low tumors showed highest ESR1, NFATC2IP, PTI1, ERBB2, and OBSL1 expressions. Fourth, results of the survival analysis showed that lower expression of HER2 was associated with improved relapse-free survival for HR-positive tumors, but not for HR-negative tumors. CONCLUSIONS: The present study highlights the unique features of HER2-low tumors in terms of their clinical characteristics as well as their gene expression profiles. HR status may influence the prognosis of patients with HER2-low expression, and patients with HR-positive/HER2-low expression may have a favorable outcome.

Effects of parameters on radiofrequency guidewire ablation: In vitro and in vivo experiments.

BACKGROUND: A novel ablation technique with guidewire has emerged as a promising approach for mapping and ablation of arrhythmias originating from left ventricular summit. However, its biophysical characteristics have not been fully clarified. METHODS AND RESULTS: In the in vitro experiment, guidewire ablation (GA) was performed in vessel models of 1.17 and 2.24 mm to determine the maximum safety power. Then with the maximum safety power, the predictive value of generator impedance (GI) drop on lesion radius was explored. In the in vivo experiment, the feasibility of the maximum safety power and lesion formation was verified in the living swine. It was found that in both groups, the incidence of steam pops increased along with the raise of ablation power, and the maximum safety power was 10 W for the 1.17-mm group and 15 W for the 2.24-mm group. There was a strong linear correlation between GI drop and maximum lesion radius (in 1.17 mm-10-W group: r = .961; in 2.24 mm-15-W group: r = .918). In the in vivo experiment, besides ventricular fibrillation happened once, no other complications were observed, and lesions were found at both 48-h and 8-week groups. CONCLUSIONS: The safety power of GA should be adjusted according to the diameter of the vessel. Besides, the GI drop can predict the lesion radius during GA.

A pathway activity-based proteomic classifier stratifies prostate tumors into two subtypes.

Prostate cancer (PCa) is the second most common cancer in males worldwide. The risk stratification of PCa is mainly based on morphological examination. Here we analyzed the proteome of 667 tumor samples from 487 Chinese PCa patients and characterized 9576 protein groups by PulseDIA mass spectrometry. Then we developed a pathway activity-based classifier concerning 13 proteins from seven pathways, and dichotomized the PCa patients into two subtypes, namely PPS1 and PPS2. PPS1 is featured with enhanced innate immunity, while PPS2 with suppressed innate immunity. This classifier exhibited a correlation with PCa progression in our cohort and was further validated by two published transcriptome datasets. Notably, PPS2 was significantly correlated with poor biochemical recurrence (BCR)/metastasis-free survival (log-rank P-value < 0.05). The PPS2 was also featured with cell proliferation activation. Together, our study presents a novel pathway activity-based stratification scheme for PCa.

Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves.

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor-ligand affinity ultrafiltration-liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid-solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.

In-depth analysis of the xanthine oxidase inhibitors of Cicer arietinum L.-based receptor-ligand affinity coupled with complex chromatography.

INTRODUCTION: Cicer arietinum L. is the choice of health food for people with diabetes, hypertension, and hyperlipidemia. As an essential source of high-nutrition legumes, it is also an important source of dietary isoflavones. OBJECTIVES: In order to improve the preparation efficiency of natural plants, a rapid biological activity screening and preparation of xanthine oxidase inhibitors from C. arietinum L. was established. METHODS: Xanthine oxidase (XOD) inhibitors were rapidly screened using ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) based on receptor-ligand affinity. The change in XOD activity was evaluated by enzymatic reaction kinetics measurement. The potential bioactive compounds were verified through molecular docking. In addition, the biological activity of ligands screened was separated and purified by complex chromatography. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Three active ingredients, namely daidzin, daidzein, calycosin with XOD binding affinities were identified and isolated from the raw plant materials via semi-preparative high-performance liquid chromatography (HPLC), 0-60 min, 5-50% B and countercurrent chromatography (CCC) (ethyl acetate/acetic acid/water [5:0.8:10, v/v/v]). CONCLUSION: This study will help to elucidate the mechanisms of action of natural plants of interest at the molecular level and could also provide more opportunities for the discovery and development of new nutritional value from other natural resources.

Modeling and optimization of the protocol of complex chromatography separation of cyclooxygenase-2 inhibitors from Ganoderma lucidum spore.

INTRODUCTION: The spores of the medicinal fungus Ganoderma lucidum possess hepatoprotective properties. The main components, triterpenes, are particularly beneficial, making the screening and preparation of active triterpenes from Ganoderma lucidum significant. OBJECTIVES: We aimed to screen and verify cyclooxygenase-2 inhibitors from G. lucidum spores, establish a rapid online hyphenated technique for the preparation of active ingredients, and analyze the structures of the active ingredients. METHODS: Ultrafiltration LC combined with an enzyme inhibition assay and molecular docking was employed to screen and evaluate cyclooxygenase-2 ligands, which were prepared by pressurized liquid extraction coupled online with countercurrent chromatography and semi-preparative LC. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Six cyclooxygenase-2 inhibitors, namely, ganoderic acids I, C2 , G, B, and A and ganoderenic acid A, were screened and evaluated. They were prepared using the online hyphenated technique, following which their structures were identified. CONCLUSION: This study provides opportunities for the discovery and development of new therapeutic drugs from other natural resources, as the present instrumental setup achieved efficient and systematic extraction and isolation of natural products compared with reference separation methods, thus exhibiting significant potential for industrial applications.