| Determine the composition of polysaccharides in Polygonatum sibiricum and the changes of flavonoids content before and after irradiation,and to study the metabolic process of Polygonatum sibiricum flavonoids extract in rats.The samples of Polygonatum sibiricum and Polygonatum odoratum are extracted after ester removal,the obtained polysaccharide is hydrolyzed by hydrochloric acid,derived by PABA,and finally determined by high performance liquid chromatography-fluorescence detector(HPLC-FLD).The monosaccharide was separated on Inertsil ODS-3 C18 column(250mm×4.6 mm,5?m),and the mobile phase consisted of 5 mmol/L ammonium formate(containing 0.3%formic acid and 20 mmol/L tetrabutyl ammonium bisulfate):acetonitrile=97:3.The Excitation wavelength(Ex)is 313 nm,and the Emission wavelength(Em)is 358 nm.The flavonoids in Polygonatum sibiricum were determined by high performance liquid chromatography(HPLC).The mobile phase consisted of0.02%phosphoric acid aqueous solution(A)-acetonitrile(B),gradient elution,column temperature was 43?and flow rate with 1.2 m L/min,the ultraviolet detection wavelength is 270 nm.The flavonoids in rat plasma were determined by high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).The mobile phase consisted of 0.1%formic acid water(A)-acetonitrile(B),73%B was eluted isocratically,the flow rate was 0.4 m L/min and the column temperature was 35?.The mass spectrometer is in negative ion mode,and multiple reaction monition(MRM)is used for quantitative analysis.The results of orthogonal experiment showed that when the derivatization time was 40 min,the derivatization temperature was 70?and the dosage of derivatization agent was 200?L,the derivatization effect of monosaccharide derived from PABA was good,and the extraction with ethyl acetate reduced the peaks of chromatogram and improved the detection sensitivity.Under the determination conditions of this method,fructose,rhamnose,galactose,glucose,mannose,xylose and galacturonic acid have a good linear relationship in their respective linear ranges,R~2>0.997.The established method has high sensitivity and strong specificity,and the recovery rate is between 97.63%and 102.52%,and the determination results are accurate.The main roots and fibrous roots of Polygonatum sibiricum and Polygonatum odoratum contain these seven monosaccharides,and the monosaccharides with higher content are mannose.The monosaccharide content in Polygonatum sibiricum is higher than that in Polygonatum odoratum,and the monosaccharide content in main root is higher than that in fibrous root.PLS-DA analysis can distinguish Polygonatum sibiricum,Polygonatum odoratum and corresponding fibrous root samples.Under the determination conditions of flavonoids in Polygonatum sibiricum,rutin,myricetin,liquiritigenin,quercetin,apigenin,kaempferol and isorhamnetin in Polygonatum sibiricum can be accurately determined under the determination conditions of this method.There is a good linear relationship(R~2>0.999)among the components in the corresponding concentration range,and the determination results are accurate,with the recovery rate of 97.95%-103.71%.There are 7 kinds of flavonoids in the Polygonatum sibiricum,and the content of flavonoids in Polygonatum sibiricum decreases with the increase of irradiation dose.The method of HPLC has the characteristics of high sensitivity and strong specificity,which can be used for the determination of flavonoids in Polygonatum sibiricum.With the increase of dosage,the bioavailability of flavonoids in rats decreased gradually.Seven flavonoids can be analyzed within 7 min,and the minimum quantitative limit is 2 ng/m L.Determination of flavonoids in rat plasma by high performance liquid chromatography-tandem mass spectrometry is fast,sensitive and accurate,which is suitable for the determination of flavonoids in rat.In this study,an innovative analytical method was used to determine the polysaccharides and flavonoids in Polygonatum sibiricum,and applied to the pharmacokinetic study.The research results can provide a feasible basis for improving the quality standard and a reference for the clinical use of Polygonatum sibiricum. |
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