| Objective:Feire Puqing Powder(FPP),a Tibetan medicine,was included in the Pharmaceutical Standard Tibetan Medicine of the Ministry of Health of the people’s Republic of China:volume I.It is composed of 14 herbs such as Carthamus Tinctorious,Carthamus Tinctorious,Glycyrrhiza Uralensis,Benzoinum,Bergenia purpurascens,etc.It has the effect of clearing lung and relieving heat and anti-inflammation.It is used for infantile pneumonia,influenza,wind-heat and fever.This study has explored the antiinflammatory effects of Feire Puqing Powder on zebrafish inflammation models and its mechanism,so as to provide experimental basis for clinical promotion and secondary development of this product.This product is a traditional crude medicine powder.Under the premise of not changing the theory and material basis of crude medicine application,it has been improved into a modern suspension agent,and studies on the prescription,process and preparation quality standard of Feire Puqing suspension agent have been carried out to provide experimental basis for pilot test and production of Feire Puqing suspension agent,as well as reference for the improvement and innovation of traditional Tibetan medicine powder.Methods:1.In the pharmacodynamics study,zebrafish 72 h after fertilization were randomly transferred into 24-well plates and divided into control group,model group and FPP low,medium and high concentration(3,10,and 30 mg/L)groups.Control group and model group were not given any medicine.After FPP was co-incubated with zebrafish for one h,except the control group,the other groups were treated with 20 μmol/L copper sulfate for two h to prepare inflammation model.The migration of inflammatory cells in transgenic strain TG(zlyz: EGFP)zebrafish was observed under fluorescence microscope.Quantitative real-time PCR(q RT-PCR)was used to detect m RNA levels of nuclear factorκB2(NF-κB2),interleukin(IL-1b),tumor necrosis factor(TNF)-α and IL-8 in wild-type zebrafish.The expression levels of NF-κB and TNF-α in wild-type zebrafish were detected by Western blotting assay.The expression of NF-κB in fish tail of each group was detected by immunofluorescence method.2.In the study of the prescription and process of suspension,according to the indexes of dispersion,sedimentation volume ratio,re-dispersion,precipitate state,viscosity and so on,the excipients such as suspending agent,wetting agent and taste correction were investigated,and the suitable kinds and dosage of excipients were selected.In the process study of suspension,the RSD value of hydroxysafflor yellow A content in intermediate powder was used as the index of mixing uniformity,and JHT-20 L double motion mixer was selected to mix cornflour and suspension medium.the effects of mixing time and mixing frequency on the mixing uniformity were investigated,and the stability of the mixing process was verified by three small-scale experiments.In the irradiation sterilization process,the commercial Feire Puqing Powder was irradiated and sterilized with different doses by the electron accelerator.The appearance of the samples and the characteristics of microscopic powder were compared.The dehydrocostus lactone and costunolide in Feire Puqing Powder were identified by thin-layer chromatography(TLC).The content of hydroxysafflor yellow A was determined by the high-performance liquid chromatography(HPLC).The total number of bacterial colonies and the total number of mold and yeast colonies were determined according to the genera rules of the Chinese Pharmacopoeia.The electron beam irradiation sterilization process of Feire Puqing Powder was studied.finally,three batches of magnifying experiments were carried out to verify the feasibility and stability of prescription,mixing process and irradiation sterilization process.3.In the study of quality standard,taking the enlarged experimental samples as the research object,the thin layer identification methods of Glycyrrhiza uralensis,Carthamus Tinctorious,Glycyrrhiza Uralensis,Benzoinum and Bergenia purpwascens for Feire Puqing Suspension were established,the HPLC method for the determination of hydroxysafflor yellow A and the GC method for eugenol content were established,and three batches of magnified samples were examined according to the quality regulations of Chinese Pharmacopoeia.To establish the quality standard of Feire Puqing Suspension(draft).Results:1.The results of pharmacodynamic study showed that in the control group,the fluorescent cells were mainly distributed in the head and the aorta,and the fluorescent cells above the aorta were rare in the trunk.The fluorescence cells in the model group were increased in all areas,showing an obvious trend of extravascular migration of inflammatory cells,and the fluorescence cell count above the lateral trunk line was significantly increased compared with the control group(P < 0.001).Compared with model group,the fluorescent cell count of trunk cadre in 10 and 30 mg/L groups was significantly decreased(P < 0.01,0.001).Compared with model group,m RNA levels of IL-1b,TNF-α and IL-8 in FPP 3,10 and 30 mg/L groups were significantly downregulated(P < 0.05,0.01),and NF-κB2 m RNA levels in 10 and 30 mg/L groups were significantly down-regulated(P < 0.01).The expression levels of NF-κB and TNF-α in10 and 30 mg/L groups were significantly decreased(P < 0.01,0.001).The white fluorescence spots of NF-κB in the trunk and fin of the model group were significantly increased compared with the control group,and the fluorescence spots in the same parts of the model group were decreased after 10 mg/L FPP treatment.2.The results showed that xanthan gum and sodium alginate were selected as suspending agent,Poloxamer 188 as wetting agent,sucralose and orange essence as flavor corrector through prescription screening,and the best mixing process parameters were mixing time 10 min and mixing frequency 50 Hz.Electron accelerator irradiation sterilization is suitable for Feire Puqing Powder.When the irradiation dose is 8 k Gy,mold,yeast and bacterial colonies are not detected and there is no significant effect on the content of effective components.Three batches of magnification experiments verified the rationality of the prescription,and the feasibility,stability and reliability of the mixing process and sterilization process.3.The results of quality standard study showed that Glycyrrhiza uralensis,Carthamus Tinctorious,Aucklandiae Radix,Dalbergiae Odoriferae Lignum and Benzoinum in the preparation were qualitatively identified by TLC.The content of hydroxysafflor yellow A in preparation was determined by HPLC method and eugenol in Feire Puqing Suspension was determined by GC method.The established method is simple,easy,accurate and operable.All the tests of three batches of samples are in line with the provisions of Chinese Pharmacopoeia on suspension and powder,and the established quality standard(draft)can effectively control the quality of samples.Conclusion:Feire Puqing powder has anti-inflammatory effects on acute inflammatory model,which can reduce the expression levels of IL-1b,IL-8,NF-κB and TNF-α m RNA,and its anti-inflammatory mechanism is related to the inhibition of NF-κB /TNF-α signaling pathway.The prescription of Feire Puqing Suspension is scientific and reasonable,the preparation process is stable and reliable,and the quality control method is simple,accurate,stable and operable,so it can be used for the quality control of this preparation. |
PDF Full Text Request