Study On Separation, Analysis, Identification And Activity Of Bioactive Compounds From Flaveria Bidentis (L) Kuntze

Flaveria bidentis (L.) Kuntze was one of the most harmful invasive weedin China. It has very strong reproductive and survival abilities, and spreadsfast. Compared with simple controlling methods, utilization of the weed inuseful purposes might give more benefits. In order to utilize and control F.bidentis, bioactive compounds from F. bidentis should be deeply studied. Themost important point is separation and purification. Counter-currentchromatography (CCC), being a support-free liquid–liquid partitionchromatography, eliminates irreversible adsorption of sample onto the solidsupport, and has been widely used in preparative separation of naturalproducts. CCC is an advanced technique that it needs minimum samplepre-treatment and cleanup procedures, and permits many elution modes. Inaddition, a series of new CCC assays and apparatus extends the applicationfields of CCC and improves the separation efficiency of CCC separation and devices. CCC was mainly used for the separation of bioactive compoundsfrom F. bidentis in this thesis.In this thesis, multiple separation and analysis methods were studied forthe bioactive compounds from F. bidentis. The optimized extraction method,CCC separation methods of10compounds, HPLC analysis of the maincompounds and fingerprint of F. bidentis leaves were established. In addition,the antioxidant properties of the main flavonoids were estimated. The mainresearch results were as follows:1. The optimized extraction method of total flavonoids from F. bidentiswas established by single factor and orthogonal design experiments: F.bidentis powder was extracted (refluxed) with80%(v/v) ethanol for75min (2times). The total flavonoids in F. bidentis samples from different origins,different parts of the plant and different growing periods were tested. Resultsshowed that the highest flavonoids contents were flower and leaf, the bloomperiod and the leaf sample from Hebei province.2. CCC and preparative HPLC (pre-HPLC) were successfully used for theseparation of four main flavonol glycosides from F. bidentis, and the fourcompounds were identified by ESI-MS,¹H NMR,¹³C NMR,HSQC andHMBC. The separation efficiency of the high-speed counter-currentchromatography (HSCCC) was compared with the high performancecounter-current chromatography (HPCCC). Using GS10AB-A HSCCC bythe two-phase solvent system ethyl acetate-methanol-water (25:1:25, v/v), about400mg of the crude extract was separated, yielding3.6mg ofpatuletin-3-O-glucoside at a purity of over97%;4.4mg of astragalin at apurity of over98%and4.2mg of a mixture of quercetin-3-O-glucoside and6-methoxykaempferol-3-O-glucoside constituting over97%of the fraction.Then the mixture was separated by pre-HPLC. Four pure flavonol wereseparated using dichloromethane-methanol-water (5:3:2, v/v) by HSCCC.3. The first preparative separation of a flavonoid sulphateisorhamnetin-3-sulphate from F. bidentis by counter-current chromatography(CCC) was presented. Two kinds of solvent systems were used. Aone-component organic/salt-containing system composed of n-butanol-0.25%sodium chloride aqueous solution (1:1, v/v) was used. As a result,2.1mg ofisorhamnetin3-sulphate with a purity of over97%has been isolated from400mg of crude extract without pre-enrichment. Compared with the conventionalorganic/aqueous system, the one-component organic/salt-containing aqueoussystem was more suitable for the separation of isorhamnetin-3-sulphate, andpurer target compound was obtained from the crude extract withoutpre-enrichment using the new solvent system.4. A simple and sensitive high performance liquid chromatography methodcoupled with photodiode array detection (HPLC-DAD) was developed forsimultaneous determination of6major constituents in F. bidentis. The contentsof the6compounds in F. bidentis samples from different origins, differentparts of the plant and different growing periods were tested. The chemical fingerprint of F. bidentis leaves was established using raw materials of12batches in China. The results indicate that this multi-component determinationmethod in combination with chromatographic fingerprint analysis is suitablefor quantitative analysis and identification of F. bidentis.5. Astragalin which content was the highest in F. bidentis was developedas a national reference material. A gram scale of astragalin was separated bycontinuous injection HSCCC. It was identified by many methods and thepurity was determined by HPLC. The uniformity and stability were inspected,and results fitted the requirement of reference material. The definite value wasfinally determined by combined value determination, and was98.49%.6. The antioxidant properties of the main flavonoids were estimated.DPPH scavenging activity, T-AOC, superoxide radicals scaving effect andhydroxyl radicals scaving effect were tested. Results showed that the5flavonoids processed a certain ability of antioxidation.