Green And Effective Extraction Of Bioactive Alkaloids From The Roots Of Stephania Tetrandra By Deep Eutectic Solvents-based Ultrasound-assisted Extraction

Green and efficient extraction of active ingredients from traditional Chinese medicine(TCM)and natural drugs plays a crucial role in the corresponding efficacy and follow-up related research.Although traditional extraction methods are simple to operate,they have disadvantages such as long extraction time,low efficiency,high solvent consumption,and degradation of active components.In recent years,a series of new extraction technologies,such as solid phase(micro)extraction,liquid phase(micro)extraction,supercritical extraction and ultrasonic/microwave assisted extraction,have achieved rapid development and successfully applied to the quality analysis of traditional Chinese medicine and natural medicine.At present,the active ingredients are mainly extracted using organic solvents such as methanol,ethanol and chloroform,most of which have the disadvantages of high toxicity,high cost,flammability,volatility and low extraction yield.Deep eutectic solvents(DESs),as an emerging group of green solvents and formed by mixing a hydrogen bond acceptor(HBA)and a hydrogen bond donor(HBD)at an appropriate molar ratio,show the significant applications in many fields because of their attractive properties such as inexpensive,easy preparation,ecofriendliness,low toxicity,less volatility,high dissolution power,feasibility of structural design,sustainability and biodegradability.DESs have been applied for the extraction,separation and purification of bioactive substances(to the best of our knowledge,especially in the extraction of phenolic components,flavonoids and polysaccharides,but alkaloids are rarely reported so far)from TCM.In this dissertation,a DESs-based ultrasonic-assisted extraction(DESs-UAE)procedure was established for highly efficient and green extraction of alkaloids from the roots of Stephania tetrandra(ST).Additionally,a four-factor and three-level Box-Behnken design(BBD),a particular pattern of response surface methodology(RSM),was used to optimize extraction conditions.Compared with traditional organic solvents(methanol and 95% ethanol)and water,,the green and efficient extraction of alkaloid active components were realized.Furthermore,a variety of chromatographic separation techniques were used to separate and purify the alkaloids from the DESs extracts of ST.As a results,a total of 5 compounds were isolated,2 of which are new compounds,and their structures were determined by spectroscopic data.The main research contents are as follows:(1)In this work,an environment-friendly DESs-based ultrasonic-assisted extraction(DESsUAE)procedure was developed for highly efficient and non-polluting extraction of alkaloids from the roots of ST.A total of fifteen different combinations of DESs(alcohol-based,amide-based,sugar-based and organic acid-based),compared with traditional organic solvents(methanol,95%ethanol and water),were evaluated for extraction of bioactive alkaloids(Fangchinoline and Tetrandrine)from ST,and the results revealed that DESs system made up of choline chloride and ethylene glycol with mole ratio of 1:2 exhibited the optimal extraction efficiency for alkaloids.It can be found that alcohol-based DESs exhibited relatively higher extraction efficiencies for Fangchinoline and Tetrandrine,followed by amide-based DESs,methanol,95% ethanol,water,and sugar-based DESs.It is worth noting that organic acid-based DESs presented the lowest extraction capacity compared with other types of HBD.According to the results of single-factor experiments(including molar ratios of DESs,ultrasonic power,extraction temperature,extraction time,liquidsolid ratios,and water contents in DESs),the optimal extraction conditions for maximizing the alkaloids were obtained with the help of response surface methodology(RSM).RSM results indicated that the maximum extraction yields of Fangchinoline,Tetrandrine,and total alkaloids were attained 7.23,13.36,20.59 mg/g,respectively,within extraction temperature of 52 °C,extraction time of 82 min,DESs water content of 23%(v/v),and liquid-solid ratio of 23 m L/g.The measured results were consistent with the predicted values.Notably,the optimized DES extraction efficiency of total alkaloids,according to the experimental data analysis,is 2.2,3.3 and 4.1 times higher than methanol,95% ethanol and water,respectively.In addition,we also discussed the effects of different types of macroporous resins(NKA-9,AB-8 and D101)on the enrichment and recovery of alkaloids from DESs extract under the optimum extraction conditions.The results showed that the enrichment recovery of AB-8 macroporous resin was the highest(89%).The above results showed that DESs,as a new type of green solvent,possessing the potential to replace traditional organic solvents and water,and can be widely used in the extraction of other active components of TCM and natural medicine.(2)Additionally,in order to further explore the structural unique and biologically interesting alkaloids,we carried out the phytochemical investigation on the total alkaloids extracted by DESs,which led to the isolation and identification of 5 alkaloids.They are Fenfangjian A(FJ-1),Fenfangjian B(FJ-2),(R)-2-propylhexyl-2H-1,2,3-triazole-4-carboxylate(FJ-3),Tetrandrine(FJ-4)and Tetrandrine(FJ-5),respectively.Among them,FJ-1 and FJ-2 are 2 new compounds,all of which are bisbenzylisoquinoline alkaloids.FJ-3 is Aspergillus fumigatus triazolyl ester A,which was isolated from the roots of ST for the first time.Based on the above phytochemical investigation,the in vitro anti-inflammatory activities of the isolates were also discussed.As a result,FJ-1 and FJ-2 displayed favorable inhibitory effects on lipopolysaccharide(LPS)-induced NO production in RAW 264.7 cells.