The bone marrow microenvironment at single-cell resolution.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.

Author Correction: The bone marrow microenvironment at single-cell resolution.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural and functional changes of anterior cingulate cortex subregions in migraine without aura: relationships with pain sensation and pain emotion.

Migraine without aura is a multidimensional neurological disorder characterized by sensory, emotional, and cognitive symptoms linked to structural and functional abnormalities in the anterior cingulate cortex. Anterior cingulate cortex subregions play differential roles in the clinical symptoms of migraine without aura; however, the specific patterns and mechanisms remain unclear. In this study, voxel-based morphometry and seed-based functional connectivity were used to investigate structural and functional alterations in the anterior cingulate cortex subdivisions in 50 patients with migraine without aura and 50 matched healthy controls. Compared with healthy controls, patients exhibited (1) decreased gray matter volume in the subgenual anterior cingulate cortex, (2) increased functional connectivity between the bilateral subgenual anterior cingulate cortex and right middle frontal gyrus, and between the posterior part of anterior cingulate cortex and right middle frontal gyrus, orbital part, and (3) decreased functional connectivity between the anterior cingulate cortex and left anterior cingulate and paracingulate gyri. Notably, left subgenual anterior cingulate cortex was correlated with the duration of each attack, whereas the right subgenual anterior cingulate cortex was associated with migraine-specific quality-of-life questionnaire (emotion) and self-rating anxiety scale scores. Our findings provide new evidence supporting the hypothesis of abnormal anterior cingulate cortex subcircuitry, revealing structural and functional abnormalities in its subregions and emphasizing the potential involvement of the left subgenual anterior cingulate cortex-related pain sensation subcircuit and right subgenual anterior cingulate cortex -related pain emotion subcircuit in migraine.

pepDESC: A Method for the Detection of Differentially Expressed Proteins for Mass Spectrometry-Based Single-Cell Proteomics Using Peptide-level Information.

Single-cell proteomics as an emerging field has exhibited potential in revealing cellular heterogeneity at the functional level. However, accurate interpretation of single-cell proteomics data suffers from challenges such as measurement noise, internal heterogeneity, and the limited sample size of label-free quantitative mass spectrometry. Herein, the author describes peptide-level differential expression analysis for single-cell proteomic (pepDESC), a method for detecting differentially expressed proteins using peptide-level information designed for label-free quantitative mass spectrometry-based single-cell proteomics. While, in this study, the author focuses on the heterogeneity among the limited number of samples, pepDESC is also applicable to regular-size proteomics data. By balancing proteome coverage and quantification accuracy using peptide quantification, pepDESC is demonstrated to be effective in real-world single-cell and spike-in benchmark datasets. By applying pepDESC to published single-mouse macrophage data, the author found a large fraction of differentially expressed proteins among three types of cells, which remarkably revealed distinct dynamics of different cellular functions responding to lipopolysaccharide stimulation.

Efficient Carrier Multiplication in Self-Powered Near-Ultraviolet γ-InSe/Graphene Heterostructure Photodetector with External Quantum Efficiency Exceeding 161.

Carrier multiplication (CM) in semiconductors, the process of absorbing a single high-energy photon to form two or more electron-hole pairs, offers great potential for the high-response detection of high-energy photons in the ultraviolet spectrum. However, compared to two-dimensional semiconductors, conventional bulk semiconductors not only face integration and flexibility bottlenecks but also exhibit inferior CM performance. To attain efficient CM for ultraviolet detection, we designed a two-terminal photodetector featuring a unilateral Schottky junction based on a two-dimensional γ-InSe/graphene heterostructure. Benefiting from a strong built-in electric field, the photogenerated high-energy electrons in γ-InSe, an ideal ultraviolet light-absorbing layer, can efficiently transfer to graphene without cooling. It results in efficient CM within the graphene, yielding an ultrahigh responsivity of 468 mA/W and a record-high external quantum efficiency of 161.2% when it is exposed to 360 nm light at zero bias. This work provides valuable insights into developing next-generation ultraviolet photodetectors with high performance and low-power consumption.

Modulating Carrier Dynamics in PdSe2: The Role of Pressure in Electronic and Phononic Interactions.

PdSe2 is a puckered transition metal dichalcogenide that has been reported to undergo a two-dimensional to three-dimensional structural transition under pressure. Here, we investigated the electronic and phononic evolution of PdSe2 under high pressure using pump-probe spectroscopy. We observed the electronic intraband and interband transitions occurring in the d orbitals of Pd, revealing the disappearance of the Jahn-Teller effect under high pressure. Furthermore, we found that the decay rates of interband recombination and intraband relaxation lifetimes change at 3 and 7 GPa, respectively. First-principles calculations suggest that the bandgap closure slows the decay rate of interband recombination after 3 GPa, while the saturation of phonon-phonon scattering is the main reason for the relatively constant intraband relaxation lifetime. Our work provides a novel perspective for understanding the evolution of the electron and modulation of the carrier dynamics by phonons under pressure.

Dual Function of the Third Component in Ternary Organic Solar Cells: Broaden the Spectrum and Optimize the Morphology.

Ternary organic solar cells (T-OSCs) have attracted significant attention as high-performance devices. In recent years, T-OSCs have achieved remarkable progress with power conversion efficiency (PCE) exceeding 19%. However, the introduction of the third component complicates the intermolecular interaction compared to the binary blend, resulting in poor controllability of active layer and limiting performance improvement. To address these issues, dual-functional third components have been developed that not only broaden the spectral range but also optimize morphology. In this review, the effect of the third component on expanding the absorption range of T-OSCs is first discussed. Second, the extra functions of the third component are introduced, including adjusting the crystallinity and molecular stack in active layer, regulating phase separation and purity, altering molecular orientation of the donor or acceptor. Finally, a summary of the current research progress is provided, followed by a discussion of future research directions.

Four-wavelength laser interferometry for the demodulation of dual-cavity fiber-optic extrinsic Fabry-Perot interferometric sensors.

A four-wavelength passive demodulation algorithm is proposed and experimentally demonstrated for the interrogation of the one cavity in a dual-cavity extrinsic Fabry-Perot interferometric (EFPI) sensor. The lengths of two cavities are adjusted to generate four quadrature signals for each individual cavity. Both simulation and experimental results are presented to validate the performance of this technique. The experimental results demonstrate that dynamic signals at frequencies of 100 Hz, 200 Hz, and 300 Hz with varying amplitude are successfully extracted from a dual-cavity EFPI sensor with initial lengths of 93.4803 µm and 94.0091 µm. The technique shows the potential application to measure dynamic signals in dual-cavity fiber-optic EFPI sensors.

All-sapphire fiber-optic sensor for the simultaneous measurement of ultra-high temperature and high pressure.

An all-sapphire fiber-optic extrinsic Fabry-Perot interferometric (EFPI) sensor for the simultaneous measurement of ultra-high temperature and high pressure is proposed and experimentally demonstrated. The sensor is fabricated based on all-sapphire, including a sapphire fiber, a sapphire capillary and a sapphire wafer. A femtosecond (fs) laser is employed to drill a through hole at the side wall of the sapphire capillary to allow gas flow. The sapphire fiber is inserted from one side of the sapphire capillary. The sapphire wafer is fixed at the other side of the sapphire capillary. The first Fabry-Perot (FP) cavity, composed of the end face of the sapphire fiber and the front surface of the sapphire wafer, is used for measuring pressure, while the second FP cavity, composed of the two surfaces of the sapphire wafer, is used for measuring temperature. Experimental results show that the sensor can simultaneously measure ultra-high temperature and gas pressure within the temperature range of 20 - 1400 °C and the pressure range of 0 - 5 MPa. The temperature sensitivity is 0.0033 µm/°C, and the pressure sensitivity decreases as the temperature increases, reaching 1.8016 µm/MPa and 0.3253 µm/MPa at temperatures of 20 °C and 1400 °C, respectively.

Monitoring pediatric CNS non-germinomatous germ cell tumors via cerebrospinal fluid circulating tumor DNA.

BACKGROUND: Accurate molecular and clinical stratification of patients with central nervous system (CNS) non-germinomatous germ cell tumors (NGGCTs) remains challenging, impeding the development of personalized therapeutic approaches. Herein, we investigated the translational significance of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) in pediatric NGGCTs to identify characteristic features of CNS NGGCTs and to identify a subset of patients for whom the presence of residual disease is a risk factor and an indicator of shorter progression-free survival (PFS) and overall survival (OS). METHODS: Medical records of patients with CNS NGGCTs between January 1, 2018 and December 31, 2022 were reviewed retrospectively. RESULTS: The cohort consisted of 11 male and six female patients. Tumor markers were elevated in four of the five people who underwent surgery. The remaining 12 patients were diagnosed with malignant NGGCTs according to elevated tumor markers. Among them, ctDNA before chemotherapy as well as ctDNA clearance were consistently associated with PFS and OS (p < .05). By setting a ctDNA positivity threshold of 6%, patients with high ctDNA (above the threshold) levels, which had limitation due to the selection based on optimal statistic from the survival analysis, had significantly inferior 5-year PFS and OS compared to those with low levels (below the threshold). ctDNA or ctDNA clearance combined with the presence of residual disease predicted significantly worse OS and PFS (p < .05). CONCLUSIONS: CSF ctDNA might allow the study of genomic evolution and the characterization of tumors in pediatric NGGCTs. CSF ctDNA analysis may facilitate the clinical management of pediatric NGGCT patients, and aid in designing personalized therapeutic strategies.

A context-dependent perspective to understand the relation between parent-child physiological synchrony and child socioemotional adjustment.

Physiological synchrony is an important biological process during which parent-child interaction plays a significant role in shaping child socioemotional adjustment. The present study held a context-dependent perspective to examine the conditional association between parent-child physiological synchrony and child socioemotional adjustment (i.e., relationship quality with parents and child emotion regulation) under different (i.e., from highly unsupportive to highly supportive) emotional contexts. One hundred and fifty school-age Chinese children (Mage = 8.64 years, 63 girls) and their primary caregivers participated in this study. After attaching electrocardiogram (ECG) electrodes, parent-child dyads were instructed to complete a 4-minute conflict discussion task. Parent-child physiological synchrony was calculated based on the within-dyad association between parents' and children's respiratory sinus arrhythmia (RSA) levels across eight 30-second epochs. Parental emotional support, child relationship quality with parents, and child emotion regulation during the discussion task were coded by trained research assistants. Supporting our hypotheses, parental emotional support moderated the relations of parent-child RSA synchrony with both child relationship quality with parents and child emotion regulation. Furthermore, the Johnson-Neyman technique of moderation indicated that the associations between parent and child RSA synchrony and child socioemotional adjustment indicators shifted from negative to positive as the parental emotional support became increasingly high. Our findings suggest that parent-child physiological synchrony may not be inherently adaptive or maladaptive, highlighting the importance of understanding the function of parent-child physiological synchrony under specific contexts. RESEARCH HIGHLIGHTS: Physiological synchrony may not be inherently adaptive or maladaptive, and the meanings of parent-child physiological synchrony might be contingent on contextual factors. Parental emotional support moderated the relations between parent-child respiratory sinus arrhythmia (RSA) synchrony and child socioemotional adjustment indicators (i.e., child relationship quality with parents and child emotion regulation). More positive/less negative parent-child RSA synchrony was associated with better child socioemotional adjustment under a supportive emotional context, whereas with poorer child socioemotional adjustment under an unsupportive emotional context. These findings highlight the significance of considering the emotional context in physiological synchrony studies.

Transmission mechanism of antibiotic resistance genes and their differences between water and sediment in the Weihe River Basin.

Antibiotic resistance genes (ARGs) are emerging microbial pollutants that are regulated by many factors and pose potential threats to aquatic environments. In this study, we used network analysis, correlation analysis, and constructed models based on metagenomic sequencing results to explore the spatial patterns, impact mechanisms, transmission risks and differences in ARGs in the water and sediment of the Weihe River Basin. The findings revealed notable disparities in ARGs, mobile genetic elements (MGEs), and bacterial communities. In the sediment, the abundance of ARGs was considerably greater than that in water. Moreover, the percentage of ARGs shared by the two components reached a value of 85.8%. Through network analysis, it was determined that the presence of 16 MGEs and 20 bacterial phyla was strongly associated with ARGs (R2 > 0.7, P < 0.05). The Mantel test showed that abiotic factors including DO, pH, nutrients, and heavy metals played important roles in the distribution of ARGs (P < 0.05). A structural equation model revealed that the key factors influencing the distribution of ARGs in water were bacterial diversity and environmental parameters (standardized effects of -0.730 and -0.667), and those in sediment were bacterial diversity and MGEs (standardized effects of -0.751 and 0.851). Neutral modeling indicated that deterministic processes played an important role in the assembly of ARGs in the water of the Weihe River Basin, and stochastic processes were dominant in the sediment. There was a highly significant positive linear correlation between ARGs and pathogens, and there was more complex co-occurrence in the water than in the sediment (R2 > 0.9, P < 0.05), with stronger migration and transmission occurring. Exploring ARGs in large-scale watersheds is immensely important for elucidating their traits and transmission mechanisms and consequently paving the way for the formulation of efficient strategies to mitigate resistance threats.

Effect of Volatile Anesthesia Versus Intravenous Anesthesia on Postoperative Pulmonary Complications in Patients Undergoing Minimally Invasive Esophagectomy: A Randomized Clinical Trial.

BACKGROUND: The effect of intraoperative anesthetic regimen on pulmonary outcome after minimally invasive esophagectomy for esophageal cancer is yet undetermined. The aim of this study was to determine the effect of volatile anesthesia (sevoflurane or desflurane) compared with propofol-based intravenous anesthesia on pulmonary complications after minimally invasive esophagectomy. METHODS: Patients scheduled for minimally invasive esophagectomy were randomly assigned to 1 of 3 general anesthetic regimens (sevoflurane, desflurane, or propofol). The primary outcome was the incidence of pulmonary complications within the 7 days postoperatively, which was a collapsed composite end point, including respiratory infection, pleural effusion, pneumothorax, atelectasis, respiratory failure, bronchospasm, pulmonary embolism, and aspiration pneumonitis. The severity of pulmonary complications, surgery-related complications, and other secondary outcomes were also assessed. RESULTS: Of 647 patients assessed for eligibility, 558 were randomized, and 553 were analyzed. A total of 185 patients were assigned to the sevoflurane group, 185 in the desflurane, and 183 in the propofol group. Patients receiving a volatile anesthetic (sevoflurane or desflurane) had a significantly lower incidence (36.5% vs 47.5%; odds ratio, 0.63; 95% confidence interval, 0.44-0.91; P = .013) and lower severity grade of pulmonary complications ( P = .035) compared to the patients receiving propofol. There were no statistically significant differences in other secondary outcomes between the 2 groups. CONCLUSIONS: In patients undergoing minimally invasive esophagectomy, the use of volatile anesthesia (sevoflurane or desflurane) resulted in the reduced risk and severity of pulmonary complications within the first 7 postoperative days as compared to propofol-based intravenous anesthesia.

G-quadruplex-guided RNA engineering to modulate CRISPR-based genomic regulation.

It is important to develop small moelcule-based methods to modulate gene editing and expression in human cells. The roles of the G-quadruplex (G4) in biological systems have been widely studied. Here, G4-guided RNA engineering is performed to generate guide RNA with G4-forming units (G4-gRNA). We further demonstrate that chemical targeting of G4-gRNAs holds promise as a general approach for modulating gene editing and expression in human cells. The rich structural diversity of RNAs offers a reservoir of targets for small molecules to bind, thus creating the potential to modulate RNA biology.

Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing.

It is important to control CRISPR/Cas9 when sufficient editing is obtained. In the current study, rational engineering of guide RNAs (gRNAs) is performed to develop small-molecule-responsive CRISPR/Cas9. For our purpose, the sequence of gRNAs are modified to introduce ligand binding sites based on the rational design of ligand-RNA pairs. Using short target sequences, we demonstrate that the engineered RNA provides an excellent scaffold for binding small molecule ligands. Although the 'stem-loop 1' variants of gRNA induced variable cleavage activity for different target sequences, all 'stem-loop 3' variants are well tolerated for CRISPR/Cas9. We further demonstrate that this specific ligand-RNA interaction can be utilized for functional control of CRISPR/Cas9 in vitro and in human cells. Moreover, chemogenetic control of gene editing in human cells transfected with all-in-one plasmids encoding Cas9 and designer gRNAs is demonstrated. The strategy may become a general approach for generating switchable RNA or DNA for controlling other biological processes.

The association between household and family composition and mental health of the elderly: mediating role of lifestyle.

BACKGROUND: Mental health in the elderly has multiple determinants, and studies indicate household and family composition, economic status, and family support are key factors. However, these are difficult to modify, and better lifestyle for the elderly can be a possible intervention. The current study examined the mediating role of lifestyle in the association between these three types of the household and family composition (living alone, living with a spouse, and living with children) and mental health in older adults. METHODS: We studied 5,407 participants (58.7% female, age 45 + years) from the Beijing Aging Brain Rejuvenation Initiative Project. All participants underwent a battery of examinations to measure degree loneliness, depression, and global cognitive function. We also surveyed personal lifestyles. We used a mediation analysis to determine the relative contribution of each lifestyle factor on mental health outcomes. RESULTS: Older adults living alone rarely participated in mental and social activities and often had irregular diets; those adults living with children spent most of their time caring for grandchildren and had irregular eating and sleeping schedules; those living with a spouse often engaged in a variety of leisure activities and had the best life habits. Mediation analyses showed that dietary and sleeping irregularity partially mediated the negative effects of living alone on mental health, and were moderated by age and gender. CONCLUSIONS: Living with a spouse was associated with benefits for the mental health of middle-aged and older adults (especially older and female individuals), through modifying better lifestyles than those of individuals with the other two types of the household and family composition.

SPATS2L is a positive feedback regulator of the type I interferon signaling pathway and plays a vital role in lupus.

Through genome-wide association studies (GWAS) and integrated expression quantitative trait locus (eQTL) analyses, numerous susceptibility genes ("eGenes", whose expressions are significantly associated with common variants) associated with systemic lupus erythematosus (SLE) have been identified. Notably, a subset of these eGenes is correlated with disease activity. However, the precise mechanisms through which these genes contribute to the initiation and progression of the disease remain to be fully elucidated. In this investigation, we initially identify SPATS2L as an SLE eGene correlated with disease activity. eSignaling and transcriptomic analyses suggest its involvement in the type I interferon (IFN) pathway. We observe a significant increase in SPATS2L expression following type I IFN stimulation, and the expression levels are dependent on both the concentration and duration of stimulation. Furthermore, through dual-luciferase reporter assays, western blot analysis, and imaging flow cytometry, we confirm that SPATS2L positively modulates the type I IFN pathway, acting as a positive feedback regulator. Notably, siRNA-mediated intervention targeting SPATS2L, an interferon-inducible gene, in peripheral blood mononuclear cells (PBMCs) from patients with SLE reverses the activation of the interferon pathway. In conclusion, our research highlights the pivotal role of SPATS2L as a positive-feedback regulatory molecule within the type I IFN pathway. Our findings suggest that SPATS2L plays a critical role in the onset and progression of SLE and may serve as a promising target for disease activity assessment and intervention strategies.

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.

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.

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.