To explore the variation laws of volatile oil during the extraction process of Artemisiae Argyi Folium and its impact on the quality of the medicinal solution, as well as to achieve precise control of the extraction process, this study employed headspace solid phase microextraction gas chromatography-mass spectrometry(HS-SPME-GC-MS) in combination with multiple light scattering techniques to conduct a comprehensive analysis, identification, and characterization of the changes in volatile components and the physical properties of the medicinal solution during the extraction process. A total of 82 volatile compounds were identified using the HS-SPME-GC-MS technique, including 21 alcohols, 15 alkenes, 14 ketones, 9 acids, 6 aldehydes, 5 phenols, 3 esters, and 9 other types of compounds. At different extraction time points(15, 30, 45, and 60 min), 71, 72, 64, and 44 compounds were identified in the medicinal solution, respectively. It was observed that the content of volatile components gradually decreased with the extension of extraction time. Through multivariate statistical analysis, four compounds with significant differences during different extraction time intervals were identified, namely 1,8-cineole, terpinen-4-ol, 3-octanone, and camphor. RESULTS:: from multiple light scattering techniques indicated that at 15 minutes of extraction, the transmittance of the medicinal solution was the lowest(25%), the particle size was the largest(0.325-0.350 nm), and the stability index(turbiscan stability index, TSI) was the highest(0-2.5). With the extension of extraction time, the light transmittance of the medicinal solution improved, stability was enhanced, and the particle size decreased. These laws of physicochemical property changes provide important basis for the control of Artemisiae Argyi Folium extraction process. In addition, the changes in the bioactivity of Artemisiae Argyi Folium extracts during the extraction process were investigated through mouse writhing tests and antimicrobial assays. The results indicated that the analgesic and antimicrobial effects of the medicinal solution were strongest at the 15-minute extracting point. In summary, the findings of this study demonstrate that the content of volatile oil in Artemisiae Argyi Folium extracts gradually decreases with the extension of extraction time, and the variation in volatile oil content directly influences the physicochemical properties and pharmacological efficacy of the medicinal solution. This discovery provides important scientific reference for the optimization of Artemisiae Argyi Folium extraction processes and the development and application of process analytical technologies.
Oils, Volatile/pharmacology* ; Artemisia/chemistry* ; Gas Chromatography-Mass Spectrometry ; Drugs, Chinese Herbal/pharmacology* ; Chlorogenic Acid/pharmacology* ; Solid Phase Microextraction ; Quality Control

AIM To analyze the metabolites of nobiletin in rats in vivo based on characteristic ions.METHODS Ten rats were assigned into administration group and control group,and given intragastric administration of the 0.5％CMC-Na suspension of nobiletin(250 mg/kg)and 0.5％CMC-Na solution,respectively,after which plasma,urine and feces were collected,solid phase extraction method was adopted in pretreatment,UHPLC-HRMS analysis was performed.The candidate metabolites were systematically described according to diagnostic product ions,chromatographic retention time,accurate molecular weight and neutral loss fragments,after which accurate metabolites were obtained in the established metabolite data set with-CH3(m/z 15)characteristic ions as a baits.RESULTS A total of 64 metabolites were identified,whose main metabolic pathways were glucuronidation,sulfation,hydrogenation and their compound reactions.CONCLUSION This experiment elucidates the metabolites of nobiletin in rats in vivo,which provides a new reference for its further development.

AIM To investigate the effects of Zuogui Jiangtang Tongmai Recipe on necroptosis pathway in a rat model of type 2 diabetes mellitus(T2DM)complicated with cerebral infarction(CI).METHODS The SD rats were randomly divided into the sham operation group,the model group,the metformin group(0.045 g/kg),and the low,medium and high dose Zuogui Jiangtang Tongmai Recipe groups(6.5,13,26 g/kg),with 9 rats in each group.In contrast to rats of the sham operation group,rats of the other groups were given 4 weeks feeding of high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin to establish a T2DM rat model with one week stable blood glucose,followed by gavage of corresponding drugs 3 days before the establishment of the middle cerebral artery occlusion(MCAO)model.After 7 days of administration,the rats had their CI injury assessed by mNSS method and TTC staining;their level of blood glucose detected by blood glucose meter;their levels of glycated serum protein,serum TNF-α and IL-1β detected by ELISA;their cerebral mRNA expressions of FADD,RIPK1,RIPK3 and MLKL detected by RT-qPCR;and their cerebral protein expressions of FADD,p-RIPK1,p-RIPK3 and p-MLKL detected by Western blot.RESULTS Compared with the sham operation group,the model group displayed increased levels of blood glucose value,glycosylated serum protein,neurological function score,cerebral infarction volume,cerebral FADD,RIPK1,RIPK3 and MLKL mRNA expressions,cerebral FADD,p-RIPK1,p-RIPK3 and p-MLKL protein expressions,serum TNF-α and IL-1β levels(P＜0.01);and more disordered and morphologically diverse neurons with smaller nucleus.Compared with the model group,the groups intervened with medium or high dose Zuogui Jiangtang Tongmai Recipe,or metformin shared improvement in terms of the aforementioned indices(P＜0.05,P＜0.01);and more neurons with regular morphology neat arrangement,and reduced cell gap.CONCLUSION Zuogui Jiangtang Tongmai Recipe can improve the neurological dysfunction of the rat model of T2DM complicated with CI,which may associate with the inhibited activation of necroptosis signaling pathway.

Although indigenous malaria has been eliminated in Wuhan since 2013,imported malaria remains a potential threat as an infectious source of local malaria transmission.The epidemiological and clinical characteristics of imported malaria are particularly important in areas where local malaria has been eliminated.This study was aimed at analyzing the epidemiological and clinical characteristics of imported malaria in Wuhan from 2012 to 2019,to provide a basis for further improving the preven-tion and control of imported malaria.Patients in Wuhan diagnosed with imported malaria from January 1,2012,to December 31,2019,were included in this study.A case-control study was con-ducted to analyze the features of patients with severe malaria.Uni-variate and multivariate logistic regression was used to identify risk factors for prolonged hospital length of stay(LOS).Among 229 imported malaria cases,212(92.6％)were in Chinese citizens,and most cases were in men(96.5％).The gender ratio is 28:1,and the age of cases is mainly concertrated between 18 and 50 years old(89.1％).More than 80％of patients were mi-grant workers,and most cases were infections from African countries(92.6％).Plasmodium falciparum(80.8％)was the dominant species.Fifty-three severe malaria cases were identified during the study period.Compared with uncomplicated cases,severe cases tended to occur in patients with no history of malaria(P=0.008),patients infected with Plasmodium falciparum(P=0.009),and patients who were initially misdiagnosed(P＜0.001).The median LOS was 6 days,and the species of infec-tion(Plasmodium falciparum),the use of antimalarial drugs(group B),antipyretic time(longer than 3 days),and the turn-around time of blood smear microscopy(longer than 3 days)were significantly associated with longer LOS(all P＜0.05).Al-though malaria has been eradicated in Wuhan for many years,imported cases continue to pose a threat.Efforts should be made to strengthen malaria knowledge education for outbound personnel.Additionally,medical institutions must enhance diagnosis and treatment capabilities for malaria,and adhere to standardized treatment processes,and the development of drug resistance and occurrence of severe malaria must be prevented.

The medicinal value of vanilla planifolia is of great interest.We analyzed the common princi-pal components of VOCs in different parts of vanilla planifolia,and the human serum albumin (HSA),β-lactoglobulin (β-La) andα-lactalbumin (α-La) were used as template proteins to establish a chain a-nalysis approach with the 'solid phase microextraction gas chromatography-mass spectrometry-multispec-troscopy-physical modelling-pharmacokinetics' (S-M-P-P ) .The mechanisms of the transport and phar-macodynamics for the common principal components of vanilla planifolia were analyzed.The results showed that the common primary VOCs in different parts of vanilla planifolia was vanillin (Van),which attenuated the endogenous fluorescence of HSA/β-La/α-La by static bursting,and formed hydrogen bonds and Van der Waals forces with HSA,and noncovalent complexes with β-La/α-La through hydro-phobic forces.Their interaction facilitates the transport of Van in vivo to intestinal and hepatic tissues and its metabolism by CYP1A2 and CYP2C9 enzymes to exert its pharmacological effects.This study provides a comprehensive and in-depth investigation of the transport mechanisms and pharmacological effects of VOCs from vanilla planifolia,which provides an important reference for understanding the medicinal po-tential of plant derived VOCs.

Aim To study the proliferation,invasion and migration ability of Quaking(QKI)in gastric cancer(GC)via elucidating the molecular mechanisms associated with QKI in the occurrence and development of GC through bioinformatics.Methods Differential expression analysis of QKI was performed across vari-ous human cancer samples by merging data from the TCGA and GTEx databases.The correlation was ana-lyzed between QKI protein expression and tumor muta-tion burden(TMB)score,microsatellite instability(MSI)score,and ESTIMATE score,and the correla-tion was also explored between QKI protein expression and overall survival(OS),disease free survival(DFS),and progression free survival(PFS).EMT related genes that could encode DECircRNAs were ob-tained through bioinformatics analysis to construct a QKI-EMT-circRNAs regulatory network.The differenti-ally expressed circRNAs and EMT related genes in TMK1 cells were verified.The proliferation,invasion and migration ability of the QKI was studied by using the knockdown system.Results QKI was differential-ly expressed in the vast majority of tumors and was closely related to TMB,MSI,and tumor microenviron-ment(TME);QKI emerged as a high-risk factor for predicting OS,DFS,and PFS in individuals with com-mon human cancers.QKI regulated the splicing of 6 EMT related gene transcripts to form eight circRNAs,all of which were significantly associated with the prog-nosis of gastric cancer patients.Cell experiments showed that compared to normal gastric epithelial cells,only hsa_ccirc_0004015,CALD1,and CDK14 were down-regulated in TMK1 cells.Knocking down QKI inhibited the proliferation,invasion and migration ability of TMK1 cells.Conclusion QKI exerts regu-latory control over the transcription of six EMT-related genes,resulting in the formation of circRNAs,thereby promoting the pathogenesis and progression of GC.QKI is highly expressed in TMK1 cells,and knock-down of QKI can inhibit the proliferation,invasion and migration ability of TMK1 cells.

Aim To explore the effect of serum contai-ning Duhuo Jisheng Decoction on the proliferation,ap-optosis and inflammation of fibroblast-like synoviocytes(FLS)induced by tumor necrosis factor-α(TNF-α)in rheumatoid arthritis(RA)and to reveal the under-lying mechanism.Methods The MH7A cells were divided into five groups:normal group(10％blank se-rum),model group(10 μg·L-1 TNF-α+10％blank serum),Duhuo Jisheng Decoction low(10 μg·L-1 TNF-α+2.5％drug-containing serum+7.5％blank serum),medium(10 pg·L-1 TNF-α+5％drug-con-taining serum+5％blank serum),high(10 μg·L-1 TNF-α+10％drug-containing serum)dose group.The concentration of serum containing Duhuo Jisheng Decoction was screened by MTT method.Cell prolifer-ation was detected using EdU staining.The expression of proliferation marker Ki67 was detected using immu-nofluorescence staining.The apoptosis rate was meas-ured by flow cytometry.The contents of IFN-γ,IL-4,IL-6 and IL-10 in each group were detected by ELISA.The mRNA and protein expression of Bax,Bcl-2,caspase-3 and TLR4/MyD88/NF-κB signaling pathway were detected by RT-qPCR and Western blot.Results Compared with the normal group,the cell prolifera-tion activity of the model group significantly increased,and the level of apoptosis decreased.The content of IFN-γ and IL-6 increased,and the content of IL-4 and IL-10 decreased.The TLR4/MyD88/NF-κB signaling pathway was activated.After the intervention of low,medium and high dose groups of serum containing Du-huo Jisheng Decoction,it could effectively improve the abnormal proliferation of cells and enhance apoptosis.At the same time,it inhibited the inflammatory re-sponse and the activation of TLR4/MyD88/NF-κB sig-naling pathway.Conclusions The serum containing Duhuo Jisheng Decoction can inhibit the abnormal pro-liferation of RA-FLS induced by TNF-α and the secre-tion of pro-inflammatory factors,and enhance apoptosis and anti-inflammatory levels.The mechanism may be related to the regulation of TLR4/MyD88/NF-κB sig-naling pathway.

Objective To investigate the clinical and genetic features of children with 3-methylcrotonyl-coenzyme A carboxylase deficiency(MCCD).Methods A retrospective analysis was conducted on the clinical manifestations and genetic testing results of six children with MCCD who attended Children's Hospital Affiliated to Zhengzhou University from January 2018 to October 2023.Results Among the six children with MCCD,there were 4 boys and 2 girls,with a mean age of 7 days at the time of attending the hospital and 45 days at the time of confirmed diagnosis.Of all children,one had abnormal urine odor and five had no clinical symptoms.All six children had increases in blood 3-hydroxyisovaleryl carnitine and urinary 3-hydroxyisovaleric acid and 3-methylcrotonoylglycine,and five of them had a reduction in free carnitine.A total of six mutations were identified in the MCCC1 gene,i.e.,c.1630del(p.R544Dfs*2),c.269A>G(p.D90G),c.1609T>A(p.F537I),c.639+2T>A,c.761+1G>T,and c.1331G>A(p.R444H),and three mutations were identified in the MCCC2 gene,i.e.,c.838G>T(p.D280Y),c.592C>T(p.Q198*,366),and c.1342G>A(p.G448A).Among these mutations,c.269A>G(p.D90G)and c.1609T>A(p.F537I)had not been previously reported in the literature.There was one case of maternal MCCD,and the child carried a heterozygous mutation from her mother.Five children with a reduction in free carnitine were given supplementation of L-carnitine,and free carnitine was restored to the normal level at the last follow-up visit.Conclusions This study identifies two new mutations,c.269A>G(p.D90G)and c.1609T>A(p.F537I),thereby expanding the mutation spectrum of the MCCC1 gene.A combination of blood amino acid and acylcarnitine profiles,urine organic acid analysis,and genetic testing can facilitate early diagnosis and treatment of MCCD,and provide essential data for genetic counseling.

Background/Aims: Despite the high efficacy of direct-acting antivirals (DAAs), approximately 1–3% of hepatitis C virus (HCV) patients fail to achieve a sustained virological response. We conducted a nationwide study to investigate risk factors associated with DAA treatment failure. Machine-learning algorithms have been applied to discriminate subjects who may fail to respond to DAA therapy. Methods: We analyzed the Taiwan HCV Registry Program database to explore predictors of DAA failure in HCV patients. Fifty-five host and virological features were assessed using multivariate logistic regression, decision tree, random forest, eXtreme Gradient Boosting (XGBoost), and artificial neural network. The primary outcome was undetectable HCV RNA at 12 weeks after the end of treatment. Results: The training (n=23,955) and validation (n=10,346) datasets had similar baseline demographics, with an overall DAA failure rate of 1.6% (n=538). Multivariate logistic regression analysis revealed that liver cirrhosis, hepatocellular carcinoma, poor DAA adherence, and higher hemoglobin A1c were significantly associated with virological failure. XGBoost outperformed the other algorithms and logistic regression models, with an area under the receiver operating characteristic curve of 1.000 in the training dataset and 0.803 in the validation dataset. The top five predictors of treatment failure were HCV RNA, body mass index, α-fetoprotein, platelets, and FIB-4 index. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the XGBoost model (cutoff value=0.5) were 99.5%, 69.7%, 99.9%, 97.4%, and 99.5%, respectively, for the entire dataset. Conclusions Machine learning algorithms effectively provide risk stratification for DAA failure and additional information on the factors associated with DAA failure.

Celastrol and wilforlide A are the main active triterpenoids of the traditional Chinese medicine Lei Gong Teng, which have anti-tumour, anti-inflammatory and immunosuppressive activities, and are the material basis for the clinical efficacy of Lei Gong Teng-related Chinese medicinal preparations. By analysing the biosynthetic pathway of active ingredients, optimizing genetic elements and utilizing "cell factory" to produce triterpenoids heterologously will be an effective way to obtain from Tripterygium wilfordii in the future in a "low-cost and high-efficient" manner. CYP712Ks are the first cytochrome P450s involved in the skeleton modification of friedelane-type and oleanane-type triterpenoids in T. wilfordii, and they can catalyse the generation of polpunonic acid from friedelin and 3-epi-katonic acid from β-amyrin by carboxylation at C-29 position. In this study, four multifunctional TwCYP712K1/2/3/5 were used to clarify the catalytic function and substrate selection preference using in vivo functional characterization in Saccharomyces cerevisiae. The spatial structure of the protein-substrate binding was clarified through homology modeling and molecular docking, and then the differential amino acids in the active pocket of the protein were mutated to clarify the crucial amino acids determining the catalytic function and the selection of substrate structure. A total of 63 mutant elements were constructed, and the amino acid sites affecting the carboxylation function of TwCYP712Ks were analyzed. In particular, the key amino acids affecting the substrate selectivity of TwCYP712K2 towards oleanane-type and friedelane-type triterpenoids were revealed and the TwCYP712K2^F127I and TwCYP712K2^A227T mutants would result in a reversal of the product ratio. In conclusion, four proteins of TwCYP712Ks were semi-rationally designed by homologous protein alignment and mutual mutation to elucidate multiple amino acid sites determining the catalytic function of the proteins, and a series of activity-enhancing or altering mutants were obtained, which provide abundant catalytic elements for the biosynthesis of active triterpenoids from T. wilfordii, and the mechanism of carboxylation in the C-29 position was initially elucidated.