Panax ginseng, a venerable herb in traditional Chinese medicine, boasts a rich history of usage in the treatment of dementia, particularly in its various manifestations. However, despite its long-standing popularity and empirical evidence of effectiveness, the precise mechanisms underlying its therapeutic actions have remained elusive. Currently, research on pharmacodynamic components and mechanisms predominantly utilizes serum pharmacology and metabolomics research methods that are based on liquid chromatography-mass spectrometry technology. However, these methods are unable to capture the spatial distribution information of relevant substances, leading to a lack of comprehensive understanding of the pharmacodynamic components and their underlying mechanisms. To bridge this gap in knowledge, a method of spatial metabolomics and air flow assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI), was used to delve into the molecular mechanisms of Panax ginseng in treating Alzheimer’s disease (AD). The pharmacodynamic results demonstrated that Panax ginseng extract can significantly improve the state of brain pathological damage and spatial learning memory ability in AD model rats. This finding highlights the potential of ginseng as a therapeutic agent in AD management. Furthermore, the metabolomics analysis revealed that Panax ginseng modulates the levels of 19 biomarkers that are intricately linked to AD. These biomarkers span across 8 key metabolic pathways, including arginine and proline metabolism, purine metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism. These pathways are essential for maintaining neuronal health and function, and their dysregulation is often associated with the pathogenesis of AD. Importantly, the study identified 7 active ginseng constituents that accumulate in brain tissue. These compounds work in a holistic manner to treat AD by modulating metabolites related to neuroinflammation, neuronal damage, energy deficits, and abnormal fatty acid metabolism. The comprehensive approach suggests that Panax ginseng may offer a multifaceted therapeutic strategy for AD. The unique capabilities of mass spectrometry imaging allow to analyze the spatial distribution of both endogenous and exogenous substances in parallel. This correspondence not only provides a deeper understanding of the specific effects of drug components, but also sheds light on how these components interact with the body’s endogenous metabolic networks to exert their therapeutic effects. In conclusion, the study provides new light on the mechanisms of action of Panax ginseng in treating AD and paves the way for future research and clinical applications of the ancient herb.

Metabolic abnormalities in mammalian joint tissues are closely linked to various diseases, including rheumatoid arthritis and osteoarthritis. The variations of small molecule metabolites within bone joint tissues can affect cell proliferation, drive the secretion of inflammatory mediators, mediate leukocyte infiltration, and consequently induce synovial inflammation and cartilage damage. Therefore, the development of novel mass spectrometry imaging (MSI) methods for visualizing metabolites in joint tissues is crucial for comprehensively understanding the metabolic characteristics of different microregions under both physiological and pathological conditions. In this study, an airflow-assisted desorption electrospray ionization (AFADESI)-MSI was used to establish an ambient MSI method for rat joint tissues. To ensure the effectiveness of the method, dynamic range, sensitivity, and imaging effect were selected as the primary evaluation criteria. The tissue preparation, section thickness, and spray solvent systems were systematically optimized. Compared to the AB glue transfer method, the tape adhesion method can obtain complete bone joint tissue sections and clearer images. A section thickness of 20 μm, as opposed to 15 μm, provides higher ion intensity in the m/z 600-900 range. Among the tested spray solvent systems, compared with methanol-water (8:2, V/V), acetonitrile-water (8:2, V/V), and the acetonitrile-isopropanol-water (6:2:2, V/V/V), the acetonitrile-isopropanol-water (4:4:2, V/V/V) prove to be the most effective, detecting the highest number of metabolites and lipids when employed as the spray solvent. The precision of this method was evaluated on six adjacent rat knee sections under positive and negative ion modes, with the relative standard deviation (RSD) for the ion intensity of representative metabolites being less than 20%. Subsequently, this method was utilized for spatially resolved metabolomics analysis of rat knee tissues. A total of 613 metabolites are annotated in the overall knee tissues. Based on the optical images, the rat knee tissues were segmented into three microregions for bone, marrow, and cartilage. 452, 418, and 451 metabolites in these microregions are identified, respectively. In bone, the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and the TCA cycle are more prominent. In bone marrow, ascorbate and purine metabolism pathways are more significant. In cartilage, arginine and proline metabolism, as well as lysine degradation, are more pronounced. These findings highlight significant metabolic heterogeneity across different microregions of rat knee joint tissues. Overall, this study develops an AFADESI-MSI method for in-depth profiling of metabolites within rat joint tissues, providing a powerful tool for in situ visualization of metabolites in metabolic studies of joint tissues.

The rapid and accurate differentiation of Angelica sinensis and Levisticum officinale is a significant challenge in the quality control of traditional Chinese medicine. Angelica sinensis and Levisticum officinale are different genera of the same family. Due to their similar appearances and partially shared chemical components, unscrupulous traders often sell counterfeit Levisticum officinale as Angelica sinensis to make exorbitant profits. In this study, a fast and easy method of ultrasonic extraction and nebulization in real-time/carbon fiber ionization mass spectrometry (UENRT/CFI-MS) was introduced for the swift discrimination of these two easily confused traditional Chinese medicines. UENRT/CFI-MS primarily consists of an online extraction and nebulization unit, an ionization unit. The online extraction and nebulization unit employ an ultrasonic nebulizer chip for real-time sample extraction and nebulization. The ionization unit comprises a 10 mm long carbon fiber rod nested in a metal tube, achieving ionization through the application of a 3 kV high voltage, and the auxiliary solvent (methanol) is continuously supplied to the carbon fiber at a rate of 5 μL/min. Slices of Angelica sinensis and Levisticum officinale from different origins with similar size, appearance, and quality (diameter less than 16 mm, thickness not exceeding 0.5 cm) were selected and placed directly on the nebulizer chip. For each analysis, 200 μL methanol was continuously added using a pipette. When the nebulizer was activated, the lower end of the nebulizer chip generated a continuous spray of tiny droplets towards the carbon fiber tip for several seconds, which were then ionized before entering the mass spectrometer for detection. This device integrates online ultrasonic extraction, nebulization, and carbon fiber ionization technologies, enabling real-time analysis of complex samples without cumbersome pretreatment. By applying this technique, a characteristic ion peak of m/z 496 is uniquely identified in Levisticum officinale, serving as a distinctive marker for differentiation of Angelica sinensis and Levisticum officinale. Subsequently, comparative analysis was conducted using sample extracts on electrospray ionization mass spectrometry (ESI-MS) and carbon fiber ionization mass spectrometry (CFI-MS). These experimental results showed that CFI-MS exhibits superior selectivity and efficiency in detecting the m/z 496 characteristic peak, which is not observable in ESI-MS analysis. This finding highlights the unique ability of carbon fiber ionization to selectively detect certain compounds. The UENRT/CFI-MS technique was applied for the direct analysis of Angelica sinensis and Levisticum officinale, and can obtain the analysis results in just a few seconds. A distinctive ion of m/z 496 is detected in Levisticum officinale, facilitating swift discrimination between Angelica sinensis and Levisticum officinale. However, further research is needed to elucidate the structure of the m/z 496 characteristic peak. Additionally, the method’s applicability to a wider range of herbal medicines and its performance in more complex matrices should be investigated. This method is expected to play a role in traditional Chinese medicine identification.

Lipids are one of the most important biomolecules, where they serve as building blocks for celluar membranes and involve in signal transduction and energy storage. The molecular imaging of lipids is critical for understanding complex biological processes, especially under pathological conditions. Spinal cord injury (SCI) always leads to a severe insult motor and sensory function impairment. The direct physical damage to spinal cord will lead to secondary injury consisted of uncontrollable oxidative stress and sever neuroinflammation, subsequently causes lipid peroxidation. The oxidative stress and lipid peroxidation in SCI result in alterations of lipid metabolism and homeostasis, thus induce the changes and heterogeneous spatial distribution of lipids. Therefore, mapping the lipid distribution in injured spinal cords is necessary for understanding the pathological microenvironment of SCI. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a powerful imaging tool for in situ visualization of numerous biomolecules simultaneously. To improve the matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) performance of lipids, developing new matrices is urged. Our previous studies have shown that the matrix of (E)-propyl α-cyano-4-hydroxylcinnamylate (CHCA-C3) exhibits excellent performance for detecting hydrophobic proteins and peptides, which has good co-crystallization performace and increased hydrophobicity. Herein, CHCA-C3 was applied for MALDI-MSI of lipids after SCI, and the changes and spatial distribution of lipids at different times after SCI were analyzed. Compared with CHCA, CHCA-C3 shows profound improvement in lipid detection, that more MS peaks are obtained with higher intensity. Using CHCA-C3, 251 MS peaks are observed in MS spectra, and 25 of them are identified as lipids by searching against database. Moreover, the difference of lipid distribution in injured spinal cords at various time after SCI was investigated. And four lipids, including cholesterol ester CE (20:4), phosphatidylcholine PC (32:1), PC (34:0) and PC (40:0), show significant difference in the spatial distribution at 12 h (acute stage), 3 day (subacute stage) and 7 day (subacute stage) after SCI. Subsequently, the segmentation model (SM) and principal component analysis (PCA) were performed to comprehensive analyzing the 251 MS peaks, and obvious differences are found at spectra obtained from injured spinal cord at different injured phases. In conclusion, this research not only expands the application of CHCA-C3 in MALDI-MSI, but also provides the changes and spatial distribution of lipids in SCI. And the result provides the in-depth understanding of lipidomic changes in SCI, as well as supports the development of new therapeutic strategies for SCI treatment.

The concoction process is a vital technique for reducing the toxicity and enhancing the efficacy of Chinese herbal medicines. To examine the alterations in the chemical compositions of Aurantii Fructus before and after stir-frying in diverse origins, a method of internal extraction electrospray ionization mass spectrometry (iEESI-MS) was developed. It combines the advantages of internal extraction and electrospray ionization mass spectrometry, and is capable of detecting target molecules in complex samples with highly efficient and sensitive manner. iEESI-MS was employed to directly analyze the Aurantii Fructus samples before and after processing without complex sample pretreatment, which can obtain their fingerprints and identify the principal difference substances present in the samples before and after processing. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used for data handling. The aforementioned screened main differences in substances were subjected to analysis in a variety of origins. The experimental conditions were optimized including the temperature of ion transport tube, spray voltage, flow rate of the extractant, proportion of methanol spray solution and distance from the spray port to the mass spectrometry port, etc. The results showed that a total of 35 substances are identified in the Aurantii Fructus samples, including flavonoids, coumarins, alkaloids, volatile oils, and amino acids. The chemical composition of Aurantii Fructus exhibites notable differences before and after preparation. Flavonoids constitute approximately two-thirds of the identified distinct compounds, and serve as crucial quality markers for differentiating the origin and quality of Aurantii Fructus. The results of the human heat map analysis of Aurantii Fructus from disparate origins in Hunan, Sichuan and Jiangxi demonstrated that the variation of the 12 distinct substances present in Aurantii Fructus from different origins is considerable. This indicated that the contents of these 12 substances are inconsistent among the different origins, which is related to the disparate quality of Aurantii Fructus from different origins in terms of its medicinal effects. This study provides a novel approach to ambient mass spectrometry for the quality identification of herbs such as Aurantii Fructus and the material basis of efficacy change before and after processing.

Semen Lepidii is the dried and mature seeds of the cruciferous plant of Descurainia Sophia or Lepidium Apetalum, being a widely-used traditional Chinese medicine for various pharmacological effects, such as antibacterial, antitumor, diuretic and improving immune function. However, the chemical components of Semen Ledidii from different regions have not been clear, limiting the further application of this herb medicine in clinic. Therefore, in this study, a method of ultra-performance liquid chromatography-electrostatic Orbital ion trap tandem mass spectrometry (UPLC-Orbitrap-MS/MS) was developed to identify the chemical components of the three Semen Lepidii, namely Descurainia Sophia 1 (DS1), Descurainia Sophia 2 (DS2) and Lepidium Apetalum (LA) from different regions, and to study the fragmentation pathways of the identified components. The methanol-water (70:30, V/V) extract of the three mashed powder samples was separated on an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm×1.7 μm), and infused into high mass resolution Orbitrap mass spectrometer for analyzing under positive and negative ion modes. MS/MS data were acquired with a Full MS/dd-MS² mode, and the chromatographic peaks were aligned and extracted by using Compound Discoverer (CD) software. Then, the accurate mass informations of the precursor and fragment ions were matched to the databases in the CD platform for identification of components in the samples, and studied the fragmentation pathways of identified compounds. A total of 39 compounds are identified in DS1, including alkaloids, flavonoids, cardiac glycosides, amides, organic acids, amino acids, and others. This method was used to analyze methanol-water extract of DS2 and LA, 37 compounds are identified in DS2, including 10 compounds which are not identified in DS1, and 42 compounds are identified in LA, including 7 compounds which are not identified in both DS1 and DS2. The results indicated that the chemical components of Semen Lepidii originated from different regions are remarkably different and may act as medicine in various ways. This study provides a fundamental for further studying the molecular mechanism of pharmacological effects of Semen Lepdii, which can promote its clinical application.

Breast cancer is a malignant tumor that predominantly affects women. The incidence of breast cancer has risen annually and is increasingly occurring in younger population, posing a significant threat to women’s health. Estrogen receptor-positive breast cancer is the most prevalent subtype, accounting for 75% of all cases. Endocrine therapy, particularly with tamoxifen, serves as the primary treatment for these subtypes. However, due to the highly heterogeneous nature of breast cancer, some patients remain at risk for recurrence even after undergoing tamoxifen treatment. Therefore, it is essential to investigate the mechanism of tamoxifen in breast cancer treatment from a single-cell perspective. In comparison to single-cell genomics and single-cell proteomics, single-cell metabolomics can offer a clearer representation of the chemical changes occurring within individual cells by measuring the end products of cellular activity—metabolites. Thus, this approach can provide a more direct reflection of the differences between cells. In this study, single-cell mass cytometry was employed to investigate metabolite alterations in MCF-7 cells upon exposure to tamoxifen, aiming to elucidate its mechanism from a single-cell perspective. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was utilized to assess the impact of tamoxifen on MCF-7 cell proliferation and established optimal conditions for subsequent therapies involving these cells. The results showed that 576 metabolites under positive ion mode and 480 metabolites under negative ion mode are identified by using single-cell mass cytometry approach. In total, 811 metabolites are detected in MCF-7 cells without tamoxifen treatment compared to 776 metabolites identified following tamoxifen administration. It demonstrated that metabolites observed under negative ion mode effectively distinguish between MCF-7 cells treated with and without tamoxifen. Exposure to tamoxifen can result in upregulation of 28 metabolites and downregulation of 59 metabolites, and these changes predominantly influence metabolic pathways including taurine-hypotaurine metabolism, caffeine metabolism, cysteine-methionine metabolism, glycine-serine-threonine metabolism, purine metabolism, starch-sucrose metabolism, and pyrimidine metabolism. The findings indicated that the intervention of the aforementioned metabolites and metabolic pathways may represent one of the mechanisms through which tamoxifen exerts its effects against estrogen receptor-positive breast cancer. This study employed single-cell metabolomics based on mass spectrometry to explore the metabolic alterations in breast cancer cells induced by tamoxifen, which is often overlooked in metabolomics studies based on bulk cells. Furthermore, it elucidates the mechanism of tamoxifen’s action against estrogen receptor-positive breast cancer from a single-cell metabolomic perspective, thereby provides valuable insights for understanding the mechanisms underlying other therapeutic agents.

Diabetic retinopathy (DR), a common microvascular complication, is the leading cause of visual impairment among the working-age population. Late-stage DR can lead to irreversible vision loss, whereas vision impairment can be prevented by controlling blood sugar in a normal level in the early stage of DR. Notably, as early-stage DR is typically asymptomatic, the majority of DR patients are diagnosed at an advanced stage, which causes a great burden to the patients. Therefore, the development of accurate and efficient biomarkers for the early diagnosis of DR is essential for the prevention of DR. In recent decades, a large number of studies have found that serum complement C3 is closely associated with the development of DR. In this study, a method for the detection of complement C3 by mass spectrometry was established by optimizing the enrichment conditions of glycopeptides using C18 materials. It was found that among volunteers with different stages of DR, the level of complement C3 galactose modification showed a tendency to rise first and then go down with the development of DR. The difference in complement C3 glycosylation was used for the diagnosis of different stages of DR, yielding an area under curve (AUC) of up to 0.761.

A method of ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-TSQ MS) was used to study the products of protopanaxadiol ginsenoside Rb₁ and protopanaxatriol ginsenoside Rg₁ in simulated digestion solution in vitro for researching the digestion characteristics of ginsenoside. The resullt showed that ginsenoside Rd, Rg₃, Rg₅, and Rk₁ are found in the digestion products of ginsenoside Rb₁. In addition, ginsenoside F₂ is found in the simulated intestinal juice, suggesting that it is the peculiar degradation product. Therefore, the degradation pathways of ginsenoside Rb₁ are as follows: ginsenoside Rb₁→ginsenoside Rd→insenoside Rg₃→ginsenoside Rg₅/Rk₁, ginsenoside Rb₁→ginsenoside Rd→ginsenoside F₂. In the digestive fluids of Rg₁, both F₁ and Rh₁ are identified. The degradation pathways of ginsenoside Rg₁ are determined to be: ginsenoside Rg₁→ginsenoside F₁ and ginsenoside Rg₁→ginsenoside Rh_1. The results showed that the contents of degradation products in simulated saliva, simulated gastric juice and simulated intestinal juice change with the digestion time. The digestion products ginsenoside Rd, Rg₃, Rg₅ and Rk₁ of ginsenoside Rb₁ are the highest after 2-6 h of simulated gastric juice digestion. Ginsenoside F₂ is only produced in simulated intestinal juice to digest, in simulated intestinal juice to digest after 4 h content to the maximum. In simulated intestinal fluid, the degradation products of ginsenosides Rb₁ and Rg₁ are the highest at 4-6 h of digestion. Ginsenoside Rg₁ is degraded in the mock digest to generate F₁ and Rh₁. Degradation products in simulated saliva, gastric juice and intestinal juice content gradually increased, in simulated gastric juice to digest its content reached the highest after 2 h. The degradation of ginsenosides in simulated saliva is milder compared to simulated gastric juice and simulated intestinal juice. However, the degradation products in simulated gastric juice are more abundant than in simulated intestinal juice. In the process of digestion, ginsenoside Rg₁ degrades more easily than Rb₁. Ginsenosides Rg₁ and Rb₁ are hydrolyzed in the digestive tract to produce a variety of small molecular saponins, which provide an important chemical and biological basis for the development and utilization of ginsenosides.

Sanhuang decoction, a traditional Chinese medicine prescription, was firstly described in Golden Chamber by Zhang Zhongjing in the Han Dynasty, and has been used clinically for thousands of years to treat diabetes and obesity. However, the specific bioactive compounds being responsible for the hypoglycemic and hypolipidemic effects of Sanhuang decoction and the underlying therapeutic mechanism remain elusive, and further research efforts are in great need. Hence, the aim of this study was to discover the potential hypoglycemic and hypolipidemic phytochemicals in Sanhuang decoction and to explore their potential mechanisms of action. Here, the in vitro α-glucosidase (α-Glu) and lipase (LP) inhibitory activities of Sanshuang decoction were determined, and the results showed that Sanhuang decoction possesses significant α-Glu and LP inhibitory activities in vitro. Then, 24, 17 and 25 potential bioactive ligands in Sanhuang decoction are successfully captured by combining affinity ultrafiltration liquid chromatography-mass spectrometry with three drug targets, namely α-Glu, α-amylase (α-Amy) and LP, respectively. These potential bioactive ingredients fished out from Sanhuang decoction exhibit various binding capacities to α-Glu, α-Amy and LP, respectively. Thereof, epicatechin-3-O-gallate possesses significant hypoglycemic and hypolipidemic effects. Finally, some representative active ligands were further verified with molecular docking assay, and the molecular docking results are consistent with those of affinity ultrafiltration. Furthermore, in addition to hydrogen bond (H-bond), there are various other interaction forces between these potential bioactive ingredients and their corresponding target enzymes, such as Pi-Sigma, Pi-Pi T-shaped and Pi-Alkyl interactions. In this study, the potential hypoglycemic and hypolipidemic constituents in Sanshuang decoction were explored and identified by using affinity ultrafiltration liquid chromatography-mass spectrometry with three drug targets tightly correlating to the metabolisms of blood sugar and lipids in the human body, and the effective material basis of Sanshuang decoction can thus be preliminarily clarified. In this way, the interaction network among these bioactive compounds and three drug targets were constructed, thus providing theoretical support for the clinical applications of Sanhuang decoction for the treatments of obesity and diabetes.

As the secondary metabolites of plants, flavonoids are very important active ingredients in natural medicinal plants and have been proved to possess the extensive biological activities. It has been reported that flavonoids can bind to plasma proteins. Lysozyme is a globular protein and can interact with many small molecules for therapeutic applications. Therefore, the studies of interactions between flavonoids and proteins are not only helpful for understanding of the biological action of small natural organic molecules, but also are beneficial for the development of novel drug candidates. Native electrospray ionization mass spectrometry has been widely applied in the studies of the interactions of proteins and small molecules. During the electrospray ionization (ESI) analysis, some organic solvents are often used as a cosolvent. However, it is not clear for the effects of addition of some organic solvents on the ESI analysis of protein-ligand complexes. The solvent dimethyl sulfoxide (DMSO) is one of cosolvents. There are few studies on the effect of DMSO on protein-small molecule interactions by electrospray mass spectrometry (ESI-MS). Here, the effects of DMSO on the ESI-MS analysis of four lysozyme-flavonoids of icariin, rutin, naringin and scutellarin complexes were investigated. The stable, labile and non-specific binding protein complexes with small molecule ligands were determined by ESI-MS, respectively. It was found that the content of DMSO affects the apparent binding constants of lysozyme and small molecule ligand complexes. The low amounts of DMSO lead to increase the apparent affinity of the labile lysozyme-flavonoid complexes to some extent. It also can stabilize the lysozyme complex with N,N',N''-triacetylchitotriose even under the high capillary temperature. The addition of DMSO cannot lead to the non-specific binding of lysozyme complex with maltose. In addition, the content of DMSO also affects the charge states of the lysozyme complexes. Compared with the sample without DMSO, the charge states of the complex initially decrease with the addition of DMSO. Then the charge states of the complex increase with the further increasing content of DMSO and even increase to higher charge states than those without DMSO. Therefore, it is indicated that the addition of DMSO can affect the ESI-MS analysis of the interaction of protein and small ligands, including the apparent binding constants and charge states of protein-ligand complexes. For the labile lysozyme-flavonoid complexes in ESI-MS, DMSO at the optimized content is shown to stabilize these complexes during ESI-MS analysis. It was suggested that the amount of DMSO used in ESI-MS should be carefully controlled.

The mitochondrial-associated endoplasmic reticulum membrane (MAM) is an essential component of eukaryotic cells. It is involved in many critical cellular functions, such as bioenergetics, mitophagy, apoptosis, and calcium signaling, regulating cellular physiological activities. The MAM proteins of mammalian cells have been extensively studied. However, as an important model organism of eukaryotic single cell, only a few MAM proteins were found in yeast, named endoplasmic reticulum and mitochondria encounter structures (ERMES). There is currently a lack of systematic analysis of MAM protein in yeast. Here, the MAM proteins in Saccharomyces cerevisiae that grow under different nutritional conditions were comprehensively identified by using Percoll gradient differential centrifugation and label-free quantification mass spectrometry technique. Finally, 1 486 MAM component proteins are obtained, including well-known MAM proteins and more new components. The functional enrichment analysis of these proteins showed that they are mainly enriched in protein synthesis and transport, glucose metabolism, and lipid metabolism. They are located primarily in the endoplasmic reticulum, mitochondria, Golgi apparatus, and other organelles. Their molecular functions are mainly related to transferase, oxidoreductase activity, proton-transporting ATPase activity, and protein and lipid binding. KEGG metabolic pathway analysis indicated that these proteins participate in sugar biosynthesis, fatty acid metabolism, steroid biosynthesis, endoplasmic reticulum protein processing, amino acid biosynthesis, and other processes. Under different nutritional conditions, the proteins related to mitochondrial division and fusion were identified, such as CAF4, FIS1, DNM1, FZO1 and UGO1. Except for regulatory effects on the structure of mitochondrial, the MAM proteins also have other functions, such as vesicular transport (SSO2, GOS1, VAM6), ion transport (RSN1, YVC1, ATP18), sterol synthesis (ERG1, ERG2, ERG3) and autophagy (ATG39, ATG15, ATG22). Through differential expression analysis, 176 differentially expressed proteins (DEPs) are found under starvation conditions, including 39 up-regulated and 137 down-regulated proteins; 158 DEPs are found under overnutrition conditions, including 155 up-regulated and 3 down-regulated proteins. By analyzing the protein-protein interaction network of DEPs, it was found that they are mainly involved in the biosynthesis of sugars, proton-transporting ATPase activity, GPI-anchored protein biosynthesis, steroid biosynthesis, protein processing in the endoplasmic reticulum, and mitochondrial redox reactions. The pathways and critical proteins involved in sterol synthesis and protein processing were determined, such as MCD4 and CNE1. This research provides significant data resources for further exploring the biological functions of MAM proteins and helps to understand their roles during the process of cellular stress.

The abuse of antibiotics has caused severe environmental issues. Photocatalytic treatment technology has the advantages of high efficiency, strong effectiveness and no secondary pollution, which presents great potential for degrading or removing many kinds of environment pollutants. However, the intermediate products formed during the photocatalytic degradation process of antibiotics may possess greater toxicity than the antibiotics themselves, and the toxicity of most of these intermediates still remains unclear. Therefore, understanding the photocatalytic degradation pathways of antibiotics and obtaining qualitative and quantitative information about the main degradation products are of vital importance for the large-scale application of photocatalytic technology in the removal of antibiotics and other pollutants. Probe electrospray ionization mass spectrometry (PESI-MS) technology, adopting a solid probe as the electrospray ionization emitter, has the advantages of rapid response, low sample consumption, high salt tolerance, and strong resistance to suspended particles. These features make PESI-MS to be an ideal technique for direct analysis of complex systems, such as biological and chemical reactions. In this study, PESI-MS was used to perform continuous real-time in situ monitoring and analysis of the photocatalytic degradation of oxytetracycline (OTC), which photocatalyzed by TiO₂ under UV irradiation for 160 min, with spectra collected at a resolution of 1 spectrum per second. The results showed that the intensity of protonated OTC ions decreases progressively during the reaction, and forms product ions at various stages of degradation. 35 Kinds of key degradation products are identified by high time-resolved spectra. Based on the changes in the extracted ion chromatograms (EIC) of these products, 7 major degradation products are screened out and 10 possible degradation pathways are proposed, two of which can lead to the degradation of OTC into lower molecular weight products. Although complete photocatalytic degradation of OTC is not achieved in the experimental conditions, the amount of degradation products observed in this study is significantly higher than those previously reported using various high performance liquid chromatography mass spectrometry (HPLC-MS). Furthermore, in the early stages of the photocatalytic process, products of combined ethyl radicals and ethoxy radicals are detected, which likely are contributed by the ethanol added intentionally in the reaction. The use of organic solvents to reduce solution surface tension during PESI operation at atmospheric pressure is inevitable. In the future, we aim to develop a high-pressure PESI ionization source to resolve this issue, which is expected to reveal a more accurate photocatalytic reaction mechanism and pathway. The findings of this study provide valuable data for understanding the kinetics of photocatalytic reactions and developing new photocatalysts, helping researchers gain a more comprehensive understanding of photocatalytic mechanisms.

American ginseng contains a variety of active ingredients. Bioactive peptides with biological functions can be obtained by protease hydrolysis, which has physiological activities such as lowering blood pressure, anti-oxidation and antibacterial. The inorganic salts, polysaccharides and free amino acids contained in the hydrolysate of protease will affect the biological activity of peptides to some extent, so it is necessary to further separate and purify the hydrolysate. At present, the commonly used peptides separation methods are time-consuming and labor-intensive, and the yield of target peptide components is low. In this study, large-scale separation and purification of proteins in a short time can be achieved by the protein purification system, which improves work efficiency and reduces costs. Biological activity and functional properties of peptides are stronger than those of protein. Therefore, it is of great significance to study the antioxidant peptides in freeze-dried American ginseng for nutritional evaluation and pharmacological effects. The amino acid composition of the peptides extracted from freeze-dried American ginseng was investigated by ultra-high performance liquid chromatography-quadrupole electrostatic field-Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap MS/MS). The crude protein in freeze-dried American ginseng was extracted by alkali-soluble acid precipitation method, and hydrolyzed by various proteases. The optimal protease for enzymatic defrosting of the crude protein was screened out using DPPH free radical clearance as an evaluation index. The crude protein was enzymolized with the best protease, and different components were obtained by 3 ku dialysis bags. The components with better anti-oxidation activity were separated by protein liquid phase analysis system. The results showed that after pepsin hydrolysis for 1 h, DPPH free radical scavenging activity is the best, and the clearance rate is (56.02±2.01)%. Further, the antioxidant activity of <3 ku components is better than that of >3 ku components or endogenous peptides. The components with good antioxidant activity (<3 ku) were separated by ÄKTA^TMpure protein liquid phase analysis system to evaluate the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of each fraction, among which F1 and F2 have strong antioxidant activities. The amino acid composition was identified by UHPLC-Q-Orbitrap MS/MS. The hydrophobic amino acids such as Gly, Val and Leu are identified in F1 and F2, which may make them have good antioxidant activities. This method can quickly and effectively identify the antioxidant peptides composition in freeze-dried American ginseng, and provides a basis for revealing the relationship between antioxidant peptides composition and antioxidant activity.

The ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q TOF MS^E) technology with UNIFI software was used to analyze and identify the chemical compositions of Aconiti Lateralis Radix Praeparata (FZ) and Descurainiae Semen Lepidii Semen (TLZ) herb pairs (FZ-TLZ). The UPLC separation was performed on a Waters ACQUITY UPLC BEH C18 column (1.7 μm×2.1 mm×150 mm), and the column temperature was 30 ℃, the injection volume was 2 μL. The mobile phase was consisted of 0.1% formic acid acetonitrile (A) and 0.1% formic acid aqueous solution (B) with the flow rate of 0.2 mL/min. UPLC-Q TOF MS^E was used to collect mass spectrum data under positive and negative ion modes. Then, the precise relative molecular mass and tandem mass spectrum information of the compounds were analyzed by UNIFI software. Combined with the mass spectrum information of standard and relevant references, a total of 142 compounds are identified from water extraction of FZ-TLZ, in addition to 3 common components, 96 components are identified in FZ, and 43 components are identified in TLZ, the identified compounds are mainly alkaloids in FZ and flavonoids in TLZ. On this basis, the mechanism and active components of FZ-TLZ in treating heart failure were studied by network pharmacology technology. Using these components as candidate compounds, potential targets for active ingredients and heart failure targets were identified using databases, such as TCMSP, SwissADME, DisGeNET, Drugbank, OMIM, TTD, TTD, and GeneCards. A total of 99 active ingredients are screened, involving 133 intersecting targets. Protein-protein interaction network was performed using the STRING database. The key network of “drug-component-target” was constructed using Cytoscape software, and 10 key targets (BCL2, IL6, STAT3, CASP3, PPARG, ESR1, EGFR, AKT1, MMP9, PTGS2) are screened using the CytoHubba plugin. Using topological parameters, 19 key active compounds are screened, including flavonoids (such as Karanjin, Kaempferol, Isohamnetin), alkaloids (such as ignavine, Deltoin) and andrographolide compounds. The DAVID database was used to predict the intersection target anti heart failure pathway, which are mainly involved in the regulation of AGE-RAGE signaling pathway in diabetic complications, EGFR tyrosine kinase inhibitor resistance and HIF-1 signaling pathway. This study not only provides data support for comprehensive understanding of the chemical composition of the compatibility of FZ and TLZ, but also is a preliminary research basis for clarifying the potential mechanism of FZ and TLZ in treating heart failure.

The fruits of Aronia melanocarpa (Michx.) Elliot have high nutritional value and medicinal value. This fruit is rich in flavonoids, anthocyanins and anthocyanins and presents good biological activity and pharmacological effects in antioxidant, anti-aging, anti-inflammatory, antibacterial, hypolipidemic, hypoglycemic, anti-cancer, antidepressant, anti-fatigue, etc. It is necessary for the development of this fruit material to explore more chemical constituents. The Global Natural Products Social (GNPS) molecular network based on mass spectrometry technology can establish an association network with the similarity and fingerprint characteristics of similar components, and quickly analyze unknown components in natural products. In this study, ultra-high performance liquid chromatography Orbitrap tandem mass spectrometry (UPLC-Qrbitrap-MS/MS) combined GNPS molecular network was developed for the rapid analysis of constituents in fruits of Aronia melanocarpa (Michx.) Elliot. Under the positive and negative ion modes, the high resolution mass data were acquired. The multiple tandem mass and fragmentation rules were summarized. The molecular network was established based on the similarity of characteristic fragmentation. According to the retention time, accurate molecular mass and fragment ions of references, the constituents in the fruits were identified and deduced. The molecular network of anthocyanins, proanthocyanidins, flavonoids and organic acids were built. Totally 42 compounds are identified in the fruits of Aronia melanocarpa (Michx.) Elliot, including 20 anthocyanins, 3 proanthocyanidins, 12 flavonoids and 7 organic acids. It was concluded that UPLC-Qrbitrap-MS/MS combined with GNPS molecular network can be used to rapidly and accurately identify the constituents in fruits of Aronia melanocarpa (Michx.) Elliot. This results provides a basis for the research of efficacy substances, and the development and utilization of Aronia melanocarpa (Michx.) Elliot fruits.

In order to screen the active ingredients of Qinbai Qingfei Concentrated Pill (QQCP) on treating pneumonia contributed by Pheretima, the extracts of QQCP with and without Pheretima were detected by ultra-high performance liquid chromatography/quadrupole-exactive Orbitrap mass spectrometry (UHPLC/Q-Exactive Orbitrap MS) after conducting the cell anti-inflammatory experiment. Compound Discoverer (CD) software was used to screen the differential ions, and the differential components were identified according to MS² spectra and standards. Further, the active ingredients and targets were predicted by carrying out network pharmacology of differential components treating pneumonia. At last, active ingredients which were identified by standards and their corresponding targets were verified by molecular docking and real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results of cell anti-inflammatory experiments showed that QQCP can exert anti-inflammatory activity by decreasing the expression of IL6, IL-1b, CXCL2 and CXCR2. After detecting the extracts of QQCP by applying UHPLC/Q-Exactive Orbitrap MS, a total of 15 components are identified on the basis of their retention time, MS/MS spectra as well as standards. After that, there are 7 components connecting and 22 core targets obtained by applying networkpharmacology. After verified by molecular docking and RT-qPCR, α-linolenic acid and adenosine are considered as the active components of QQCP treating pneumonia. This study develops a method of UHPLC/Q-Exactive Orbitrap MS uniting network pharmacology to screen the contributing active components from one medicine in Chinese medicine compounds, which provides a new idea for the study on active components of Chinese medicine compounds.

Flos Carthami (FC) has a good therapeutic effect on chronic alcoholic liver injury (CALI) in clinical practice, but the treatment mechanism is not very clear. Therefore, elucidating the molecular mechanism of action of FC in treating CALI is of great significance for the further development and application of drugs. Male Wistar rats were as the research object. The model group rats were orally administered with 8 mL/kg alcohol for 28 days to establish a CALI model, while the treatment group rats were orally administered with high (4.2903 g/kg), medium (1.4301 g/kg), and low (0.4767 g/kg) doses of FC extract. Potential biomarkers related to CALI were identified using rat serum metabonomics analysis methods combined with ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) technology, and the regulatory mechanisms of FC on these biomarkers were investigated. The BP neural network model was established by MATLAB software to deal with the classification problem of omics data. The H&E staining experiment found that after gavage, high-dose FC extract can reduce the degree of liver cell damage. Compared with the model group, the expression levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the high-dose FC group decrease, indicating that the high-dose FC extract has liver protective effect. The classification accuracy of the BP neural network model is 95.8%, and the classification effect is good. Through volcanic map analysis, a total of twenty biomarkers related to CALI are identified, and FC can have a callback effect on these biomarkers. The results indicated that FC may exert therapeutic effect on CALI by regulating the metabolism of triglycerides, fatty acids, phospholipids, bile acids, amino acids and Vitamin E. This study provides a theoretical foundation for the promotion and clinical application.