| Tuberculosis is one of the most prevalent epidemic diseases in China,and has become the main disease of human death caused by infectious diseases.Especially in recent years caused by environmental pollution caused by immunization,HIV and hepatitis B infection,anti-TB drugs side effects,multidrug resistance,extensive multidrug resistance and other factors,so that the incidence of tuberculosis re-emergence of the trend.The main approach to the clinical treatment of tuberculosis is mainly chemotherapy,but with the pathogen Mycobacterium tuberculosis resistance increased,just rely on direct killing of Mycobacterium tuberculosis has been difficult to effectively control the tuberculosis,and chemotherapy drugs face a serious challenge.Thus,with multiple targets,multi-role mechanism of traditional Chinese medicine compound in the treatment of drug-resistant tuberculosis has recently been widespread concerned.The mid-term clinical study of anti-tuberculosis Yifei Tongluo Granule which was supported by the "Eleventh Five-Year" and "Twelfth Five-Year" "infectious disease prevention'',found that this Chinese medicine compound combined with chemotherapy drugs and other treatment programs shows better absorption rate of lung lesions than the simple application of chemotherapy drugs,and can significantly reduce the side effects of chemotherapy drugs.Although the clinical efficacy of Yifei Tongluo Granule is significant,the substance basis and mechanism in vivo are not clear.The structure-activity relationship and dose-effect relationship are also unknown.The lack of these basic studies seriously limits clinical application of this product.In this study,we selected the exact effect of anti-tuberculosis as the research object,and comprehensively used various international frontier immunotherapy theories and advanced techniques to carry out the preliminary study on the mechanism,substance basis and quality control of the drug the study.The main research contents of this subject include:(1)Evaluate the pharmacological and immunoregulatory mechanisms of the total extract and polysaccharide components of Yifei Tongluo Granule by the established model of immunoassay in mice;(2)Study the substance basis of Yifei Tongluo Granule by the establishment of fingerprints;(3)Study the dynamic changes of the pharmacokinetic substance basis in vivo;(4)The main active ingredients and anti-inflammatory mechanism of Bletilla striata were studied,and the anti-inflammatory material base and mechanism of Bletilla striata were described.The research of pharmacological mechanism of Yifei Tongluo Granule includes the in vitro drug susceptibility test of the extraction,the establishment of the immunoregulation model in vivo,the evaluation of the immunoregulatory mechanism of the small molecule components and the polysaccharide.The results showed that all the extracts did not show the effect of anti-Mycobacterium tuberculosis in the range of possible concentration.Thus,the focus of our research was transferred to the immunotherapy role of Yifei Tongluo Granule in vivo and vitro based on the relationship between tuberculosis and immune as well as the clinical related reports.The extract was isolated to the fraction of small molecules and the fraction of polysaccharide components and study their immunoreactivity and antioxidant activity in vivo and vitro The experimental results show that,in vitro,the two fractions both have good free radical scavenging ability and also have obvious protective effect on macrophages,and can regulate the intmacrophage-related immune function and inflammatory response to a certain degree.In vivo,the effect of immunoregulation and anti-oxidation of the compound was proved for the first time.The study of Pharmacological mechanism provides a strong basis for the treatment of drug-resistant tuberculosis through the immune regulation of the compound.The basic research of small molecule active constituents of Yifei Tongluo Granule includes the establishment of fingerprints,the structural identification and quantitative analysis of the main active ingredients.The two-wavelength fingerprints(272 and 300 nm)of Yifei Tongluo Granule were established by HPLC-DAD technique.The characteristic peaks were unidirectionally assigned and analyzed by the method of "Chinese medicine Fingerprint Similarity Evaluation System(2004A)Evaluation,for the preparation of compound ingredients laid the foundation for analysis.A total of 19 chemical constituents in Yifei Tongluo Granule were identified by LC-HRMS.The relative contents of coumarin,phenols,flavonoids,alkaloids and malate were higher,Jun medicine Baiji,one hundred,minister medicine dwarf tea,purple to the small,thistle,adjuvant and so on.The results showed that the chlorogenic acid,chlorophyll acid,chlorogenic acid,pyrithione,chlorogenic acid B,chlorogenic acid A,chlorogenic acid A,Militarine,Mongolian glycosides and thalidomides were 0.121 ± 0.018,0.318 ± 0.039,0.096 ± 0.019,0.100 ± 0.018,0.207±0.031,0.085 ± 0.021,0.058 ± 0.014,0.103 ± 0.020,2.152 ± 0.195.0.091 ± 0.014,0.183± 0.028 mg/g.Study on the pharmacokinetic behavior of the main components of Yifei Tongluo Granule.The results showed that the three components of Bergenin,Esculetin and Rutin were different from those previously reported,and their half-life T1/2 in rats doubled.The pharmacological characteristics of the compound preparation Yifei Tongluo granule further revealed the complexity of the traditional Chinese medicine compound system,and provided the scientific basis for the clinical drug monitoring and drug safety of Yifei Tongluo Granule.According to the study of Chinese herbal preparations Yifei Tongluo Granules in the above chapters,we found that the highest content of the compound prescription is Militarine which belongs to the component of Bletilla striata.In the preliminary study,it has shown that the liposoluble constituents are superior to polysaccharides of Bletilla striat in antioxidant capacity,so we think that it is possible that liposoluble components play a major role on its efficacy.In vitro,we also confirmed that the BM60 treatment reduced the production of NO in LPS-induced inflammatory response model of NR8383 macrophages,which indicated that BM60 might have a potential anti-inflammatory effect.However,due to the huge differences in the environment both inside and outside the body,we need to build rat ALI Imodel by intratracheal instillation of lipopolysaccharide which could evaluate the activity in vivo to show wether or not BM60 has the same anti-inflammatory effect in LPS?induced ALI.The pathological morphology of the model group showed that neutrophils were infiltrated in the lung tissue,and the pulmonary septum was thickened and edema,and the alveolar structure was destroyed in some fields of view.After treatment with BM60,the alveolar structure of the rats was significantly restored,the Pulmonary edema and infiltration of inflammatory cells in the alveolar cavity was also reduced.In addition,the BM60 treatment suppressed LPS-induced nuclear factor-KB(NF-KB)activation and the infiltration of inflammatory cells and tumour necrosis factor(TNF-a),interleukin-1(IL-1?),and interleukin-6(IL-6)production.BM60 has potential therapeutic effect on chronic inflammation,which may owe to the polyphenols of Bletilla striat.Therefore,the BM60 was then qualitatively analyzed by comparing its molecular formula and unsaturation which were known by HPLC-ESI-HRMS technology,as well as commercial standards and literature.Finally,23 compounds were detected from BM60,6 bibenzyls,8 bibenzyls,4 dihydrophenanthrenes,1 biphenanthrenes,1 flavonoid and 3 glucosyloxybenzyl 2-iso-butylmalates,among which biphenyl and phenanthrene compounds were higher content and have more types.This method characterizes the main components of BM60 in Bletilla striat and provides a basis for the development of new antioxidant active components.In summary,the subject of this study basically defined the immune mechanism of Yifei Tongluo and pharmacodynamic material basis through the fingerprints and the establishment of quantitative components.The mechanism of this study on the mechanism and material basis of Yifei Tongluo Granule provides a theoretical and experimental basis for further improving the anti-tuberculosis efficacy and the wider clinical application of the drug. |
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