Studies On Separation And Purification And Annual Dynamics Of Echinacoside From Penstemon Barbatus (Can.) Roth And Bioengineering Of The Enzymes Involved In The Biosynthesis Of Echinacoside

Echinacoside was known not only as a caffeic acid derivative, but also as a phenylethanoid glycoside. It possesses a spectrum of beneficial activities, such as antioxidation, anti-inflammatory, antineoplastic, neuroprotective and hepatoprotective activities. Moreover, this compound can boost the immunoprotective efficacy. Echinacoside was firstly reported to be extracted from Echinacea angustifolia DC with a content around 0.2% and subsequently prepared from the aerial part of Penstemon crandallii A. Nels with a content of 0.09%. Currently, echinacoside is largely extracted from Cistanche tubulosa, a perennial parasitical plant on the roots of Haloxylox ammodendron and H. persicum, with the content of no less than 1.0%. The content of echinacoside is the only quality standard for the herbal medicine of C. tubulosa in Pharmacopoeia of the People's Republic of China. C. tubulosa is a slow-growing plant endemic to arid lands and deserts and recalcitrant to domestication. Increasingly demands for the soaring medication value of echinacoside further endangered C. tubulosa. New alternative source other than C. tubulosa for echinacoside is urgent both for commercial demands and environmental protection.Taking the adventage of high performance liquid chromatography, this research was focusing on the screening of the alternative source that was abound in echinacoside. High-speed counter-current chromatography was imployed for the rapid separation and purification of echinacoside. The structure of the purified echinacoside was confirmed by IR,¹H NMR and ¹³C NMR. In order to enhance the content of echinacoside, the secondary metabolism of the biosynthetic pathway of echinacoside was studied by means of molecular biology as well. The main results are listed as follows:1. The annual dynamics of echinacoside in the leaves and the roots of P. barbatusBy screening, echinacoside was firstly reported to be isolated from P. barbatus in this study. The content of echinacoside varied within one year and the peak contents of it were 9.09±0.32 mg/g in the leaves and 7.25±0.36 mg/g in the roots annually. The result indicated that the content of echinacoside in P. barbatus was almost the same as that in C. tubulosa. Potentially, it might be an ideal source for preparation of large scale of echinacoside.2.42.0 mg echinacoside with a purity of 96.3% was isolated from the root of P. barbatus by recycling high-speed counter-current chromatographyEchinacoside was isolated from P. barbatus by HSCCC for the first time. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography and recycling high-speed counter-current chromatography with the solvent system n-butanol-water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR,¹H NMR and ¹³C NMR is identical to the standard sample. The result demonstrated that echinacoside could be rapidly and efficiently isolated from P. barbatus by conventional high-speed counter-current chromatography and recycling high-speed counter-current chromatography.3. Cloning and expression characterization of 3-dehydroquinate synthase cDNA from E. AngustifoliaTotal RNA was extracted from tissue cultures of E. Angustifolia with Trizol method. A cDNA fragment of 289 bp was amplified by RT-PCR with a pair of degenerated primer designed according to the reported conserved amino acid sequence of 3-dehydroquinate synthase (aroB). The cDNA of aroB, named EanaroB (GenBank No. EU293857), of E. Angustifolia was obtained by Rapid amplification of cDNA ends and sequence spliced based on the known cDNA fragment. Sequence analysis indicated that EanaroB is 1424 bp and contains a 1326 bp open reading frame encoding a 442 predicated amino acid residues which shared arround 80% identity with other plant aroB, such as Lycopersicon esculentum, Vitis vinifera, Fagus sylvatica and Arabidopsis thaliana. Analysis of semi-quantitative RT-PCR indicated that aroB gene was transcribed in leaf and flower, less in root and stem.4. Construction of plant high efficient expression vector pCAMBIA2301 G-EaC4Hl and the obtainment of bioengineering agrobacteriumpMD18-T--EaC4H1 and intermediate vector pCAMBIA2301G were digested by Sac I and Xba I. Since there was a restriction endonuclease site within the coding region of EaC4Hl,4 bands of pMD18-T-EaC4H1 were obtained by incomplete degistion. The target gene EaC4Hl of 1612 bp and the large fragment of pCAMBIA2301G of 12 kb were recovered and ligated. The recombinant plasmid was transformed into Agrobacterium tumefaciens strain LBA4404. This work was approved by PCR and DNA sequencing. The pCAMBIA2301G-EaC4H1-carrying LBA4404 could be then used for plant transformation. This work underlay the first step for studing the biosynthetic pathway of caffeic acid derivatives and improving the content of echinacoside in E. angustifolia and P. barbatus.