| Object:In this study the quality standard of Mesona chinensis was studied for its quality evaluation.At the same time,extraction of Mesona chinensis was optimized and purification process were studied with macroporous resin for its active substances.And the effects of the ability of degrade lipid in vitro could be investigated preliminarily.This research will offer a new strategy for the scientific development of the use of Mesona chinensis resources.Method:1.Microscopic identification was used to identify the pharmacological characteristics of Mesona chinensis powder.And TLC method was adopted to simultaneously identify oleanolic acid and ursolic acid in a thin-layer plate.High Performance Liquid Chromatography-Secondary Array Detector was employed to establish a fingerprint of Mesona chinensis from different areas.Determinated of Six Components in Mesona chinensis by High Performance Liquid Chromatography and Ultraviolet-Spectrophotometer.Cluster analysis and principal component analysis were used to analyze the data.2.Nuclear gene ITS1,ITS2 sequence and Chloroplast psbA-trnH,rbcL,matK sequence were used as DNA barcod to analyse genetic relationship of Mesona chinensis from different resources and identify its adulterants.3.Orthogonal experiment-single factor method was used toextract total flavonoids from Mesona chinensis.The purification method of macroporous resin was optimized,and the antioxidant activity of the purified product was determined.4.3T3-L1 preadipocytes cell was induced differentiation with cocktail method in vitro.The drug group treated by different concentrations of Mesona chinensis extracts intervention in the process of cell differentiation.Lipid droplets cells were stained with oil Red 0,PPAR y,C/EBP a,ACS-L1,SREBPS,AMPK in total protein were determinate by Western blot analysis.Result:1.Quality standard research:Mesona chinensis powder's microscopic characteristic was clear.The TLC spots showed the same colors to the reference substances in the corresponding position.The water content was in the range of 6.14%-9.45%.And the content of ethanol extracts were in the range of 20.82%-30.69%.The fingerprint of Mesona chinensis was established with calibration 9 peaks.Mesona chinensis ingredients from different origins have different characteristics.This study clustered the sample into 4 categories.3 main ingredients are extracted,reached to 81.073%.20 samples of similarity were between 0.829-0.997.The results of the three methods are mutual corroboration.This method was proved to be simple,accurate and repeatable,principal components were extracted and the cumulative contribution was reached to 87.979%.This function is following Total falconoid?phenolic acid and sugar were play an important role in the quality evaluation of Mesona chinensis.2.Molecular identification:MatK sequence amplification success rate is low;ITS1 sequence has 2 single change points and the success rate is low.ITS2,rbcL and psbA-trnH all have not variable site,and they have a high rate in amplifying and Sequencing.Their lengths ranged from 233 to 671 bp.The NJ tree constructed by ITS2 sequence was capable to identify the Mesona chinensis and its adulterants.While the other sequences have some confusion when they are distinguished.The ITS2 barcode can be considered as the preferred sequence to identify Mesona chinensis.3.The results showed that the optimal conditions of the ethanol extraction were as followed:ethanol concentration for 50%,solid-liquid ratio for 1:50 and time for 1.5h.Under these conditions,we could get 13.31%of the total flavonoids.Meanwhile,X-5 type of macroporous resin was chosen for the separation of total flavonoids,The rates of adsorption is 12.73mg/g and the desorption rate is 58.84%.When the X-5 was adsorbed with the sample solution containing 0.3 mg/mL extractive at a pH of 2-4,and deported with 0%-40%of ethanol for gradient elute at a flow rate of 2BV mL/h.Extracted total flavonoids had strong scavenging capacity on DPPH radical,The crude extract inhabitant concentration 50(IC50)value is 80 ?g/mL,The purified extract inhabitant concentration 50(IC50)value is 21 ?g/mL.4.Study on the lipid-lowering effects in vitro:Mesona chinensis can significantly reduce lipid content in 3T3-L1 cells.The expression of PPAR-?,C/EBP and ACS-L1 was significantly decreased and AMPK protein was increased,while the expression of SREBP protein had no obvious effect in the treatment group during differentiation process,draw the conclusion:The Mesona chinensis can interfere 3T3-L1 differentiated into generation and accumulation of fat in the process,possibly through inhibition of fatty acid synthesis pathway,temporarily unable to prove and sterol synthesis.Conclusion:This quality control methods can be used for quality control of grass Mesona chinensis standards.The establishment of the study on substances purification method can be used for Mesona chinensismaterial base,its results can serve as a theoretical basis of in vivo an in vitro. |
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