Studies On The Material Basis Of The Active Fraction Of Qing-Luo-Fang

Treatment with traditional Chinese medicinal composite is one of the most important characteristics of traditional Chinese medicine(TCM).Studying material basis of TCM composite contributes a lot to modernizing the industry of Chinese medicinal herbs.In the present study,a herbal formula used to treat Rheumatoid Arthritis,Qing-Luo-Fang,is employed to address this key point.This dissertation divides into five parts to study the material basis of the active fraction of Qing-Luo-Fang.The first part:summary of the related literatureThe clinical applications and modern researches of Qing-Luo-Fang were concluded in the preface.The chemical constituents and pharmacological activities of Sophora flavescens Ait.,Sinomenium acutum,Phellodendron amurense Rupr.,which constitute Qing-Luo-Fang were summed up.213 references were cited.The second part:studies on the preparative technology of the active fraction of Qing-Luo-Fang and the preliminary evaluation of it's pharmacodynamic actionFirstly,the extraction technology of Qing-Luo-Fang was perferred by using total alkaloids contents as the evaluation quota.Secondly,for remaining the varieties of active constituents from the herbal formula,after investigating the purification results of several kinds of resins and the rationality of the process,an optimal process flow of preparation was obtained.And the active fraction of Qing-Luo-Fang was acquired by this preparative technology.The technology was tested and verified through 3 batched of samples.The results showed that the preparative technology of the active fraction was reasonable,steady and repetitive.The quantitative method of total alkaloids in the active fraction of Qing-Luo-Fang was established.The results showed that these methods were accurate and had nice recovery rates and good repetition,which could be used to control the quality of the active fraction.Also,the preliminary evaluation of it's pharmacodynamic action was studied.Firstly, two models of croton oil induced mouse ear edema and acetic acid induced writhing were selected as the models to evaluate the anti-inflammatory and analgesic effects of the active fraction of Qing-Luo-Fang respectively.The results showed that the active fraction exhibited good anti-inflammatory and analgesic effects.Secondly,the effect of the active fraction on rats with collagen-induced arthritis was observed.It indicated that the joint swelling of rats was meliorated obviously,and the quantity of collagen antibodyâ…¡in serum decreased.The results indicated that the active fraction had an obvious therapeutic effect for this model and a certain function of anti-angiogenesis.The third part:research on the chemical constituents of the active fraction of Qing-Luo-Fang and the flavonoid fraction of Sophora flavescens Ait.The chemical constituents of the active fraction of Qing-Luo-Fang and the flavonoid fraction of Sophora flavescens Ait.were researched.Several purification and separation methods and many kinds of structure elucidation and identification technologies,such as UV,IR,MS,¹H-NMR,¹³C-NMR,were used.26 compounds were identified.17 compounds were isolated from the active fraction of Qing-Luo-Fang for the first time.And the 17 compounds included 9 alkaliods,2 phenylpropanoids,2 lactones,2 sterols, 1 flavonoid glycoside and 1 long-chain alkanoic acid.2 compounds,shihulinmonin A and hexacosanoic acid,were isolated from Sophora flavescens Ait.,or Sinomenium acutum,or Phellodendron amurense Rupr.for the first time. According to the structure style and biogenesis,shihulinmonin A was likely to come from Phellodendron amurense Rupr..And hexacosanoic acid came from Sophora flavescens Ait..The remained 15 compounds had already been isolated from Sophora flavescens Ait., Sinomenium acutum,or Phellodendron amurense Rupr..Among them,matrine, sophocarpine,oxymatrine,oxysophocarpine and triforlirhizin came from Sophora flavescens Ait.;sinomenine,acutumine and acutumidine came from Sinomenium acutum; berberine,palmatine and obaculactone came from Phellodendron amurense Rupr.;syringin and syringaresinol-4,4'-O-bis-β-D-glucoside were found in Sinomenium acutum and Phellodendron amurense Rupr.;β-sitosterol and daucosterol were found in the three Chinese meteria medicas.Sophora flavescens Ait.was the monarch drug of Qing-Luo-Fang.The chemical constituents of the flavonoid fraction of Sophora flavescens Ait.were researched.It was conducted to obtain the monomeric compounds for it's activity screening and the establishment of the fingerprint of Qing-Luo-Fang.9 compounds were isolated from the flavonoid fraction of Sophora flavescens Ait.. Among them,hexacosanoic acid were isolated from Sophora flavescens Ait.for the first time.The other compounds were kurarinone,nor-kurarinone,triforlirhizin,formononetin, kushenol O,β-sitosterol,daucosterol and sucrose.The fourth part:Quantitative determination of kurarinone and nor-kurarinone in Sophora flavescens Ait. Kurarinone and nor-kurarinone were two typical flavonoids in Sophora flavescens Ait.. In this part,an HPLC method for the quantitative determination of kurarinone and nor-kurarinone in Sophora flavescens Ait.was established.It could provide a reference for the quality control and evaluation of Sophora flavescens Ait.and Chinese materia medica preparation.Before this study,the quantitative determination of kurarinone in Sophora flavescens Ait.had not been reported.The determination was carried out with Phenomenex Gemini C 18 column(250 mm×4. 6 mm,5μm),using acetonitrile - water(50:50) as mobile phase,at a flow rate of 1.0 mL/min and detected at the wavelength of 290 nm.The results showed that the linear ranges of kurarinone and nor-kurarinone were 0.3186～1.5930μg(r = 0.9998),0.1128～0.5640μg(r = 0.9999),with the average recoveries(n = 6) were 100.56%(RSD = 0.93%), 96.66%(RSD = 0.85%)for Sophora flavescens Ait.respectively.The method was simple, accurate,and with a good reproducibility.It was found that the quantitative of kurarinone and nor-kurarinone in Sophora flavescens Ait.from different regions was differing sharply.The fifth part:initial studies on the constituents absorbed into blood after the administration of the active fraction of Qing-Luo-FangHPLC-MS method was employed in the analysis of the constituents absorbed into blood after the oral administration of the active fraction of Qing-Luo-Fang.After comparing the HPLC-MS information of plasma containing drug,blank plasma and the active traction of Qing-Luo-Fang,8 new constituents was found in rabbit blood.6 of them was identified and they were oxymatrine,oxysophocarpine,matrine,sinomenine,sophocarpine and palmatine.The remained 2 constituents with the mass number of 594.8 and 780.2 were undetectable in the active fraction of Qing-Luo-Fang.These 2 constituents were speculated to be the transforms in vivo of other constituents of the active fraction,or even the endogenous substances of rabbit blood stimulated by the active fraction.According to the results of qualitative analysis,variations of the concentration of 5 constituents in vivo were investigated.It provided references for further studies of it's pharmacokinetics and clinical pharmacology.