| ObjectiveRehmannia rehmannii is one of the most commonly used tonic herbs.According to statistics,its frequency of use ranks in the top 10 in the prescriptions of traditional Chinese medicine.It is the most frequently used and main direct traditional Chinese medicine.Different processing technology of rehmannia Huang Zhongmao core cost indican 5-HMF results wavelength of fingerprint and chemical composition difference of rehmannia glutinosa polysaccharide and other main ingredient rehmannia glutinosa LC-MS identification,through its main ingredients for rat ovarian granular cells after determination of biological activity,on the one hand,for the different processing technology provide theoretical basis for the efficacy of rehmannia glutinosa differences,on the other hand in clinical medical guarantee the quality of rehmannia glutinosa yinpian has a certain reference value.Methods1.Study on different processing technology of rehmannia glutinosal.In related literatures and compare various provinces and regions on the basis of Chinese traditional medicine preparation,combined with the early stage of the laboratory of rehmannia glutinosa processing methods are discussed and formulated the method of three kinds of processing technology of rehmannia glutinosa,respectively is nine steam nine sun processing method(wine),nine steam nine drying processing method,the method of one-time steamed method(modern processing),as standard for further researches on the composition of rehmannia glutinosa.2.Establishment of determination method for polysaccharide content and monosaccharide composition of rehmannia rehmannii.Rehmannia glutinosa polysaccharides were extracted by water extraction and alcohol precipitation.The content of polysaccharides was determined by classical phenol-sulfuric acid method,and the methodology was investigated.The monosaccharide composition of the prepared Rehmannia glutinosa polysaccharide was analyzed by gas chromatography.The heating procedure was as follows:keeping the temperature for 3 minutes at 120 C,raising the temperature to 180 C at 3 C/min,and then raising the temperature to 210 C at 2 C/min for 4 minutes.Sample volume was 1.0μl,gas flow rate was 1.0ml/min,inlet temperature was 250 C,detector temperature was 280 C,column temperature was 210 C,air,nitrogen and hydrogen flow rates were 400,25 and 30 ml/min,and shunt ratio was 1:20.A method for the determination of monosaccharide composition of Rehmannia glutinosa polysaccharides was established and the methodology was investigated.3.Analysis and determination of main components of rehmannia rehmanniae by multi wavelength fingerprint method.The water extract of cooked Rehmannia glutinosa was prepared by modern method with 5.OOg of cooked Rehmannia glutinosa processed by one steaming,one drying,one drying and nine steaming and nine drying respectively.The interference of sugar was removed by adding anhydrous ethanol to 80%alcohol content.The components of cooked Rehmannia glutinosa were determined by high performance liquid chromatography with different processing techniques.The innovative dual-wavelength fingerprint analysis method(205nm,284nm)was used to maximize the presentation.The chromatographic conditions are as follows:mobile phase acetonitrile(phase A)and 0.02%formic acid water(phase B)gradient elution,gradient elution procedure is 0-5 min,1%-1.5%A,5-12 min,1.5%-3%A,12-17 min,3%-7%A,17-50 min,7%-9.5%A,50-70 min,9.5%-18%-100 min,18%-21%A,flow rate is 1 ml/min,and preparation of pilose and florin.The standard curve of pentahydroxymethyl furfural was used to determine its content.The characteristic peaks of fingerprint were analyzed by LC-MS,and the methodological study was carried out.4.Study on the activity of granulosa cells in ovary of rats treated with rehmannia rehmannii.The total polysaccharides of Rehmannia glutinosa prepared by nine steaming and nine drying,nine steaming and nine baking and modern processing were extracted respectively.Establishment of ovarian granular cell model in,female rats,and the gradient polysaccharide solutions of 25,50,75,100,150,200,250,300,400 μg/ml prepared by different processing methods were given to the cells respectively.MTT method was used to detect the effects of different processing methods on the proliferation of ovarian granular cells after 24 hours of culture.SPSS 20.0 statistical software was used for analysis,and single factor analysis of variance showed that P<0.05 was the significant difference.Results1.Study on different processing technology of rehmannia glutinosal.According to the previous experimental study and consulting the relevant literature,three processing methods were formulated to prepare Rehmannia glutinosa.This experiment used nine steamed and nine sun-cured Rehmannia glutinosa as water-proof and clear steaming for 6 hours,and the next day from 10 a.m.to 5 p.m.as one-time sun-drying.The purpose of nine steamed and nine sun-cured Rehmannia glutinosa was achieved eight times Referring to "Guangdong Provincial Code for Processing Traditional Chinese Medicine 2005 Edition":take raw Rehmannia glutinosa,wash it,moisten it,steam it for 12 hours,take it out by cease fire,bake 80%of it,slice it thickly and dry it.In addition,this study combines the ancient and modern methods of processing,for the first time,nine steamed and nine baked Rehmannia glutinosa,six hours water-proof steaming of the raw land,and four hours baked in a 60 temperature oven as a steaming bake,so eight times to nine steamed and nine baked Rehmannia glutinosa.2.Establishment of determination method for polysaccharide content and monosaccharide composition of rehmannia rehmannii.The content of total polysaccharides in Radix Rehmanniae processed by nine steaming and nine drying was determined by classical phenol-sulfuric acid method.The content of total polysaccharides in Radix Rehmanniae processed by nine steaming and nine drying was the highest,accounting for 1.625%of the total content of medicinal materials(20.00g extraction).According to the conditions of gas chromatography,the separation degree of each chromatographic peak is good,the number of theoretical trays is more than 5000.All three polysaccharides contain arabinose,mannose,glucose,galactose,galacturonic acid and rhamnose.However,the molar ratio of monosaccharides in the prepared Rehmannia glutinosa polysaccharides with different processing technologies is different.The components of monosaccharides in the nine-steamed and nine-sun-dried Rehmannia glutinosa polysaccharides are relatively balanced A:Ara:Man:Glc:Gal:GalA=0.62:0.41:0.72:1.00:3.83:0.61.3.Analysis and determination of main components of rehmannia rehmanniae by multiwavelength fingerprint method.According to the above chromatographic conditions,the characteristic fingerprints of prepared Rehmannia glutinosa were constructed by selecting one peak at 205 nm and 284 nm respectively according to the analysis of high performance liquid chromatography.The fingerprint data were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Edition".The S1 sample spectrum was used as the reference spectrum for calculating and correcting the similarity.The median method was used to generate the contrast spectrum.The similarity calculated at 205 nm wavelength ranged from 0.507 to 0.954,and at 284 nm,from 0.002 to 0.999,the similarity between steaming,drying and nine-steaming and nine-steaming at 205 nm was between 0.201 and 0.970,and between 0.001 and 0.999 at 284 nm.It can be seen that the chemical composition of raw Rehmannia glutinosa during the processing of nine-steaming,nine-steaming and nine-steaming is dynamic.The difference of chemical composition before and after processing is significant.Secondly,the wavelength is between 0.001 and 0.999.The variation of 284 nm was more significant than that of 205 nm.In the main content determination,the content of 5-HMF gradually increased to a stable change in the process of making cooked Rehmannia glutinosa from processed Rehmannia glutinosa to nine steamed Rehmannia or nine steamed Rehmannia glutinosa.The content of 5-HMF in nine steamed Rehmannia glutinosa and nine steamed Rehmannia glutinosa was nearly twice as much as that in modern processed Rehmannia glutinosa.The content of another ingredient,Mulberry Anthocyanin,decreases gradually after different processing methods of raw Rehmannia glutinosa.The content of Mulberry Anthocyanin in modern method and Jiuzhai Jiuxiao Rehmannia glutinosa is in line with Pharmacopoeia regulations.It belongs to Jiuzhai Jiuxiao Rehmannia glutinosa.The content of Mulberry Anthocyanin in Jiuzhai Jiuxiao baked Rehmann In order to further confirm the chemical constituents of common peaks in the extracts of Radix Rehmanniae,the structures and properties of common peaks in the fingerprint of Radix Relumanniae were obtained by UPLC-AB Sciex Triple TOF 5600+time-of-flight liquid chromatography-mass spectrometry using negative ion scanning mode.The ten common peaks selected by double wavelength were levulinic acid,Cistanoside F,rehmannin C,5-HMF,respectively.Capsaicin,echinarin,pyrogalloside A,pilose glycoside,pyrogalloside B,isopilose glycoside.4.Study on the activity of granulosa cells in ovary of rats treated with rehmannia rehmannii.The results of MTT assay showed that ovarian granular cells proliferated differently under the action of different components of ripe Rehmannia glutinosa polysaccharides,showing a dose-effect relationship.When the concentration was 150μ/mL,the proliferation effect was the most remarkable,and the nine-steamed,nine-steamed,nine-baked,nine-steamed,ripe and raw Rehmannia glutinosa polysaccharides at the concentration of 100,150,200 μg/mL were compared with the control.On the other hand,when the concentration was 200 μg/mL,there was a significant difference between Jiuzhaijiuzhaoji polysaccharide and modern method of Jiuzhaijiuzhaoji polysaccharide(p<0.05),which indicated that Jiuzhaijiuzhaojiuzhaoji polysaccharide had a better processing effect,because different molecular weight polysaccharides entered the body’s digestion and metabolism,and the larger molecular weight polysaccharide was difficult to absorb.The results showed that the effect of Polysaccharides from Jiuzhai Jiuzhao Radix Rehmanniae was more remarkable,which also reflected that Jiuzhai Jiuzhao Radix Rehmanniae had better material and pharmacodynamic basis. |
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