ObjectiveTo compare the contents and relative molecular weight distributions of proteins and polypeptides in Naoxuekang dropping pills， Huoxue Tongmai capsules and Maixuekang capsules of Hirudo single medicinal preparations， to evaluate the in vitro anticoagulant， antiplatelet and fibrinolytic activities of the three preparations， and to investigate the effects of temperature， pH and digestive enzymes on the anticoagulant activities of the three preparations. MethodsThe contents of soluble proteins and polypeptides in the three preparations were determined by bicinchoninic acid assay（BCA） and Bradford method， and the relative molecular weight distributions of the three preparations were determined by electrophoresis combined with gel chromatography. The antithrombin activity of the three preparations was evaluated by fibrinogen-thrombin time（Fibg-TT） method， and their anticoagulant activities were further assessed by the elongations of activated partial thromboplastin time（APTT）， prothrombin time（PT） and thrombin time（TT）. The antiplatelet aggregation activities of the three preparations were measured by turbidimetry and the fibrinolytic activities were measured by fibrin plate method. Relative TT was used as index to investigate the effects of temperature， pH and digestive enzyme buffer on anticoagulant activities of the three preparations. ResultsAt the lowest single dosage， the contents of proteins and polypeptides were in the order of Maixuekang capsules>Huoxue Tongmai capsules>Naoxuekang dropping pills. Both Huoxue Tongmai capsules and Maixuekang capsules had 11 electrophoretic bands between 4.0 kDa and 90 kDa， the bands of Maixuekang capsules were more clear in the range of >25 kDa， and there was 1 obvious band at 14 kDa for the two capsules. Huoxue Tongmai capsules had one specific band at 9.0 kDa and Maixuekang capsules had one specific band at 48.0 kDa. Naoxuekang dropping pills only had 2 electrophoretic bands at 6.5 kDa and 8.5 kDa， primarily containing peptides below 2 kDa， most of which were oligopeptides. The anticoagulant activity concentrations of the three preparations exhibited a certain dose-dependent effect. At the lowest single dosage， The anticoagulant activity concentrations were ranked as Naoxuekang dropping pills>Huoxue Tongmai capsules>Maixuekang capsules. The prolongation effect of the three preparations on coagulation time was dose-dependent. At the same concentration， the prolongation effect of Naoxuekang dropping pills and Huoxue Tongmai capsules was APTT prolongation rate>TT prolongation rate>PT prolongation rate， whereas for Maixuekang capsules， the sequence was TT prolongation rate>APTT prolongation rate>PT lengthening rate. At the single minimum dosage， the order of APTT prolongation rate was Maixuekang capsules>Huoxue Tongmai capsules≈Naoxuekang dropping pills， the order of PT prolongation rate was Naoxuekang dropping pills≈Maixuekang capsules>Huoxue Tongmai capsules， and the order of TT prolongation rate was Maixuekang capsules>Huoxue Tongmai capsules>Naoxuekang dropping pills. The three preparations showed dose-dependent effects on platelet aggregation induced by adenosine diphosphate（ADP） and arachidonic acid（AA）， and the effect induced by ADP was stronger than that induced by AA. The anti-platelet aggregation effect of Naoxuekang dropping pills was significantly stronger than that of Maixuekang capsules（P<0.01）， whereas Huoxue Tongmai capsules had the effect of promoting platelet aggregation. None of the three preparations had the ability to dissolve fibrin. The anticoagulant activity of Naoxuekang dropping pills was least affected by heating， while the activities of the two capsules decreased significantly within 5 min above 80 ℃， and continued to decrease within 2 h. Compared with pure water， the anticoagulant activities of the three preparations could be increased by 1-3 times under strong acidity（pH 1-3）. In the pepsin buffer， the anticoagulant activity of Naoxuekang dropping pills could be increased by 1-3 times， while the anticoagulant activities of Huoxue Tongmai capsules and Maxuekang capsules were significantly decreased， the lowest levels were about 60% and 20%， respectively. In trypsin buffer， the anticoagulant activities of Naoxuekang dropping pills， Huoxue Tongmai capsules and Maixuekang capsules decreased significantly， and the lowest levels decreased to about 41%， 41% and 35%， respectively. ConclusionThe contents of proteins and polypeptides and relative molecular weights of the preparations derived from lyophilized fresh Hirudo powder， dried Hirudo powder and reflux extract of Hirudo decrease sequentially， and the anticoagulant activity decrease gradually， but the anticoagulant pathway is different. And the anti-platelet aggregation activity of the reflux extract is significantly enhanced. The heat resistance and gastrointestinal stability of the three preparations increase successively， and the first two are suitable for enteric-soluble preparations， while the latter is suitable for routine oral administration. The above results can provide data reference for the rationality of different preparation methods， active substances， pharmacodynamics and mechanism of Hirudo preparations.

OBJECTIVE To characterize paclitaxel nanoparticles （PTX-PLGA-NPs） and evaluate their in vitro inhibitory effect on Lewis lung cancer cells. METHODS The PTX-PLGA-NPs prepared by the emulsion-solvent evaporation method were characterized in terms of particle size， polydispersity index （PDI）， Zeta potential， microscopic morphology， encapsulation efficiency， drug loading， ultraviolet-visible absorption characteristics and stability. Using mouse Lewis lung cancer cells as the subjects and paclitaxel reference substance as the control， the cytotoxicity and in vitro killing activity of PTX-PLGA-NPs were detected using CCK-8 method and Calcein-AM/PI double staining method， respectively. The effects of PTX-PLGA-NPs on cell apoptosis and cell cycle were assessed by Annexin Ⅴ-FITC/PI staining method and PI staining method， respectively. RESULTS PTX-PLGA-NPs were spherical with an average particle size of （172.03±0.95） nm， PDI of 0.098±0.012， and Zeta potential of （－1.76±0.02） mV. The encapsulation efficiency and drug loading were （52.32±0.66）% and （7.07±0.18）%， respectively， and the ultraviolet-visible absorption characteristics were not affected by the carrier polylactic-co-glycolic acid. When stored in the dark at 4 °C for 7 days， no significant change was noted in particle size， and the average PDI （after 1， 2， 4 and 7 days of storage） was under 0.3. Compared with the paclitaxel reference substance group， the PTX-PLGA-NPs group had more cells in a state of death， the survival rate （at the PTX concentration of 11.2 μg/mL） was significantly decreased， and both the apoptosis rate and the proportion of G2 phase cells were significantly increased （P＜0.05）. CONCLUSIONS The prepared PTX-PLGA-NPs indicate homogeneity in particle size， uniform dispersion， stable properties， and stronger in vitro killing effect on lung cancer cells than PTX.

ObjectiveTo establish a high performance liquid chromatography（HPLC） fingerprint of Yanghetang benchmark sample， and evaluate its quality with chemometric methods， so as to provide a reference for the quality control of this benchmark sample. MethodHPLC was used to establish the fingerprint of Yanghetang benchmark sample with ZORBAX SB-C₁₈ column（4.6 mm×250 mm， 5 μm）， the mobile phase was consisted of acetonitrile（A） -0.05% phosphoric acid aqueous solution （containing 0.05% triethylamine solution）（B） for gradient elution（0-5 min， 2%-3%A； 5-15 min， 3%-5%A； 15-65 min， 5%-30%A； 65-90 min， 30%-70%A）， the flow rate was 1.0 mL·min^-1， the column temperature was 35 ℃， and the detection wavelength was 210， 260 nm. Traditional Chinese Medicine（TCM） Chromatographic Fingerprint Similarity Evaluation System （2012 edition） combined with cluster analysis， principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to evaluate the quality differences between different batches of Yanghetang benchmark samples， and to find the main chemical components responsible for the quality differences. ResultHPLC fingerprint of Yanghetang benchmark sample was established， 13 common peaks were identified and attributed to each common peak， including peaks 2 and 8 from Rehmanniae Radix Praeparata， peaks 10 and 11 from Cinnamomi Cortex， peaks 1， 3-6 from fried Sinapis Semen， peak 13 from Ephedrae Herba， and peaks 7， 9， 12 from Glycyrrhizae Radix et Rhizoma. Eight of them were identified by comparing with control substance， which were 5-hydroxymethylfurfural（peak 2）， sinapine thiocyanate（peak 4）， glycyrrhizin（peak 7）， verbascoside（peak 8）， cinnamic acid（peak 10）， cinnamaldehyde（peak 11）， glycyrrhizic acid（peak 12） and ephedrine hydrochloride（peak 13）. The similarities of the HPLC fingerprints of 15 batches of Yanghetang benchmark samples with the control fingerprint were all greater than 0.80. The three chemometric methods could classify the samples into two categories. Eight differential components were screened out， among which 5-hydroxymethylfurfural， sinapine thiocyanate， verbascoside and ephedrine hydrochloride were identified. ConclusionThe established fingerprint analysis method is accurate， stable and reproducible， which basically reflects the overall chemical composition of Yanghetang benchmark sample， and can provide a basis for establishment of quality standards for compound preparations of this famous classical formula.

OBJECTIVE To design paclitaxel prodrug nanoparticles with dual reactive oxygen species/glutathione response (ProPTX-SS-NPs), providing new ideas and methods for the application of paclitaxel. METHODS The optimal preparation method and process of prodrug nanoparticles was investigated by using particle size and PDI as indicators; the morphology of prodrug nanoparticles was observed through electron microscopy and their particle size, potential, encapsulation efficiency, drug loading capacity, etc were investigated; the in vitro release characteristics of nanoparticles in reactive oxygen species and glutathione environments were investigated; the in vitro cytotoxicity and cellular uptake of prodrug nanoparticles were investigated through cell experiments. RESULTS The nano particle size prepared by the optimal process was (130.20±2.18)nm, with the PDI of 0.12±0.01, the Zeta potential of (-8.45±0.01)mV, the drug load of (10.27±1.36)%, and the encapsulation rate of (93.22±2.20)%. The prodrug nanoparticles showed reactive oxygen species and glutathione dual responsive release ability, and could significantly inhibit the proliferation of MCF-7, HepG2, and MDA-MB-231. Its inhibitory effect on MDA-MB-231 cells was the most significant. The IC50 of prodrug nanoparticles on MDA-MB-231 cells was (0.71±0.11)μmol·L, while the IC50of PTX was (22.38±3.27)μmol·L. CONCLUSION ProPTX-SS-NPs have excellent tumor microenvironment response performance and significant anti-tumor activity, which is a highly potential and promising anti-tumor nanosystem.

AIM: To study the inhibitory effect of Xihuang Pill on H

Objective To identify the content of tubeimoside I in Paniculate Bolbostemma from 16 differ-ent locations and determine the high-containing locations and influencing factors.Methods The quanti-tative determination method of tobeimoside I in Tubeimu(Paniculate Bolbostemma, Rhizoma Bolbostem-atis)in Chinese Pharmacopoeia(2015 Edition)was used to determine the content of tobeimoside I in 16 batches of Tubeimu for deciding producing areas with higher content of Tubeimu and analyzing the influ-encing factors.The origin producing areas was classified by using group-average clustering analysis meth-od taken content of tobeimoside I as the index.Results The results showed that the content of tobeimo-side I was in a range from 1.234%to 3.174%in different producing areas,which was accorded with the requirement of Chinese Pharmacopoeia.The clustering analysis classified 16 producing areas into 4 groups,and content of tobeimoside I was the highest in Tubeimu material produced from Shangluo and Hanzhong.Conclusion The temperature and humidity had influence on Tubeimu material, and content of tobeimoside I was higher in the producing areas with higher temperature and humidity.

This study was aimed to search anti-platelet aggregation effectors from Gardenia jasminoides extract with the employment of platelet affinity extraction method coupled with HPLC, in order to provide pharmacological experi-mental evidences of the selected effectors to verify its feasibility. Under physiological conditions, washed rat platelets were added into G. jasminoides extract and then a mixture was gained. Consequently, some components from G. jas-minoides extract were combined to the platelets in the mixture while some were not owing to their special chemical structures and properties. Firstly, the uncombined components were washed off from the mixture. Secondly, the com-bined components in the leftover was washed down and collected, respectively, right after destroying the occupied platelets' structures. Thirdly, different collected eluents were analyzed, respectively, by HPLC established in the pre-vious work to search the effectors. Fourthly, pharmacological experiments were implemented for confirmation. The re-sults showed that dominant effective components from G. jasminoides extract acting on anti-platelet aggregation were identified as geniposide. Further evident was provided as well by pharmacological experiment that geniposide exhibit-ed significant inhibitory effect on anti-platelet aggregation in rats induced by ADP, rat tail collagen and thrombin(P< 0.01). It was concluded that the platelet affinity extraction-HPLC method proposed in this paper can be utilized to analyze the correlation of effectors from G. jasminoides extract and its pharmacological effects. Moreover, there are some correlations between screened chemical substances and their pharmacological effects.

Objective To establish a high purity primary culture methods of human nasal epithelial cells (HNEC) in vitro and to provide a successful primary culture model for evaluation experiments of the nasal preparation .Methods Primary culture of human nasal epithelial cells were performed with enzymatic dissociation of isolated tissue and cultured in serum-free medium .HNEC were separated through magnetic field by immunomagnetic beads .We determined the purity of the separated cells by light microscopy and flow cytometry .The morphology of HNEC was observed with a scanning electron microscope .Results Under an inverted phase microscope ,the cells morphology was paving stone shaped .Under the scanning electron microscopy ,abundant microvilli and cilia differentiation were observed .Flow cytometry showed the epithelial cells accounted for 99% .Conclusion The highly purified HNEC can be directly isolated by the magnetic cell sorting system .The cell model can be used for the basic research of nasal cavity preparation .

This study was aimed to develop an ICP-MS method to determine contents of six types of heavy metals, which include Cr, Pb, Cd, As, Hg and Cu, in Pian-Zai-Huang Gan-Bao (PZHGB) tablets. The samples were pro-cessed by microwave digestion. And then, they were determined by ICP-MS. The results showed that the working curves of Cr, Cu, As, Cd, Hg and Pb had good linear relationships with the correlation coefficient which was more than 0.9993. The average recovery rate of Cr was 101.2%. The relative standard deviation (RSD) was 7.6%. The av-erage recovery rate of Cu was 96.4%, with the RSD of 3.5%. The average recovery rate of As was 101.3%, with the RSD of 1.8%. The average recovery rate of Cd was 100.2%, with the RSD of 0.3%. The average recovery rate of Hg was 100.5%, with the RSD of 4.2%. The average recovery rate of Pb was 104.2%, with the RSD of 5.6%. It was concluded that the method was stable and reliable, with high sensitivity and repeatability. This method can be used in the content determination of six types of heavy metals, which include Cr, Pb, Cd, As, Hg and Cu in PZHGB tablets.

Objective To optimize the matrix formulation of the extract of Chuanxiong Rhizoma cataplasm. Methods The optimal proportion of matrix formulation was selected by uniform design and response surface method. The primary adhesion force, lasting adhesion force, peel strength and residuals on the anti-sticking paper were used as test parameters. The optimal ratios of NP-700, glycinate aluminum, glycerin, tartaric acid, the concentration of drug, and the volume of drug were acquired. Results The matrix formulation of the cataplasm was made with NP-700∶glycinate aluminum∶glycerin∶tartaric acid∶concentration of drug∶volume of drug=1.1∶0.08∶7∶0.04∶4∶5. Conclusion Uniform design combined with response surface method can be used for the optimization of the matrix formulation of cataplasm. The optimal matrix prescription has higher drug-loading, with good spreadability, moisture-retention capability and adhesive force.