Safety,efficacy and quality control are the three essential elements for drugs,including traditional Chinese medicine(TCM). Currently,rapid development of life sciences,medicine and phar?macy provides new prospects for TCM. Considering the long history of TCM application,modern phar? macology and toxicology, global standards and expectations on the safety of TCM, how to evaluate the safety of TCM scientifically has become a key to the research and development of TCM. This review aims to discuss current technical requirements of non-clinical safety study of TCM as well as the challenges.

Objective To evaluate the role of Restriction fragment length polymorphism (RFLP) analysis in detection of the fragment of GEF1α/a gene which are both located at ct and a mating type loci in identification of species, varieties, genotypes and mating types of Cryptococcus neoformans species complex(Cryptococcus neoformans and Cryptococcus gattii). Methods The GEF1α/a gene was selected from 20 genes which both located at α and a mating type loci for RFLP analysis, according to the requirements of sequence similarities and primer design in PCR-RFLP analysis. Primer pair was designed from the conserved regions of GEF1α/a genes of distinct genotypes and mating types of reference strains to amplify a fragment of GEF1α/a gene from Cryptococcus neoformans and Cryptococcns gattii strains tested. Sequence alignment,restriction maps analysis, endonucleases selection and electrophoresis stimulation were conducted by using DNAMAN and Vector NTI software. EeoT14 Ⅰ and Hap Ⅱ endonucleases were selected for RFLP analysis of the GEF1α/a fragments amplified from 125 isolates of Cryptococcns neoformans and Cryptococcus gattii. Results An approximate 1 300 bp fragment was amplified from total 82 Cryptococcus neoformans and 43 Cryptoceccus gattii isolates. However, negative PCR results were found in the reference strains of Cryptococcus laurentii, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krnsei,Candida glabrata, Trichosporon asahii, Aspergillus fumigatns and Aspergillus flavus. RFLP analysis successfully identified the species, varieties, genotypes and mating types of total 125 isolates of Cryptococcus neoformans and Cryptococcns gattii tested in this study. Condusion PCR-RFLP analysis of the GEF1α/a fragment has the potential value in identification of species, varieties, genotypes and mating types of Cryptococeus neoformans species complex simultaneously and rapidly, and may be a useful tool in molecular epidemiological analysis.

Objective To study the effects of Qingjinkangkuoyin (QJKKY) on TNF-α and NE in rats with bronchiectasis.Methods Models were established by intrabronchially injecting with pseudomonas aeruginosa,and divided into 5 groups by random:the QJKKY high dose treatment group (given high dose of QJKKY into stomach),the QJKKY low dose treatment group (given low dose of QJKKY),the levofloxacin group (given levofloxacin),the model group (given normal saline),and the normal contrast group (given normal saline).After 2 weeks of treatment,the histopathology of lung tissue,the levels of TNF-α and inflammatory cells in peripheral blood and NE in rats' lung tissue were detected.Results Compared with the model group (160.425±9.9293)ng/L,QJKKY could decrease the level of TNF-α in blood significantly [high dose of QJKKY treatment group was (137.133±6.1646)ng/L,P＜0.05]; the expression of inflammatory cells in serum were decreased significantly by QJKKY [high dose of QJKKY treatment group was (1.106± 0.3580) 109/L,P＜0.05].Low dose of QJKKY treatment group was (1.086 ±0.2433) 109/L,(P＜0.05) ; the expression of NE in lung tissue were decreased remarkably by QJKKY [high dose of QJKKY treatment group(80.697 ±4.5877)ng/L,P＜0.05]; low dose of QJKKY treatment group is (80.747±3.6925)ng/L,(P＜0.05); and the histopathologic change of lung tissue in QJKKY treatment groups were ameliorated under light microscope by HE staining.Conclusion Qingjinkangkuoyin could cure bronchiectasis by decreasing the expression of TNF-αin peripheral blood and NE in rats' lung tissue.

Objective To investigate the role of prokinetic agent itopride in colonic preparation before colonoscopy examination in patients with constipation.Methods A total of 115 outpatients with history of chronic constipation who requested colonoscopy were collected.According to colonic preparation proposal,patients were divided into three groups.Group A (39 cases) took standard dosage of PEG-E solution six hours before colonoscopy examination.Group B (38 cases) took 150 mg itopride 30 minutes before administration of lavage solution.Group C (38 cases) took itopride 150 mg at 7 am,12 am and 8 pm the day before the examination and on the examination day took the same medicine as that of group B.The blood pressure,heart rate and blood electrolytes were monitored before and after taking medicine in the patients of three groups.Quality of colon cleansing of each group was observed and side effects were also observed.One-way analysis of variance (least significant difference,LSD) test was performed for pairwise comparisons among the three groups.Chi-square test was applied for count data.Results Both group A and group B excluded one patient because of malignant carcinoma with colon stricture under colonoscopy,and 113 patients completed the whole colon examination.There was no significant difference in the baseline patients' data of three groups.The colon cleaning score of group C (7.28±1.11) was higher than those of group A and B (6.55±1.18 and 6.51±1.16,LSD test,both P＜0.05).The frequency of bowel movements defecation of group C (8.31± 1.32) was more than those of group A and group B (7.11± 1.41 and 6.94± 1.51,LSD,test,both P＜0.05).There was no significant difference in terms of intestinal bubble scores,blood pressure,heart rate,blood electrolytes the uncomfortable degree of colonic preparation and rate of side effects of the three groups.Conclusion The colonic preparation can be safely and effectively improved by taking high dose of itopride one day before and on the day of colonoscopy examination.

Objective To explore clinical observation of the effect of severe blepharoptosis correction with modified frontalis muscle suspension.Methods Thirty three cases (41 eyes) with congenital severe blepharoptosis were treated with modified frontalis muscle suspension,and the operative effect was analyzed retrospectively.Double eyelid incision and concealauxiliary incision on partial-bitamporal of the superciliary arch were adopted.After taking the frontal muscle flap crossed through the subcutaneous tunnel between two incisions and fixed on the superior tarsus.Closed palpebral fissure with suture method after adjustment was satisfied.Results At 1 ～ 24 (9.76 ± 5.15) months post-operatively,all incisions of 33 cases were primary healing,eyelid radian satisfaction and no corneal exposure complication occurrence.The early postoperative reaction was mild,while only 1 case discovered subcu-taneoushematoma in superciliary arch.The satisfactory corrections were 30 cases,which undercorrections were 2 cases and 1 case was over correction.No palpebral and exposed keratotitislong-tern complication was found.Conclusions The operation of modified frontalis muscle suspension is satisfactory,safe and effective with little complications and less injury in intraoperative.

Objective To identify the AD hybrid strains and its hybrid types within Cryptococcus neoformans.Methods Difierent hybrid types of AD strains were analyzed by PCR 0f STE20 and MF genes within MAT locus and CIA4 and GPal genes out of MAT locus.The PCR-RFLP analysis of g6341 gene was also performed.Results The mating types of 18 AD strains were precisely identified by PCR of STE20 gene,whereas those of H strain were not identified.CL44 gene was better than the GPal gene in PCR identification of the AD hybrids.In the RFLP analysis of g6341 gene,AD strains were grouped into 2 distinct RFLP patterns based on the mating type on serotype A allele.The mating types of AD strains were not identified by the molecular analyses based on the CL44,GPal and g6341 genes.Conclusion It is necessary to use multi-locus analyses of genes within and out of the MAT locus in precise identification of the AD strains and their hybrid types of Cryptococcus neoformans.

Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands.

Objective To investigate the molecular epidemiology of clinical cryptococcal isolates from China by analyzing the constituents and distributions of the varieties,genotypes and mating types (MAT)of them.Methods (1)PCR fingerprinting and PCR amplification were performed by using the minisatellite-specific core sequence of wild-type phage M13 as single primer.Genotypes of the 110 cryptococcal isolates from China were assigned by comparison with the reference strains of the 8 major molecular types loaded on gel.(2)Identification of the varieties and mating types was carried out by PCR using the specific primers of the varieties and mating types.Results Of the 110 clinical cryptococcal isolates,strains of Cryptococcus neoformans var.grubii with genetype VNⅠ and mating type MATα were the most representative ones(89.1%)followed by strains of C.neoformans var.gattii(8.2%)including isolates of genotype VG I,mating type MATα(7.3%)and genotype VGⅡ,mating type MATα(0.9%);AD hybrids with the genotype VNⅢ,mating type MAT-/α and genotype VN Ⅲ,mating type MATα/-(1.8%);and isolate of C.neoformans var.neoformans with the genotype VNⅣ and mating type MATa(0.9%).Conclusion Of the clinical isolates from China,all three varieties and AD hybrids are found.The vast majority(>99%) of strains possess the α allele in MAT locus and most of them are C.neoformans vat.grubii with the genotype VN I,which accord with the data of most studies of clinical molecular epidemiology in other geographic areas.However.no genotype of VNⅡ.VGⅢ and VGⅣ isolates are found in this study.

Objective To analyze the composition and distribution of pathogens from 1366 superficial candidiasis cases in Shanghai.Methods Candida species identification was carried out for 1366 adults or children with superficial candidiasis by using CHROMagar Candida plates,API20C AUX system,etc.Pal's agar,Xylose assimilation and the test for growth at 45 ℃ were utilized to differentiate Candida dubliniensis.Newly identified pathogenic Candida species including Candida orthopsilosis,Candida metapsilosis,Candida fermentati,Candida nivariensis and Candida bracarensis were differentiated by molecular biological methods.Finally,the composition and distribution of pathogens in superficial candidiasis cases were statistically analyzed.Results A total of 1366 Candida strains,included 2 Candida orthopsilosis strains and 4 Candida metapsilosis strains,were isolated from these cases.Among these isolates,Candida albicans predominated(79.0％),followed by Candida parapsilosis(9.5％),Candida tropicalis(2.9％)and Candida guilliermondii(1.9％).The composition of Candida species was significantly different between child and adult patients(x2 =196.46,P ＜ 0.01),with the isolation rate of non-albicans Candida species being 14.4％ and 45.8％ respectively in child and adult patients.Conclusions Candida albicans is still the dominant pathogen of superficial candidiasis.Candida orthopsilosis and Candida metapsilosis can cause superficial candidiasis.The isolation rote of non-albicans Candida species is higher in adult patients than in child patients.

Objective To establish a multiplex PCR targeting the intergenic spacer regions (IGS) for the identification of Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii.Methods Primers were designed by using the software ClustalW2 and Oligo 6 based on the sequence of IGS1 region,which shows high sequence variability in the genome of Cryptococcus neoformans and Cryptococcus gattii.,for the multiplex PCR.Then,the developed multiplex PCR was performed to identify 51 Cryptococcus neoformans strains representing genotypes VNI-VNIV and VNB as well as 41 Cryptococcus gattii strains representing genotypes VGI-VGIV.The identification results were compared with those from common PCR by using primers GPA1A,CLA4D and SODlgattii specific to Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii,respectively,as well as with those from the canavanine-glycine-bromothymol blue (CGB) medium-based culture.Results The developed multiplex PCR successfully identified the 92 Cryptococcus neoformans and Cryptococcus gattii.strains,and yielded negative results from the other tested pathogenic yeasts,which revealed a high specificity of the designed primers.False positive results were observed in the identification of two Cryptococcus gattii strains with GPA1A primer-based PCR,one Cryptococcus gattii strain with CLA4D primer-based PCR,one var.grubii strain and one var.neoformans strain with CGB culture,while no false negative results were observed in the detection of these Cryptococcus strains by any of these methods.Conclusions The developed multiplex PCR in this study can rapidly and accurately identify Cryptococcus neoformans var.grubii,var.neoformans,AD hybrid,and Cryptococcus gattii,with superior performance in comparison with common PCR and CGB medium-based culture.