Objective To isolate and purify flavanomarein from Coreopsis tinctoria, and to study its anti-platelet aggregation effect. Methods The dry inflorescence of Coreopsis tinctoria was extracted by refluxing using 55% ethanol to obtain ethanol extract(ETE), and then the aqueous layer from ETE was extracted by ethyl acetate to obtain ethyl acetate extract(AR)after gathering by macroporous resin. Furthermore, Flavanomarein of flavonoids was isolated from ETEP by polyamide chromatography column. The blood sample of healthy subjects was collected via vein and randomly divided into blank control group(saline), ETE group(high dose of 900 μg·mL-1, medium dose of 300 μg·mL-1, low dose of 100 μg·mL-1), flavanomarein group(high dose of 90 μg·mL-1, medium dose of 30 μg·mL-1, low dose of 10 μg·mL-1), aspirin(acetylsalicylic acid, ASA)group(500 μg·mL-1). A platelet aggregation model induced by adenosine diphosphate(ADP)and thrombin was established. Born turbidimetric method was used to determine the platelet aggregation inhibition rate by observing its anti-platelet aggregation effect. Results Flavanomarein with purity of more than 92% was successfully isolated from the dry inflorescence of Coreopsis tinctoria. Compared with the blank control group, the medium and high dose groups of ETE and the high dose group of flavanomarein could inhibit ADP-induced platelet aggregation(P<0.01). Compared with the blank control group, the medium dose group of ETE and the medium dose group of flavanomarein could inhibit thrombin-induced platelet aggregation, and the differences were statistically significant(P<0.01). The high dose of AR group and the high dose of flavanomarein group could inhibit thrombin-induced platelets aggregation, and the difference was statistically significant(P<0.01). Conclusion Flavanomarein in the Coreopsis tinctoria was one of the main active substances for anti-platelet aggregation.
Objective To observe the protective effect and mechanism of daurinoline on oxygen-glucose deprivation(OGD)in rat cortical neurons. Methods Using MTT, flow cytometry, 2, 7'- dichlorofluorescein diacetate(DCFH-DA)fluorescent probe, and cationic fluorescent dye JC-1, the reactive oxygen species(ROS)generation and mitochondrial membrane potential(MMP)in rat cortical cultures were detected. Results Daurinoline(0.1, 1, 10 μg·mL-1)could reduce the injury of OGD reperfusion on the cortical neurons of rats. And it could decrease the intracellular ROS generation and stabilize mitochondrial membrane potential after OGD reperfusion. Conclusion Daurinoline markedly protected rat primary cortical neurons against OGD-reoxygenation-induced toxicity in vitro. The possible mechanism underlying daurinoline protective effects involved regulating ROS generation, stabilizing membrane potential, and protecting mitochondrial function.
Objective To investigate the protective effect of peonidin on mice with type 2 diabetes mellitus(T2DM)and non-alcoholic fatty liver disease(NAFLD), and to explore its underlying mechanism. Methods Forty-eight male C57BL/6 mice were divided into normal control group, model control group, peonidin high-dose group, middle-dose group, and low-dose group. The T2DM mice with NAFLD model was induced by intraperitoneal injection of streptozotocin combined with high fat diet. The levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total cholesterol(TC), triglyceride(TG), blood glucose and insulin(INS)in serum were tested. The levels of inflammatory cytokines, tumor necrosis factor-a(TNF-α)and interleukin-6(IL-6)in serum were determined, and malondialdehyde(MDA)and superoxide dismutase(SOD)in liver tissues were also detected. The histopathological changes of liver tissue were observed by HE staining. The NF-κB protein expression in liver was measured by western blot. Results The levels of ALT, AST, TC, TG, blood glucose, and INS were higher in model control group than in normal control group(P<0.05). Compared with the normal control group, the levels of inflammatory cytokines TNF-α and IL-6 in serum, MDA and NF-κB expression in liver tissues was significantly increased in model control group(P<0.05), and the level of SOD in liver tissues was greatly decreased(P<0.05). Compared with the model control group, the levels of serum ALT, AST, TC, TG, blood glucose and INS in peonidin groups were decreased(P<0.05), so were the levels of inflammatory cytokines TNF-α and IL-6 in serum, as well as the MDA and Nrf2 expression in liver tissues(P<0.05). The level of SOD in liver tissues was greatly increased in peonidin groups than in the model control group(P<0.05). HE staining showed that histopathological changes in liver tissue were significantly alleviated in peonidin groups. Conclusion Peonidin showed the protective effect on T2DM mice with NAFLD. It could significantly inhibit inflammation and oxidative stress. The mechanism might be related with the downregulation of NF-κB expression.
Objective To optimize the extraction process of total flavonoids from Cyclocarya paliurus, and to investigate the intervention effect of total flavonoids on oxidative stress and glycolipid metabolism disorder in obese mice model. Methods Based on α-glucosidase inhibition rate and DPPH free radical scavenging rate, Box-Behnken design-response surface method combined with the hierarchical analysis were used to optimize the extraction process of total flavonoids from Cyclocarya paliurus. The in vivo activity of total flavonoids from Cyclocarya paliurus was investigated on obese mice induced by high fat diet. Results The IC50 value of total flavonoids from Cyclocarya paliurus on the α-glucosidase inhibition was(101.22±4.94)μg·mL-1, and the IC50 value of DPPH free radicals scavenging activity was(23.75±1.93)μg·mL-1. Total flavonoids could reduce body mass, liver index, fat content, transaminase, blood lipid, and glucose level in obese mice, and ameliorate lipid peroxidation. Conclusion The optimized extraction process was simple and feasible. Total flavonoids had antioxidant, lipid lowering, and hypoglycemic effects. And they could improve oxidative stress and metabolism disorder of glycolipid level in obese mice. The activity was positively correlated with the mass concentration of total flavonoids.
Patients infected with SARS-CoV-2 show elevated levels of inflammatory cytokines and disorders of clot-related indicators.Whether there is a higher risk of thromboembolism has not been confirmed in those patients.However,based on previous research on SARS-CoV,we infer that SARS-CoV-2 may promotes the formation of blood clots by increasing the expression of inflammatory cytokines in infected epithelials.These cytokines can enhance coagulation cascade,disturbethe physiological balance of the blood coagulation and anticoagulation,and then progress the body into high coagulation state.The review of the relationship between SARS-CoV-2 and coagulation state plays an important role for those infected patients’ clinical management.
The prognosis of novel coronavirus pneumonia is closely related to host's immune response.Immunity system plays a vital role in controlling and eliminating virus infections.Thymosin immunomodulators can enhance the body's cellular immune function.Based on existing evidences,this paper reviews the sources and differences of thymosin drugs,of which the immunomodulatory effects in different populations and application in viral infection are also summarized.In addition,we discuss the roles of thymosin drugs in the prevention and treatment of new coronavirus infections,and put forward suggestions for drug selection and pharmaceutical care,in order to provide reference for clinical decision-making related to new coronavirus pneumonia and improve its prevention and treatment effects.
Objective To analyze the clinical characteristics of 2019 novel coronavirus pneumonia(COVID-19) cases of different clinical subtypes. Methods Fifty-four COVID-19 patients were enrolled from Hubei No.3 People’s Hospital of Jianghan University from Jan 24 to Feb 8,2020.Patients were divided into normal patient group,severe patient group,and death group according to clinical diagnoses.The epidemiology,clinical symptoms,test results,imaging data,and treatment data of the three groups of patients were retrospectively collected and analyzed. Results The positive rate of nucleic acid detection was 45.5% in all confirmed COVID-19 patients.Median age of normal patient group,severe patient group,and death group were 49,60,and 70 years old,respectively,with significant differences(P<0.05).There were significant difference in chronic medical illness(P<0.01) among normal patient group(13.0%),severe patient group(80.0%) and death group(66.7%),while significant difference in comorbid conditions was also observed(P<0.01) in these three groups(4.4%,40.0% and 100.0%).Hospitalization days of death group was 6.5 days,which was significantly lower than that in the normal group(11 days,P=0.026)or the severe group(21 days, P=0.045).In all patients,common symptoms included fever 47(87.0%),cough 41(75.9%),fatigue 29(53.7%),and muscle pain 25(46.3%).On day of admission,all patients had median of lymphocyte count at 0.7×109·L-1(IQR 0.6~1.2),C-reactive protein at 46.4 mg·L-1(IQR 12.7~84.3),serum amyloid at 373.5 ng·mL-1(IQR57.1~961.7),and erythrocyte sedimentation rate at 42.7 mm·h-1(IQR17.3~74.5).In the death group,median of troponin was 0.6 μg·L-1(IQR 0.5~0.6) and lactate dehydrogenase was 842.0 U·L-1(IQR 556.5~1127.5).Imaging examination showed that 68.5% patients had patchy lung shadow.Current treatment of COVID-19 included antiviral drugs(100.0%),antibacterial drugs(100%),immune-boosting drug(51.9%),and corticosteroids(85.2%). Conclusion Positive outcomes were relatively lower in nucleic acid test.Clinical symptoms,laboratory indicators,and imaging data could be reasonably used for diagnosis of COVID-19.
The novel coronavirus outbreak,which was declared a public health emergency of international concern, has attracted wide attention in the world.The positive role of Traditional Chinese Medicine has been emphasized in the “Diagnosis and Treatment of Novel Coronavirus Pneumonia(Trail Version 6)”,and some Chinese patent medicines(CPMs)that contain Huoxiang Zhengqi capsules(pills,liquid,oral solution),Jinhua Qinggan granules,Lianhua Qingwen capsules(granules),Shufeng Jiedu capsules(granules),Xiyanping injection,Xuebijing injection,Reduning injection,Tanreqing injection,Xingnaojing injection,Suhexiang pills,Angong Niuhuang pills,Shenfu injection,Shengmai injection,and Shenmai injection are recommended to treat Corona Virus Disease 2019(COVID-19).The pharmacological effect,clinical application,and adverse reaction of above fourteen CPMs in symptomatic or supportive treatment of viral infection,pneumonia,and other related diseases were reviewed through searching and summarizing Chinese literatures published during 2003-2020.It may provide a guide for clinicians to rationally use the CPMs.
There is still no specific medicine for novel coronavirus(SARS-CoV-2)pneumonia(COVID-19).Here,we summarized the pharmacology properties and mechanisms of remdesivir and interferon-α,and made a theoretical assessment of the combined use of both drugs in the treatment of COVID-19 through the epidemiology and pathogens analysis of COVID-19.Remdesivir is a nucleoside analogue with a broad-spectrum antiviral effect,and interferon-α is an immune regulatory protein with a broad-spectrum antiviral effect.Both of them exert antiviral effects through different actions,so it is rational to apply them in combination to improve the antiviral effect.In addition,interferon-α exhibits an anti-inflammatory effect at the site of infection,thereby the combination with remdesivir could be used for both etiological and symptomatic treatment of COVID-19.In this article,we provide a theoretical basis for guiding clinical therapy for fighting against COVID-19,although a large amount of clinical trials is still in need of exploring for the clinical use of combining remdesivir and interferon-α in the treatment against COVID-19.
Objective To evaluate the antiviral therapies on severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) rapidly and systematically,and to explore the clinical feasibility of subcutaneous use of interferon-α in the treatment of SARS-CoV-2. Methods Systematically retrieved relevant clinical studies published in Chinese and English from databases,such as CNKI,VIP,Wangfang,CBM,PubMed,Cochrane Library,et al.In addition,the web site of WHO,Chinese Center for Disease Control and Prevention,Centers for Disease Control and Prevention of United States,and National Health Commission of the People’s Republic of China were retrieved,so as to search the relevant diagnosis,treatment programsand the relevant references.According to evidence-based pharmacy,evidence evaluation and data extraction were carried out. Results A total of 1502 articles were retrieved and 11 studies of them were included finally.The result showed:① The early use of subcutaneous interferon-α should be within 48 hours after the diagnosis of coronavirus infection.With the prolongation of medication,cure rate will decrease,so the treatment should be as early as possible.②There was no significant difference in therapeutic effect among different types of interferon.③Interferon-α combined with ribavirin has been proved to be effective in some studies,but their adverse reactions need to be vigilant.Most of the adverse reactions were tolerable,including hemoglobin decreasing,transaminase increasing and amylase increasing.And most of them did not affect the treatment. Conclusion Interferon-α can be used as an experimental therapy in the antiviral treatment of SARS-CoV-2,especially in the newly infected patients.Therefore,randomized controlled trials and real world clinical studies should be carried out as soon as possible to further explore the clinical effect of interferon-α on SARS-CoV-2.
SARS-CoV-2 belongs to the new type of the genus betacoronavirus,which has strong infectivity and transmission power.The epidemic situation has attracted wide attention in the whole world.Because the academic background of most clinicians of respiratory department or infectious disease department were western medicine,their knowledge of traditional Chinese medicine is relatively lacking.Based on the guidelines of the fifth edition of "pneumonia diagnosis and treatment program for novel coronavirus infection",make a reference to the clinicians about the rational use of the Chinese patert medicine for the novel coronavirus infectious pneumonia.
Objective To detect and analyze safety signals of α-interferon (IFN-α) through data mining methods based on FAERS,in order to provide reference for clinical safe use of IFN-α. Methods Data of FARES from 2004Q1 to 2019Q4 (64 quarters) were downloaded.After drug names standardized by MedEx and adverse events classified by MedDRA,the adverse event report cases with IFN-α as the primary suspect drug was extracted,and the safety signal detection was conducted using PRR and ROR methods. Results A total of 2889 adverse event reports with IFN-α as the first suspected drug were gathered.Among them,the male/female ratio was 1.32,and the median age was 55 years old.Reports from doctors accounted for 39.91%.Reports from United States accounted for 58.81%.The main routes of administration reported were subcutaneous,intravenous,and intramuscular,accounting for 43.89%.More than half of the cases did not report the route of IFN-α administration.The number of reported cases of “general disorders and administration site conditions”,“investigations”,“blood and lymphatic system disorders” was relatively large.Among the top 20 signal detected events,“hypophosphataemia” and “blood creatine phosphokinase increase” have not been included in IFN-α specification yet. Conclusion Some high frequency adverse events of IFN-α are similar to the symptoms of novel coronavirus pneumonia.Compared with the traditional routes of administration,the incidence of adverse events of IFN-α atomization inhalation may be lower in theory,however,it is necessary to monitor and judge the related drugs and events in clinical practice.
Objective To explore the efficacy and safety of ribavirin in novel coronavirus pneumonia based on previous coronavirus studies. Methods Systematically retrieve relevant clinical studies from Chinese and English databases such as CNKI, VIP, Wangfang Data, PubMed, Web of Science, Embase, EBSCO retrieving cohort studies and case reports on the efficacy of ribavirin in the treatment of SARS and MERS. The retrieval time range is from January 2003 to January 30, 2020. Results The effectiveness study showed that some patients with SARS and MERS received ribavirin, the condition was controlled and improved, and the cure rate and clinical treatment outcome were also good.Safety studies have shown a high incidence of adverse reactions to ribavirin, mainly anemia and arrhythmia, and other adverse reactions have been reported. Conclusion Ribavirin may be effective in the treatment of COVID-19, but further clinical trials are needed to confirm it. At the same time, due to the high incidence of adverse reactions, all indicators should be carefully evaluated before clinical use and closely monitored during treatment.
Objective To detect and analyze safety signals of chloroquine phosphate,which is one of the latest recommended drugs for the treatment of novel coronavirus pneumonia (NCP) due to COVID-19,through the data mining methods based on FAERS database,in order to provide references for clinical safe use of chloroquine phosphate. Methods FARES Data from 2004Q1 to 2019Q4 (64 quarters) were downloaded.After drug names standardization by MedEx and adverse events classification by MedDRA,the adverse event reports were extracted using the chloroquine phosphate as the primary suspected drug,and the ADR signal detection was conducted using PRR and ROR methods. Results A total of 357 cases with chloroquine phosphate as the primary suspected drug were gathered.Among them,the male/female ratio was 0.60,and the median age was 39 years old.Reports from “other health-professional” accounted for 49.86%,and from “doctors” accounted for 23.35%.The United States reports the most,accounting for 20.17%.The main routes of administration were oral,accounting for 35.01%.A total of 148 ADR signals were detected by ROR method,while 147 ADR signals were detected by PRR,of which 147 signals are identical.The results of ADR signal detection showed that the number of ADE reports of “cardiac disorders” was the largest,of which was 372,accounting for 20.36% with 28 signals detected.Then,the number of the ADR signals of “all kinds of abnormalities”,“nervous system disorders” and “psychiatric disorders” were relatively larger in turns. Conclusion Before using chloroquine phosphate,med; cation evaluation should be performed well,especially when patients have basic diseases such as cardiac diseases,nervous diseases or mental illness etc.,and monitoring should be strengthened to further reduce and evaluate the risk of ADR.
Objective To provide a reference for the safe use of ribavirin in clinic,adverse drug events signals of ribavirin were detected and analyzed through data mining methods based on FAERS database. Methods Sixty-four quarterly data of FARES database from 2004 to 2019 were downloaded.After drug names standardization by MedEx and adverse drug events classification by MedDRA,the safety signal reports setting ribavirin as the primary suspected drug was searched and detected,using proportional reporting ratio(PRR) and reporting odds ratio(ROR) methods. Results A total of 9854 ribavirin related death events were gathered.Among them,51.00% of reports were males,and the male/female ratio was 1.32,and the average age was 54.01 years old.The reporting countries are mainly the United States,France,Australia,the United Kingdom,and Germany.Most of the routes of administration were oral administration,followed by transplacental,subcutaneous,intravenous,and inhalation.There are many adverse events reported in systemic and administration site reactions,gastrointestinal system events,various examination abnormalities,blood and lymphatic system events,and nervous system events. Conclusion To avoid drug withdrawal or misdiagnosis of disease progression,adverse drug events and blood routine monitoring should be monitored when ribavirin is used in the treatment of novel coronavirus pneumonia.
In Wuhan,the hardest hit area of the epidemic novel coronavirus pneumonia (NCP),the number of confirmed cases continues to increase,and the number of people admitted to hospitals has reached saturation.To this end,on February 2,2020,Wuhan quickly launched the construction of a “square cabin hospital” to specifically treat patients with mild illness.There are a large number of patients in the square cabin hospital,among which there are many patients with chronic diseases when the original diseases are superimposed by with NCP resulting in a great increase in the complexity of drug use,pharmaceutical security and drug intervention need to be solved urgently.This paper mainly discusses how to guarantee the drug supply and ensure the safety and reasonableness of drug use in the situation of lack of pharmacists in square cabin hospital.
Novel coronavirus pneumonia(NCP) broke out in Wuhan. As the hardest hit area of Wuhan,the growing number of confirmed cases need better therapy.On January 25,2020,the Sino-French New City Branch,Tongji Hospital of Huazhong University of Science and Technology was transformed into designated hospital for NCP.A total of 18 medical teams nationwide carried out treatment work here.In a short period of time,it was quickly transformed into a designated hospital for critical patients.Because most patients were critically ill,the demand for medication increased dramatically.How to guarantee the need of medication supply and pharmaceutical care became a urgent problem to be resolved .Our article mainly discussed how to ensure the supply of drug in designated hospital for critical patients,and how to ensure the safety and rationality of medication use.
Objective To establish a method for the determination of trans-π-oxocamphor concentration in rat plasma and investigate its pharmacokinetic profile in rat. Methods The plasma samples were processed by a liquid-liquid extraction, and the samples were analyzed by GC-MS using toluene as an internal standard. The determination was performed on a HP-5MS capillary column using helium as the carrier gas at a flow rate of 1.11 mL·min-1. The sample size was 1 μL under the programmed temperature control. Electron bombardment ion source was used for positive ion scanning in a single ion monitoring pattern. Plasma of six rats was sampled and tested after intravenous administration of trans-π-oxocamphor, and DAS 2.1.1 was used to evaluate the pharmacokinetic parameters. Results The linear range of trans-π-oxocamphor concentration was in 0.1~10.0 μg·mL-1 with LLOQ of 0.1 μg·mL-1. The average absolute recovery rate was 76.94%~85.31% with matrix effects of 94.42%~98.50%. The RSDs of intra-day and inter-day ranged from 1.45% to 10.36%. The pharmacokinetics study showed the two-compartment model was suitable for profiling the concentration-time curve of trans-π-oxocamphor, which demonstrated its quick elimination profile. The main pharmacokinetic parameters were obtained, including AUC(0-t)=20.459±2.034 mg·L-1·min, t1/2z =6.938±3.093 min, CLz=0.175±0.017 L·min-1·kg-1, and Cmax=5.604±0.641 mg·L-1. Conclusion The established method was proved to be simple, sensitive and accurate, which could be used to determine the plasma concentration of trans-π-oxocamphor in rat. And it can provide a reference for the study of pharmacokinetics in human.
Objective To determine and explore the effective concentration of catalpol on the proliferation and osteogenic differentiation of mouse osteoblast precursor cells MC3T3-E1. Methods CCK8 method was used to determine the effects of catalpol on the safety and proliferation of MC3T3-E1 cells at different time points(1, 3, 6 days). Quantitative fluorescence real-time PCR was used to detect the mRNA expression levels of osteogenic differentiation key genes, such as Runx2, Bglap, and Col1α1. Aminoantipyrine-phenol method was used to analyze the alkaline phosphatase activity in cell culture supernatants at different time points(4 and 8 days). Alizarin red staining was used to detect the changes of cell mineralization levels. Results Catalpol at different concentrations did not promote the proliferation of MC3T3-E1 cells. Even when the concentration of catalpol reached 4000 mg·L-1, it would not cause cell death. Catalpol at the concentration of 500 mg·L-1 could significantly increase the activity of ALP, promote cell mineralization, and enhance the expression levels of osteogenic differentiation marker genes. Conclusion Catalpol had the effect of promoting osteogenic differentiation and mineralization on mouse osteoblast precursor cells MC3T3-E1, and had a wide range of safe concentration for the cells.
Objective To study the effect of gambogic acid on serum metabolic homeostasis in mice, and to explore its effective molecules in vivo. Methods Eleven female mice were randomly divided into two groups, and they were injected with gambogic acid(10 mg/kg)and blank solution via tail veins, respectively. Liquid chromatography-mass spectrometry(LC/MS)and gas chromatography-mass spectrometry(GC/MS)were used as the integrated approach for the characterization of the mice serum metabolic fingerprints in two groups. Partial least squares-discriminant analysis(PLS-DA)and(non-)parametric tests were performed for pattern recognition and statistical analysis. Differential metabolites were screened and identified, and then the related metabolic pathways were focused. Results Compared with control group, the serum levels of 14 metabolites, such as threonine, serine and glutamic acid, were increased significantly in the gambogic acid treated group, while the levels of 7 metabolites such as PC and LysoPE were decreased significantly, involving amino acid metabolism, fatty acid metabolism, tricarboxylic acid cycle and other pathways. Conclusion Gambogic acid can significantly affect the amino acid metabolism, phospholipid metabolism and energy metabolism, suggesting that its effects may be related to these pathways. This study provides a reference for further revealing the mechanism of action of gambogic acid.
Objective To establish an ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method for the determination of imatinib in human plasma, and to evaluate the relative bioavailability and bioequivalence of imatinib mesylate tablets in post-prandial conditions in Chinese healthy volunteers. Methods Twenty four healthy volunteers were randomly divided into two groups. Each group received a single oral dose of 400 mg imatinib mesylate tablet as a test or reference drug in post-prandial conditions. UPLC-MS/MS was used to determine the concentration of imatinib in plasma. The pharmacokinetic parameters were calculated by the WinNonlin 6.4 statistical software, and the bioequivalence of the two preparations was evaluated by the SAS 9.2 software. Results After oral administration of test or reference preparation, imatinib in plasma reached peak concentration(2 308.3±873.59)and(2 119.6±597.20)ng·mL-1 at 3.47(1.98-5.98)h and 2.97(1.98-6.00)h, respectively. AUC0-t were(39 724.7±18 670.30)and(35 294.4±7 991.97)h·ng·mL-1; AUC0-inf were(40 111.0±19 014.95)and(35 595.0±8 048.28)h·ng·mL-1. The 90% confidence intervals of Cmax, AUC0-t and AUC0-inf for two preparations after a logarithmic transformation were 95.52%-119.59%, 97.07%-119.35%, and 97.10%-119.39%, respectively, which were all within the limits of 80.00%-125.00%. Conclusion It could therefore be concluded that the test preparation of imatinib mesylate tablets was bioequivalent to that of Gleevec tablets(reference).
Objective To establish an LC-MS/MS method for simultaneous determination of hydrocortisone and 6β-hydroxycortisol concentrations in human urine. Methods Hydrocortisone-d4 and 6β-hydroxycortisone-d4 were selected as internal standards(IS), and the urine samples were treated with methanol containing IS for analysis. The chromatographic column was a CORTECS® C18+ column(2.1 mm×50 mm,2.7 μm), and the mobile phase was methanol and water containing 0.1% formic acid. A gradient elution was applied with a flow rate of 0.6 mL·min-1. MS/MS detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 363.2→121.1 for cortisol, m/z 367.2→121.1 for hydrocortisone-d4, m/z 379.2→325.2 for 6β-hydroxycortisol, and m/z 383.2→328.2 for 6β-hydroxycortisol-d4, respectively. Results The detecting linearity for hydrocortisone and 6β-hydroxycortisol was good within the range of 1.00~300 ng·mL-1 and 5.00~1500 ng·mL-1, respectively, and the lower limit of quantification was 1.00 ng·mL-1 and 5.00 ng·mL-1, respectively. The precision within and between batches was less than 15.0%, and the accuracy was within the range of -6.9% to 5.7%. The matrix effect was 95.6%~106.0%, and the recovery was 97.2%~108.3%. The urine sample was stable at 25 ℃ for 10 hours, or in 3 circles of freezing and thawing, or at -80 ℃ for 4 months. And the supernatant after preparation was stable at 10 ℃ for 24 hours in the sampler. Conclusion In this study, a sensitive, accurate, convenient, and rapid method for simultaneous determination of hydrocortisone and 6β-hydroxycortisone concentrations in human urine was established, which could be applied for the evaluation of human CYP3A activity.
Objective To establish a rapid, efficient, sensitive, stable, and reliable pre-column derivatization-ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the simultaneous determination of 11 ultra-trace estrogens in human serum, including estrone(E1), estradiol(E2), estriol(E3), 2-hydroxyestrone(2-OHE1), 16 α - hydroxyestrone(16α-OHE1). Methods Liquid-liquid extraction using tert-butyl methyl ether was selected for the treatment of human serum samples, and then the derivatization process was performed using dansyl chloride. The isotopically labeled estrone-13C was used as an internal standard. The chromatographic analysis was carried out on a Waters ACQUITY UPLC BEH C18 column(2.1 mm×50 mm,1.7 μm)using acetonitrile and water containing 0.1% formic acid as the mobile phase. The flow rate was set at 0.3 mL·min-1 and the column temperature was controlled at 40 ℃. The multiple reaction monitoring(MRM)in positive electrospray mode was employed for the detection and quantification of various estrogens in serum, and the analysis time was set at 5 min. Results Dansulyl chloride derivatization could significantly improve the ionization efficiency of various estrogens under ESI source. The linear range of 11 estrogens in serum was 20-2000 pg·mL-1, with a lower limit of quantification at 20 pg·mL-1. The recovery rate could reach more than 70%. The precision and accuracy within and between batches were all within 15%. The serum samples, which were placed at room temperature for 24 hours, or were repeatedly frozen and thawed for three times, or were placed in sample tray at -4 ℃ for 24 hours, or were placed in 70 ℃ for 1 month, were stable. The stability met the requirements for biological sample determination. Conclusion This method has the advantages of short analysis time, high sensitivity, good stability and strong specificity. It can be used for the quantitative analysis of estrogen metabolism network in clinical serum samples.
Objective To determine the effects of seven hepatotoxic traditional Chinese medicine monomers on rat hepatocyte uptake transporters Oct1 and Oatp1b2 by establishing a rat primary hepatocyte model. Methods The mRNA expressions of uptake transporters Oct1 and Oatp1b2 on adherently cultured rat primary hepatocytes were measured at 4, 8, 16, and 24 h. The probe substrates of the two transporters, ranitidine and rosuvastatin, were used to evaluate the function of the transporters. LC-MS/MS was used to measure the contents of the substrates in hepatocytes, and the effects of substrate concentration and incubation time on transporter uptake function were examined. Using the primary hepatocyte model, seven traditional Chinese medicine monomers(high and low doses)and two transporter inhibitors(verapamil and rifampicin)were incubated with primary hepatocytes respectively, to observe the effect of the tested traditional Chinese medicine monomers on the uptake of transporter. Results The mRNA expressions of the two transporters were down-regulated with the increase of culture time within 24 h, and the uptake of ranitidine and rosuvastatin decreased with the increase of substrate concentration, and the uptake reached saturation with the increase of time. In the validated primary hepatocyte model, the high and low doses of celastrol, glycyrrhetinic acid, and saikosaponin D could significantly inhibit the uptake function of Oct1 and Oatp1b2. The high dose of parietic acid could slightly inhibit the uptake of Oatp1b2, while the low dose of triptonide could induce the uptake of Oct1. Conclusion The rat primary hepatocyte model was successfully established. Hepatotoxic traditional Chinese medicine monomers, including celastrol, glycyrrhetinic acid, saikosaponin D, parietic acid, and triptonide, could inhibit or induce the uptake of Oct1 and Oatp1b2, which may provide a new way to study the mechanism of hepatotoxicity.
Objective To investigate the effect of Zhike Dingchuan decoction combined with montelukast on inflammatory factors and airway responsiveness in patients with cough variant asthma, and to analyze its clinical efficacy. Methods 120 patients who met the CVA diagnostic criteria were selected as the research objects. And according to the random number table method, the patients were divided into experimental group who was treated with Zhike Dingchuan decoction combined with montelukast(n=40), control group 1 who was treated with montelukast sodium chewable tablet(n=40), and control group 2 who was treated with Zhike Dingchuan decoction(n=40). The treatments continued for 3 months and the serum IL-1β, IL-6, TNF-α, serum IgE levels, pulmonary function, FeNO, ACT, and AQLQ were monitored and compared. Results The serum levels of IL-1β, IL-6, TNF-α, and IgE in the experimental group were significantly lower than those in the treatment group of Zhike dingchuan decoction alone and montelukast alone(P<0.05), and the pulmonary function indexes FEV1, FEV1 / FVC, FEV1 %, FEV1 improvement rate, FeNO, ACT score, AQLQ score were significantly better in the experimental group than those in the control group 1 and 2, and the differences were statistically significant(P<0.05). Conclusion The treatment of cough variant asthma with Zhike Ding chuan decoction combined with montelukast could effectively relieve the inflammatory response, enhance the immunity, improve the lung function of the patients, and control the patient's asthma, which have great clinical application values.
Objective To observe the efficacy, adverse events and the influence on carcino embryonic antigen (CEA) and traditional chinese medicine (TCM) syndromes of Kangai injection combined with chemotherapy in the treatment of patients with advanced lung adenocarcinoma of spleen-lung qi deficiency type. Methods 91 patients with advanced lung adenocarcinoma of spleen-lung qi deficiency type were enrolled and divided into treatment group and control group. The treatment group was treated with Kangai injection combined with a pemetrexed/cisplatin (PP) regimen for chemotherapy, while the control group was treated with PP regimen alone for 21 days as a cycle and received 2~6 cycles of treatment. The short-term therapeutic effects were evaluated, the occurrence of adverse reactions were recorded, serum CEA levels before and after treatment were detected, and the improvement of TCM syndromes were observed. Results The disease control rates of the treatment group and control group were 85.71% and 83.33%, respectively; the objective response rates were 42.86% and 30.95%, respectively, without statistical significance (P>0.05). The incidence of adverse events in the treatment group was lower than that in the control group (P<0.05). CEA levels in both groups decreased after treatment, but there was no statistical difference between the two groups (P>0.05).The total effective rate of TCM syndromes in the treatment group was 97.62%, and the improvement was better than 73.80% in the control group (P<0.05). Conclusion Kangai injection can reduce the adverse events caused by chemotherapy and improve the TCM syndromes of patients with advanced lung adenocarcinoma.