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OBJECTIVE： To provide a theoretical basis for the development of the following products of Coptis chinensis- Zingiber ojjicinale couplet medicine （“Coptis chinensis-Zingiber ojjicinale” for short） prescription. METHODS： The prescriptions containing Coptis chinensis-Zingiber ojjicinale were collected from the Dictionary of TCM Prescription and input into TCM inheritance support platform software （V2.5） to establish the database. The frequency of major diseases and compatibility medicinal materials were analyzed statistically. The core combination of medicinal materials in the prescriptions containing Coptis chinensis-Zingiber ojjicinale were analyzed statistically with association rule Apriori algorithm （support degrees were 15％， 20％， 25％， confidence was 0.90）. Top 2 main diseases in the list of frequency， compatibility medicinal materials for dispelling internal cold and medicine for clearing heat with highest compatibility frequency were selected and analyzed in respect of prescription rules. RESULTS： A total of 492 prescriptions containing Coptis chinensis-Zingiber ojjicinale were screened， 9 kinds of major diseases （frequency≥15）， such as dysentery， diarrhea， accumulation， fullness. There were 21 commonly used compatibility medicinal materials （frequency≥55）， including Angelica sinensis-Panax ginseng， Magnolia officinalis， Aconitum carmichaelii， Scutellaria baicalensis， etc. There were 19 commonly used medicinal materials combinations， including Coptis chinensis-Zingiber ojjicinale-Panax ginseng， Coptis chinensis-Zingiber ojjicinale- Magnolia officinalis， Coptis chinensis-Zingiber ojjicinale-Angelica sinensis. There are 5 kinds of core medicinal materials commonly used in treating dysentery with Coptis chinensis-Zingiber ojjicinale，and 9 kinds of core medicinal materials for treating dysentery. There are 8 kinds of core medicinal materials in Coptis chinensis-Zingiber ojjicinale compatible with medicine for dispelling internal cold Aconitum carmichaelii prescription.and 7 core medicinal materials in compatible with medicine for clearing heat Scutellaria baicalensis prescription. CONCLUSIONS： The major diseases treated with prescriptions containing Coptis chinensis-Zingiber ojjicinale are mainly digestive tract diseases. It can treat different diseases being compatible with different medicinal materials， this study aslo can provide theoretical basis for the development of subsequent products.
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Objective To study the optimum technological conditions of macroporous resin separation and purification of polyphenol composition. Methods Fingerprint of Ilex paraguariensis was established by HPLC method. The best resin, loading conditions and elution requirements were screened with chlorogenic acid and elution rate of total active constituents served as the index. Results The optimum technological conditions were as follows:the separation carrier was D101 macroporous resin,the concentration of the sample liquid was 0.10 g?mL-1 ,the resin adsorption quantity of crude drugs was 1.5 g?g-1 ,the impurity was removed by four times of amount of water at first,and then eluted with four times the amount of 70％ ethanol in a gradient manner.After purification,the elution rate of chlorogenic acid was up to 82.83％,and the elution rate of total effective component was about 72. 48％. The resemblance of HPLC fingerprint was over 0. 98, and the components were balancedly recoveried. Conclusion The purification process of total polyphenols in Ilex paraguariensis by D101 macroporous resin was simple,reliable,and can be used for large-scale production.
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Objective To establish a stable and reliable mouse model as an alternative to the traditional model of impaired glucose tolerance induced by calorie restriction and its effect on glucose homeostasis.Methods Forty 16-week-old SPF C57BL/6J mice (half male and half female) were randomly divided into four groups by sex and the way of feeding.The mice in the ad libitum (AL) group had free access to basic diet, while the mice in the intermittent fasting (IF) group had normal diet and fasting on alternate days, with free access to water on the fasting days.The changes of body weight and blood glucose concentration in each group were monitored, and intraperitoneal glucose tolerance test and insulin tolerance test in mice were performed before and after the 12-week IF treatment.Results At 12 weeks after IF treatment, the body weight and blood glucose concentration of mice did not show significant difference.After i.p.injection of glucose, the blood glucose concentration of IF mice was less increased than the AL group, and after the insulin injection, the blood glucose concentration was more decreased.Compared to the AL group, the areas under the curve of tolerance test in the IF group were significantly decreased (P < 0.05).Conclusions After IF treatment, the mice show an enhanced sensitivity to insulin and improved glucose tolerance.This establishment method of mouse model of intermittent fasting is easy and simple, therefore, can be used as an effective alternative to traditional calorie restriction model of impaired glucose tolerance.
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Objective:To obtain the prokaryotic expression product for the group 6 allergen of Dermatophagoides farine ( Der f 6) and detect its IgE-binding rates with sera from asthmatic children. Methods: By enzyme digestion of pET28a (+)-Der f 6 with BamHⅠ plus XhoⅠ,the target gene Der f 6 was obtained and linked into the vector pET32a (+) to construct the recombinant plasmid pET32a(+)-Der f 6, which was then transfected into E. coli BL21 cells for expression, induced with isopropyl-β-D-thiogalactoside ( IPTG) ,purified by affinity chromatography and identified by SDS-PAGE,Western blot and AMLDI-TOF,and tested by ELISA for IgE reactivity with sera from asthmatic children. Results:The plasmids pET32a(+)-Der f 6 were constructed,transformed into E. coli BL21 and expressed successfully. SDS-PAGE of the purification product showed a specific band,Western blot showed the successful binding between the purification product and the His-tag in the plasmids,and MALDI-TOF/TOF identified the identical structure to the allergen Der f 6. Using the ELISA method developed with the recombinant proteins as coating antigen,the positive rate was 41. 3% (19/46) in asthmatic children allergic to dust mite. Conclusion: The plasmids pET32a (+)-Der f 6 were constructed successfully,expressed in E. coli BL 21 (DE3). The recombinant fusion protein has a good reactivity with sera from asthmatic children.
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ObjectiveComparingfourgroupsofimmunosuppressivemousemodelsestablishedby cyclophosphamide administration in different doses and periods , we used partial least squares ( PLS) analysis to evaluate the immunosuppressive mouse models comprehensively and select the appropriate way to establish this model .Methods In this experimental study, 58 male KM mice were randomly divided into five groups: normal group (10 mice) were given normal saline daily by i.p.injection, model group 1 (12 mice) was given 40 mg/kg CTX daily by i.p.injection for 10 days, model group 2 (12 mice) was given 80 mg/kg CTX daily by i.p.injection for 3 days, model group 3 (12 mice) was given 40 mg/kg CTX daily by i.p.injection twice for a week, model group 4 (12 mice) was given 50 mg/kg CTX daily by i.p.injection for 7 days.After the injection of cyclophosphamide , the daily metabolic activities were detected everyday such as body weight , water intake , and food intake , organ index and immunological indexes such as blood RT of the model mice were measured as well .Using partial least squares ( PLS) analysis to the models and analyzing the final weight , final anal temperature , organ index , and blood routine examination in order to evaluate the immunosuppressive mouse models comprehensively .Results Compared with the normal group , different dosages of CTX reduced the weight and anal temperature of model mice (P<0.05), the food intake and water intake (P<0.01), and the spleen index and thymus index ( P<0.01 ) .Besides , the amount and percentage of basophilic granulocytes decreased ( P <0.05 ) , and the percentage of MCHC , macrophage went up , as well as the absolute value of WBC and lymphocytes were decreased in the model groups 1, 2, and 4 (P<0.05).According to the PLS analysis, there were significant differences among models 1, 2, and 4 when compared with the normal group , especially the most different in the model group 1.Conclusions After the PLS analysis of different indexes , the optimal way to establish immunosuppressive mouse models is the procedure with 40 mg/kg CTX daily injected i .p.for ten days .Our findings provide experimental evidence for the establishment of immunosuppressive mouse models .
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Objective:To compare structure and function of mite Der f 3,Der p 3 and Eur m 3.Methods: Obtained mite allergens amino acid sequences from the International Union of Immunological Societies nomenclature database .Then physiochemical characterization,sequence alignment,secondary structure,three dimensional (3D) structure and epitopes of three proteins were analyzed by bioinformatics methods.Results:According to results of alignment ,Der f 3,Der p 3 and Eur m 3 displayed 88.51%sequence iden-tity.Der f 3,Der p 3 and Eur m 3 all contained three active sites and two trypsin functional domains ,which showed high identity of amino acid residues.Active sites of three proteins ,which closing to each other in three dimensional (3D) structure,constituting the active center of the enzyme.Secondary and 3D structure of three proteins all contains α-helices,β-sheets and random coils.Epitopes analysis revealed that Der f 3,Der p 3 and Eur m 3 all have 5 main potential epitopes located in random coils.Epitope sequences of Der f 3,Der p 3 and Eur m 3 overlapping in three domains (peptides of 79-81aa,129-135aa and 172-174aa),but the residues in these three domains were not identical.Conclusion:These studies lay the foundation for biochemical and genetic analysis of these 3 allergens,and may contribute to vaccine development for allergen-specific immunotherapy.