OBJECTIVE:To explore the effects of the different compatibility ratios of rutin with quercetin on the pharmacoki-netics of rutin in rats in vivo. METHODS:30 rats were randomly divided into rutin group(rutin-quercetin molar ratio of 4:0,the same below),quercetin group(rutin-quercetin ratio of 0:4),BL1 group(rutin-quercetin ratio of 3:1),BL2 group(rutin-quercetin ratio of 2:2)and BL3 group(rutin-quercetin ratio of 1:3),6 rats in each group,all group was administrated 10 mg/kg(calculat-ed by quercetin core of rutin and quercetin) intragastrically. The blood sample 0.3 mL was respectively taken from tail vein after 0.25,0.5,0.75,1,1.5,2,3,4,6,8,10,12,16,20,24 h of administration,the plasma concentration of quercetin(rutin me-tabolite) was determined by HPLC. DAS 2.0 software was used to calculate the pharmacokinetic parameters. RESULTS:The AUC0-24 h in rutin group,quercetin group,BL1 group,BL2 group and BL3 group were (4.908 ± 0.877),(8.382 ± 3.671), (8.473 ± 0.709),(4.366 ± 2.297),(8.914 ± 2.642)μg·h/L;MRT0-24 h were (9.675 ± 1.359),(3.142 ± 0.489),(3.517 ± 1.128), (3.376 ± 1.046),(4.494 ± 1.653) h;tmax were (5.726 ± 5.645),(1.375 ± 0.703),(1.125 ± 1.438),(1.417 ± 2.300),(1.292 ± 0.954) h;and cmax were (0.609 ± 0.202),(2.341 ± 0.539),(2.425 ± 1.217),(1.464 ± 0.677),(3.325 ± 2.425)μg/L. Compared with rutin group,AUC0-24 h and cmax in quercetin group,BL1 group and BL3 group were significantly increased(P<0.05),tmax and MRT0-24 h were significantly decreased(P<0.05);cmax in BL2 group was significantly increased(P<0.05),tmax and MRT0-24 h were significantly decreased(P<0.05). Compared with quercetin group,except AUC0-24 h was significantly decreased in BL2 group(P<0.05),there were no significant differences in AUC0-24 h,MRT0-24 h,tmax or cmax in BL1 group,BL2 group and BL3 group(P>0.05). CONCLUSIONS:Quercetin can inhance the related indexes of rutin in rats in vivo.

OBJECTIVETo develop a quantitative analysis of multi-components by single marker(QAMS) method for simultaneous determination of three coumarins in Angelica dahurica var. formosana, by using one standard substance.

METHODWith imperatorin as internal reference standard, and 2 relative correction factors (RCF) to imperatorin were calculated within certain ranges. Their contents in 20 batches of samples, collected from different areas, were determined by both external standard method and QAMS. The method was evaluated by comparison of the quantitative results between the two methods.

RESULTNo significant differences were found in the quantitative results of three coumarins in 20 batches of A. dahurica var. formosana determined by the two methods (RSD < 5%).

CONCLUSIONQAMS is feasible and suitable for quality control of A. dahurica var. formosana.

Angelica ; chemistry ; Chromatography, High Pressure Liquid ; Coumarins ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Furocoumarins ; chemistry ; Reference Standards

OBJECTIVETo prepare pravastatin sodium-loaded chitosan microspheres to allow sustained drug release.

METHODSThe drug-loaded chitosan microspheres were prepared by using genipin as the cross-linker. The influences of molecular weight of chitosan, volume ratio of oil and water, reaction temperature, and stirring speed on the formation of chitosan microspheres were investigated. The morphology of the microspheres was observed using scanning electron microscopy. The encapsulation efficiency, swelling ratio under different pH conditions, and in vitro drug release were measured.

RESULTSThe in vitro release of pravastatin sodium could last for at least 31 days. The drug release rate varied with the reaction condition. The drug entrapment efficiency of the microsphere was 54.7%. The optimal processing conditions were as follows: chitosan viscosity of 200-400 mPa·s, oil-water proportion of 10:1, stirring speed of 850 r/min, and reaction temperature at 40 degrees celsius;.

CONCLUSIONThe pravastatin sodium-loaded microspheres show good sustained drug release property, and the drug release rate can be modified by controlling the cross-linking time.

Chitosan ; Cross-Linking Reagents ; Delayed-Action Preparations ; Iridoids ; Microscopy, Electron, Scanning ; Microspheres ; Pravastatin ; chemistry

Objective:To explore the effect of high-frequency and low-frequency transcranial magnetic stimulation (rTMS) on the unaffected pharyngeal motor cortex of dysphagic stroke survivors.Methods:Forty-two stroke survivors with dysphagia were enrolled and randomly divided into a high-frequency stimulation group ( n=14), a low-frequency stimulation group ( n=13), and a sham group ( n=15). All received conventional swallowing training. The high- and low-frequency stimulation groups additionally received 250 pulses of 5Hz or 1Hz rTMS over the cortical representation of the mylohyoid muscle on the unaffected side daily for 2 consecutive weeks. In the sham group, sham rTMS was applied with identical protocols. Before and after the intervention, all subjects were subjected to a videofluoroscopic swallowing study and surface electromyography (sEMG). They were also evaluated using the fuctional dysphagia scale (FDS) and the penetration aspiration scale (PAS). Results:After the intervention, a significant improvement was observed in the average PAS, FDS and sEMG results in both rTMS groups compared with the sham control group. The average FDS score of the high-frequency stimulation group had improved significantly more than that of the low-frequency group.Conclusions:rTMS of the contra-lesional cortical representation of the mylohyoid muscle at either 5Hz or 1Hz can effectively improve dysphagia post-stroke. The higher frequency gives superior results.

Objective:To observe the effect of contralateral controlled functional electrical stimulation (CCFES) on the recovery of upper limb motor function after a stroke.Methods:Stroke survivors 1 to 6 months after onset were randomly divided into a CCFES group (14 cases, group A), a CCFES intensive group (14 cases, group B) and a neuromuscular electrical stimulation group (15 cases , group C). In addition to routine rehabilitation training, groups A and B received contralateral controlled functional electrical stimulation, while Group C received routine neuromuscular electrical stimulation. Group B was treated twice daily, while the other two groups were given 1 session each day, 5 times a week for 3 weeks. Each session lasted 20 minutes. The Fugl-Meyer assessment (FMA), the Modified Barthel Index (MBI), surface electromyography and the active range of motion for wrist dorsiflexion were used to evaluate the subjects′ upper limb function before and after the treatment.Results:There was no significant difference in any of the measurements among the three groups before the treatment. After the treatment, all of them had improved significantly, with the improvements in groups A and B significantly greater than in group C, on average.Conclusions:Both normal and intensive contralateral controlled functional electrical stimulation have significant advantages over neuromuscular electrical stimulation in promoting functional recovery of the upper limbs. Intensive CCFES training is superior to routine training in improving muscle strength and range of motion.

Objective:To compare using a rat model of post-stroke depression (PSD) the effect of 28 days of high-intensity interval training (HIIT) with that of medium-intensity continuous training (MICT) on inflammation and neuron apoptosis induced by phosphatase and the deletion of a tensin homolog on chromosome ten (PTEN).Methods:Male Wistar rats were randomly divided into a SHAM, a PSD, an MICT and an HIIT group. Except in the SHAM group, blood flow in the middle cerebral artery was blocked for 90 minutes followed by reperfusion. Repeated but unpredictable mild stimulation was then applied to induce depression. The rats in the HIIT and MICT groups started 28 days of training 24 hours after the successful modeling. The running platform speed was adjusted according to the lactic acid threshold and the maximum speed was measured weekly. In the SHAM group the common carotid artery, internal carotid artery and external carotid artery were only separated without occlusion and there was no depressive stimulation. Any improvements in depression were detected using the sucrose preference test and the forced swimming test. The expression of PTEN, nuclear factor kappa B (NF-κB) and nod-like receptor protein 3 (NLRP3) were detected using western blotting. The expression of cysteine-containing, aspartate-specific proteases (caspase-3) was detected immunohistochemically.Results:Compared with the SHAM group, the PSD group′s average immobility time in the swimming test was significantly longer and its average sucrose consumption was significantly less. Compared with the PSD group, the rats in the MICT and HIIT groups showed, on average, significantly more sucrose consumption and shorter immobility time, indicating that their depression was ameliorated. The expression of PTEN, NF-κB and NLRP3 in the MICT and HIIT groups was, on average decreased significantly compared with the PSD group. The HIIT group′s averages were at the same time significantly lower than those of the MICT group. Average caspase-3 levels in the dentate gyrus of the MICT and HIIT group rats were significantly lower than in the PSD group, with the HIIT group′s average significantly lower than that of the MICT group.Conclusions:Both high- and medium-intensity interval training show neuroprotective effects. They inhibit activation of the PTEN/NF-κB/NLRP3 pathway, reducing the expression of apoptotic proteins in the hippocampus. Higher intensity training has a more obvious antidepressant effect.

Objective:To prepare the anti-programmed death-ligand 1 (PD-L1) nanoantibody P3C8-C3Fab by ligating with C3Fab and to investigate its role in plasma half-life.Methods:The C3Fab peptide derived from protein G was molecularly fused with the nanobody P3C8 by DNA recombination technology. The nanoantibody P3C8-C3Fab was inducibly expressed and purified in the E. coli BL21 strain, and the binding of it to PD-L1 protein, mouse IgG, and PD-L1-expressing tumor cells was detected by enzyme-linked immunosorbent assay (ELISA). The residual P3C8-C3Fab was detected in mouse serum at different times using double-antibody sandwich ELISA to assess the prolongation of the plasma half-life of PD-L1 nanobodies by C3Fab. Results:The nanoantibody P3C8-C3Fab was successfully constructed, and it could efficiently express itself in soluble form in BL21. The purified NbP3C8-C3Fab protein was obtained with a mass fraction of about 90% at a yield of 7.18 mg/L. The affinity of P3C8-C3Fab for PD-L1 protein and mouse IgG gradually increased with increasing mass concentration and showed a concentration correlation. The binding of P3C8-C3Fab to lung cancer A549 cells showed a concentration correlation. The concentration standard curve of P3C8-C3Fab in mouse serum showed a typical S-shape with a concentration correlation. The plasma half-life of P3C8 was only 0.44 h, while the plasma half-life of P3C8-C3Fab was 21.27-fold higher, up to 9.36 h.Conclusions:The linkage of C3Fab to the nanobodies of P3C8 can significantly prolong the plasma half-life of P3C8, which is valuable for the improvement of in vivo nanobody effects.

Background: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing’s syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Methods: Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected.A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing–Bablok regression and Bland–Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Results: Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r = 0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r = 0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LCMS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8–1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Conclusions Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Objective:To investigate the ability of separate and combined biopsy methods to distinguish clinically significant prostate cancer(csPCa)from clinically insignificant prostate cancer(incsPCa),we assessed diagnostic positive rates for patients undergoing transperineal pro-state systematic biopsy(SB),cognitive fusion targeted biopsy(CF-TB),and combined biopsy(CB)(i.e.SB combined with CF-TB)under intra-venous anesthesia.Methods:We analyzed clinical data from 151 patients with prostate-specific antigen(PSA)≤50 ng/mL undergoing their first prostate biopsy in Cancer Hospital of Huanxing Chaoyang District Beijing and National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College from January 2019 to November 2021.The 3.0 Tesla standard prostate multi-parametric magnetic resonance imaging(mpMRI)examinations found 161 lesions with prostate ima-ging reporting and data system(PI-RADS)scores≥3.With patients under intravenous anesthesia and indwelling catheter,2-4 needle CF-TB biopsies were performed using transperineal ultrasound guidance,followed by 12 needle SB.Patients who underwent SB,CF-TB,and CB were each analyzed by stratification for their respective csPCa and incsPCa detection rates,age,PSA,CF-TB needle count,PI-RADS score,and digital rectal examination results.Results:The median PSA value for all patients was 11.50(0.52-49.37 ng/mL).In total,161 lesions with PI-RADS score≥3 points were found.All 151 patients received 12 needles of SB,while 47,52,and 52 patients received 2,3,and 4 needles of CF-TB,respectively.The respective positivity rates of SB,CF-TB and CB in diagnosing csPCa were 54.3%(82/151),53.0%(80/151)and 58.9%(89/151).Statistical results indicate that the difference in positivity rate between CB and SB is significant(P=0.016)as is the difference between CB and CF-TB positivity rates(P=0.004).The respective positivity rates of SB,CF-TB,and CB in diagnosing incsPCa were 7.9%(12/151)、9.3%(14/151),and 11.3%(17/151).The positivity rate of CB was not significantly different than that of SB or CF-TB(all P>0.05).Stratification plane analysis with age,PSA value,number of CF-TB needles,PI-RADS score,and digital rectal examination results showed that the 2-needle CF-TB scheme was inferior to CB in diagnosing csPCa(P=0.031).There was no significant difference in the csPCa positivity rates of 3-needle and 4-needle CF-TB relative to CB.Conclusions:CB achieves a higher csPCa diagnosis rate without increasing de-tection of incsPCa under transperineal ultrasound guidance.CF-TB with 3-needles per lesion was highly effective in diagnosing csPCa.

OBJECTIVE： To prepare puerarin microemulsion with phase Ⅰ metabolic regulation （R-PR-ME） and to study pharmacokinetic characteristics of rats in vivo. METHODS： R-PR-ME and Puerarin microemulsion without metabolic regulation （NR-PR-ME） were prepared by Shah method. Pseudo-ternary phase diagram was used to optimize microemulsion formula using drug loading amount as index. The particle size and PDI of microemulsion were characterized by using a laser particle size analyzer. Rats were used as animal models， and HPLC method was used to determine the blood concentration of puerarin before and 5， 10， 15， 20， 30， 45， 60， 90， 120， 180， 240， 360， 480， 600 min after intragastric administration of R-PR-ME， NR-PR-ME and puerarin suspension （PR-SP） at puerarin dosage of 120 mg/kg. The pharmacokinetic parameters were calculated by using DAS 2.0 software. SPSS 19.0 software was used for statistical analysis. The relative bioavailability of R-PR-ME was calculated with NR-PR-ME as reference preparation. RESULTS： The formula of R-PR-ME included that oleoyl polyoxyl-6 glyserides （oil phase）-polysorbate 20 （emulsifier）-glycerides （co-emulsifier） mass ratio of 2 ∶ 4 ∶ 4； drug-loading amount of 67.50 mg/g， particle size was （22.59±0.53） nm （n＝3） and PDI was 0.182±0.017 （n＝3）. The formula of NR-PR-ME included that soybean oil （oil phase）-polysorbate 80 （emulsifier）- glycerol （co-emulsifier） mass ratio of 1 ∶ 4.5 ∶ 4.5， drug-loading amount of 61.32 mg/g， particle size of （15.45±1.06） nm（n＝3） and PDI of 0.156±0.012 （n＝3）. Pharmacokinetic parameters of R-PR-ME， NR-PR-ME and PR-SP included that AUC0-600 min were （134.187±37.152）， （65.145±18.762） and （49.623±12.143） μg·min/mL； cmax were （1.316±0.306）， （1.082±0.294） and （0.425±0.106） μg/mL； MRT were （155.068±33.204）， （100.264±27.683）， （60.524±14.086） min； t1/2β were （365.880±101.250）， （283.280±80.940）， （80.063±21.189） min （n＝6）， respectively. Compared with PR-SP， AUC0-600 min， cmax， MRT and t1/2β of R-PR-ME and NR-PR-ME were increased significantly （P＜0.05 or P＜0.01）. Compared with NR-PR-ME， AUC0-600 min， MRT and  t1/2β of R-PR-ME were more higher （P＜0.05）. The relative bioavailability of of R-PR-ME was 205.98％. CONCLUSIONS： R-PR-ME is prepared successfully with high drug-loading amount， and can significantly increase the bioavailability of puerarin in rats， compared with PR-SP and NR-PR-ME.