| [Objective]:To study the molecular characterization of the traditional Chinese herbal medicine Oldenlandia diffusa and Lobelia chinensis,these two kinds of medicinal materials respectively based on DNA barcoding technology and high resolution melting analysis technology.At the same time,study the compound pharmacodynamics of adriamycin nephropathy in rats.[Method]:The ITS2 sequence was obtained by the extraction of the original plant DNA of Oldenlandia diffusa and its adulterants,and the related sequences were obtained by GenBank.The data were analyzed by CodonCode Aligner software,MEGA software was used for mutation analysis,genetic distance calculation and construction(NJ)phylogenetic clustering tree.(NJ)system clustering tree was used to identify the species and identify the authenticity of the phytoplankton.Real-time PCR was performed with Rotor-Gene Q.The ITS2 sequence was used to analyze the high-resolution melting curve of Lobelia chinensis and its common adulterants and related genera species.At the same time,40 parts of Lobelia chinensis on sell were identified.Whether the melting curve coincides with the base plant coincides the identification species,and the non-coincident species will identify the species after sequence alignment of the PCR products.48 rats were divided into blank group,model group,positive drug group and compound Lobelia chinensis injection group with low,medium and high dose groups.Urine,serum and kidney tissues were collected and immunohistochemistry was performed by HE staining and TNF-? and IL-6.The related indexes were analyzed.[Result]:The intraspecific genetic distances of Oldenlandia diffusa were lower than the interspecific genetic distances between Oldenlandia diffusa and its adulterants,the NJ tree showed that the original plant of Oldenlandia diffusa could be distinguished from its mixed products.29 kinds of authentic herbs in the market are genuine 8 samples counterfeit.Scutellaria barbata and Poly gala is a newly discovered counterfeit goods.There were significant differences in the melting curves of Lobelia chinensis and its six common mixed products and their three different haplotypes,and 25 samples were found belong to Lobelia haplotype A,both Lobelia haplotype B and C found 5 samples,2 parts of Scutellaria barbata,and another three samples did not coincide with the known base plant curve,PCR product sequencing to barcode identification system.Three new counterfeit goods are Artemisia annua and Digitaria sanguinalis.The high dose group could significantly reduce the urinary protein content in the urine,and the serum creatinine,blood urea nitrogen,total cholesterol,triglyceride,total protein and serum albumin were significantly improved compared with the model group.Group relative effect is not significant.The results of HE staining showed that the effect of IL-6 and TNF-a on the expression of IL-6 and TNF-a increased with the increase of the expression of IL-6 and TNF-a.[Conclusion]:DNA barcoding and HRM technique can be used to identify the traditional Chinese herbal medicine Oldenlandia diffusa and Lobelia chinensis,and provide reference and specification for the identification of other raw materials of Chinese herbal medicine.The raw materials of traditional Chinese medicine are solved from the source,Do not use the phenomenon of occurrence,to ensure the safety of medication.At the same time,the compound injection on the pathological changes of adriamycin nephropathy rats,the drug dose reaches a certain extent with a clear therapeutic effect,follow-up in clinical research provides a theoretical basis. |
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