Purpose: To evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of SHR2285, the first oral coagulation factor XIa (FXIa) inhibitor developed in China in combination with aspirin, clopidogrel or ticagrelor in healthy subjects.Methods: This study was a single-center, randomized, double-blind, placebo-controlled (only SHR2285) design (NCT04945616). A total of 52 healthy subjects, 29 male and 23 female, were completed in this study. The subjects were divided into three groups: A, B and C, 16 subjects in group A [aspirin + clopidogrel + placebo or SHR2285 200 mg bid ] 16 subjects in group B [aspirin + clopidogrel + placebo or SHR2285 300 mg bid ] and 20 subjects in group C ), respectively. All groups were administered orally for six consecutive days. Safety, tolerability, pharmacokinetics and pharmacodynamics parameters were assessed.Results: 1) SHR2285 was well tolerated, and all adverse events were mild. There was no evidence of an increased risk of bleeding. 2) After 6 days of twice-daily administration, SHR2285 could reach a steady state. The mean half-life of SHR2285 in group A, group B and group C was 13.9 h, 14.5 h and 13.8 h, respectively. 3) SHR2285 markedly inhibited FXI activity and prolonged activated partial thromboplastin time (APTT). In group A, group B and group C, the mean maximum inhibition rate of FXI activity was 84.8%, 89.3% and 92.2% and the mean maximum prolongation of APTT was 2.08-fold, 2.36-fold and 2.26-fold, respectively.Conclusion: These data suggest that SHR2285, a potential oral FXIa inhibitor, is expected to become a novel, safe and effective anticoagulant when combined with aspirin, clopidogrel or ticagrelor. With the aging of the population and the change in people’s lifestyles and habits, thromboembolic diseases have increasingly become a major global health problem and the top-ranked cause of global population death . ThromboThe short-term use of dual antiplatelet therapy (DAPT) based on aspirin and platelet P2Y12 receptor inhibitors (commonly clopidogrel and ticagrelor used in China) is the cornerstone of the treatment of cardiac and systemic ischemic events in patients with coronary heart disease with an elevated risk of ischemia . DAPT haIn recent years, drugs targeting coagulation factors have become a new research hot spot for anticoagulant therapy. Non-vitamin K antagonist oral anticoagulants (NOACs) are also known as novel oral anticoagulants directly inhibiting Xa  or factor IIa (such as dabigatran) are widely used in clinical . The mosHuman FXI is a dimer composed of 607 amino acids . It beloFXIa inhibitors currently under clinical development include monoclonal antibodies [Osocimab [BAY-1213790] , AbelaciSHR2285 is a small molecule compound that selectively inhibits human FXIa, independently developed by Jiangsu Hengrui Pharmaceutical Co., Ltd. It is the first oral inhibitor of coagulation factor XIa (FXIa) developed in China and a new potential antithrombotic drug. At present, systematic preclinical pharmacology, pharmacokinetics and toxicology studies have been completed, and animal tests have confirmed that it selectively inhibits human FXIa, reduces thrombosis weight, prolongates APTT and reduces bleeding risk. Up to now, SHR2285 has completed three clinical trials , all of which are phase I trials in healthy subjects .Triple antithrombotic therapy with warfarin plus two antiplatelet agents is the standard of care after percutaneous coronary intervention (PCI) for patients with a trial fibrillation. Recently, most guidelines recommended both anticoagulation and dual antiplatelet therapy (triple therapy) . HoweverThis study (NCT04945616) was consensus on ethical principles based on international ethical guidelines, including the declaration of Helsinki, the international ethical guidelines of the Council for International Organization of Medical Sciences (CIOMS), the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, and other applicable laws and regulations. The study was reviewed and approved in writing by the ethics committee of Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, and the research plan, plan amendment, informed consent and other relevant documents such as recruitment advertisement were provided to the ethics committee. This trial strictly followed the good clinical practice (GCP) issued by National Medical Products Administration (NMPA). All subjects provided written informed consent.The overall study was a single-center, randomized, double-blind, placebo (SHR2285 only) control design. A total of 52 healthy subjects were completed in this study, including 29 males and 23 females. The subjects were divided into groups A, B and C. 16 subjects were randomised according to a Randomization and trial supply management (RTSM) by a study doctor using a SAS software program in group A and group B , 20 subjects in group C . Each subject can only participate in the trial of one dose group.For group A, the dosing regimen was aspirin + clopidogrel + placebo or SHR2285 200 mg. Specifically, aspirin: 100 mg was taken orally every morning from day 1 to day 6; clopidogrel: 300 mg in the morning of the first day, 75 mg in the morning of the second day to the sixth day; placebo or SHR2285 (1:3): 200 mg orally every morning and evening until the morning of the sixth day.For group B, the dosing regimen was aspirin + clopidogrel + placebo or SHR2285 300 mg. The use of aspirin and clopidogrel was the same as that of group A; Placebo or SHR2285: added the amount to 300 mg based on group A.For group C, the administration regimen was aspirin + ticagrelor + placebo or SHR2285 300 mg. The use of aspirin, placebo or SHR2285 was the same as that in group B. Clopidogrel in group B was adjusted to ticagrelor: 180 mg orally in the morning of the first day, 90 mg in the morning and evening from the second day to the morning of the sixth day.The subjects were administered continuously from day 1 to day 6 by oral administration with about 240 ml of normal temperature water. They were required to fast for at least 8 h before administration in the morning and take it 1 h (±10 min) before breakfast; the administration time in the evening is 12 h (+15 min) from the administration in the morning. The administration of group B and group C was initiated after the researchers and the sponsor evaluated the safety data of all subjects in group A at 48 h after the last dosing. During the whole trial period, it was forbidden to take tea, coffee and other beverages containing coffee and alcohol, eat grapefruit or grapefruit juice, and smoke. Required work and rest regularly and avoided strenuous exercise.SHR2285 was identical to the placebo except for the drug number. The blind state must be maintained from the enrollment of subjects, the recording and evaluation of study results, the inspection of the study process to data management. All subjects, researchers, the sponsor’s investigator participating in the clinical evaluation, and the supervisors (CRA) participating in the study were not informed about which drugs the subjects received for the study. The blind remained unbroken.PK blood collection points: the blood of each group was collected in the morning of day 1 before administration (within 1 h before administration), 4 h and 12 h after administration (before administration in the evening); in the morning of day 3, day 4, and day 5 before administration (within 1 h before administration), within 1 h before administration on the morning of the sixth day, and 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 15 h, 24 h, 36 h, and 48 h after administration. About 7 ml of venous blood was collected, and the plasma was collected for concentration determination of aspirin and its active metabolites  and clopyridine , ticagrelor and its active metabolite AR-C124910XX (group C), SHR2285 and its active metabolite SHR164471.PD blood collection points: the blood of each group was collected in the morning of the first day before administration (within 1 h before administration) and 4 h and 12 h after administration (before evening administration); the morning of the third, fourth, and fifth days before administration (within 1 h before administration), before administration on the morning of the sixth day (within 1 h before administration), and 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h after administration. About 6 ml of venous blood was collected for FXIa (about 3 ml) and coagulation function testing (about 3 ml).2, the male body weight was ≥50.0 Kg and <90.0 Kg and the female body weight was ≥45.0 Kg and <90.0 Kg; No smoker within 1 month before screening; The average daily alcohol intake in a week was less than 15 g. Strict contraception should be used during the trial and for 90 days after administration. The subjects could understand the research procedures and methods, voluntarily participate in the trial and sign written informed consent.Subjects were healthy volunteers aged 18–55 years, both male and female . The body mass index (BMI) of the subjects was 19–28 kg/mThe subjects should not have any allergy to the suspected study drug or any related components, or have a history of abnormal coagulation function, high-risk bleeding risk and affecting drug absorption. Subjects were not allowed to take any prescription drugs, over-the-counter drugs and Chinese herbal medicine within 14 days before taking the study drug.etc.The primary objective of this study was to evaluate the safety and tolerability of SHR2285 tablets combined with aspirin, clopidogrel or ticagrelor in healthy subjects, that is, the incidence and severity of adverse events , including subjects’ main complaint, physical examination, vital signs, laboratory examination and ECG, The secondary objective was to evaluate 1) the pharmacokinetic (PK) of aspirin and its active metabolite salicylic acid, clopidogrel, SHR2285 and its active metabolite SHR164471 in group A and group B. In group C, the PK of aspirin and its active metabolite salicylic acid, ticagrelor and its active metabolite AR-C124910XX, SHR2285 and its active metabolite SHR164471 were measured. 2) The pharmacodynamics (PD) of aspirin plus clopidogrel or ticagrelor with or without SHR2285 tablets in healthy subjects. PD indexes included coagulation factor XIa (FXIa) activity, activated partial thromboplastin time (APTT), prothrombin time (PT), and international normalized ratio (INR). On the first day before administration, the measured values of coagulation FXI activity and coagulation function  were taken as the baseline.Full analysis set (FAS), safety analysis set: all subjects who have received the study drug at least once. PK concentration and parameter analysis set: all randomized subjects who have used the study drug and have at least one PK concentration or parameter data. PD analysis set: all randomized subjects who have used the test drug and have a pre-medication baseline and at least one post-medication PD evaluation data.The classified data are summarized by frequency and percentage. Continuous data are summarized by subject number, mean, standard deviation, median, maximum and minimum. Blood drug concentration data and PK parameters were summarized by subject number, mean, standard deviation, median, maximum, minimum, coefficient of variation, geometric mean, geometric standard deviation and geometric coefficient of variation.Based on the PK concentration analysis set, the blood drug concentration was listed and summarized by descriptive statistics according to the medication group at the planned time. The mean and median blood concentration-time curved and semi-logarithmic diagrams were drawn according to the medication. In addition, the actual blood sample collection time was used to draw the blood concentration-time curve and semi-logarithmic diagram of individual subjects.max, AUCtau, AUC0-last, AUC0-inf, Tmax, t1/2, CLss/F, Vss/F, Ctrough and Caverage. The main pharmacokinetic parameters such as AUC0-last, AUC0-inf and Cmax were tabulated and summarized by descriptive statistics.Based on the actual sampling time, the PK parameters of each component were calculated as a non-compartmental model by WinNonlin . The pharmacokinetic parameters after multiple administrations were investigated: Css and Cmax of each component in the SHR2285 group (aspirin + clopidogrel/ticagrelor + SHR2285) and placebo group (aspirin + clopidogrel/ticagrelor + placebo) were statistically analyzed by ANOVA model. The medication group was used as the independent variable. The geometric mean ratio of the corresponding PK parameter (SHR2285/placebo group) and the estimated value of its 90% confidence interval was obtained by taking the exponential conversion from the mean difference estimated by the model (SHR2285 group-placebo group) and its 90% confidence interval. SHR2285 and its metabolite SHR164471, aspirin/salicylic acid, clopidogrel, ticagrelor and its metabolite AR-C124910XX were determined by Frontage Laboratories Co., Ltd.  using high-performance liquid chromatography-tandem mass spectrometry . The chromatographic column is WATERS CORTECS®T3 , column temperature is set at 40°C. Mobile phase A is ultrapure water (containing 0.1% formic acid), and mobile phase B is acetonitrile/water . The elution gradient is: 0–0.4 min, A-B (50:50), 0.4–0.8 min, A-B (5:95), 0.8–2 min, A-B (5:95), 2–2.4 (50:50), 2.4–3.5 min, A-B (50:50). The total time was 3.5 min and the flow rate was 1.0 ml/min. The ion source was ESI source at positive ion mode. The heating capillary temperature was 400°C; CAD was nine; curtain gas was 30; GS1 (N2) was 50; GS2 (N2) was 50; The method used was multiple reaction monitoring (MRM), and the ion reactions used for quantitative analysis were m/z 545.1→408.0 (SHR2285) and m/z 721.2→408.1 (SHR164471), respectively. The calibration ranges of SHR2285 and SHR164471 were 5 ng/ml (lower limit of quantification) to 5,000 ng/ml. The calibration model was linear regression, quality control samples  were determined with accuracies of 98.0%–104.5% and 100.9%–104.2%, precision of ≤6.2% and ≤6.6%, and recovery of 98.7%–103.6% and 97.3%–102.1%, respectively for both analytes.After logarithmic conversion, the AUCThe absolute value and relative baseline change percentage of coagulation factor XI (FXI) activity, APTT, PT, INR  and planned sampling time points of healthy subjects after administration were summarized and plotted (if applicable). FXI activity was measured by the Institute of Blood Transfusion, Chinese Academy of Medical Sciences of China using coagulation method. APTT was measured by Guangzhou Kingmed Center For Clinical Laboratory Co., Ltd. (China) using the coagulation method. PT was counted on the coagulation analyzer, and INR was calculated through parameter calculation. PD parameters APTT, PT, INR and FXI activity were measured with validated assays at Guangzhou KingMed Diagnostics Group Co., Ltd. and the Institute of Blood Transfusion, Chinese Academy of Medical Sciences of China. APTT and PT was evaluated using Dade Actin FSL activated APTT reagent and Tromborel S reagent respectively, automated on the instrument Sysmex CS5100 with a within-batch and inter-batch precision of both not high than 5%, and INR was calculated based on PT test results. FXI activity was measured using a clotting method by Dade actin activated cephaloplastin reagent, automated on Sysmex CS 2000i Coagulation analyser. The result data are presented as a percentage of normal, and its calibration range was 6.25%–150.0%.The study was conducted from 16 July 2021 to 16 November 2021. In group A and group B, all 32 subjects completed the study; in group C, all 20 subjects completed the study . All subAt least one treatment emergent adverse event (TEAE) occurred in 26 of 37 (70.3%) healthy subjects who received SHR2285 and 10 of 15 (66.7%) subjects who received placebo. The TEAEs of subjects are shown in Gingival bleeding and/or epistaxis occurred in three subjects in the SHR2285 dose group (A and B), but not in the placebo group. TEAE that occurred in or more of healthy subjects receiving SHR2285 compared with subjects treated with placebo included subcutaneous hemorrhage (2.7% vs. 13.3%), ecchymosis (13.5% vs. 20.0%), oral ulcer (8.1% vs. 6.7%), abdominal burning sensation (2.7% vs. 0), scratch (2.7% vs. 0), diarrhea (5.4% vs. 0), constipation (2.7% vs. 0), rash (2.7% vs. 0).We also observed abnormalities in clinically relevant laboratory values in the SHR2285 dose group compared with the placebo group, including decreased hemoglobin (8.1% vs. 6.7%), fecal occult blood positive (5.4% vs. 13.3%), urine occult blood positive (0 vs. 6.7%), elevated alanine aminotransferase (2.7% vs. 0), elevated total bilirubin (2.7% vs. 6.7%), elevated triglycerides (27% vs. 13.3%), elevated serum uric acid (21.6% vs. 20.0%), elevated blood calcium (2.7% vs. 0), hypercholesterolemia (2.7% vs. 0), elevated creatine kinase (0 vs. 6.7%), electrocardiogram T wave abnormal (8.1% vs. 26.7%), prolonged QTc interval of electrocardiogram (2.7% vs. 0).All TEAE in healthy subjects were mild. AEs in the SHR2285 group did not increase apparently compared with the placebo group, suggesting that the AEs may be caused by the combination of aspirin, clopidogrel or ticagrelor, indicating that the combination of SHR2285 with dual antiplatelet treatment did not increase the TEAE. The challenge of FXIa inhibitor combined with dual antiplatelet therapy is the risk of bleeding. In this trial, three subjects with transient mild gingival bleeding and epistaxis in SHR2285 dose group A and B compared with the placebo group. The AE of bleeding mentioned in SHR2285 300 mg dose group C was only ecchymosis, which occurred in five of 12 subjects, compared with three of eight subjects with placebo. No higher risk of bleeding was observed in group C than in the placebo group. These data suggest that it may be an adverse reaction of aspirin combined with clopidogrel or ticagrelor, and less likely to be related to SHR2285. Importantly, there was no evidence of a severe hemorrhage risk or serious adverse event (SAE). All events were resolved without treatment. None of the healthy subjects died or stopped taking TEAE.Combined with aspirin, clopidogrel or ticagrelor, three SHR2285 dose groups were administered 200 mg or 300 mg SHR2285 bid for six consecutive days, SHR2285 and its active metabolite SHR164471 in plasma reached a steady-state . The meamax was about 1.5 h. The mean t1/2 for SHR2285 after Day 6 was roughly the same (13.8–14.5 h). The peak of SHR164471 in plasma was later than the prototype, Tmax was 2.5–4 h, and the t1/2 was 12.3–14.3 h  increased by 1.3- and 1.1- fold, respectively  were similar, which were 4.5 h, 6.0 h, 7.0 h, 8.0 h and 4.0 h, respectively  of salicylic acid in the SHR2285 group and placebo group were similar, and no apparent difference was found between the two groups  of group A and placebo were similar and slightly lower than that of group B with extremely high exposure in some individuals  of the SHR2285 dose group and placebo group were similar, and no noticeable difference was found between the groups  combined with dual antiplatelet drugs showed a trend of reducing thrombotic events , but it was also accompanied by an increased risk of bleeding, which was limited in clinical application (Previous studies have suggested that anticoagulant drugs (including vitamin K antagonists, factor Xa inhibitors, lication . TherefoIn conclusion, SHR2285 significantly inhibited the activity of FXI in a dose-dependent manner. When combined with aspirin, clopidogrel or ticagrelor, it was well tolerated in healthy subjects without apparent safety or tolerance issues. There was no significant effect on PT or INR, and there was no evidence of an increased risk of bleeding. These results suggest that the FXIa inhibitor represented by SHR2285 is expected to be an effective and safe oral anticoagulant for combined dual antiplatelet therapy.


The influence of the GL of four meal types  was also determined. From the prospective Three-City study, 377 participants with plasma Aβ measurements, and who completed the Food Frequency Questionnaire, were selected. The association between plasma Aβ and GL was tested using an adjusted linear regression model. Lunch GL was associated with a lower plasma Aβ42 concentration  and lower Aβ42/Aβ40 ratio  in the model adjusted for center, age, sex, education level, APOE4 status, energy intake, serum creatinine, total cholesterol, and Mediterranean-like diet. No significant association was found with the GL of the other meal types. These results suggest that dietary GL may independently modulate the plasma Aβ of the APOE4 status. The mechanism underlying diet, metabolic response, and Aβ peptide regulation must be elucidated.Previous studies have highlighted links between a high-glycemic-load (GL) diet and Alzheimer’s disease in apolipoprotein E ε4 (APOE4) carriers. However, the impact of high-GL diet on plasma amyloid-β (Aβ), an Alzheimer’s disease hallmark that can be detected decades before clinical symptomatology, is unknown. This study examined the association between plasma Aβ peptides (Aβ Dementia affects 50 million people worldwide and represents the main cause of dependency and disability in older adults . To datehe brain . Moreovetia risk ,9. Howevtia risk . In additia risk ,11, suggtia risk , suggesttia risk .40 and Aβ42 concentration, as well as Aβ42/Aβ40 ratio. We also evaluated the potential interactive effect of APOE4 carrier status and the effect of the daily, breakfast, lunch, afternoon snack, and dinner GL values on this association.Based on these findings, we hypothesized that a high-GL diet might be associated with plasma Aβ peptide levels. Therefore, we used data from the prospective Three-City (3C) cohort study to examine the association between GL and plasma AβThe 3C study is a population-based, prospective study on the relationship between vascular factors and dementia. The study started in 1999–2000 and included participants, aged 65 years or older, from three French cities: Bordeaux, Dijon, and Montpellier . The Conwww.glycemicindex.com (accessed on 11 June 2019)). GL items are defined as “low” when the GL value is below 10 and as “high” when the value is above 20 [The 148-item semi-quantitative FFQ was divided into breakfast, lunch, dinner, and snacks between meals. The GL is an indicator of the cumulative exposure to postprandial glycemia and reflects insulin demand, induced by the carbohydrate intake . GL valuabove 20 . As prevabove 20 . In the above 20 ,22. The 40 and Aβ42 concentrations were expressed in pg/mL, and the Aβ42/Aβ40 ratio was calculated.The plasma Aβ peptide assay is described elsewhere . BrieflyAt the 2-, 4-, 7-, 10-, 12- and 15-year follow-up visits, a neurologist examined participants with suspected dementia on the basis of their neuropsychological test scores. Then, an independent committee of neurologists evaluated all potential cases of dementia to reach a consensus on the diagnosis and etiology based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition .ε4 allele. Diabetes was defined as treated diabetes, fasting glycemia > 7 mmol/L, and self-reported diabetes. Serum creatinine (mmol/L) was measured with the Jaffe method [Education level was defined as no school, primary school, high school, or graduate level. APOE genotyping was performed as previously described . APOE4 ce method . Total ce method ,27.n = 4 for Aβ40, n = 3 for Aβ42, and n = 2 for Aβ42/Aβ40) by looking at the graphs and using the Bonferroni method with the ‘car’ package in R [p value < 0.05 was considered statistically significant.Linear regression models were used to evaluate the association between GL and Aβ peptide concentration/ratio. The models were first adjusted for energy intake. Then, multicollinearity was tested using the variance inflation factor . As GL sage in R . For theAt FFQ completion, the mean age of the 377 participants was 76.1 (±5.2) years and 60.2% were women . ParticiTo evaluate the association between GL and plasma Aβ peptide concentration and ratio according to APOE4 status, the GL × APOE4 interaction was tested. As no significant interaction was detected between GL and APOE4 status, the results are presented for the whole sample.40, but not after additional adjustments in model 2. Lunch GL tended to be associated with lower Aβ42 in model 1, and the association between lunch GL and lower Aβ42 and ratio became significant after the adjustment for serum creatinine, total cholesterol, and a Mediterranean-like diet (model 2). The predicted Aβ42 and Aβ42/Aβ40 ratio values as a function of the lunch GL are presented in After removing outliers, no significant association between daily GL and plasma Aβ peptide concentration and ratio was detected in both models . In moden = 51) were older than those who remained dementia-free. They were more likely to be women and APOE4 carriers. They also had a lower education level and a lower Mediterranean-like diet score than dementia-free participants , fat, and protein , which cOur results suggest that high-GL diets could negatively affect plasma Aβ biomarkers. A possible biological explanation is that high GL indirectly participates in the mechanisms involving Aβ burden. First, a high-GL diet could promote insulin resistance through inflammation and oxidative stress ,37. ThisIn this study, we did not find an interactive effect of APOE4 status. Yet, higher amyloid deposits were detected in the brain of APOE4 carriers with AD or with normal cognition ,45. MoreThis study presents some limitations. First, Aβ peptide concentration was measured in the early 2000s. Since then, more sensitive and effective methods have been developed ,48. More42 concentration and Aβ42/Aβ40 ratio independently of the APOE4 carrier status, suggesting that AD biomarkers can be modulated by diet. As AD biomarker changes can be detected a long time before cognitive symptom appearance, these findings support the idea that long-term dietary choices constitute a critical environmental factor in the AD causal pathway. These findings pave the way for identifying new therapeutic targets and preventive measures because diet is a potentially modifiable risk factor. Experimental studies are now required to determine the underlying mechanism linking refined carbohydrate consumption and plasma Aβ peptides with dementia development.Dietary GL is associated with plasma Aβ


Angelicae pubescentis Radix (APR) in normal and arthritis rat plasma in which chloramphenicol and daidzein were used as the internal standards. After protein precipitation with acetonitrile, separation was carried out on a Thermo Hypersil GOLD C18 column using gradient elution with 0.1% formic acid aqueous and acetonitrile consisting as the mobile phase at a flowing rate of 0.3 mL/min. A Thermo TSQ QUANTIS triple quadrupole mass spectrometer was used to detect 14 compounds in positive/negative ion exchange mode and this study was the first to investigate the pharmacokinetic changes of the active compounds in rats under the pathological state of arthritis. The method was verified and the results showed that the intra- and interday precision, accuracy, matrix effect, and extraction recovery were all acceptable, and the analytes were stable under different storage conditions. In addition, the pharmacokinetic behaviors of the 14 compounds were significantly different in model rats compared with normal rats. This indicated that the pharmacokinetic behavior of drugs will vary with the pathological state of the body, which suggested that individualized and reasonable drug administration plans should be formulated for different pathological states in clinical practice. This study provided a scientific basis and data support for better understanding the pharmacodynamic substance basis and clinical application of APR against arthritis.An ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to simultaneously determine 14 compounds of  Arthritis generally refers to the inflammatory diseases caused by inflammation, infection, degeneration, trauma, or other factors that occur in the joints and surrounding tissues of the human body . The cliAngelicae pubescentis Radix (APR), a member of the Apiaceae family, is the dry root of Angelica pubescens Maxim. f. biserrata Shan et Yuan. It has the functions of removing wind and dehumidification and relieving paralytic pain [As is known to all, traditional Chinese medicine (TMC) plays an important role in clinical treatment because of its abundant resources, suitable price, stable pharmacological action, low toxicity, and few complications, and some TCMs with antiarthritic effects have been gradually approved for clinical use and have convincing efficacy –12. Angetic pain . Clinicatic pain . In additic pain  and conttic pain . Among ttic pain –18. In atic pain . Howevertic pain , 20, 21.tic pain . Chang etic pain . Howevertic pain , 14.vivo can it exert its efficacy, and the plasma drug concentration is not only related to the dosage but is also affected by the process in vivo. Moreover, the pharmacokinetic studies of TCMs are mostly carried out based on healthy experimental animals, ignoring the physiological and pathological changes, which may cause significant changes in relevant pharmacokinetic parameters [Pharmacokinetics of TCMs is an indispensable link in the modernization of Chinese medicinal materials and an important means of integrating TCMs with the world . Only whrameters , 26. Sturameters –31. The Therefore, a UPLC-MS/MS method was established for simultaneous determination of phenolic acids and coumarin compounds in plasma. This is the first study to investigate the pharmacokinetic change rule of active ingredients of APR in rats with arthritis, so as to provide reference for rational use of drugs in clinical treatment of arthritis.HPLC grade methanol and acetonitrile were obtained from Fisher Scientific (USA). HPLC grade formic acid was purchased from Dikma Co. (USA), and Wahaha purified water was obtained from Hangzhou Wahaha Group (China). Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B , isochlorogenic acid A , isochlorogenic acid C , bergapten, columbianetin acetate, isoimperatorin, osthol, and columbianadin were obtained from Chengdu Must Biotechnology Co., LTD (China), and columbianetin, xanthotoxin, psoralen, and chloramphenicol were obtained from Shanghai Nature Standard Biotechnology Co., LTD (China), and daidzein was obtained from Chengdu Purechem-Standard Biotechnology Co., LTD (China). The purity of the involved standards and reagents were over 98%, with their chemical structures exhibited in Angelica pubescens Maxim. f. biserrata Shan et Yuan and a voucher specimen were deposited at department of Chinese herbal medicine processing of Heilongjiang University of Chinese Medicine, Harbin, China (voucher numbers: APQH201904-7).The APR samples was obtained from Hubei Province  picked in April 2019, and identified by Professor Lianjie Su from Heilongjiang University of Chinese Medicine, as the dry root of μg/g for neochlorogenic acid, 3006.87 μg/g for chlorogenic acid, 75.08 μg/g for cryptochlorogenic acid, 72.07 μg/g for 3,4-diCQA, 341.27 μg/g for 3,5-diCQA, 112.30 μg/g for 4,5-diCQA, 369.02 μg/g for columbianetin, 3375.11 μg/g for psoralen, 345.41 μg/g for xanthotoxin, 241.69 μg/g for bergapten, 10280.43 μg/g for columbianetin acetate, 25.03 μg/g for isoimperatorin, 7943.10 μg/g for osthol, and 1442.12 μg/g for columbianadin, respectively.One hundred grams of the APR sample was extracted by reflux with 800 mL 50% ethanol/water (v/v) for 3 times (2 hours each time), then combined with the filtrate and concentrated under vacuum with heat, and the concentration of APR extract was 0.37 g/mL. All the sample solutions were stored at 4°C before analysis. The contents of 14 compounds were 61.85 Male Sprague-Dawley rats weighing 240–260 g were obtained from the Experimental Animal Center of Heilongjiang University of Chinese Medicine 2020-0924), and were housed under a room at temperature of 25 ± 2°C and humidity of 50 ± 15%, as well as light/dark circulation, ad libitum access to food and water for more than a week. Moreover, the rats fasted overnight before the experiment. Moreover, the study was authorized by the Ethics Regulations of Heilongjiang University of Chinese Medicine .n = 6) and named as the model group and the normal group, respectively. The arthritic model was established by injecting 0.1 mL of complete Freund's adjuvant in rats. As a control, 0.1 mL of saline was injected. After 7 days, the plasma levels of MDA and TNF-α in normal and model rats were measured by an enzyme-linked immunosorbent assay kit to determine whether the modeling was successful. The results are shown in α in model rats were significantly higher than those in normal rats, indicating that the arthritis model was successfully established. Normal and model rat blood samples were collected from the angular vein at 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h into 1.5 mL heparinized tubes after administration the APR extract at a dose of 4.1 mL/kg. All samples were immediately centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was transferred to another EP tube and stored at −80°C until analysis.Twelve SD rats were randomly divided into two groups , which was maintained at 35°C. The mobile phase consisted of 0.1% formic acid aqueous (solvent A) and acetonitrile (solvent B) and the following gradient was run: 7–8% B at 0–4 min, 8–19% B at 4–5 min, 19–19% B at 5–12 min, 19–60% B at 12–15 min, and 60–100% B at 15–18 min, in which the flow rate was maintained at 0.3 mL/min.The ultrahigh performance liquid chromatography ) was used for the chromatographic analysis. UPLC separation was achieved on a Thermo Hypersil GOLD Cm/z 353.0⟶190.9, chlorogenic acid m/z 353.0⟶126.9, cryptochlorogenic acid m/z 353.0⟶178.9, 3,4-diCQA m/z 515.0⟶353.0, 3,5-diCQA m/z 515.0⟶191.0, 4,5-diCQA m/z 515.0⟶172.9, columbianetin m/z 247.0⟶215.0, psoralen m/z 187.0⟶143.0, xanthotoxin m/z 217.0⟶161.0, bergapten m/z 217.0⟶174.0, columbianetin acetate m/z 289.0⟶229.0, isoimperatorin m/z 271.0⟶239.0, osthol m/z 245.0⟶188.9, columbianadin m/z 329.0⟶229.0, chloramphenicol m/z 321.0⟶257.0, and daidzein m/z 254.8⟶198.9. The following parameters were used: sheath gas: 30 Arb; aux gas: 10 Arb; ion transfer tube temp: 325°C; and vaporizer temperature: 350°C.The UPLC was interfaced to a Thermo TSQ QUANTIS triple quadrupole mass spectrometer and equipped with an electrospray ionization (ESI) probe working in positive and negative ion exchange scanning mode. The mass spectrum parameters of 14 compounds and their internal standard (IS) compounds were neochlorogenic acid μL plasma sample and 100 μL mixed standard solution and 20 μL ISs solution were blended with 780 μL acetonitrile, vortexed for 1 min, and the sample solution was immediately centrifuged and separated at 12,000 × g for 15 min at 4°C. At last, the supernatant was removed and placed in another EP tube, where it was dried by nitrogen at 45°C, the residue was dissolved with 100 μL 50% aqueous methanol for UPLC-MS/MS analysis.The standard stock solution of 14 compounds was prepared by dissolving 1 mg of the standard material in 1 ml of methanol, respectively. Then, calibration curve samples were prepared by diluting with different volumes of 50% methanol aqueous to obtain the mixed standard solution. For analysis, aliquots of 100 Similarly, the three different concentrations of QC samples were obtained at the final concentrations of 10, 252, 504 ng/mL for neochlorogenic acid, 10.6, 1060, 3180 ng/mL for chlorogenic acid, 11.7, 117, 234 ng/mL for cryptochlorogenic acid, 11.2, 56, 112 ng/mL for 3,4-diCQA, 12.5, 312.5, 625 ng/mL for 3,5-diCQA, 20.4, 102, 204 ng/mL for 4,5-diCQA, 39, 2260, 4520 ng/mL for columbianetin, 5.05, 252.5, 505 ng/mL for psoralen, 11.2, 560, 1120 ng/mL for xanthotoxin, 13, 340, 680 ng/mL for bergapten, 54, 2160, 4320 ng/mL for columbianetin acetate, 8.9, 445, 890 ng/mL for isoimperatorin, 5.5, 2200, 5500 ng/mL for osthol, and 12.2, 1220, 2440 ng/mL for columbianadin. In addition, ISs solution with a concentration of 100 ng/mL was used for further analysis, and all the standard solutions, calibration standards, and QC samples were stored at −80°C until further analysis.μL aqueous methanol solution and 20 μL ISs solution were added to aliquots of 100 μL of plasma sample following mixing with 780 μL of acetonitrile, and they were centrifuged at 12,000 × g for 15 min at 4°C after being vortexed for 1 min to obtain the supernatant. Then, the supernatant was dried in a nitrogen environment at 45°C, and the remaining residue was recombined with 100 μL 50% methanol aqueous and vortex for 1 min. Finally, 2 μL supernatant was injected into the UPLC-MS/MS system for analysis.Acetonitrile was used as a precipitation solvent, and protein precipitation was used to handle with the plasma samples. First, 100 The established UPLC-MS/MS method for the determination of 14 compounds in rat plasma has been validated according to FDA Bioanalytical Method Validation guidelines and the following parameters were evaluated, including selectivity, linearity, sensitivity (LLOQ), precision, accuracy, matrix effect, recoveries, and stability.Six different individual blank plasma samples not treated with APR, six blank plasma samples with spiked of standards and ISs and six rat plasma samples after oral administration of APR extract 5 min were analyzed by comparison of their corresponding chromatograms to evaluate the selectivity of the method.χ2) least square linear regression. The lower limit of quantification (LLOQ) was used to represent the sensitivity of the analytical method. Relative error (RE%) and relative standard deviation (RSD%) were used to describe the accuracy and precision, respectively, and both were permitted to be less than ± 20%.Calibration curves were established by plotting the ratio of peak area of the individual compound to IS versus different levels of compound with a weighted , freeze-thaw cycle's stability (three cycles of freeze-thaw), long term stability (storage at −80°C for 30 days), and the posttreatment stability (auto-sampler at 4°C for 24 h).Cmax), area under concentration-time curve (AUC0–t and AUC0-∞), half-time (t1/2), time to reach the maximum concentration (tmax), mean retention time (MRT), and clearance rate (CL) were calculated by DAS 2.0 software  in a noncompartment model.The pharmacokinetic parameters of 14 compounds including the maximum plasma concentration . Statistical software GraphPad Prism 5.0 was used for plotting and statistical analysis, and μm) and Waters Acquity UHPLC HSS T3 column  were used for analysis. The results showed that Thermo Hypersil GOLD column  could detect all compounds in APR with a good peak shape and good separation. Because the coumarin compounds were small polar compounds, and the elution ability of acetonitrile solution was stronger than methanol solution, when methanol solution was used as the mobile phase, the elution time would be prolonged, while acetonitrile solution was used as the mobile phase, greatly shorten the elution time and save solvent. Therefore, acetonitrile was selected as the mobile phase for gradient elution. As neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were isomers and difficult to separate, 7% acetonitrile was selected as the initial mobile phase to achieve the best separation effect.The chromatographic column, mobile phase composition, and elution gradient were optimized in this study in order to achieve the ideal results of the chromatographic behaviors, like good peak symmetry, efficient separation, and short chromatographic retention time. Thermo Hypersil GOLD column  had a better response in negative mode, and bergapten, columbianetin acetate, isoimperatorin, osthol, columbianadin, columbianetin, xanthotoxin, psoralen, and daidzein (IS2) had a better response under positive mode.In addition, the mass spectrometry conditions of 14 compounds and ISs compounds were optimized, and the results showed that c, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, and chloramphenicol , while the MRT0-t of psoralen, bergapten, and columbianetin acetate was shortened (P < 0.01), which reflected the increased bioavailability of these compounds. The reasons may be (1) under pathological conditions, the activities of metabolic enzymes and transporters related to drug metabolism and transport in the body were changed, which significantly affected the CL of drug metabolized by the liver. (2) The active ingredients could prevent the diffusion of inflammation by affecting the binding of active ingredients to plasma proteins, inflammatory factors, and related receptors in the lesion of model rats, thus affecting the pharmacokinetics process of drugs in vivo. (3) The active ingredients were distributed to other tissues. (4) The active ingredients were transformed into other components under the action of gut bacteria and drug metabolism enzymes. In addition, for bergapten, the first peak appeared at 5 min after administration of APR and the second peak appeared at 4 hours, we speculated that the possible reason was as follows: xanthotoxin, an isomer of bergapten, which could transform to bergapten in a certain way in vivo so as to make the bergapten peak appeared again after dosing 4 h. For columbianetin acetate, the double peak phenomenon occurred in 4 h after administration of APR, which may be caused by the conversion of columbianetin into columbianetin acetate in vivo through liver metabolic enzymes or gut bacteria, or the hepatointestinal circulation. Furthermore, compared with normal rats, the AUCt0-, AUC0-∞, Cmax of columbianetin decreased while CL increased and MRT shortened in model rats, which was consistent with our speculation.For coumarin compounds, the 14 components could be detected in both normal and model rats 5 min after administration APR. The AUChe blood . CompareIn conclusion, compared with normal rats, the pharmacokinetic parameters of 14 compounds in normal and model rats were significantly different. This indicated that the pharmacokinetic behavior of drugs will vary with the pathological state of the body. This study found that the pharmacokinetic behavior of 14 components in APR was correlated with the pathological state of arthritis. This study provided important scientific information for better understanding the mechanism and clinical application of APR. At the same time, the abovementioned results also provide a scientific basis and data support for 14 compounds as a pharmacodynamic substance basis of APR against arthritis.In this study, a UPLC-MS/MS method was established for simultaneous determination of 14 components in plasma and used to investigate the influence of arthritis induced by the complete Freund adjuvant on the pharmacokinetics of the14 components after oral administration of APR. The results showed that the established method had good accuracy, precision, recovery, and stability and was suitable for studying the pharmacokinetics of 14 components in rat plasma. In addition, there were significant differences in the pharmacokinetics of 14 compounds between normal and model rats. This suggested that it was necessary to formulate individualized and reasonable drug administration schemes according to different pathological states, which could not only effectively improve the safety and effectiveness of drugs but also reduce the probability of adverse reactions.


The study was aimed at evaluating the bioequivalence and safety of oseltamivir phosphate for suspension, provided by Shenzhen Beimei Pharmaceutical Co. Ltd. and manufactured by Hetero Labs Limited, and the reference product TAMIFLU® in healthy Chinese subjects.A single-dose, randomized, two-phase, self-crossed model was adopted. Among 80 healthy subjects, 40 subjects in the fasting group and 40 subjects in the fed group. Subjects in the fasting group were randomized into two sequences according to the proportion of 1:1, each given 75 mg/12.5 mL of Oseltamivir Phosphate for Suspension or TAMIFLU®, and cross-administered after 7 days. Postprandial group is the same as fasting group.max of TAMIFLU® and Oseltamivir Phosphate for Suspension in the fasting group were 1.50 h and 1.25 h, which in the fed group were both 1.25 h. Geometrically adjusted mean ratios of the PK parameters of Oseltamivir Phosphate for Suspension along with TAMIFLU® under fasting and postprandial conditions were in the range of 80.00–125.00% at the 90% confidence interval (CI). The 90% CI of Cmax, AUC0-t, AUC0-∞ for fasting group and postprandial group were , ,  and ,,. Among the subjects on medication, a total of 18 subjects reported 27 adverse events, all of which were treatment-emergent adverse events (TEAEs), six of these TEAEs were rated as grade 2 in severity and the rest were as grade 1. The number of TEAEs in the test product and the reference product were 14,13 respectively.The TTwo Oseltamivir phosphate for suspensions are safe and bioequivalent.The online version contains supplementary material available at 10.1186/s40360-023-00646-1. Influenza, a contagious respiratory disease, which induced by the influenza virus that resulting in high morbidity and mortality worldwide. Outbreaks of influenza are usually earliest detected in children with fever illness . The infCurrently, four classes of antiviral drugs are approved for influenza treatment in several countries, mainly including adamantane, neuraminidase inhibitors, membrane fusion inhibitors, and RNA-dependent RNA polymerase inhibitors . Among tOseltamivir carboxylate, which is a neuraminidase inhibitor (NAI), is produced by hydrolysis of oseltamivir through the ester bond and inhibits the isolation of mature influenza virus from host cells by inhibiting neuraminidase, thereby inhibiting virus transmission in humans , 9. OselTAMIFLU® was jointly developed by Roche Registration Ltd and Gilead Sciences, Inc. The capsule was first approved for marketing by the FDA in October 1999, and the oral suspension was approved for marketing by the US FDA in December 2000. It has been marketed in many countries and regions around the world, and is recognized as one of the most effective drugs against influenza in the world. The purpose of this study was to assess the bioequivalence of oseltamivir phosphate for suspension  and TAMIFLU®  in Healthy Chinese volunteers.http://www.chinadrugtrials.org.cn/clinicaltrials.searchlist.dhtml.This study was developed in healthy subjects in the fasting or postprandial state, using a single-dose, randomized, open-label, two-period, two-sequence, self-crossover design. It authorized by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College. The ethical approval process complies with the requirements of Good Clinical Practice (GCP), the Declaration of Helsinki and relevant Chinese laws and regulations. Registration number CTR20200764 at 26-Apr-2020. max of oseltamivir was in the range of 21.16–31.87%, and the AUC was in the range of 7.24–7.35%. Assuming one-sided α = 0.05, β = 0.2, %CV of 31.87%, a mean ratio F between the test product and reference product of 1, and the bioequivalence interval of 80.00–125.00%, the sample size of the two-period, crossover study design is 35 cases, calculated by PASS software. Considering the results of subject dropout and sample size estimation, 40 subjects were planned to be enrolled in this fasting trial and fed trial respectively, and the subjects' random numbers are K001-K040 and C001-C040 respectively. Screening examinations are performed in healthy subjects from 14 days prior to dosing to 2 days prior to dosing. Eligible healthy subjects were randomized in a 1:1 ratio into two groups for a single dose of 75 mg/12.5 mL of test and/or reference product, followed by a 7-day post-wash period and then crossover dosing. Subjects in the postprandial group had a high-fat breakfast calorie setting of 800–1000 kcal, with about 50% fat calories, 150 kcal of protein, and about 250 kcal of carbohydrates. The main food composition was milk, toast, peanuts, butter, sausage, and lettuce. Each high-fat meal was accurately weighed after a developed meal recipe. The interval time between the start of the high-fat meal and the start of dosing was 30 min, and all subjects finished the meal within 30 min. The sampling points are designed as follows: 0 h before administration (within 1 h before administration) and 5 min, 15 min, 30 min, 45 min, 1 h, 1.25 h, 1.5 h, 1.75 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 24 h, 36 h, and 48 h after administration. The concentrations of oseltamivir and oseltamivir carboxylate in human plasma are established by liquid chromatography-mass spectrometry (HPLC–MS/MS). The quantitative linear range of oseltamivir is 0.500–200 ng/mL, and the LLOQ is 0.500 ng/mL. The quantitative linear range of oseltamivir carboxylate is 4.00–800 ng/mL, and the LLOQ is 4.00 ng/mL.According to "the Guiding Principles for Human Bioavailability and Bioequivalence test of Pharmaceutical Preparations (2015 Edition)", "the Guiding Principles for Human Bioequivalence Research of Chemical Drug Imitations with Pharmacokinetic Parameters as the End Point Evaluation Indicators", and recommended dose in Tamiflu® instructions, the single oral dose of 75 mg/12.5 mL was determined for the reference preparation and test preparation of this study. According to the data in the relevant literature , the int2 ; subjects had no history of chronic or serious cardiovascular, such as hepatobiliary, renal, hematological and lymphatic, immunological, central nervous, neurological, gastrointestinal system diseases, etc., likewise health condition is well.Subjects participating in the study: all aged between 18 and 65 years old , with an appropriate gender ratio of males and females; men and women need to meet the standard weight of not less than 50 kg and 45 kg respectively, as well body mass index (BMI) ranged from 19.0–26.0 kg/mSubjects with the following situations need to be excluded: women who are breastfeeding, or subjects and their partners who are planning to become pregnant or donating sperm or eggs, unwilling or unable to voluntarily use effective contraception from the screening date to 6 months after the end of the trial; or subjects with history of psychiatric or neurological related diseases such as epilepsy or depression or family history of genetic diseases; or subjects taking any drugs or food supplements or herbal medicines within 14 days before screening or not longer than their 5 times half-life.max, Tmax, AUC0-t, AUC0-∞, t1/2, λz, as well as %AUCex for oseltamivir and oseltamivir carboxylate were included. The bioequivalence analysis of two oseltamivir phosphate for suspension using Cmax, AUC0-t and AUC0-∞ of oseltamivir in addition to oseltamivir carboxylate after natural logarithm transformation, was carried out by SAS® 9.4 software. If the 90% confidence interval (CI) of the ratio of the geometric mean of the pharmacokinetic parameters of the two products is within the range of 80.00–125.00% , the test product is considered bioequivalent to the reference product. Tabular and descriptive statistical analyses were performed on subject distribution, demographic and other baseline characteristics, and other PK parameters.Pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate were calculated using Phoenix WinNonlin  based on individual blood concentrations and sampling time  using non-compartmental analysis (NCA), CAdverse events (AEs), serious adverse events (SAEs), concomitant medications, clinical laboratory tests, vital signs, 12-lead electrocardiogram and physical examination were recorded.A total of 205 subjects were screened, of which 125 failed in screening for not meeting the inclusion criteria/for meeting exclusion criteria. Among the 80 qualified subjects, there were 40 in the fasting group and 40 in the fed group. In the fed trial, 2 subjects withdrew early. Among them, Subject S107 (C001) voluntarily withdrew early from the trial during the washout period after completing the first period of blood sample collection; and subject S152 (C021) voluntarily withdrew early from the trial 4.5 h after the first period of dosing. In the fasting trial, one subject voluntarily withdrew early. Subject S082 (K033) withdrew early from the trial due to an adverse event of severe vomiting with high volume, which occurred 1.75 h after the second period of dosing. The baseline characteristics of the subjects are shown in Table The mean oseltamivir and oseltamivir carboxylate in the fasting and fed groups had similar plasma concentration–time profiles Figs.  and 2.Fimax of oseltamivir in the fasting group was 1.50 h and 1.25 h, with Cmax of 49.41 ng/mL and 48.79 ng/mL for the reference product and test product, respectively; Tmax of oseltamivir carboxylate was both 5.00 h, with Cmax of 257.95 ng/mL and 247.69 ng/mL, respectively. The Tmax of oseltamivir of reference product and test product in the fed group were both 1.25 h, and Cmax was 43.86 ng/mL and 44.16 ng/mL, respectively; the Tmax of oseltamivir carboxylate was 6.00 h and 5.00 h, respectively, and Cmax was 204.64 ng/mL and 194.13 ng/mL, respectively. The 90% CIs for the geometrically adjusted mean ratios of the PK parameters  of the test product and the reference product of oseltamivir phosphate for oral suspension in the fasting group were (92.39–106.50) %, (94.26–100.67) %, and (94.32–100.89) %, respectively. The 90% CIs for the geometrically adjusted mean ratios of the PK parameters  of the test product and the reference product of oseltamivir phosphate for suspension in the fed group were (93.61–105.83) %, (95.64–100.19) %, and (96.06–102.66) %, respectively.The pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate in fasting or fed administration subjects included in the pharmacokinetic parameter analysis are shown in Table max of the same group of subjects after the first period of dosing, which may have an effect on PK parameters. Therefore, sensitivity analysis was performed on the bioequivalence of oseltamivir and oseltamivir carboxylate based on the original bioequivalence analysis without excluding subject S048 (K018). The results of the sensitivity analysis showed that the 90% confidence intervals for the geometrically adjusted mean ratios of the PK parameters  of the test product and the reference product of oseltamivir phosphate for suspension were all within the range of 80.00–125.00%, which was consistent with the conclusion of the bioequivalence analysis , positive urinary leukocyte esterase, urine sediment detection, abnormal urinalysis, elevated gamma-glutamyl transferase, elevated alanine aminotransferase, increased human chorionic gonadotropin, abnormal electrocardiogram QRS wave complex, and nausea. Of which 3 cases in 3 subjects were grade 2 in severity and the rest were grade 1 in severity. Adverse events of severity grade 2 are mainly elevated blood triglycerides. Vital signs and physical examination were all normal and no clinically significant. For the 12-lead ECG, 1 subject showed abnormalities with clinical significance; for laboratory tests, 4 subjects showed abnormalities with clinical significance after taking the drug for blood biochemistry tests, 3 subjects showed abnormalities with clinical significance after taking the drug for routine urine tests, 1 subject showed abnormalities with clinical significance after taking the drug for blood pregnancy tests, and no abnormalities with clinical significance after taking the drug for routine blood and coagulation tests. There were no abnormalities and clinically significant blood tests. Of the 27 cases of TEAEs in this trial, 14 were cured, 12 had an unknown outcome, 1 had a persistent outcome, and there were no serious adverse events , who had normal pregnancy test during the screening period and two-period enrollment examination, was suspected to be pregnant based on human chorionic gonadotropin (β-HCG) of 435.62 IU/L during the second-period exit examination on July 01, 2020, and was asked whether sexual intercourse has occurred, the subject explained that she once had sexual intercourse and took contraception before enrollment. The β-HCG doubled two days later and pregnancy was highly suspected. Pregnancy was confirmed by gynecological color Doppler ultrasonography, and the pregnancy event was reported and followed up. On March 12, 2021, the subject delivered a female infant at 39-week gestation, and the newborn was born naturally in good condition with an Apgar score of 10.The more frequently reported adverse events after subjects took the test product and the reference product, ranked by system organ classification (SOC), were mainly elevated blood triglycerides in various tests, positive bacterial tests and positive urinary leukocyte esterase. The main gastrointestinal adverse event was nausea , this includes elevated C-reactive protein, decreased mean cell volume, elevated neutrophil count, nausea, and, as in the postprandial group, also elevated blood triglycerides, abnormal urinalysis, and vomiting. Of which 3 cases in 3 subjects were grade 2 in severity and the rest were grade 1 in severity. Vital signs, physical examination, and 12-lead electrocardiogram were all normal and no clinically significant; in terms of laboratory tests, after medication, 6 subjects showed abnormal and clinically significant blood biochemical tests, 2 subjects showed abnormal and clinically significant routine blood tests, 2 subjects showed abnormal and clinically significant routine urine tests, and pregnancy tests and coagulation tests were normal and clinically significant. Of the 13 cases of TEAEs in this trial, 8 were cured, 5 had an unknown outcome, and there were no serious adverse events  and 3.67 h (58.52% CV), respectively; the Tmax of oseltamivir carboxylate were both 5.00 h, and t1/2 were 7.05 h (16.70% CV) and 6.85 h (14.31% CV), respectively. The Tmax of oseltamivir of reference product and test product in the fed group were both 1.25 h, and t1/2 were 6.18 h (88.47% CV) and 7.23 h (88.69% CV), respectively; the Tmax of oseltamivir carboxylate were 6.00 h and 5.00 h, respectively, and t1/2 were 7.88 h (28.04% CV) and 8.28 h (23.09% CV), respectively. The 90% CIs for the geometrically adjusted mean ratios of Cmax, AUC0-t, and AUC0-∞ of oseltamivir phosphate under fasting and fed conditions were (92.39–106.50)%, (94.26–100.67)%, (94.32–100.89)%, and (93.61–105.83)%, (95.64–100.19)%, (96.06–102.66)%, respectively, all within the range of 80.00–125.00%, which met the criteria for determining bioequivalence, and both were bioequivalent when administered under fasting and fed conditions. Based on the pharmacokinetic results after feeding and fasting states, we can also see that food causes changes in the values of the pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate, where the AUC0-∞ of oseltamivir increases after feeding and the AUC0-∞ of oseltamivir carboxylate decreases, but this change has no effect on the clinical effect of oseltamivir. The Cmax, AUC0-t of oseltamivir carboxylic acid were lowered under fed conditions, and some literature suggests that this may be related to an interaction with the carboxylesterase enzyme responsible for prodrug conversion [In this study, the oseltamivir phosphate for suspension produced by Hetero Labs Limited was compared with the originator TAMIFLU® to evaluate the absorption rate and degree of absorption in healthy humans, and to investigate the human bioequivalence of the two drugs. The difference between the test product and the reference product TAMIFLU® under fasting and fed conditions in the pharmacokinetic parameters between was not significant, which was similar to the results of previous studies , 18. TheThis study confirmed the interchangeability of the test product and TAMIFLU® by verifying their bioequivalence, and that food intake did not affect the pharmacokinetics and bioequivalence of the two products. For patients, it is possible to achieve the same therapeutic effect by choosing a drug with a relatively good price.In terms of adverse reactions, the most common side effects of oseltamivir phosphate were nausea (incidence 10%), vomiting (2–15%), abdominal pain, diarrhea, headache, insomnia and dizziness. Other side effects included conjunctivitis, rhinorrhagia, allergy, cardiac arrhythmias, gastrointestinal bleeding, erythema multiforme, toxic epidermal necrolysis, confusion, hepatitis, epilepsy, and neuropsychiatric events, but the incidence was < 1%. Oseltamivir is generally well tolerated –22. In tThe limitation of this study is as follows, the study is a bioequivalence trial in healthy Chinese subjects, the pharmacokinetic parameter data express the absorption distribution and elimination in Chinese subjects, and the bioequivalence in other races has not been evaluated yet, so it is not known whether there are differences between different races, which will be further verified in later trials.This study showed that oseltamivir phosphate for suspension  and TAMIFLU®  met bioequivalence criteria in the matter of the absorption rate and the degree of absorption of oseltamivir phosphate and oseltamivir carboxylate, and had good safety. Food had no effect on the pharmacokinetics and bioequivalence of oseltamivir phosphate for suspension and TAMIFLU® in healthy subjects.Additional file 1. Bioequivalence analysis results of oseltamivirAdditional file 2. Summary table of adverse eventsAdditional file 3. Summary of all treatment emergent adverse events(TEAEs) based on system organ class(SOC), preferred terminology(PT)


Salvia miltiorrhiza Bunge (Danshen), had potent effects on cell proliferation and migration. Especially for human osteosarcoma U−2 OS cells, tanshinones significantly enhanced the cell adherence, implying a possible role in cell adhesion and cell migration inhibition. In this work, therefore, we aimed to provide a new insight into the possible molecule mechanisms of dihydrotanshinone I, which had the strongest effects on cell adhesion among several candidate tanshinones. RNA−sequencing-based transcriptome analysis and several biochemical experiments indicated that there were comprehensive signals involved in dihydrotanshinone I-treated U−2 OS cells, such as cell cycle, DNA replication, thermogenesis, tight junction, oxidative phosphorylation, adherens junction, and focal adhesion. First, dihydrotanshinone I could potently inhibit cell proliferation and induce cell cycle arrest in the G0/G1 phase by downregulating the expression of CDK4, CDK2, cyclin D1, and cyclin E1 and upregulating the expression of p21. Second, it could significantly enhance cell adhesion on cell plates and inhibit cell migration, involving the hyaluronan CD44−mediated CXCL8–PI3K/AKT–FOXO1, IL6–STAT3–P53, and EMT signaling pathways. Thus, the increased expression of CD44 and lengthened protrusions around the cell yielded a significant increase in cell adhesion. In summary, these results suggest that dihydrotanshinone I might be an interesting molecular therapy for enhancing human osteosarcoma U−2 OS cell adhesion and inhibiting cell migration and proliferation.In the screening of novel natural products against cancer using an in vitro cancer cell model, we recently found that tanshinones from a traditional Chinese medicine, the rhizome of  Cancer ranks as a leading cause of death and an important barrier to increasing life expectancy in every country of the world . For exaNatural products are still thought of as important resources of drug discovery and pharmaceutical agents due to their diversity in structure and bioactivities ,9. DurinSalvia miltiorrhiza Bunge (Danshen) [S. miltiorrhiza [As shown in Danshen) ,16, a faDanshen) . PreviouDanshen) ,19 indiciorrhiza ,20. Seveiorrhiza , anti-oxiorrhiza . For exaiorrhiza , induce iorrhiza , and arriorrhiza ,26. Tansiorrhiza . Cryptotiorrhiza . In addiiorrhiza ,22,29 aliorrhiza  through iorrhiza , STAT3-Ciorrhiza , PI3K/AKiorrhiza , and theiorrhiza . Howeveriorrhiza ,37,38,39In view of the significant roles of cell adhesion in cancer progression and metastasis ,41,42, t50 value for U−2 OS cells for 24 h of treatment was 3.83 ± 0.49 μM, while that for 48 h of treatment was 1.99 ± 0.37 μM, which provided the reference of dosage for subsequent experiments.Our previous studies  showed tIt should be pointed out that without treatment of DS−1, common U−2 OS cells as the control grew rapidly, showing a smooth cell surface and clear cell boundaries. However, after being treated with DS−1 at 2.5 and 5.0 µM for 24 and 48 h, the cell surface was no longer smooth; the morphology became irregular. The most important thing was that cell adhesion of DS−1-treated cells on the cell culture plate became significantly stronger than the untreated ones, and cells were tightly attached onto the cell plate, implying the possible roles of DS−1 in cell adhesion and cell migration inhibition ,41,42.50 of 15.48 ± 0.98 µM  pathway enrichment analysis and Gene Ontology (GO) term analysis were performed, and the results are illustrated in As shown in CDK2 was downregulated to 0.85 ± 0.08-fold, 0.80 ± 0.16-fold, and 0.66 ± 0.07-fold of the control group; CDK4 was downregulated to 0.53 ± 0.06-fold, 0.37 ± 0.02-fold, and 0.29 ± 0.05-fold of the control group; cyclin D1 was downregulated to 0.27 ± 0.06-fold, 0.29 ± 0.06-fold, and 0.20 ± 0.04-fold of the control group; cyclin E1 was downregulated to 0.52 ± 0.04-fold, 0.48 ± 0.08-fold, and 0.36 ± 0.04-fold of the control group. The downregulation of cyclin D1 and cyclin E1 was further confirmed by immunoblotting  ,52. EMT ectively D.snail was downregulated to 1.06 ± 0.06-fold, 0.46 ± 0.05-fold, and 0.39 ± 0.03-fold of the control group, and the expression of slug was downregulated to 1.06 ± 0.22-fold, 0.58 ± 0.13-fold, and 0.45 ± 0.07-fold of the control group , which localize on the cell surface and mediate the activation of progelatinase A  . Accordi heatmap G, and alectively H. These To validate the pivotal role of CD44 in increasing cell adhesion, CD44 in U−2 OS cells was largely knocked down by siRNA . As showPrevious studies ,59 indicAfter CD44 knockdown by siRNA, another cell digestion experiment was conducted at 25 °C again. In the control group, U−2 OS cells were totally digested with trypsin after about 120 s, while in the DS−1 group, U−2 OS cells were totally digested within 150 s F,G. CompCXCL8 was upregulated to 1.49 ± 0.10-fold, 3.34 ± 0.49-fold, and 3.71 ± 0.53-fold  and phosphatidylinositide 3-kinase (PI3K)/Akt , which iIL6 was downregulated. After treatment with 2.5, 5.0, and 7.5 µM DS−1, the expression of IL6 was downregulated to 0.81 ± 0.09-fold, 0.51 ± 0.07-fold, and 0.42 ± 0.12-fold  is a key step toward cancer metastasis. EMT plays an important role in the migration inhibition of U−2 OS cells. FASN is the starting point protein, which promotes the phosphorylation and degradation of E−cadherin and then reduces intercellular contact . DS−1 deSince the migration of U−2 OS cells was significantly inhibited, we posited that another pathway related to migration is regulated by DS−1. Previous studies suggest that MT-MMP-1 cleaves CD44 and promotes cell migration ,67. The DS−1 also induced the changes in chemokines, including CXCL8 and IL6, following with the decrease of the phosphorylation of AKT and STAT3. As a result, the cell proliferation and cell cycle were inhibited. In Xiaoqing Wang’s work, the proliferation and migration of ovarian cancer cells were inhibited by dihydrotanshinone I via transcriptional repression of the PIK3CA gene . AlthougAccording to these results, we proposed a new and systematic signal pathway in U−2 OS cells induced by DS−1. As illustrated in Salvia miltiorrhiza [As described above, tanshinones are a group of bioactive abietane diterpenes in the rhizome of iorrhiza ,22,29. Tiorrhiza . Dihydroiorrhiza . Dihydroiorrhiza . Cryptotiorrhiza . Tanshiniorrhiza . Thus, S1H NMR), were isolated and purified from the extracts of the root of S. miltiorrhiza Bunge using the gradient counter-current chromatography method in our lab [50 of DS−1, smaller doses of 2.5, 5.0, and 7.5 µM were used in most of the following experiments, and doses of 3.125, 6.25, and 12.5 µM were used for the experiments regarding FOXO1 in order to collect more surviving cells (at least >15%) for further biochemical experiments. We are very sorry for the inconsistency of some experimental doses because of nontargeted step-and-step investigation of the molecular mechanisms during a long time (more than 3 years). It is indeed a flaw in this work, but does not affect the whole conclusion.Five pure tanshinone compounds, DS−1 to DS−5 -2,5-diphenyl-2-H-tetrazolium bromide (MTT), phosphatase inhibitors, phenylmethylsulphonyl fluoride (PMSF), and ammonium persulfate (APS), (TEMED) were purchased from Beyotime Biotechnology . Defatted milk powder was purchased Dawen biological Co., Ltd. . Triton X-100 was purchased from Sangon Biotech Co., Ltd. . The BCA protein assay kit was purchased from Thermo Electron Co.  SuperEnhanced chemiluminescence detection reagents and RIPA lysis buffer were purchased from Applygen Technologies Inc. . Antifade mounting medium with DAPI and RNAeasy™ Animal Long RNA Isolation Kit were purchased from Beyotime Biotechnology .Human osteosarcoma cells U−2 OS, human cervical cells HeLa, and normal rat kidney cells NRK−49F were brought from the cell bank of the Chinese Academy of Sciences .The effects of tanshinones (DS−1 to DS−5) on the proliferation of U−2 OS cells, HeLa cells, and NRK−49F cells were measured by the classical MTT method with minor modifications as follows. Cells were seeded at 5000 per well in 96-well plates, with each group being repeated in 5 wells, and were incubated for 12 h. DS−1 to DS−5 were added at gradient concentrations, then incubated for 24 and 48 h, respectively. Then, 20 μL MTT solution  was added to every well, and the cells were further incubated at 37 °C for 4 h until a purple precipitate was visible. The plate was centrifuged at 1000 rpm for 1 min; the supernatant was suctioned, and 150 μL of DMSO was added per well; the plate was shaken on a shaker protected from light for 15 min. We placed the plate in an enzyme marker and measured the absorbance values at a 490 nm wavelength.Sample preparation: U−2 OS cells were seeded in a 10 cm culture dish, and we cultured them to the logarithmic growth phase, suctioned the previous medium, and added new medium with DS−1 at 0 and 6.25 μM; each group was repeated two times. After 24 h of culture, the dish was washed with PBS reagent 3 times. One milliliter of TRIzol reagent was added to every dish for cell lysate and then collected in centrifuge tubes.Transcriptome sequencing and analysis were carried out by Novogene Bioinformatics Technology Co., Ltd. .Raw data in the fastq format were firstly processed through in-house Perl scripts. In this step, clean data were obtained by removing reads containing adapter or containing ploy-N and low-quality reads from raw data. All the downstream analyses were based on the clean data with high quality.https://github.com/DaehwanKimLab/hisat2, accessed on 4 May 2022), and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. FeatureCounts v1.5.0-p3  was used to count the reads numbers mapped to each gene. Then the Fragments PerKilobase of transcript sequence per Millions base (FPKMs) of each gene were calculated based on the length of the gene and reads’ count mapped to this gene.Reference genome and gene model annotation files were downloaded from the genome website directly. The index of the reference genome was built using Hisat2 v2.0.5 . DESeq2 provides statistical routines for determining differential expression in digital gene ex-pression data using a model based on the negative binomial distribution. The resulting p-values were adjusted using the Benjamini and Hochberg approach for controlling the false discovery rate. Genes with an adjusted p-value < 0.05 found by DESeq2 were assigned as differentially expressed.Differential expression analysis of two groups  was performed using the DESeq2 R package v1.16.1 .Gene Ontology (GO) enrichment analysis and KEGG enrichment analysis of differentially expressed genes were implemented by the ClusterProfiler R package v3.4.4 (−ΔΔcT) determined the relative expression of mRNA.Total RNA within cells was extracted by the RNAeasy™ Animal Long RNA Isolation Kit with Spin Column ; the concentrations and OD260/OD280 and OD230/OD260 values were measured by NanoDrop. Five micrograms of RNA was used for reverse transcription; each sample was diluted to 16 μL, and 4 μL of synthesized premix solution (5×) was added, then the mixtures were placed in a 42 °C water bath for 10 min, then an 80 °C water bath for 10 min to deactivate reverse transcriptase. After reverse transcription, each sample was diluted to 140 μL. We designed the primer sequence of a gene on the primer3 web version 4.1.0, and the primers are shown in U-2 OS cells were seeded in 6-well plates at a density of 200,000 per well and cultured to 90% abundance after 18 h. The cells were scratched with a 10 μL pipette tip along the ruler; each well was scratched 3 times in parallel, and the line was drawn perpendicular to the scratch on the back for fixed-point photography. We suctioned the previous medium and washed the cells with a PBS reagent. Then, we added 2% FBS medium with DS−1 and cultured the cells at 37 °C for different times. After 0, 24, and 48 h, the plate was taken out, and the scratches were observed and photographed by the inverted microscope, while the distance of scratches at different times was measured.Cell mobility = (pre-migration distance – post-migration distance)/pre-migration distance × 100%. The cell migration rate of blank control was set as 100%, and the relative cell migration rate = cell migration rate/cell migration rate of control group × 100%Coverslip preparation: we cultured U−2 OS cells on sterile glass coverslips in 24-well plates and then treated them with DS−1 at different concentrations.Fixation and staining: We washed the cells with PBS 3 times and fixed the cells with 200 μL 4% paraformaldehyde/PBS solution at 4 °C for 15 min. We then rinsed 3 times with PBS and permeabilized with 200 μL 0.2% TritonX-100 at room temperature for 30 min. Then, we blocked with 200 μL blocking agent at 37 °C for 1 h and diluted the primary antibody in dilution buffer as recommended in the specific products’ datasheet. We overlaid 150 μL of diluted antibody and kept slips covered overnight at 4 °C or for 1 h at 37 °C. We rinsed 3 times with PBS, 5 min for each wash, diluted fluorescent secondary antibody in dilution buffer, and incubated for 1 h at room temperature. Then, we rinsed 3 times with PBS again, 5 min for each wash. We took out the slips and laid them on the slides, dropping 3 μL of antifade mounting medium with DAPI onto them. Then, we covered the slips with cover glass. We then observed and photographed the samples with two-photon laser confocal microscopy (Lsm710nlo).Sample preparation: U−2 OS cells seeded in 10 cm Petri dishes were collected and washed with ice-cold PBS and RIPA lysis buffer with PMAF; phosphatase inhibitor was added, and the tubes were placed on ice for 30 min and vortexed for 30 s at intervals of 10 min. The samples were centrifuged at 12,000 g at 4 °C for 15 min, and the supernatants were collected in new centrifuge tubes. The protein samples were placed on ice, and the concentration of each protein sample was measured by the BCA kit (Thermo). The protein concentration was adjusted to the same by milli Q ultrapure water.Loading and running the gel: Equal amounts of protein were loaded into the wells of the 10% SDS-PAGE gel, along with molecular weight markers, and 15 μg of total protein from the cell lysate was loaded. We then ran the gel for 45 min at 200 V.Transferring the protein: We prepared the transfer stack as follows: negative plate, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and positive plate. The transfer was for 90 min at 200 mA.Antibody staining: We blocked the membrane for 1 h at room temperature using 5% blocking solution, then incubated the membrane with appropriate dilutions of the primary antibody in the blocking solution overnight at 4 °C. We then washed the membrane with TBST reagent 3 times, 5 min each. Then, we incubated the membrane with the recommended dilution of labeled secondary antibody in blocking solution at room temperature for 1 h and washed the membrane with TBST reagent 3 times, 5 min each. For signal development, super-enhanced chemiluminescence detection reagents were added to the surface of the membrane for 1 min, then excess reagent was removed, and the membrane was covered in transparent plastic wrap. Images were acquired using darkroom development techniques for chemiluminescence and analyzed by Image J.5 cells were plated in 2 mL of growth medium in a 6-well plate and cultured for 12 h until 70–90% confluence was obtained. To transfect each well, 100 pmol siRNA was diluted in 200 µL of opti-MEM without serum, and 5 µL of HighGene Transfection reagent  was also added. Then, the solution was mixed by vortexing. SiRNA and HighGene Transfection reagent complexes were added to each well and shaken gently. Half the amount of the used medium was displaced by fresh medium after 6 h of cell transfection in order to reduce the toxicity of the reagents to the cells. After 24 h, the transfected cells were treated with DS−1 or harvested for further use.Small interfering RNA (siRNA) oligonucleotide was synthesized by Sunya Biotechnology Co., Ltd . The siRNA sequences were as follows: sense (5′–3′), UAUUCCACGUGGAGAAAAAtt; antisense (5′–3′), UUUUUCUCCACGUGGAAUAca . A totalAntibodies against FOXO1, phospho-FOXO1 (Ser256), and AKT were purchased from Abcam Biotechnology . Antibodies against CD44, Phospho-AKT (S473), Bcl-2, p53, phospho-p53 (S20), p21, cyclin D1, and cyclin E1 were purchased from Abclonal Biotechnology Co., Ltd. . Antibodies against E-cadherin, vimentin, actin, and GAPDH were purchased from Beyotime Biotechnology. Peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (H + L), FITC-conjugated goat anti-rabbit IgG (H + L), and Cy3-conjugated goat anti-rabbit IgG (H + L) were purchased from Dawen biological Co., Ltd. .t-test was used to evaluate the significance between the two groups, while multiple-group comparisons were evaluated by one-way ANOVA followed by Bonferroni’s test. A value of p < 0.05 was considered statistically significant, and asterisks show significant differences between groups: * p < 0.05, ** p < 0.01, *** p < 0.005.Statistical analysis was performed using Graphpad Prism 5.0 . Results are expressed as the mean ± standard deviation (SD) from at least three separate experiments. Student’s In this study, DS−1 was found to possess potent effects in enhancing cell adhesion and inhibiting cell proliferation and migration on U−2 OS cells. The transcriptome results indicated that DS−1 could regulate a comprehensive signal with multiple biological processes. Further biochemical experiments confirmed that DS−1 could arrest the cell cycle, inhibit cell proliferation and migration, and enhance cell adhesion, mainly through the hyaluronan CD44-mediated CXCL8-PI3K/AKT-FOXO1, IL6-STAT3-P53, and EMT signaling pathways. Therefore, all the results indicated that dihydrotanshinone I (DS−1) could be an interesting molecule for enhancing cell adhesion and inhibiting cell proliferation and migration of U−2 OS cells. To the best of our knowledge, this is the first report that DS−1 can induce CD44-mediated U−2 OS cell adhesion and proliferation inhibition, although more work is required to explore the therapeutic roles using multiple cells and animal models in vitro and in vivo. This is very useful for future osteosarcoma research and drug development.


Chinese Materia Medica and Jiangsu New Medical College record that Radix Veratri root is Liliaceae Veratrum taliense Loses. f. and the root of Veratrum stenophyllum Diels. According to traditional Chinese medicine (TCM) example, Radix Veratri is a Liliaceae plant Veratrum taliense. Another literature pointed out that the aliases of Veratrum taliense and Veratrum angustifolia are both Radix Veratri, and their effects are basically the same. The main active ingredient of Veratrum is veratramine, of which veratramine and Jervine are higher in content, reaching 24.60% and 21.28% of the total alkaloids, respectively. Veratrum alkaloids are both toxic and effective ingredients. In addition to its good clinical efficacy, attention should also be paid to its pharmacokinetic characteristics in vivo. It is particularly important to study the pharmacokinetic characteristics of veratramine and Jervine in vivo.  The goal of this study was to develop a simple and effective method for measuring veratramine and Jervine in rat plasma at the same time. This method was used to study the pharmacokinetic characteristics of veratramine and Jervine in the alcohol extract of Radix Veratri in rats, to provide a reasonable basis for the clinical use of Radix Veratri. m/z, Jervine quantitative ion pair was 426.2⟶114.1m/z, and Bullatine B (IS) quantitative ion pair was 438.2⟶420.1m/z. In the positive ion mode, the multireaction monitoring (MRM) mode was used to determine the blood concentration of veratramine and Jervine. DAS 3.3.0 was used to calculate the relevant pharmacokinetic parameters.  Eighteen SD rats were randomly assigned into three groups, half male and half female, and were given 0.04 g/kg, 0.08g/kg, and 0.16 g/kg Radix Veratri alcohol extract, respectively. Blood samples were collected at different time points and were analyzed by LC-MS/MS after protein precipitation. Bullatine was set as the internal standard; the plasma samples were extracted with ethyl acetate. After the sample was processed, acetonitrile-10 mM ammonium acetate, whose pH was adjusted to 8.8 with ammonia water, was taken as the mobile phase. Veratramine quantitative ion pair was 410.1⟶295.1r of three consecutive batches of the standard curve was greater than 0.995. Veratramine's lower quantification limit was 0.745 ng/mL, Jervine's was 1.11 ng/mL, and precision and accuracy were both less than 15%. The accuracy of veratramine was between 88.96% and 101.85%, and the accuracy of Jervine was between 92.96% and 104.50%. This method was adopted for the pharmacokinetic study of alcohol extracts of Radix Veratri. The results showed that only Cmax of veratramine female rats did not show linear kinetic characteristics in the dose range of Radix Veratri alcohol extract from 0.04 g/kg to 0.16 g/kg. For AUCt0‐ and Cmax of veratramine and Jervine, it could not determine whether the Radix Veratri alcohol extract showed linear kinetic characteristics within the dosage range of 0.04 g/kg~0.16 g/kg. Veratramine and Jervine showed obvious gender differences in the absorption and elimination stages. The absorption rate of veratramine and Jervine by male mice was about 10 times higher than that of female mice, and the elimination rate of male mice is about 20 times lower than that of female mice. It was suggested that the clinical application of the steroidal alkaloids veratramine and Jervine in Radix Veratri required rational use of drugs based on gender.  Veratramine had a good linear relationship in the concentration range of 0.0745~18.2 ng/mL, and that of Jervine was 1.11~108 ng/mL. The correlation coefficient  An LC-MS/MS analysis method suitable for the pharmacokinetic study of veratramine and Jervine in Radix Veratri in SD rats was established to provide a basis for in vivo pharmacokinetic studies. The pharmacokinetic characteristics of veratramine and Jervine in the alcohol extract of Radix Veratri were significantly different in female and male rats. During the clinical use of Radix Veratri, it should pay close attention to the obvious gender differences that may occur after the medication. Radix Veratri is also known as small Veratri, Veratrum, small brown bag, human hair, etc. The taste is bitter, cool in nature, and highly toxic. It has the effects of dispelling blood stasis and swelling, analgesic and hemostasis, lowering blood pressure –3, expecThe paper's organization paragraph is as follows: The materials and methods are presented first. Second are experimental results of the proposed work. Thirdly, it consists of the results and discussion sections. Finally, the research work is concluded.Veratramine and Jervine reference substances' purity was no less than 98%, which were purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. . Bullatine B  was purchased from the Beijing Shengshikangpu Chemical Technology Research Institute (batch number: N-019-150906). Heparin sodium  was purchased from Aladdin (batch number: C162031). Ammonium acetate  was purchased from Aladdin (batch number: k1616041). Methanol  was purchased from MERCK (batch number: I0887507 717). Sinopharm Chemical Reagent Co., Ltd. provided analytical pure ethyl acetate (batch number: 20170824). Sichuan Xilong Chemical Co., Ltd. provided the ammonia  (batch number: 150324). The water was ultrapure water made by the Drug Safety Evaluation Center of Yunnan Institute of Materia Medica.μL. The mass spectrum parameters were as follows: veratramine m/z: 410.1⟶295.1, Jervine m/z: 426.2⟶114.1, and Bullatine B m/z: 438.2⟶420.1. Ion source parameters were as follows: CUR: 20.00 psi, IS: 5,500.00 V, TEM: 500.0°C, GS1: 50.0 psi, GS2: 50.0 psi, CAD: medium, and interface heater(ihe): on.The researchers used an API-3200 triple quadrupole tandem mass spectrometer and an electrospray ionisation ion source (ESI). Multiple reaction monitoring (MRM) was used to quantify the sample, which was separated on an Agilent ZORBAX Extend C18 column. The mobile phase was A (10 mmol/L ammonium acetate) and B (acetonitrile), 0-0.2 min, 10% B; 0.2-2 min, 10%-90% B; 2-5 min, 90% B; 5-5.1 min, 90%-10%B; and 5.1-7.5 min, 10%B. The flow rate was 0.4 mL/min, the column temperature was set to 20°C, and the measurement solution was 10 μg/mL, 553 μg/mL, 496 μg/mL, and 513 μg/mL. Diluted with methanol, the concentrations of veratramine were 0.745, 2.24, 6.70, 20.2, 60.5, and 182 ng/mL; those of Jervine standard working fluid were 11.1, 27.8, 69.5, 174, 434, and 1,080 ng/mL; those of veratramine were 0.745, 2.70, 25.6, and 143 ng/mL; and those of Jervine quality control working solution were 1.11, 31.6, 194, and 724 ng/mL. Quality control samples were used for precision, accuracy, matrix effect, recovery, and stability investigations. The same method was used to prepare Bullatine B (IS) internal standard working solution with a concentration of 128 ng/mL, and all working solutions were stored in a refrigerator at 2-8°C.5.45 mg and 5.53 mg of veratramine reference substance and 4.96 mg and 5.13 mg Jervine reference substance were taken, two copies each. Methanol was diluted to stock solutions with concentrations of 545 μL internal standard working solution was added into a 2 mL EP tube, blown and dried with nitrogen at room temperature, and added with 100 μL of plasma sample mixed in vortex for 2 min. Then, 40 μL 25% ammonia water was added and mixed in vortex for 1 min, and 1 mL ethyl acetate was added and mixed in vortex for 3 min and centrifuged at 6,000 r/min at room temperature for 10 min. 600 μL of the supernatant was taken and blown and dried with nitrogen at room temperature. It was reconstituted with 150 μL methanol and mixed in vortex for 2 minutes and centrifuged at 15,000 r/min at room temperature for 10 minutes, and the supernatant was taken for analysis.10 It was fully verified according to the US Food and Drug Administration's bioanalytical method verification guidelines, as illustrated in Figures y = 0.291x + 17.1 × 10−4 (r = 0.9965). The Jervine standard curve was y = 0.0462x − 5.51 × 10−3 (r = 0.9981). The lower limits of quantification of veratramine and Jervine were 0.0745 and 1.11 ng/mL, respectively, and those of RSD% were 5.49% and 0.12%, respectively.The standard curve was obtained by measuring no less than six points for each compound. The veratramine concentrations were 0.0745, 0.224, 0.670, 2.02, 6.05, and 18.2 ng/mL, respectively. The Jervine concentrations were 1.11, 2.78, 6.95, 17.4, 43.4, and 108 ng/mL, respectively. The lowest detectable concentration was used to define the lower limit of quantification. It was also repeated six times to ensure precision and accuracy. The veratramine standard curve was n = 6) and intraday (n = 6) were determined after four concentrations of quality control samples were measured, those of veratramine were 0.745, 2.70, 25.6, and 143 ng/mL, respectively, and those of Jervine were 1.11, 31.6, 194, and 724 ng/mL, respectively (The accuracy and precision of intraday (ectively . The absn = 6) was veratramine of 2.70, 25.6, and 143 ng/mL, respectively, and Jervine of 31.6, 194, and 724 ng/mL, respectively. The extraction recoveries of the two compounds were veratramine of 81.22%~94.42% and Jervine of 81.65%~87.98%  2015-030, and the laboratory animal quality certificate number was No. 51203500004128. There are 9 females and 9 males each, 7-8 weeks, the weight range of female rats was 200-230 g, and the weight range of male rats was 200-230 g. The weight difference of the grouped animals was not more than 20% of the average weight of all animals, and they were rolled into low, medium, and high dose groups, with half males and half females. Samples were collected at 0.15, 0.5, 1, 3, 6, 9, 18, 30, 48, 72, and 96 h before intragastric administration (0 h) and after administration. 0.3 mL of blood was collected from the rat's retroorbital venous plexus, placed in a centrifuge tube soaked in 2 mL of heparin sodium, and centrifuged  for 15 min, and the plasma was frozen and stored at -20°C for testing.t0‐, AUC0−∞, and T1/2. Cmax and Tmax adopted actual measured values, and other pharmacokinetic parameters adopted statistical moment parameters.The pharmacokinetic parameters were calculated using the DAS 3.3.0 pharmacokinetic program to calculate the main pharmacokinetic parameters AUCα · Dβ  was used for multidose linear relationship analysis. α, β, and the 90% confidence interval (90% CI) of the β value in the power function model were calculated using the DAS 3.3.0 pharmacokinetic program. The linear judgment interval after the criterion was changed using the DAS 3.3.0 pharmacokinetic program was 0.839-1.161, based on the bioequivalence criterion of 0.8 to 1.25. When the 90% CI of the β value fell within the linear judgment interval, the linear relationship was considered to be established; otherwise, the linear relationship did not hold.Power function model PK = The absorption rate of male mice was higher than that of female mice, and the elimination rate of male mice was lower than that of female mice, showing obvious gender differences in veratramine  and JervPower Model in the hypothesis test method was used according to the standard of 0.8~1.25, and the linear interval calculated by 1 ± ln(θL)/ln(DH/DL) < β < 1 ± ln(θH)/ln(DH/DL) should be between 0.839 and 1.161. The results of determinations of whether veratramine and Jervine in the alcohol extract of Radix Veratri showed linear kinetic characteristics within the dose range of 0.04 g/kg to 0.16 g/kg are presented in DAS 3.3.0 was employed, the In the initial selection of internal standards, the first selected compounds were aconitine, hypaconitine, Bullatine A, Wilforgine, and Bullatine B. The response of aconitine and hypaconitine could not reach the linear medium concentration, and the response was extremely unstable. Although Bullatine A and Wilforgine could achieve the required response, their chromatographic peaks were far from the target peak, and the analysis time was long, which increased the experimental cost. The peak shape of Bullatine B was good, it can be completely separated from the target peak, and it can be better dissolved in the sample. After testing, there was no Bullatine B in Radix Veratri, so Bullatine B was selected as the internal standard.Cmax of veratramine female rats did not show linear kinetic characteristics within the dosage range of 0.04 g/kg~0.16 g/kg of Radix Veratri alcohol extract. For others, it cannot be judged whether AUCt0‐ and Cmax were linear kinetic characteristics within the dosage range of 0.04 g/kg~0.16 g/kg of Radix Veratri alcohol extract.t0‐ and average Cmax of males were much higher than those of females. Female rats have considerably greater CLz/F clearance rates of veratramine and Jervine than male rats. However, there was no discernible variation in the half-life of veratramine in male and female rats, while the half-life of Jervine was considerably different in female and male rats. This was due to the fact that female rats had a considerably higher volume of veratramine dispersion than male rats, and Jervine was removed faster in female rats than in male rats. The above situation showed that after administration of the alcohol extract of Radix Veratri, veratramine and Jervine were more widely distributed in female rats than in male rats, but the clearance rate was much lower than that in female rats. In addition, at the dosage designed in this experiment, the average AUCt0‐ and average Cmax of males were much higher than that of females. The drug exposure in male rats was much higher than that of female rats, but the clearance rate was much lower than that of female rats. Male rats were more prone to accumulate toxicity in the course of long-term administration, and the symptoms of toxicity were greater than that of females.In the low-dose group, middle-dose group, and high-dose group, the average AUCThe blood drug concentration and main pharmacokinetic parameters of the Radix Veratri alcohol extract group were analyzed. The absorption rate of veratramine and Jervine by males was higher than that of female rats. Although Bullatine A and Wilforgine were able to provide the desired response, their chromatographic peaks were distant from the intended peak, and the analysis time was considerable, increasing the expense of the experiment. Male mice had a lower elimination rate than female mice, indicating clear gender differences. Finally, the clinical usage of the steroidal alkaloids veratramine and Jervine in Radix Veratri necessitates gender-based medication selection.