OBJECTIVE:To establish a method for the simultaneous determination of chlorogenic acid,vitexin glucoside,vi-texin rhamnoside,vitexin,rutin and hyperoside in Crataegus pinnatifida. METHODS:With reference peak of vitexin glucoside, HPLC was conducted to calculate the relative correction factor(RCF)of chlorogenic acid,vitexin glucoside,vitexin rhamnoside, vitexin,rutin and hyperoside,then the contents of above-mentioned 5 components in C. pinnatifida were calculated. The column was Agilent ZORBAX SB C18 with mobile phase of 0.1% formic acid-acetonitrile-tetrahydrofuran (gradient elution) at a flow rate of 1.0 ml/min,the detection wavelength was 350 nm,column temperature was 30 ℃,and the injection volume was 10 μl. RE-SULTS:The linear range was 12.50-400.0 μg for chlorogenic acid(r=0.999 8),25.00-800.0 μg for vitexin glucoside(r=0.999 9), 31.25-1 000.0 μg for vitexin rhamnoside(r=0.999 9),6.470-260.0 μg for vitexin(r=0.999 9),2.50-80.0 μg for rutin(r=0.999 8) and 9.375-300.0 μg for hyperoside(r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 2.0%;re-coveries were 99.2%-103.9%(RSD=1.6%,n=6),97.9%-100.8%(RSD=1.2%,n=6),99.2%-100.8%(RSD=0.5%,n=6), 97.3%-101.3%(RSD=1.5%,n=6),98.0%-103.0%(RSD=1.9%,n=6)and 95.5%-101.5%(RSD=2.2%,n=6). RCFs of vitex-in glucoside with chlorogenic acid,vitexin rhamnoside,vitexin,rutin and hyperoside were 1.119,1.009,0.706,1.063 and 0.830, respectively. CONCLUSIONS:The method is simple with good precision,stability and reproducibility,and it can be sued for the simultaneous determination of 6 components in C. pinnatifida.

Objective To prepare the Env-pseudotyped subtype B HIV-1 with enhanced green fluorescent protein ( EG-FP) gene,explore HIV-1 infection mechanisms and develop feasible methods of identification .Methods The Env-pseudo-typed viruses were packaged in HEK293T cells by cotransfection, and the reporter gene and P24 protein were detected by PCR, Western blot and ELISA .Reporter gene amplification , viral titration assay and a single round of infection assay were performed after the env-pseudotyped viruses infected HIV-1 permissive cell .Results and Conclusion A generation and identification method of the pseudotyped HIV-1 was established . The Env-pseudotyped subtype B HIV-1 has been prepared, which is able to infect SupT1 and TZM-bl cells through infection assay .

Objective:To explore the effect of hydrogen sulfide (H 2S) on modulating the subunit Kir6.2 of adenosine triphosphate sensitive potassium channels via the cyclic guanosine monophosphate-dependent protein kinase (cGMP/PKG) signaling pathway in epileptic rat models. Methods:Sixty adult male SD rats were randomly divided into the following six groups (10 rats in each group) by random number table method: control, epileptic, H 2S donor, H 2S donor+epileptic, KT5823 (one inhibitor of the cyclic guanosine monophosphate-dependent protein kinase)+H 2S donor+epileptic, and glibenclamide (one inhibitor of the adenosine triphosphate sensitive potassium channels)+H 2S donor+epileptic groups. Except the control group, SD rats were intraperitoneally injected with plentylenetetrazole to make the kindling models and their behaviours were recorded including the latency period, the grade, and the duration of the first epileptic seizure according to the Racine′s standard. The waveforms of electroencephalogram (EEG) in hippocampus were also recorded during the seizure. The mRNA and protein levels of PKG and Kir6.2 in hippocampus were evaluated by Western blotting and quantitative real-time polymerase chain reaction, and the hippocampal concentrations of cGMP and phosphorylation of cyclic guanosine monophosphate-dependent protein kinase (p-PKG) were detected by enzyme linked immunosorbent assay. Results:Rats in the epileptic group showed Ⅳ-Ⅴ grade of epileptic seizure [4.500 (4.000, 4.875)], short latency period [(10.37±8.21) min] but long duration [(69.50±24.37) s] of seizure. Compared to the epileptic group, rats in the H 2S donor group showed Ⅱ-Ⅲ grade of epileptic seizure ( P=0.004), significantly longer latency period ( P<0.001), and shorter duration of seizure ( P<0.001). Compared to the H 2S donor+epileptic group, rats in the KT5823+H 2S donor+epileptic group showed Ⅲ-Ⅳ grade of epileptic seizures, significantly shorter latency period ( P<0.001), and longer duration of seizure ( P<0.001). The results of EEG showed that the wave patterns in the epileptic group were spike or sharp waves and the amplitudes were largest [(190.570±23.590) μV]. Compared with the epileptic group, amplitudes were reduced ( P<0.001) in the H 2S donor+epileptic group. PKG mRNA and PKG protein were expressed differently among all groups (PKG mRNA: n=5, H=26.714, P<0.001; PKG protein: n=5, F=30.597, P<0.001). Compared with the control group, the expression of both PKG mRNA and PKG protein was decreased (PKG mRNA: 1.000±0.001 vs 0.782±0.064, P=0.023; PKG protein: 0.550±0.037 vs 0.145±0.020, P=0.042) in the epileptic group. Besides, Kir6.2 mRNA and Kir6.2 protein were expressed differently among all groups (Kir6.2 mRNA: n=5, H=27.761, P<0.001; Kir6.2 protein: n=5, F=60.659, P<0.001). Compared with the control group, the expression of both Kir6.2 mRNA and Kir6.2 protein was decreased (Kir6.2 mRNA: 1.000±0.001 vs 0.897±0.033, P=0.004; Kir6.2 protein: 0.384±0.035 vs 0.215±0.016, P=0.024) in the epileptic group. And the concentrations of cGMP and p-PKG were decreased (cGMP: P<0.001; p-PKG: P<0.001) in the epileptic group. The results in the H 2S donor+epileptic group were up-regulated (PKG mRNA: P=0.047; PKG protein: P<0.001; Kir6.2 mRNA: P=0.011; Kir6.2 protein: P<0.001; cGMP: P<0.001; p-PKG: P<0.001) compared with the epileptic group. However, the results in the KT5823+H 2S donor+epileptic group were down-regulated (PKG mRNA: P=0.015; PKG protein: P=0.027; Kir6.2 mRNA: P=0.013; Kir6.2 protein: P=0.017; cGMP: P=0.005; p-PKG: P<0.001) compared with the H 2S donor+epileptic group. Conclusion:A possible mechanism is that H 2S prevents the epileptic seizure from modulating the subunit Kir6.2 of ATP sensitive potassium channels via the cGMP/PKG signaling pathway.

Objective To investigate the association and mechanism between glutathione S-transferase P1(GSTP1) and radiation-induced lung injury.Methods Two effective GSTP1 siRNAs were designed and synthesized.The normal lung epithelial cell line BEAS-2B cells were transfected with GSTP1 siRNA (experimental group,siRNA-1,siRNA-2) and negative control siRNA (negative control group,NC).Western blot was performed to detect the expression levels of GSTP1 protein and EMT-related proteins.CDNB was adopted to evaluate the activity of GSTs.DCFH-DA probe was used for incubation.Flow cytometry was conducted to detect the median fluorescence intensity (MFI) and cellular apoptosis.Annexin-v/PI staining was utilized for incubation.MTT assay was performed to measure the proliferation of BEAS-2B,and the growth curve was drawn based on the results.Results After radiation,compared with the NC group,the ROS level and MFI were significantly higher in experimental group (6774.66±399.60 vs.8759.00±256.96 vs.9967.67±735.11,P＜0.05).In the experimental group,the percentage of cellular apoptosis was remarkably higher than that in the NC group (12.3± 1.16 vs.17.38± 1.65 vs.22.88± 1.20,P＜0.05).MTT assay demonstrated that the OD values in the experimental group were significantly lower than that in the NC group everyday.Further more,the level of EMT process is higher in the experimental group.Conclusions Interfering with the GSTP1 expression in lung epithelial cells can increase the intracellular ROS level,increase the percentage of cellular apoptosis,and reduce the cell proliferation rate following γ-radiation.Besides,it can also promote the epithelial mesenchymal transition in lung epithelial cells.The down-regulation of GSTP1 protein expression level probably aggravates the radiationinduced lung cell injury and promotes the epithelial mesenchymal transition.

OBJECTIVE To determi ne the contents of total fla vonoids i n Scutellaria barbata standard decoction ，evaluate in vitro antioxidant activity ，establish the fingerprint and conduct chemical pattern recognition analysis. METHODS The contents of total flavonoids in S. barbata standard decoction （calculated by scutellarein ）were determined by ultraviet-visible spectrophotometry. In vitro antioxidant activity of S. barbata standard decoction was investigated by free radical scavenging tests of 1，1-diphenyl- 2-trinitrophenylhydrazine（DPPH）and 2，2′-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ）ammonium salt （ABTS）；HPLC method was adopted. Using scutellarin as reference ，the fingerprints of 16 batches of S. barbata standard decoction were drawn and evaluated by Similarity Evaluation System of TCM Chromatogram Fingerprint （2004 A edition ），and the common peaks were determined；Pearson correlation analysis was carried out by using SPSS 24.0 software to screen substances with in vitro antioxidant activity. Taking them as variables ，cluster analysis and principal component analysis were carried out by using SPSS 24.0 and SIMCA 14.1 software. RESULTS The linear range of total flavonoids were 2.106-21.06 μg/mL（R2＝0.999 3）；RSDs of precision ， reproducibility and stability tests （120 min）were all lower than 2％；the recovery was 100.62％（RSD＝0.55％，n＝6）；the contents of total flavonoids were 0.634-1.053 mg/mL. Median inhibitory concentration （IC50） of DPPH radical scavenging experiment ranged 1.120-3.602 mg/mL，and IC 50 of ABTS radical scavenging e xperiment range d 0.684-1.327 mg/mL. The results of correlation analysis showed that the content of total flavonoids Δ 基金项目 ：河北省高校省级重点学科建设项目 （No.冀教 in S. barbata standard decoction was negatively correlated 高〔2013〕4号）；承德医学院自然科学研究计划项目（No.201824） ＊讲师，硕士。研究方向：中药质量控制 。电话：0314-2291186。 with the IC 50 of DPPH free radical and ABTS free radical E-mail：duyilongww@sina.com scavenging experiment ，and the correlation coefficients were # 通信作者 ：教授，硕士。研究方向 ：中药质量控制 。电话： －0.976 and －0.940 respectively（P＜0.01）. There were 18 0314-2291186。E-mail：phf2301@163.com common peaks in the fin gerprints of 16 batches of S. barbata 中国药房 2022年第33卷第4期 China Pharmacy 2022Vol. 33 No. 4 ·425· standard decoction ；the s imilarities were 0.964-0.997. A total of 4 common peaks were identified ，such as scutellarin （peak 8）， scutellarein（peak 14），luteolin（peak 15），apigenin（peak 17）.In the HPLC fingerprints of S. barbata standard decoction ，the peak areas of peak 3-4，8-9，12-15 and 17 were significantly negatively correlated with the IC 50 of DPPH free radical and ABTS free radical scavenging experiment （P＜0.05）. The results of cluster analysis showed that 16 batches of S. barbata standard decoction could be clustered into two categories ，of which S 2，S7-S8 and S 14-S16 were clustered into one category ，S1，S3-S6 and S 9-S13 were clustered into one category. By principal component analysis ，16 batches of S. barbata standard decoction were divided into two categories ，of which S 2，S4，S7 and S 14-S16 were clustered into one category ，and S 1，S3，S5-S6 and S 8-S13 were clustered into one. The comprehensive scores were high in the samples of S 4，S13，S15. CONCLUSIONS Established HPLC fingerprint and chemical pattern recognition analysis method can be used to evaluate the quality of S. barbata standard decoction ； peak 3-4，8-9，12-15 and 17 and total flavonoids are the potential material basis for S. barbata standard decoction to scavenge DPPH free radical and ABTS free radical.

OBJE CTIVE To establish a method for quality evaluation of Xin ’an capsule by combining fingerprint ， multi-component quantitative analysis and chemical pattern recognition analysis. METHODS High performance liquid chromatography（HPLC）combined with Similarity Evaluation System of TCM Chromatogram Fingerprint （2012 edition）were used to establish the fingerprints of 24 batches of Xin ’an capsules and evaluate the similarity. The common peaks were determined. The contents of glucosylvitexin ，rhamnosylvitexin，vitexin，hyperoside and isoquercetin in Xin ’an capsules were determined by the same HPLC method. Taking the common peak area of fingerprint as the variable ，MetaboAnalyst 5.0 tool was used to draw the cluster analysis （CA）heat map. SIMCA 14.1 software was used to perform principle component analysis （PCA）and partial least squares-discriminant analysis （PLS-DA）. RESULTS Twelve common peaks were identified with the similarity greater than 0.97. Six common peaks were identified as chlorogenic acid ，glucosylvitexin，rhamnosylvitexin，vitexin，hyperoside and isoquercetin.The linear range of glucosylvitexin ，rhamnosylvitexin，vitexin， hyperoside and isoquercetin were 2.36-151.35，9.15-585.20， 1.20-76.50， 0.68-43.20， 0.44-27.90 µg/mL（all r＞0.999）.RSDs of precision ，repeatability and stability （24 h）tests were 163.com all less than 2.00％ . The average recoveries were 95.80％（RSD＝0.96％ ，n＝6），102.10％ （RSD＝0.93％ ，n＝6）， 103.26％（RSD＝1.28％，n＝6），103.89％（RSD＝0.73％，n＝6） and 102.09％（RSD＝1.79％，n＝6），respectively. The contents of the five components were 0.988 8-1.559 1，4.336 6-11.220 1， 0.065 1-0.830 5，0.043 8-0.692 5 and 0.023 2-0.427 2 mg/grain，respectively. The results of CA and PCA showed that 24 batches of samples could be divided into three categories ，i.e. S 1-S15，S16-S18 and S 19-S24. PLS-DA showed that variable importance in projection values of the corresponding component of peak 6 and glucosylvitexin （peak 7），rhamnosylvitexin（peak 8），hyperoside （peak 10） and isoquercetin （peak 11） were greater than 1. CONCLUSIONS The established HPLC fingerprint and multi-component quantitative method are simple and feasible. Combined with chemical pattern recognition analysis ，it can be used for the quality control of Xin ’an capsules. Glucosylvitexin ，rhamnosylvitexin and other components may be differentital markers affecting the quality of each batch of samples.

OBJECTIVE To establish the fingerprint and multi-component content determination method of Crataegus pinnatifida leaves from different producing areas， and to evaluate the quality of C. pinnatifida leaves and screen the differential markers. METHODS Seventy-eight batches of C. pinnatifida leaves were collected from Chengde of Hebei Province， Huludao of Liaoning Province， Yuncheng of Shanxi Province and Linyi of Shandong Province. High-performance liquid chromatography （HPLC） and Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints （2012 edition） were used to draw the fingerprints and conduct similarity evaluation. Grey correlation analysis， cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were performed by using SPSS 19.0， MetaboAnalyst 5.0 and SIMCA 14.1 software. The differential markers affecting the quality of C. pinnatifida leaves were screened with variable importance in the projection （VIP） value greater than 1 and the error line not exceeding the origin as the criterion. Using vitexin rhamnoside as an internal reference， the contents of chlorogenic acid， glucosylvitexin， hypericin and isoquercetin in 78 batches of C. pinnatifida leaves were determined by the same HPLC combined with quantitative analysis of multi- components by single-marker （QAMS）， and the results were compared with external standard method. RESULTS Eight common peaks were calibrated in the fingerprints for 78 batches of C. pinnatifida leaves from 4 producing areas. Five known components were identified， including chlorogenic acid （peak 1）， glucosylvitexin （peak 3）， vitexin rhamnoside （peak 4）， hypericin （peak 7） and isoquercetin （peak 8）； their similarities ranged from 0.871 to 0.998. Average relative correlations of samples from Chengde of Hebei Province， Huludao of Liaoning Province， Yuncheng of Shanxi Province and Linyi of Shandong Province were 0.538， 0.528， 0.462 and 0.435， respectively. CA and PCA showed that the samples from Chengde of Hebei Province and Huludao of Liaoning Province were roughly classified into one category， while the samples from Linyi of Shandong Province and Yuncheng of Shanxi Province were roughly classified into one category； VIP values of peak 1， 2， 3 and 5 were all greater than 1. By QAMS， the relative correction factors of chlorogenic acid， glucosylvitexin， hypericin and isoquercetin were 0.401， 0.993， 1.670 and 1.615 （RSD＜2%）. Compared with external standard method， except for isoquercetin in the two batches of samples （S39 and S41）， there was no significant difference in the content of each component in other batches of samples （the relative deviations≤ 5%）. CONCLUSIONS The established fingerprint and QAMS method are simple to operate and can be used to evaluate the quality of C. pinnatifida leaves. The sample from Chengde of Hebei Province is relatively good in quality. Chlorogenic acid （peak 1）， glucosylvitexin （peak 3）， and the corresponding components of peaks 2 and 5 may be differential markers affecting the quality of C. pinnatifida leaves.