The scavenging activities of Ophiopogon japonicus for superoxide anion (O_2~(·-)) radicals by HPLC-DAD coupled with chemiluminescence(CL) was studied and the active constituents in it were screened.The aqueous extracts of Ophiopogon japonicus were divided into the ethyl ether fraction and the n-butanol fraction.Their activities were investigated respectively and the reduction of CL with the pyrogallic acid 3-amino-phthalhydrazid (luminol) Na_2CO_3/NaHCO_3 buffer (pH 11.0) system worked as an activity index of the antioxidant constituents.Major active components in Ophiopogon japonicus could be identified by HPLC-DAD-MS/MS and the reference substances.The antioxidant activities of different ingredients were evaluated by standard potency equations using quercetin as positive control.The experimental data indicated that the scavenging activities of Ophiopogon japonicus for superoxide anion (O_2~(·-)) radicals are mainly from homoisoflavone in the ethyl ether fraction.It has shown that the method for on-line detection is an advantageous tool to rapidly determinate antioxidants from complex herbs.

AIM: To study the scavenging activities of Xiaoaiping Injection (Marsdenia tenacissima (Roxb) wight et Arn) for H2O2 and O2?- radicals by HPLC coupled with chemiluminescence and to trace the active constituents on line. METHODS: Solution of Xiaoaipin Injection was passed through HPLC to enter into the mixing tees (contained the luminol hydrogen peroxide solution and luminol pyrogallol solution,respectively),and recorded chemiluminescence signals were recorded,and their chemical structures were identified in combination with LCMS/MS. RESULTS: Three phenolic acids were identified,3-Caffeoylquinic acid,5-Caffeoylquinic acid,4-Caffeoylquinic acid,having scavenging activities for free radicals. CONCLUSION: Phenolic acids in Xiaoaiping Injection make a significant contribution to its scavenging activities for H2O2 and O2?-. It is shown that the method for on-line detection is an advantageous tool to rapidly determine antioxidants from traditional Chinese medicine.

Aim: To investigate the enhancement of enhancers on matrine in vitro and in vivo. Methods: The permeability of matrine across rat intestine was determined by non-everted intestinal sacs model in vitro, and the in vivo activity was assayed in a model of acute liver injury in mice. Sodium deoxycholate (SDC) was selected as the enhancer Candidate. Results: Among the tested absorption enhancers, only polysorbate 80 (Tween 80) and SDC showed to be capable of increasing the transport of matrine through small intestinal mucosa in rats.Enhancement ratios of 1.6 was observed for 0.5% SDC solutions with the best absorption enhancement for matrine in vitro, it was chosen to conduct hepatoprotective study in vivo. In mice with acute liver injury, combination of matrine with 0.5% SDC demonstrated considerably more activity than matrine along after oral administration. Conclusion: The results indicated that the absorption of matrine could be significantly improved by the absorption enhancer.

Objective To investigate the effect of luxS inactivation on the oxidative stress of Streptococcus mutans and perform preliminary analysis of potential mechanism.Methods Strains were grown to mid-logarithmic phase and divided into three groups,one was used as control and inoculated into normal TPY medium,and the other two groups were experimental groups,and there were separately inoculated into TPY containing 58.8 mmol/L hydrogen peroxideor TPY containing 58.8 mmol/L hydrogen peroxide and 0.1 mmol/L 2,2'-dipyridyl.The survival rate of strain was calculated at 0.5,1,and 2 h.All the data were statistically analyzed.Results Compared with the control group,the survival rate of luxS mutation was always higher than standard strain at all pre-determined time inexperimental groups (P＜0.05),and compared with experimental group without iron chelator,the survival rate of strains was not raised with the added of iron chelator (P＞0.05).Conclusion luxS gene is involved in oxidative stress tolerance of Streptococcus mutans,and the oxidative stress tolerance is not achieved by avoiding the toxic effects of the Fenton reaction

Objective: To study the difference between the acid resistance of Streptococcus mulans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Solutions of Streptococcus mulans standard strain and LuxS mutant strain with same density were prepared and cultured at pH 3. 5 to 7. 0 BH1 liquid for same period. Terminal growth situation was compared. After being acidized in pH 5.5 BHI liquid, the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared. Results; (DAt pH 6.0 to 7. 0, the difference of growth between Streptococcus mulans standard strain and LuxS mutant strain was not significant at the same pH value, and the differences of bacterial growth situation under three different pH values were not significant. (1)At pH 4.5 to 5.5, the difference of growth between the two strains was significant. (2)At pH 3.0,the survival rate of LuxS mutant strain(0.006 5% )was significantly lower than the standard strain (0.078% ). (3)At pH 5.5, the survival rate of LuxS mutant strain(0.747% ) was lower than the standard strain(8.65% )by about 10 times after the pre-acidification. Conclusion; (4)At sub-lethal pH value, there is significant difference of aciduricity between Streptococcus mu-tans standard strain and LuxS mutant strain. The acid resistance of standard strain is stronger than that of LuxS mutant strain. The two strains both display the capability of acid tolerance responses. LuxS mutant strain is more sensitive to acid inactivation, but the capability of acid tolerance responses still exists.

Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

Objective To knock out the entire LuxS gene of Streptococcus mutans UA159 strain via homologous recombination and construct a LuxS-deleted mutant strain of S.mutans.Methods The erythromycin resistance gene(Eymr)was inserted between the two DNA fragments located in the upper and downstream of LuxS gene that had been amplified by PCR.Then the two DNA fragments along with the inserted Eymr were engineered into pUCl9 plasmid to construct the recombination plasmid pUCluxKO.Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in the isolation of erythromycin resistant S.mutans,transformants,which was then subjected to polymerase chain reaction,Vibrio harveyi BBl70 luminescence bioassay and sequencing analysis.Results Restriction endonuclease analysis showed that pUCluxKOmutant vector had been successfully recombined.The deletion of LuxS of S. mutans mutants was confirmed bv PCR with primers specific for the genes of LuxS and the erythromycin resistance.S.mutans mutant could not induce bioluminescence.indicating the mutant had been successfully recombined.The constructed Chinese S.mutans showed good stability after 20 generations of cultivation.Conclusion The S.mutans gene allelic exchange plasmid is constructed correctively and a LuxS-negative mutant of S.mutans has been constructed.which can be helpful for further study of the role of LuxS in the pathogenesis of S.mutans.

AIM: To determine the total saponins in Shengmai Powders. METHODS: After comparing three chromogenic method, perchloric acid choosed as the chromogenic agent to determine total saponins. RESULTS: The content of total saponins in Shengmai Powder preparation Xindekang Granules was 6.14%. CONCLUSION: The method of perchloric acid is simple, relatively precise and reproducible. It is a reliable method for evaluating the quality of Shengmai Powders.

Objective:To investigate the effects of Origin-Preserving Decoction on the proliferation of marrow hematopoietic cells in aplastic anemia mice. Methods:Based on experimental AA models induced by Benzene and 60Co radiation in mice, the experiments including semi-solid colony culture (CFU-GM、CFU-E、BFU-E) and liquid culture (MTT Assay) were undertaken to investigate the effects of Origin-Preserving Decoction on the proliferation of marrow hematopoietic cells in AA mice. Results:Origin-Preserving Decoction (3g/kg) (ig.) could improve the outputs of CFU-GM, CFU-E and BFU-E in AA mice damaged by benzene and 60Co radiation: MTT assay showed that Origin-Preserving Decoction could stimulate the hematopoeisis of bone marrow of AA mice radiated by 60Co. Conclusions: Origin-Preserving Decoction has a good future for AA therapy.

Objective To explore the proper proportion of effective fractions in Shengmaisan(saponins of Radix ginseng,saponins of Radix ophiopogonis,Lignans of Fructus schisandrae)in different anoxia models.Methods Acute cerebral hypoxia was induced by sodium nitrite and decapitation in mice,and the orthogonal design was used in these two models to find the proper proportion of three effective fractions.The gasping time of the mice,which were decapitated was observed to compare the anti-anoxia effects of XZF with other clinical drugs,and cerebral ischemic reperfusion injury model of mice was also used to study the effect of XZF on related biochemical index.Results XZF(the proportion of saponins of Radix ginseng,saponins of Radix ophiopogonis,Lignans of Fructus schisandrae as 7 ∶ 2 ∶ 6)significantly prolonged the gasping time,and decreased brain nitrogen monoxidum(NO)content after reperfusion of the mice at dosages of 50 mg/kg and 150mg/kg.Meanwhile,XZF also reduced malondialdehyde(MDA)content and increased superoxide dismutase(SOD)activity at the higher dosage.Conclusion XZF obtained by experimental screening exerts a significant protective effect on cerebral ischemia injury in mice,which provide some pharmacological evidence for further development of new modern Chinese drug composed with effective fractions for cerebral vascular diseases.