Extraction,Isolation,Purification And Antioxidant Activity Of Flavonoids From The Forest Of P. Kingianum Coll.

Polygonatum kingianum Coll.et Hemsl.is a plant of the lily family and its rhizome is a medicinal herb used for both food and medicine.The excavation and evaluation of the active ingredients in Polygonatum kingianum Coll.is an important tool to enhance the comprehensive utilization of PK.In this study,the extraction and purification process of total flavonoids was optimized,and nine flavonoid components were isolated and identified by spectral mass spectrometry.The in vitro antioxidant and hypoglycaemic activities were further clarified,providing theoretical and technical support for the further development and utilisation of the total flavonoids of PK.The main findings are as follows:（1）Optimization of the extraction process of total flavonoids from PKCombining the single-factor test with Box-Behnken response surface design method,the effects of four factors,namely,ethanol volume fraction,extraction temperature,extraction time and material-liquid ratio,on the extraction rate of total flavonoids were investigated and found to be material-liquid ratio>ethanol concentration>extraction temperature,respectively.The best extraction process and parameters for the total flavonoids of PK were determined:ethanol concentration of 79%,extraction temperature of 61℃,extraction time of 2 h,material-liquid ratio of 1:20（g/m L）,and the extraction rate reached 0.29%.Based on the optimum extraction process,the total flavonoid content of 11 different regions of Yunnan,including Baoshan and Wenshan,was determined.The results showed that the highest total flavonoid content of 3.307±0.036mg/g was obtained from Kunming PK（Safflower）,which was therefore selected for further purification and identification studies.（2）Purification and structural identification of total flavonoids from PKThe optimum process parameters were screened by optimization of the separation and purification parameters.The adsorption and desorption rates were studied,and it was found that among the seven macroporous resins D101,AB-8,NKA-II,NKA-9,HPD-400,HP-20,X-5 and polyamide,AB-8 resin was the best choice for the adsorption（63.02±1.50%）and desorption（89.62±0.24%）of the total flavonoids of PK.The optimum conditions for static adsorption and desorption were determined as follows:adsorption equilibrium time of 240 min,adsorption temperature of 30℃,upper sample concentration of 40mg/m L;desorption equilibrium time of 30 min and ethanol elution concentration of 70%.The optimal process parameters for dynamic adsorption and desorption of AB-8 macroporous resin were determined as follows:loading flow rate of 1.0 m L/min,loading volume of 70 BV,elution flow rate of 1.5 m L/min and 5BV 70%ethanol for elution of the resin.The total flavonoid content of PK was the highest with a purity of5.31%,which was 11.8 times higher than the purity of the crude extract.A combination of UV absorption spectroscopy,IR absorption spectroscopy and ultra performance liquid chromatography high resolution tandem mass spectrometry identified the purified nine flavonoid components as（6αR,11αR）-10-Hydroxy-3,9-dimethoxy-pterocarpan,2’,7-Dihydroxy-3’,4’-dimethoxy-isoflavan,Neoliq-uiritin,3’-Methoxydaidzein,Neoisoliquiritin,Myricetin,Liquiritigenin,Isoliquiritigenin and Kaempferol.The content of six flavonoids was quantified by high performance liquid chromatography（HPLC）at an absorption wavelength of 275 nm,with Neoliquiritin（7.12）,Neoisoliquiritin（11.776）,Myricetin（1.16）,Liquiritigenin（371.41）,Kaempferol（30.14）,Isoliquiritigenin（1.91）μg/g.Further activity evaluation studies were carried out on the purified total flavonoids.（3）Evaluation of antioxidant and hypoglycemic activities of total flavonoids from PKIn this experiment,the in vitro antioxidant activity of the flavonoids in PK was evaluated by measuring the clearance of DPPH,ABTS,hydroxyl and superoxide anion antioxidant indexes using Vc as control.The clearance rates were:82.44%,86.22%,73.25%and 75.69%respectively.The results of comparing the scavenging ability of flavonoids and polysaccharides of PK at the same concentration showed that the scavenging rates of DPPH and ABTS by the purified extract of PK were 79.44%and 75.22%with IC₅₀values of 2.152 mg/m L and 6.94 mg/m L,respectively;the scavenging rates of DPPH and ABTS free radicals by the polysaccharides of PK were 68.41%and The scavenging rates of flavonoids on DPPH and ABTS radicals were found to be higher than those of PK.The total flavonoids and polysaccharides of PK were rationed and the highest radical scavenging activity of DPPH and ABTS was observed at the same concentration when the ratio of polysaccharides to flavonoids purified was 1:9,with the scavenging rate of76.94%and 68.60%respectively,and the scavenging rate of free radicals by purified C.elegans flavonoids was much higher than that before purification,with IC₅₀values of 3.00 mg/m L,5.61 mg/m L,5.61 mg/m L and 5.61 mg/m L respectively,The IC₅₀values were 3.00 mg/m L,5.61 mg/m L,4.80 mg/m L and 3.51mg/m L,respectively.The inhibition rates ofα-glucosidase andα-amylase activities were determined and the IC₅₀values of purified and crude extracts of PK were found to be 1.09 mg/m L and 1.23 mg/m L,respectively.The results of this study indicate that the purified flavonoids of PK extract have higher antioxidant activity and hypoglycaemic activity,and the findings of this study provide data reference and technical support for the evaluation of the activity of PK extract.