AIM　Taxane diterpenoid compounds are expected to be isolated from Taxus chinensis cell cultures. METHODS　Cell cultures were extracted with 95% alcohol, distributed by different solvents, isolated via column chromatography on silica gel and purified by crystallization. RESULTS AND CONCLUSION　Two taxane diterpenoid compounds were isolated from Taxus chinensis cell cultures for the first time.Their structures were identified as 14β-(2-methylbutyryl)oxy-2α,5α, 10β-triacetoxytaxa-4(20),11-diene(1) and 5α-(cinnamoyl)oxy-7β,9α,10β,13α-tetraacetoxytaxa-4(20),11-diene(2) respectively.

Under physiological conditions, many vital functions of the body are controlled by transient release of bioactive substances at a specific time and site. Based on the circadian rhythm character of disease and chronotherapeutic conceptions, pulsatile delivery system has been designed to achieve optimal therapeutic effect and reduce the toxic and side-effect. In recent years, more and more studies are focused on the pulsatile multiparticulate drug delivery system. Pulsatile multiparticulate system possesses many benefits, such as no risk of dose dumping, predictable gastric emptying, flexible release patterns and increased bioavailability. Based on these premises, the aim of this review is to summarize the major design methods of pulsatile multiparticulate and the research progress.

Object To study the inhibitory effect and mechanism of extract from cultured cell of Taxus chinensis (Pilg.) Rehd. on hepatocarcinoma cell line SMMC 7721. Methods Trypan blue stain assay was used to determine tumor cell growth curve, flow cytometry and Giemsa staining were used to analyze cell cycle and cell apoptosis. Results Inhibitory effect happened on tumor cell line SMMC 7721, G 2/M stage of tumor cells increased with the addition of T. chinensis cell extract by carrene and apoptosis happened. Conclusion The extracts from cultured cell of T. chinensis have inhibitory effect on SMMC 7721 cells and can further induce cell apoptosis.

Objective:To optimize the overall desirability of orthogonal design sith multi index evaluations. Methods:Four multi index evaluations were applied to optimize the overall desirability of an orthogonal design,and their results wer analyzed. Results:According to the optimum conditions,there was no difference among the four multi index evaluations. Conclusion:multi index test can be used to represent the most desirable variables in experimental design.

To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of epirubicin hydrochloride (EPI) in rat plasma, daunorubicin hydrochloride was used as internal standard. The plasma samples were deproteinated with methanol, and separation was performed on a reversed-phase CAPCELL PAK C18 column (3.0 mm x 50 mm, 3 microm). The mobile phase contained methanol-0.1% formic acid (80:20). Detection was carried out by multiple reaction monitoring on a HP1200-6410 QQQ LC/MS system. Different preparations of EPI solution, EPI-LIP (EPI-liposome) and EPI-LTSL (EPI-thermosensitive liposome) was administered in rats by i.v with the same dosage (12 mg kg(-1)). The pharmacokinetic model and parameters were fitted and calculated by the DAS ver2.0 software. The calibration curve was linear in the range of 0.01-50 microg mL(-1). The limit of quantification was 0.01 microg mL(-1). RSDs of intra- and interbatch precisions were all less than 11.9%. The average extract recovery was 89.3% and 92.1%, respectively. The pharmacokinetics of EPI in rats with all preparations were fitted to three compartments, which all fast distributed and slowly eliminated. The t1/2 alpha, t1/2 beta, t1/2 gamma, AUC(0-infinity), and MRT(0-infinity) of EPI-LTSL group were 7.5, 1.3, 12.6, 12.9, 3.7 times those of EPI solution group; and 1.6, 1.4, 12.3, 2.9, 2.6 times those of EPI-LIP group. Moreover, the CL of the latter two groups was about 13.4 times of the former EPI-LTSL group. EPI-LTSL can significantly improve AUC and prolong the circulation time of EPI in rat plasma.

Objective:To evaluate the in vitro release behavior of doxorubicin(Dox)-loaded microspheres and the stability of Dox during encapsulation process and in vitro release.Methods: Dox-loaded microspheres were prepared by double emulsion(W/O/W) method with poly(lactic-co-glycolic acid)(PLGA) as the carrier material.The physical and chemical characteristics of microspheres,including the mean diameter,morphology,drug entrapment efficiency and loading rate,were evaluated.The in vitro release behavior and its influencing factors were determined by ultraviolet spectrophotometry.Dox stability was evaluated by HPLC method during the encapsulation process and in vitro release.Results: The prepared microspheres had a complete spheric shape and dispersive quality.The mean diameter of the microspheres was 85 ?m;the drug entrapment efficiency was 95.1%;and the loading rate was 14.8%.Releasing rate of the microspheres slowed down with the increase of PLGA concentration and the decrease of W/O value.The encapsulation process had no obvious effect on the stability of Dox,while Dox degraded during in vitro release as the prolongation of time.On day 10,the peak area of degraded material accounted for 2.46%.Conclusion: Dox can be encapsulated in the microspheres by double emulsion method and different release rates of Dox can be achieved by adjusting PLGA concentration and W/O volume ratio.

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.

Pharmaceutical preparations can directly affect the administration methods and therapeutic effects of drugs , which is a priority for the research and development of the military specialized medicament .Foreign armies started pharma-ceutical formulation research very early , and some of their research concepts and strategies are worth learning from .In this paper , dosage forms were used as the classification factor and several formulations with distinct military characteristics were described in detail .The features of military specialized medicament were analyzed from the perspective of pharmaceutics , based on which future development in the formulation of military specialized medicament was predicted .

Despite tre mendous research efforts have been devoted to the analysis of nanoparticles (NPs)biohazard,the potential mechanism for nanotoxicity has not yet been syste mati-cal y elucidated.This review intends to point out the confusions about nanotoxicity in the field and tries to look into the mecha-nism from a new perspective.Currently,there are three puzzles:① no relationship between dose and toxicity could be observed in nanotoxicity;②there is a theory for the″size effects″,however, it cannot explain some cases contrary to the doctrine;③ NPs made of different materials with various sizes could have the same toxic effects through sti mulating oxidative stress.In fact, human body is co mposed of various biological molecules,and the biological function of a living syste m is reflected by the inter-actions and conversions of those molecules.NPs,on the other hand,are the invader of human body which has no ability to transport or convert or digest the foreigner.Thus,NPs could cause celldamage due to the physical blockage of micro-circula-tion,celldestruction due to membrane rando m insertion,and celldysfunction due to physical contacting with big biological mole-cules.The physical damages caused by various NPs could be divided into three categories:adhesion lesion,card inlay and puncture.Above al ,by analyzing wide spectrum of NPs varying in co mposition,shape and size,the author draws a conclusion that physical damage is the origin of nanotoxicity.