| Eucommia ulmoides Oliv. is a kind of aboundant resources in China. The main active chemical constituents are flavonoids, iridoids, lignans and phenylpropanoids, which are valuable in appliaction. Recent research shows that the valency of flavone aglycone is higher than flavone glycosides. In this dissertation, based on the establishment of the rapid detection method of HPLC, the leaf of Eucommia ulmoides Oliv. was used to prepare the flavone aglycones by using acid hydrolysis. Then their were enriched with polyamides through two-steps. At the same time, the interaction of flavonoids with Bovine Serum Albumin(BSA) were analyzed systematicly in the absence and presence of metal ions. Furthermore, the activities of flavonoids and flavone of Eucommia ulmoides Oliv. were explored in proliferation to splenocytes and production of cytokine. The main contents were as follows:(1)The HPLC method for simultaneous determination of flavonoid aglycones of Eucommia ulmoides Oliv. was established, and the content of flavonoid aglycones of leaf and Eucommin were measured before and after hydrolysis. The optimized determination condition of HPLC was: Diamonsil C18 column(4.6mm×250mm,5μm)as the stationary phase, methanol-0.2% aqueous phosphoric acid solution(63∶37) as the mobile phase at a flow rate of 1.0 mL/min, and the detection wavelength was set at 370 nm, the column temperature was 30℃. Quercetin and kaempferol showed good relationship at the range of 0.5~80μg/mL and 0.5~18μg/mL, respectively. The linear equation of quercetin and kaempferol were Y=41375X-76112, R=0.9997, and Y=9694.9X-67684, R=0.9996. Methodology study results showed that the precision, stability, repeatability and recovery were good enough. The content of quercetin and kaempferol were only 0.021% and 0.005% in the samples of Eucommia ulmoides Oliv.. But after the acid hydrolysis, the contents increased 20 times and 7 times, respectively.(2)The preparation of flavonoid aglycone from the extract of Eucommia ulmoides Oliv. leaves was studied by using acid hydrolysis. And their enriching method by using polyamides through two-steps was established. The optimal hydrolysis parameters were: 6 mol/L of hydrochloric acid hydrolysis for 3h at 80 ℃, and the solid-liquid ratio was 2∶1(mg/mL). Under this condition, the content of flavonoid aglycones reached 2.512%. The content of flavonoid aglycones increased to 12.87% after the first-step of static adsorption by polyamide resin. Then the content of flavonoids glycosides can reach up to 63.19% through the second-step of enriching by polyamide resin. Further more, the content of quercetin could reach 85.27% by column chromatography.(3)The interactions between five flavonoids of Eucommia ulmoides Oliv. with BSA were investigated in the absence and presence of Cu2+、Zn2+、Pb2+、Fe3+ and Mn2+ by using simple spectra techniques under the simulative physiological condications. The results showed that the intrinsic fluorescence of BSA was quenched remarkedably by rutin, quercetin, kaempferol, astragalin and quercitrin. The quenching mechanisms were static quenching procedure. The binding constants’ s order were kaempferol> quercetin> rutin> quercitrin> astragalin, meanwhile the high affinity binding sites number approximated to 1 in different systems, respectively. The thermodynamic analysis suggested that hydrogen bonding force, Vander Waals force and hytrophobic interaction played an important role in different systems. however, the negative sign for ΔG indicated the interaction process was spontaneous in differents systems. Based on the theory of F?rster energy transfer, the energy was transferred by non-radioactive, and the binding distance between quencher and BSA were rutin> quercitrin> astragalin> kaempferol> quercetin. The conformational changes of BSA was observed from UV-Vis spectra, synchromous fluorescence spectra and three-dimensional fluorescence spectra. It revealed polypeptide backbone structure and mico-envrionment of anmio acids had been changed at different degrees, especially for tryptophan. There were no obviously changes for quenching mechanisms and binding sites when the metal ions coexist. But they could change bingding constants, interaction forces and binding distances at different degrees.(4)The methods of MTT and ELISA were used to investigate the effects of flavonoids extracts of Eucommia ulmoides Oliv. on the prolifeation and production of IL-2 and IFN-γ cytokine production of the mouse lymphocytes. The results showed that flavonoid extract of Eucommia ulmoides Oliv. and quercetin could siganificantly upregulate prolifeation and production of cytokines of IL-2 and IFN-γ in lymphocytes stimulated by ConA or LPS. Along with the extension of time, compareing with the blank group, the experimental results changed significantly. The results of multiple significance analysis showed that there was certain dose concentration dependence. However, kaempferol could effectively inhibit proliferation and production of cytokine in a certain concentration range. |
PDF Full Text Request