Study On Extraction Process And Quality Standard Of Active Ingredients Of Rheum Tanguticum Maxim.ex Balf.

R.tanguticum Maxim.ex Balf.is the general term for perennial herbs of the genus rhubarb in the Polygonaceae family,the traditional medicinal parts for its roots and rhizome,bitter cold smell,has the effect of eliminating the old and bringing forward the new,purging heat through the intestine,and cooling blood detoxification,mostly used for human and animal stool constipation,blood heat,gastrointestinal stagnation,wet heat syndrome,heat burn and scald and phlegm accumulation edema and other symptoms.The most abundant chemical in R.tangguticum Maxin.ex Balf is anthraquinone,which accounts for about 5%.There are about 40 kinds of rhubarb anthraquinone ingredients known,which are index ingredients controlled by pharmacopoeias.In this study,the extraction of total components of anthraquinone in R.tangguticum Maxin.ex Balf was optimized by reflux extraction method combined with Box-Behnken response surface method,followed by the simultaneous determination of the content of six anthraquinone components in rhubarb by high performance liquid chromatography,and finally,the secondary metabolites of rhubarb in five different regions of the alpine meadows of the Qinghai-Tibet Plateau were analyzed by ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS),and the differential metabolites in rhubarb were fully covered.The unbiased analysis,analysis and discussion of the influence of major environmental factors on the accumulation of secondary metabolites of rhubarb in the five places have reference significance for further research on the relationship between secondary metabolites of rhubarb and their habitat conditions,and provide certain theoretical guidance and technical basis for the screening of suitable living environments for rhubarb cultivation and the determination of excellent germplasm resources.The following results were obtained in this study area:(1)The data and charts obtained by the comprehensive Dsign Expert 10.0.1response surface analysis software show that the single-factor size order affecting the extraction of active ingredient content in R.tangguticum Maxin.ex Balf is as follows:crushed particle size> ethanol volume fraction> feed-liquid ratio> Extraction time;The optimal extraction conditions of this experiment were: crushing particle size 200 mesh,ethanol volume fraction of 70%,material-liquid ratio of 20:1m L/g,extraction time of1.6h,and the anthraquinone content in the final R.tangguticum Maxin.ex Balf was1.74%,which was similar to the software prediction value.(2)In this study,the separation and purification conditions of the main components of rhubarb extract(sennoside A,aloe emodin,emodin,chrysin acid,chryphanol and emodin methyl ether)were systematically optimized.The following conclusions were drawn: 1)HP-20 adsorption resin has the strongest ability to adsorb the main components of rhubarb extract;2)When the sample is shaken for 30 or 60 minutes,the HP-20 adsorption resin reaches the maximum adsorption capacity when the sample volume does not exceed 10 m L;3)The ethanol-to-water ratio was between 0-90%,and there was no obvious difference in the adsorption capacity of HP-20 adsorption resin to adsorb the main components of rhubarb.However,when the ethanol-to-water ratio is70%,the adsorption capacity of HP-20 adsorption resin to emodin decreases.Therefore,the elution ratio finally determined in this study is 50% ethanol water;4)There was no significant difference in the ability of HP-20 adsorption resin to adsorb the main components of rhubarb extract with different flow rates.A flow rate of 8 min/20 m L was used for elution in this study.The optimized high performance liquid chromatography method in this study realized the determination of the content of seven index components in rhubarb,which provided a reference method for improving the quality control level of rhubarb.(3)The metabolites of Guoluo and Haibei rhubarb fruits were similar,while the metabolites of Yushu and Hainan rhubarb fruits had obvious regional differences.The contents of flavonoids,organic acids,isoflavones,purine nucleotides and sugars in fruits in different regions of rhubarb are affected by the external environmental conditions of plants.In addition to the influence of genetic factors,the synthesis and accumulation of secondary metabolites in rhubarb fruit are closely related to its environmental factors such as soil,climate,terrain and water quality.In general,temperature has a significant effect on the formation of active ingredients in rhubarb fruit,and temperature may affect the antioxidant activity and biochemical reaction rate of enzymes in rhubarb plants,thereby affecting the production and accumulation of metabolites of plant organic matter.In this study,the extraction process of rhubarb components was optimized by reflux method,Box-Behnken response surface method and high performance liquid chromatography,which provided certain scientific data for a series of subsequent studies of rhubarb.The differences of secondary metabolites and the enrichment of differential metabolites in five representative production areas of the Qinghai-Tibet Plateau were compared and analyzed by non-targeted metabolomics technology,which provided data support and reference for further study of the mechanism of response mechanisms of secondary metabolites and environmental factors in rhubarb in different production areas from the molecular level.