Study On The Accumulation Path Of Polyunsaturated Fatty Acids In Oviductus Ranae And Preparation Process Of Solid Dispersion Formulation

Objective: Collecting Rana temporaria chensinensis David(R.chensinensis)in different locations and at different times.The composition and content of polyunsaturated fatty acids(PUFAs)in R.chensinensis were determined.Transcriptome sequencing of Oviductus Ranae.The synthetic route of PUFAs of Oviductus Ranae is obtained.And obtain a fatty acid dehydrogenase gene.Real-time fluorescence quantitative determination of Oviductus Ranae.To explore the expression level of Oviductus Ranae in different periods,so as to obtain high quality Oviductus Ranae.Further,the obtained high-quality Oviductus Ranae is prepared into a dosage form by a solid dispersion technique.And establish the quality standards of the preparation.The stability and acute toxicity of the formulation were investigated.And the immunoassay of the dosage form is verified.Methods: Samples of R.chensinensis from different locations and at different times were collected.Take the fallopian tube of R.chensinensis,and keep it in the refrigerator at-80.0 °C.The PUFAs in the Oviductus Ranae are extracted and methylated.The content of PUFAs was determined by gas chromatography.The composition and relative content of PUFAs were analyzed by American StatView software.And analyze the variation of PUFAs,explore the best quality area of Oviductus Ranae and the best collection period.In order to determine the accumulation pathway of PUFAs in Oviductus Ranae.Transcriptome sequencing of Oviductus Ranae,generation of databases,analysis and statistics.The synthetic pathway of PUFAs in Oviductus Ranae and the fatty acid dehydrogenase gene affecting PUFAs in Oviductus Ranae were obtained.The RNA in the Oviductus Ranae was extracted by the Trizol method.RNA concentration and quality were measured using a Nanodrop 2000 ultra-micro UV/visible spectrophotometer.Total RNA integrity was determined by 1.0% agarose gel electrophoresis.The total RNA in the Oviductus Ranae was reverse transcribed into cDNA using a Takara kit.Real-time quantitative PCR was used to detect the expression of fatty acid dehydrogenase gene in different periods of Oviductus Ranae.High-quality Oviductus Ranae was obtained in comparison with the determination of the content of PUFAs in Oviductus Ranae in each period.Further,the obtained high-quality Oviductus Ranae is prepared into a dosage form by a solid dispersion technique.And establish the quality standards for the best dosage form.Conduct stability and acute toxicity studies.The mouse animal experiment was used to verify the immunoassay of the best preparation of Oviductus Ranae.Results: Gas chromatography was used to determine the PUFA content of Oviductus Ranae in different locations and different periods of R.chensinensis.The results showed that the PUFA content of Oviductus Ranae in the source of R.chensinensis breeding plant in Sandaohu Town,Jingyu County,Jilin Province,China.It was also found that the content of PUFAs in Oviductus Ranae reached the highest in the scattered hibernation period(November to December).Analysis of the sequencing results of the Oviductus Ranae transcriptome.The synthetic pathway of PUFAs in Oviductus Ranae and the fatty acid dehydrogenase gene affecting the content of PUFAs in Oviductus Ranae were found.Real-time quantitative PCR was used to determine the expression of Oviductus Ranae in different periods.It was found to have the highest expression during the scattered hibernation period(November to December).The obtained high-quality Oviductus Ranae was prepared into a pellet by a solid dispersion technique.The substrate is polyethylene glycol 4000.The ratio of drug to polyethylene glycol 4000 is 1:3.The condensate is methyl silicone oil(100 mm 2/s): light liquid paraffin(1:1).The drug and matrix melting temperature was 70 °C.Established the quality standard of Oviductus Ranae dropping pill,in line with the 2015 edition of the Chinese Pharmacopoeia standard,and the stability test meets the requirements.It is proved that the hazelnut oil drop pills are actually non-toxic.Oral administration of different doses of Oviductus Ranae drop pills for 30 days can enhance the delayed type hypersensitivity in mice,indicating that Oviductus Ranae dropping pill can enhance cellular immune function.And the test results show that it can improve the carbon clearance ability of mouse monocyte-macrophages,indicating that Oviductus Ranae drop pills can enhance mononuclear-macrophage function.Conclusion: The PUFAcontent in the Oviductus Ranae was highest in the scattered hibernation period of China(November to December).The results of real-time quantitative PCR were determined to determine the expression levels of different concentrations of Oviductus Ranae.And determine the pathway of biosynthesis of PUFAs in Oviductus Ranae,and find key enzyme genes.The obtained high-quality Oviductus Ranae was prepared into a pellet by a solid dispersion technique.The quality standards are in compliance with the 2015 edition of the Chinese Pharmacopoeia.The immunoassay proves that Oviductus Ranae dropping pill can improve immunity.