Study on the Relationship between Cerebral Blood Perfusion, Neuronal Cytokines and Cognitive Function in Patients with Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by the progressive emergence of multiple cognitive deficits. Early diagnosis is of great significance for the intervention and treatment of AD. The objective of this study is to explore the relationship between cerebral blood perfusion, neuronal cytokines and cognitive function in patients with AD. METHODS: AD patients admitted to the 903 Hospital of the People's Liberation Army Joint Logistics Support Force from June 2020 to January 2023 were retrospectively selected as the study objects, and 65 healthy people who underwent physical examination during the same period were included in the control group. Subjects in both groups underwent 3.0 T magnetic resonance imaging (MRI) to observe their cerebral blood perfusion parameters. The level of cognitive function in both groups was assessed using the Montreal Cognitive Assessment (MoCA). Venous blood was collected from both groups, and the serum levels of brain-derived neuronal factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) were measured by enzyme-linked immunosorbent assay (ELISA). The correlation of serum BDNF and GDNF levels with cerebral blood perfusion parameters and MoCA score in the AD group was analyzed using Spearman analysis. RESULTS: The cerebral blood flow signal intensity of the left frontal lobe, right frontal lobe, left temporal lobe, right temporal lobe, left parietal lobe, right parietal lobe, left occipital lobe, and right occipital lobe of the observation group was significantly lower than that of the control group (p < 0.001). The visuospatial, executive functions, naming, attention, language function, abstract generalization ability, memory ability, orientation, and total MoCA scale scores were significantly lower than those of the control group (p < 0.001). The serum levels of BDNF and GDNF in the observation group were significantly lower than those in the control group (p < 0.001). The results of Spearman analysis showed that cerebral blood perfusion parameters of the left frontal lobe, right frontal lobe, left temporal lobe, right temporal lobe, left parietal lobe, right parietal lobe, left occipital lobe, and right occipital lobe were positively correlated with cognitive function scores in AD patients, serum BDNF and GDNF levels were positively correlated with cognitive function scores in AD patients, and the correlation was statistically significant (p < 0.05). CONCLUSION: In AD patients, blood perfusion parameters and serum BDNF and GDNF levels were significantly lower than those of healthy people. Cerebral blood perfusion parameters of the left frontal lobe, right frontal lobe, left temporal lobe, right temporal lobe, left parietal lobe, right parietal lobe, left occipital lobe, and right occipital lobe, and BDNF and GDNF levels were positively correlated with cognitive function scores in AD patients.

The pathogenesis and potential therapeutic targets in sepsis.

Sepsis is defined as "a life-threatening organ dysfunction caused by dysregulated host systemic inflammatory and immune response to infection." At present, sepsis continues to pose a grave healthcare concern worldwide. Despite the use of supportive measures in treating traditional sepsis, such as intravenous fluids, vasoactive substances, and oxygen plus antibiotics to eradicate harmful pathogens, there is an ongoing increase in both the morbidity and mortality associated with sepsis during clinical interventions. Therefore, it is urgent to design specific pharmacologic agents for the treatment of sepsis and convert them into a novel targeted treatment strategy. Herein, we provide an overview of the molecular mechanisms that may be involved in sepsis, such as the inflammatory response, immune dysfunction, complement deactivation, mitochondrial damage, and endoplasmic reticulum stress. Additionally, we highlight important targets involved in sepsis-related regulatory mechanisms, including GSDMD, HMGB1, STING, and SQSTM1, among others. We summarize the latest advancements in potential therapeutic drugs that specifically target these signaling pathways and paramount targets, covering both preclinical studies and clinical trials. In addition, this review provides a detailed description of the crosstalk and function between signaling pathways and vital targets, which provides more opportunities for the clinical development of new treatments for sepsis.

Dracorhodin targeting CMPK2 attenuates inflammation: A novel approach to sepsis therapy.

BACKGROUND: Despite all modern advances in medicine, an effective drug for treating sepsis has yet to be found. The discovery of CMPK2 spurred hopes for the treatment of sepsis. However, CMPK2-untapped target inhibitors are still an enormous obstacle that has hindered the CMPK2-centric treatment of sepsis. METHODS: Here, we found that the CMPK2 gene is highly expressed in the whole blood of sepsis patients by RNA-Seq. First, recombinant CMPK2 was purified by a eukaryotic expression purification system, and the activity of recombinant CMPK2 was detected by the ADP-GLO assay. Second, we developed an affinity MS strategy combined with quantitative lysine reactivity profiling to discover CMPK2 ligands from the active ingredients of Chinese herbs. In addition, the dissociation constant Kd of the ligand and the target protein CMPK2 was further detected by microscale thermophoresis technology. Third, we used this strategy to identify a naturally sourced small molecule, dracorhodin (DP). Using mass spectrometry-based quantitative lysine reactivity profiling combined with a series of mutant tests, the results show that K265 acts as a bright hotspot of DP inhibition of CMPK2. Fourth, immune-histochemical staining, ELISAs, RT-qPCR, flow cytometry and immunoblotting were used to illustrate the potential function and related mechanism of DP in regulating sepsis injury. RESULTS: Our results suggest that DP exerts powerful anti-inflammatory effects by regulating the NLRP3 inflammasome via the lipopolysaccharide (LPS)-induced CMPK2 pathway. Strikingly, DP significantly attenuated LPS-induced sepsis in a mouse model, but its effect was weakened in mice with myeloid-specific Cmpk2 ablation. CONCLUSION: We provide a new framework that provides more valuable information for new therapeutic approaches to sepsis, including the establishment of screening strategies and the development of target drugs to provide a theoretical basis for ultimately improving clinical outcomes for sepsis patients. Collectively, these findings reveal that DP is a promising CMPK2 inhibitor for the treatment of sepsis.

Safety and immunogenicity of heterologous boosting with orally aerosolised or intramuscular Ad5-nCoV vaccine and homologous boosting with inactivated vaccines (BBIBP-CorV or CoronaVac) in children and adolescents: a randomised, open-label, parallel-controlled, non-inferiority, single-centre study.

BACKGROUND: Heterologous booster immunisation with orally administered aerosolised Ad5-nCoV vaccine (AAd5) has been shown to be safe and highly immunogenic in adults. Here, we aimed to assess the safety and immunogenicity of heterologous booster immunisation with orally administered AAd5 in children and adolescents aged 6-17 years who had received two doses of inactivated vaccine (BBIBP-CorV or CoronaVac). METHODS: We did a randomised, open-label, parallel-controlled, non-inferiority study to assess the safety and immunogenicity of heterologous booster immunisation with AAd5 (0·1 mL) or intramuscular Ad5-nCoV vaccine (IMAd5; 0·3 mL) and homologous booster immunisation with inactivated vaccine (BBIBP-CorV or CoronaVac; 0·5 mL) in children (aged 6-12 years) and adolescents (aged 13-17 years) who had received two doses of inactivated vaccine at least 3 months earlier in Hunan, China. Children and adolescents who were previously immunised with two-dose BBIBP-CorV or CoronaVac were recruited for eligibility screening at least 3 months after the second dose. A stratified block method was used for randomisation, and participants were stratified by age and randomly assigned (3:1:1) to receive AAd5, IMAd5, or inactivated vaccine. The study staff and participants were not masked to treatment allocation. Laboratory and statistical staff were masked during the study. In this interim analysis, adverse events within 14 days and geometric mean titre (GMT) of serum neutralising antibodies on day 28 after the booster vaccination, based on the per-protocol population, were used as the primary outcomes. The analysis of non-inferiority was based on comparison using a one-sided 97·5% CI with a non-inferiority margin of 0·67. This study was registered at ClinicalTrials.gov, NCT05330871, and is ongoing. FINDINGS: Between April 17 and May 28, 2022, 436 participants were screened and 360 were enrolled: 220 received AAd5, 70 received IMAd5, and 70 received inactivated vaccine. Within 14 days after booster vaccination, vaccine-related adverse reactions were reported: 35 adverse events (in 13 [12%] of 110 children and 22 [20%] of 110 adolescents) in 220 individuals in the AAd5 group, 35 (in 18 [51%] of 35 children and 17 [49%] of 35 adolescents) in 70 individuals in the IMAd5 group, and 13 (in five [14%] of 35 children and eight [23%] of 35 adolescents) in 70 individuals in the inactivated vaccine group. Solicited adverse reactions were also reported: 34 (13 [12%] of 110 children and 21 [10%] of 110 adolescents) in 220 individuals in the AAd5 group, 34 (17 [49%] of 35 children and 17 [49%] of 35 adolescents) in 70 individuals in the IMAd5 group, and 12 (five [14%] of 35 children and seven [20%] of 35 adolescents) in 70 individuals in the inactivated vaccine group. The GMTs of neutralising antibodies against ancestral SARS-CoV-2 Wuhan-Hu-1 (Pango lineage B) in the AAd5 group were significantly higher than the GMTs in the inactivated vaccine group (adjusted GMT ratio 10·2 [95% CI 8·0-13·1]; p<0·0001). INTERPRETATION: Our study shows that a heterologous booster with AAd5 is safe and highly immunogenic against ancestral SARS-CoV-2 Wuhan-Hu-1 in children and adolescents. FUNDING: National Key R&D Program of China.

Different software processing affects the peak picking and metabolic pathway recognition of metabolomics data.

In untargeted liquid chromatography‒mass spectrometry (LC‒MS) metabolomics studies, data preprocessing and metabolic pathway recognition are crucial for screening important pathways that are disturbed by diseases or restored by drugs. Here, we collected high-resolution mass spectrometry data of serum samples from 221 coronary heart disease (CHD) patients under two different chromatographic columns (BEH amide and C18 column) and evaluated the three commonly used software programs (XCMS, Progenesis QI, MarkerView) from four aspects (including signal drift, peak number, metabolite annotation and metabolic pathway enrichment). The results showed that the data preprocessed by the three software programs have different degrees of signal drift, but the StatTarget could improve the data quality to meet the data analysis requirement after correction. In addition, XCMS surpassed other software in detection of real chromatographic peaks and Progenesis QI was the best performer in terms of the number of metabolite annotation. XCMS and Progenesis QI showed different performance in pathway enrichment. However, metabolic pathways based on the combination of XCMS and Progenesis QI had a high coincidence with Progenesis QI. In addition, we also reported that C18 and amide columns were highly complementary and have great potential for cooperation in the context of metabolic pathways. In this study, the effects of different chromatographic columns and software pretreatments on metabolomics data were evaluated based on clinical large cohort samples, which will provide a reference for the metabolomics of clinical samples and guide subsequent mechanistic research.

Cyclophilin effector Al106 of mirid bug Apolygus lucorum inhibits plant immunity and promotes insect feeding by targeting PUB33.

Apolygus lucorum (Meyer-Dur; Heteroptera: Miridae) is a major agricultural pest infesting crops, vegetables, and fruit trees. During feeding, A. lucorum secretes a plethora of effectors into its hosts to promote infestation. However, the molecular mechanisms of these effectors manipulating plant immunity are largely unknown. Here, we investigated the molecular mechanism underlying the effector Al106 manipulation of plant-insect interaction by RNA interference, electrical penetration graph, insect and pathogen bioassays, protein-protein interaction studies, and protein ubiquitination experiment. Expression of Al106 in Nicotiana benthamiana inhibits pathogen-associated molecular pattern-induced cell death and reactive oxygen species burst, and promotes insect feeding and plant pathogen infection. In addition, peptidyl-prolyl cis-trans isomerase (PPIase) activity of Al106 is required for its function to inhibit PTI.Al106 interacts with a plant U-box (PUB) protein, PUB33, from N. benthamiana and Arabidopsis thaliana. We also demonstrated that PUB33 is a positive regulator of plant immunity. Furthermore, an in vivo assay revealed that Al106 inhibits ubiquitination of NbPUB33 depending on PPIase activity. Our findings revealed that a novel cyclophilin effector may interact with plant PUB33 to suppress plant immunity and facilitate insect feeding in a PPIase activity-dependent manner.

Apolygus lucorum effector Al6 promotes insect feeding performance on soybean plants: RNAi analysis and feeding behaviour study with electrical penetration graph.

The mirid bug Apolygus lucorum, a dominant mirid species in northern China, is a notorious polyphagous pest with more than 200 hosts, including several major crops such as cotton and soybean, resulting in massive economic loss. Studies of insect salivary effectors may provide a novel control strategy for A. lucorum. An A. lucorum effector, that is, Al6, that inhibits plant immunity by using glutathione peroxidase to repress reactive oxidase accumulation was previously identified. In this study, we further explored the molecular functions of Al6 associated with feeding behaviour and insect survival on soybean, a major host of A. lucorum, using RNA interference and electrical penetration graph (EPG) techniques. We initially observed the injury symptom of this mirid bug and characterized feeding behaviour on soybean leaves using EPG. Our results revealed that A. lucorum preferred to feed on young plant organs such as tender leaves, shoots and buds. This mirid bug used cell rupture as a feeding strategy to ingest cell contents from plant tissues. Subsequently, we silenced the Al6 gene using RNAi and investigated the feeding behaviour, honeydew excretion, body weight, and survival rates of A. lucorum on soybean after Al6 knockdown. Our results demonstrated that silencing of Al6 significantly reduced feeding duration, amount of honeydew secretion, body weight, and survival rates of A. lucorum. Thus, our findings provide a novel molecular target of plant-mediated RNAi for the control of A. lucorum.

Integrated microbiome and metabolome analysis reveals the potential therapeutic mechanism of Qing-Fei-Pai-Du decoction in mice with coronavirus-induced pneumonia.

Current studies have shown that gut microbiota may be closely related to the severity of coronavirus disease 2019 (COVID-19) by regulating the host immune response. Qing-Fei-Pai-Du decoction (QFPDD) is the recommended drug for clinical treatment of patients with COVID-19 in China, but whether it exerts a therapeutic effect by modulating the immune response through gut microbiota remains unclear. In this study, we evaluated the therapeutic effects of QFPDD in pneumonia model mice and performed 16S rRNA sequencing and serum and lung tissue metabolomic analysis to explore the underlying mechanisms during the treatment. Then, Spearman correlation analysis was performed on gut microbiome, serum metabolome, and immune-inflammation-related indicators. Our results suggest that QFPDD can restore the richness and diversity of gut microbiota, and multiple gut microbiota (including Alistipes, Odoribacter, Staphylococcus, Lachnospiraceae_NK4A136_group Enterorhabdus, and unclassified_f_Lachnospiraceae) are significantly associated with immune-inflammation-related indicators. In addition, various types of lipid metabolism changes were observed in serum and lung tissue metabolome, especially glycerophospholipids and fatty acids. A total of 27 differential metabolites (DMs) were significantly correlated with immune-inflammation-related indicators, including 9 glycerophospholipids, 7 fatty acids, 3 linoleic acid, 2 eicosanoids, 2 amino acids, 2 bile acids, and 2 others. Interestingly, these DMs showed a good correlation with the gut microbiota affected by QFPDD. The above results suggest that QFPDD can improve the immune function and reduce inflammation in pneumonia model mice by remodeling gut microbiota and host metabolism.

Feeding Behavior of Riptortus pedestris (Fabricius) on Soybean: Electrical Penetration Graph Analysis and Histological Investigations.

Riptortus pedestris (Fabricius) is a major agricultural pest feeding on soybean pods and seeds. The large populations occur during seed maturity stages from pod filling to harvest. Its infestation results in shriveled and dimpled seeds while vegetative structures (leaflet and stem) remain green, known as "Stay Green" syndrome. Additional evidence also demonstrates that soybean pods and seeds are required for Riptortus pedestris development. However, the feeding behavior strategies employed by this stink bug to feed on soybean plants are still not clear. In the present study, the feeding behaviors of R. pedestris on soybean plants were recorded by electropenetrography (EPG), and a waveform library was created for this species. A total of five phases of waveforms-nonprobing, pathway (Rp1), xylem sap ingestion (Rp2), salivation and ingestion (Rp3), and interruption (Rp4)-were identified. Non-probing waveforms Z and NP and pathway (Rp1) were found in all tested plant structures (leaflet, stem, cotyledon, and pods). Waveform Rp2 (xylem sap ingestion, xylem ingestion) was primarily recorded during R. pedestris feeding on leaflets and stems, while Rp3 (salivation/ingestion) was only observed during feeding on cotyledon and pods. Histological examinations confirmed that correlation between Rp2 and stylet tip positioning in the xylem vessel in leaflets and stems. Stylet tips end in the tissues of cotyledon and pods when Rp3 is recorded. Taken together, our results demonstrate that R. pedestris ingests xylem sap from vegetative tissues of soybean (leaflet and stem) via a salivary sheath strategy to obtain water. It mainly acquires nutrients from soybean pods and/or seeds using cell-rupture tactics. This study provided insightful information to understand the field occurrence patterns of "Stay Green" syndrome, which may have important implications for pest control.

Metastasis patterns and prognosis of octogenarians with metastatic breast cancer: A large-cohort retrospective study.

BACKGROUND: Breast cancer may differ biologically in patients aged over 80 years. The objective of the current study was to analyze the metastasis patterns and prognosis of elderly patients with metastatic breast cancer (MBC) and compare it to patients of other ages. METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was utilized to select MBC patients from 2010 to 2015. Chi-squared test was used to compare clinicopathological characteristics among different aged groups. The Kaplan-Meier method and multivariate Cox model were utilized for survival analysis. RESULTS: A total of 10479 MBC patients were included, among which 1036 (9.9%) patients were aged over 80 years. Compared with other aged group, the elderly patients tended to have a higher proportion of HR+/Her2- subtype, white race, lower tumor differentiation, and receive less treatment, including surgery, chemotherapy and radiotherapy (P< 0.001). MBC patients with different age presented with distinctive metastatic patterns. The older patients were more likely to have lung metastasis, but less likely to have bone, brain, liver and multiple sites metastasis than the younger group (P <0.001). The proportion of TNBC subtype increased substantially in the older patients with brain metastasis, compared to the younger and middle-aged group. The old age was demonstrated to significantly associate with worse prognosis of MBC patients. Additionally, our findings also showed that older MBC patients could achieve dramatical overall survival benefit from surgery (HR = 0.58; P <0.001) and chemotherapy (HR = 0.59; P <0.001), but not the radiotherapy (HR = 0.96; P = 0.097). CONCLUSION: The elderly MBC patients presented with distinctive metastatic patterns, clinical characteristics, and prognostic outcomes compared with younger patients. Our findings could assist clinicians in making appropriate therapeutic decision.

The role of 20E biosynthesis relative gene Shadow in the reproduction of the predatory mirid bug, Cyrtorhinus lividipennis (Hemiptera: Miridae).

Cytorhinus lividipennis is a natural enemy of rice planthoppers and leafhoppers. Improving the fecundity of C. lividipennis will be helpful to improve its control effect on pests. However, little is known about the hormonal regulatory mechanism of reproduction in C. lividipennis. In the current study, we examined the role of 20-hydroxyecdysone (20E) biosynthesis relative gene Shadow in the reproduction of C. lividipennis. The complementary DNA sequence of ClSad is 2018 -bp in length with an open reading frame of 1398-bp encoding 465 amino acid residues. ClSad was readily detected in nymphal and adult stages, and highly expressed in the adult stage. ClSad was highly expressed in the midgut and ovaries of adult females. Moreover, RNA interference-mediated knockdown of ClSad reduced the 20E titers and ClVg transcript level, resulting in fewer fully developed eggs and a decrease in the number of eggs laid by dsSad-injected adult females within 15 days. These results suggest that ClSad plays a critical role in the reproduction of C. lividipennis. The present study provides insights into the molecular mechanism of the ClSad gene for the reproduction of C. lividipennis.

Miniaturized solid-phase extraction using a mesoporous molecular sieve SBA-15 as sorbent for the determination of triterpenoid saponins from Pulsatilla chinensis by ultrahigh-performance liquid chromatography-charged aerosol detection.

A simple, sensitive and efficient solid-phase extraction method, combined with ultrahigh-performance liquid chromatography-charged aerosol detection, was applied to the pre-concentration and determination of four triterpenoid saponins from Pulsatilla chinensis (P. chinensis) ultrasound extract samples. Mesoporous molecular sieve Santa Barbara Amorphous 15 (SBA-15) displayed higher selectivity compared to Mobil Composition of Matter 41 (MCM-41), NH2-SBA-15 and mesoporous carbon when it comes to being used in pretreatment methods. It was applied as an effective sorbent in the SPE for the enrichment of the target analytes. Additionally, several key experimental parameters including the kinds of sorbents, the amount of SBA-15, the elute pH and types of elution solvent were investigated in detail. Under the optimized conditions, the satisfactory linearity (r2 ≥ 0.9940) was acquired and the limits of detection reached 0.461-0.976 µg/mL for the target analytes. The recoveries ranged from 95.1%-103.2%. The experimental results showed that SBA-15 was a candidate material for the purification and concentration of target triterpenoid saponins from complex P. chinensis samples. The study provided theoretical support for the application of mesoporous materials in the field of drug separation and provided references for the extraction and determination of trace compounds in the complex systems of traditional Chinese medicine.

Distinguishing moral hazard from access for high-cost healthcare under insurance.

CONTEXT: Health policy has long been preoccupied with the problem that health insurance stimulates spending ("moral hazard"). However, much health spending is costly healthcare that uninsured individuals could not otherwise access. Field studies comparing those with more or less insurance cannot disaggregate moral hazard versus access. Moreover, studies of patients consuming routine low-dollar healthcare are not informative for the high-dollar healthcare that drives most of aggregate healthcare spending in the United States. METHODS: We test indemnities as an alternative theory-driven counterfactual. Such conditional cash transfers would maintain an opportunity cost for patients, unlike standard insurance, but also guarantee access to the care. Since indemnities do not exist in U.S. healthcare, we fielded two blinded vignette-based survey experiments with 3,000 respondents, randomized to eight clinical vignettes and three insurance types. Our replication uses a population that is weighted to national demographics on three dimensions. FINDINGS: Most or all of the spending due to insurance would occur even under an indemnity. The waste attributable to moral hazard is undetectable. CONCLUSIONS: For high-cost care, policymakers should be more concerned about the foregone efficient spending for those lacking full insurance, rather than the wasteful spending that occurs with full insurance.

Integrated strategy for accurately screening biomarkers based on metabolomics coupled with network pharmacology.

Screening diagnostic biomarkers can be challenging due to the complexity of traditional Chinese medicine (TCM) and ambiguous pharmacological mechanisms. In this study, we reported an integrated strategy for accurately screening diagnostic biomarkers based on metabolomics coupled with network pharmacology. First, a feasible pharmacological model was established through systems pharmacology and based on metabolomics-based techniques to explore diagnostic biomarkers. While the components satisfying the q-value < 0.05, fold change (FC) ≥ 1.2 or FC ≤ 0.8, coefficient of variance (CV) ≤ 30%(QC) and the variable importance in the project (VIP) value > 1 are considered to be diagnostic biomarkers. Second, the ingredients were retained only when oral bioavailability (OB), Caco-2 permeability, drug half-life, TPSA and drug likeness (DL) satisfied the criteria (OB ≥ 40%; Caco-2 ≥ -0.4; HL ≥ 4 h; TPSA˂140; DL ≥ 0.18) suggested by the TCMSP database. Moreover, ingredients that exhibit extensive biological activity in TCM are also retained. Third, the effect targets of TCM were screened using the TCMSP database, Swiss Target Prediction and STICH online software. Disease targets were gathered from the therapeutic target database (TTD), PharmGkb and TCMSP database. Hub genes were screened by potential protein-protein interaction (PPI) network pharmacology analysis. Finally, a metabolic network pathway is established between the diagnostic biomarker and the hub gene. In the network analysis of metabolic pathways, most of the genes involved in this pathway are the second-step-obtained hub genes, which can explain the accuracy of the identified biomarkers. The proposed integrated strategy was successfully applied to explore the mechanism of action of Pulsatilla decoction (PD) in the treatment of acute ulcerative colitis (UC). Based on this integrated strategy, 23 potential biomarkers of acute UC treated with PD were identified. In conclusion, the integrated strategy provides novel insights into network pharmacology and metabolomics as effective tools to illuminate the mechanism of action of TCM.

Characterisation of hederacoside C metabolites using ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry based on automatic fragment ion search.

INTRODUCTION: Hederacoside C (HDC) is a bioactive natural triterpenoid saponins constituent originating from traditional Chinese medicines, playing an important role in the treatment of acute respiratory infections and chronic inflammatory bronchitis. Meanwhile, it is recognised by Korea as a botanical drug. OBJECTIVES: In order to develop an integrated template approach to analysing screening and identification of the metabolites of traditional Chinese medicines. This study will provide available information for further pharmaceutical studies of HDC and other triterpene saponins. METHODOLOGY: An analysis strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) technique combined with automatic fragment ion search (FISh) was firstly exploited for the characterisation metabolites of HDC in vivo and in vitro. Accurate full mass scan combined with an on-line FISh annotations approach was developed to rapidly identify all the potential metabolites of HDC. Furthermore, FISh accurately located the structure of the target compound in a large number of mass spectral data. RESULTS: A total of 34 metabolites were detected and tentatively identified by analysing comprehensive biological samples. The results clearly demonstrated that HDC underwent general metabolic reactions including dealkylation, reduction, oxidation, desaturation, dehydration, cysteine conjugation, GSH conjugation, taurine conjugation, and glycine conjugation to produce 26 phase I and eight phase II metabolites. CONCLUSION: In the present study, UHPLC-Q-Exactive Orbitrap MS technique combined with FISh provided a rapid and efficient platform to characterise metabolites of HDC in vivo and in vitro. The proposed method could develop an integrated template approach to screen and identify the constituents and metabolites of traditional Chinese medicines.

Safety assessment and antioxidant evaluation of betulin by LC-MS combined with free radical assays.

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

A high-throughput metabolomics approach for the comprehensive differentiation of four Pulsatilla Adans herbs combined with a nontargeted bidirectional screen for rapid identification of triterpenoid saponins.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

Study on the metabolites of betulinic acid in vivo and in vitro by ultra high performance liquid chromatography with time-of-flight mass spectrometry.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

UPLC-Q-TOF-MS/MS-guided dereplication of Pulsatilla chinensis to identify triterpenoid saponins.

INTRODUCTION: Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. OBJECTIVE: To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. METHODOLOGY: In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. RESULTS: As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. CONCLUSION: This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

UHPLC-Q-TOF-MS/MS based screening and identification of the metabolites in vivo after oral administration of betulin.

Betulin is an active natural pentacyclic triterpene ingredient with valuable anti-cancer and anti-HIV efficacies. In the present study, an efficient approach was developed to screening and identification of metabolites and to assess the metabolic profiles of betulin in vivo using UHPLC-Q-TOF-MS/MS system based on multiple mass defect filter data acquisition (MMDF) and multiple data processing techniques. Based on the proposed method, 56 phase I and 6 phase II metabolites were detected in rats after oral administration of betulin. The main biotransformation routes of betulin were identified as demethylation, dehydroxylation, deoxidization, dehydration. Conjugation with sulfate, taurine, cysteine and N-acetylcysteine groups produced 6 phase II metabolites. This study not only provided useful information for further study of the pharmacology and mechanism of betulin in vivo, but also provided essential data for further pharmaceutical studies of other pentacyclic triterpenes.