Ultrasonic-assisted Ionic Liquid Extraction Of Biflavonoids From Selaginella Tamariscina (Beauv.) Spring And Ginkgo Biloba L. And Their Antioxidant Activity

Objective:Selaginella tamariscina（Beauv.）Spring and Ginkgo biloba L.,two traditional Chinese medicines,contain a variety of bioactive components such as biflavonoids,phenolic acids.Biflavonoids are one of the active ingredients in S.tamariscina and G.biloba,have anti-oxidant,anti-tumor and anti-inflammatory effects.In this paper,the aim of this study is to establish an analytical method for simultaneous determination of various biflavonoids in two plants,to optimize the extraction process of biflavonoids with ionic liquid（IL）as a green solvent and investigate the antioxidant activities of the two extracts,which provide the basis for better application of S.tamariscina and G.biloba.Methods:1.High performance liquid chromatography（HPLC）was adopted to detect the content of amentoflavone（AME）and hinokiflavone（HIN）in S.tamariscina,and bilobetin（BIL）,ginkgetin（GIN）,isoginkgetin（IGIN）and sciadopitysin（SCI）in G.biloba.2.Six ILs were synthesized by one-step method using benzoxazole and six different types of acids or acid salts as raw materials.The structure of the synthesized ILs were characterized by elemental analysis,infrared spectroscopy and nuclear magnetic resonance,and the thermal stability of the ILs were evaluated by thermogravimetric analysis.3.In this paper,the optimal extraction process was explored of AME and HIN from S.tamariscina and BIL,GIN,IGIN and SCI from G.biloba by ultrasonic-assisted ionic liquid extraction（UAE-IL）.Firstly,the effects of six benzoxazoles,six pyridines and five imidazoles ILs on the extraction of major biflavonoids from two plants were screened simultaneously,and the best ionic liquid in the extraction was selected.Then,single factor experiments were used to optimize the concentration of ionic liquid,solid-liquid ratio,ultrasonic power,ultrasonic time and extraction times.After the evaluation of several parameters,the main influencing factors on the extraction rate of biflavonoids were further analyzed by response surface analysis（RSM）.Moreover,the comparison with traditional extraction methods,and the recovery and reuse of IL were explored in detail.4.After evaluating the optimized extraction process,the antioxidant activity of the two extracts obtained in the two plants were actively investigated by four different antioxidant methods（DPPH radical-scavenging,ABTS radical-scavenging,metal ion chelating method and iron ion reduction method）in vitro.Results:1.Observing the chromatograms of the extracts of S.tamariscina and G.biloba,we found that the chromatographic peaks of the main biflavonoids had reached baseline separation,and the retention time were basically the same as that of their standards;the reference standards of AME,HIN,BIL,GIN,IGIN and SCI were in the range of 0.041⁰.244 mg/mL,0.040⁰.243 mg/mL,0.040⁰.239 mg/mL,0.041⁰.245 mg/mL,0.041⁰.245 mg/mL and 0.041⁰.247 mg/mL,respectively.The linear relationship between mass concentration and peak area was good,R² were more than 0.999;the RSD of the experimental results based on methodology investigation were all less than 2.0%,and the average recovery rates were 97.15%,97.73%,97.52%,97.17%,98.33%,respectively.It showed that this analytical method could be applied to the determination of the major biflavonoids in S.tamariscina and G.biloba.2.Six benzoxazole ILs were synthesized for the first time:[HBox]CH₃SO₃,[HBox]CF₃SO₃,[HBox]p-Ts,[HBox]PF₆,[HBox]Br and[HBox]CF₃COOH;Thermogravimetric analysis was used to evaluate the thermal stability of six ILs.It was found that the thermal stability of ILs were not related to the benzoxazole ring,but to the anion species.the order of thermal stability was:p-Ts^->CHSO₃^->CF₃HSO₃^->PF₆^->Br^->CH₃COO^-.3.In order to optimize the analytical conditions of UAE-IL for major biflavonoids from the two plants,the best IL with the best extraction effect should be selected first,[Bpy]BF₄was the best for S.tamariscina,and[Epy]BF₄ was the best for G.biloba;The optimal level of each influencing factor was investigated by single factor experiments and response surface analysis.The optimal extraction conditions of two biflavonoids from S.tamariscina were as follows:IL concentration was 0.15 mol/L,solid-liquid ratio was 1:12g/mL,ultrasonic power was 280 W,ultrasonic time was 30 min,and extraction times were3 times.The optimal extraction conditions of four biflavonoids from G.biloba were as follows:IL concentration was 0.15 mol/L,solid-liquid ratio was 1:14 g/mL,ultrasonic power was 280 W,ultrasonic time was 25 min,and extraction times were 3 times.The extraction rates of AME and HIN in S.tamariscina were 13.51 mg/g and 6.74 mg/g respectively.The extraction rate of BIL,GIN,IGIN and SCI in G.biloba were 2.44 mg/g,4.33 mg/g,6.50 mg/g and 13.97 mg/g,respectively.The experimental results of recovery of IL on the extraction of biflavonoids showed that the recovered IL could repeatedly extract biflavonoids for six times and the extraction rate were about 90%,which indicated that the IL could be effectively recovered and reused.4.The antioxidant activities of S.tamariscina and G.biloba extracts were investigated by four antioxidant methods.Among them,IC₅₀ of DPPH radical-scavenging from S.tamariscina and G.biloba were 39.95μg/mL and 35.05μg/mL,respectively,and the maximum scavenging rates were 88.435%and 88.53%,respectively.IC₅₀ of ABTS radical scavenging from the two plants were 45.59μg/mL and 46.18μg/mL,respectively,and the maximum scavenging rates were 95.71%and 91.29%,respectively.The maximum absorbance of the extracts of S.tamariscina and G.biloba by the iron ion reduction method were 1.757 and 1.653,EC₅₀ were 50.39μg/mL and 46.35μg/mL,respectively.The chelation rates of metal ions of the extracts from the two plants were94.49%and 97.54%,respectively.The results showed that the two extracts had certain free radical scavenging,metal ion chelation and reduction power.Conclusion:In this study,a method was established for the determination of major biflavonoids in S.tamariscina and G.biloba.Six benzoxazole ILs were synthesized and have good thermal stability.The single factor experiments and response surface methodology were used to optimize the extraction process of major biflavonoids from S.tamariscina and G.biloba by UAE-IL and the optimal extraction conditions were obtained.Furthermore,the recycling of IL was realized.Finally,antioxidant experiments showed that the extracts of S.tamariscina and G.biloba had certain antioxidant activity.