Studies On Quality Evaluation And Germplasm Resouece Utilization Of Rehmannia Based On HPLC Fingerprints

The purpose of the research is to establish and improve HPLC fingerprint technology ofthree kind of rehmannias, i.e. fresh rehmannia, dry rehmannia and prepared rehmannia, and toassess and control the quality of the crude medicine, as well as to assess the value of the closerelative species of Rehmannia glutinosa for medicine and to breed new varieties with thefingerprint technology.The contents of the research include method exploring of rehmannia HPLC fingerprints,establishing the HPLC fingerprints for fresh, dry and prepared rehmannia. Quality assessmentof the three types of rehmannia, the difference of fingerprint among them, quality control andnew ways to processing prepared rehmannia are also concerned. Chemical fingerprint,usability and biologic characters of close relative species of Rehmannia glutinosa andbreeding new materials are discussed in the end.In order to establish and improve HPLC fingerprint method, single-factor analysis wasadopted. Preparation methods of sample solution and HPLC condition for fingerprint werestudied. The methods were gradually set up. After being soaked in methanol, the sample wasabstracted by ultrasonic. The solution was dried. Residues were dissolved with a little lowconcentration methanol solution and the concentrated sample solution resulted. Acetonitrileand phosphoric acid solution was used as mobile phase for linear gradient elution. HPLCfingerprints at205nm for fresh rehmannia and dry rehmannia, fingerprints at205nm ordouble wavelength fingerprints at205nm and284nm for prepared rehmannia were acquired.At the same time, methods for content determination of catalpol,5-hydroxymethyl furfural(5-HMF) and acteoside, the major constituents in criteria, at single or multiple wavelengthswith fingerprint determination were established. HPLC fingerprints of fresh rehmannia canaccurately reflect species characters. Besides species characters, dry remannia fingerprint canalso show information of climate of producing areas, cultivation mode and storage status etc.But prepared rehmannia fingerprints mainly carry processing information. They reflect theprepared degree of processing.To assess the quality of rehmannia, Well-established HPLC fingerprint technology andsynthesis value of major components in principal component analysis (PCA) were applied toevaluate the quality of dry rehmannia, prepared rehmannia and fresh rehmannia. The resultindicated that the principal components type in PCA of fresh rehmannia and dry rehmanniawas classified on chemical property of chromatographic peaks. While in prepared rehmannia,it was related to the variation characters of peaks in processing. In PCA, dry rehmanniasscored high in synthesis value, i.e. with high quality, were almost from the major producing areas of rehmannia, Henan and Shanxi, or the materials freshly processed. And the synthesisvalue of major components was related to content of catalpol. But it was related to5-HMFand acteoside in prepared rehmannia and fresh rehmannia respectively. And also the methodfor assessing integrated quality of rehmannia was simplified. Only a few of peaks includingprescriptive constituents in criteria were needed to assess integrated quality of rehmannia.The medical nature is different among fresh rehmannia, dry rehmannia and preparedrehmannia. To unveil the essence of the difference, the HPLC fingerprints of three typerehmannia were compared and variation of fingerprint in processing prepared rehmannia wasstudied. The results showed that fingerprint differences between fresh rehmannia and dryrehmannia were mainly in posterior part. It was the area of phenethanol glycosides. Thecontents of phenethanol glycosides were signaficantly decreased from fresh rehmannia to dryrehmannia. Meanwhile, most other secondary components also declined and a few ofderivative peaks appeared and increased. Fingerprint differences between dry rehmannia andprepared rehmannia were almost in forepart. During processing prepared rehmannia, catalpoland some other iridoid glycosides declined or disappeared, while the derivatives such as5-HMF increased. These changes might be one of the reasons for that the medical natureshifted from cold of fresh rehmannia to warm of prepared rehmannia.In order to make the best use of germplasm resource of genus Rehmannia, chemicalfingerprint characters of a part of close relatives of R. glutinosa in the genus, exceptR．chingii, was studied with fresh rehmannia fingerprint technology, as well as pollinationcharacter of species and cross compatibility between them with common cross method. Theresult indicated that it was difficult to determine the relationship in the genus by chemicalfingerprint for their intimate relations. The content of catalpol in the root of three relatives islower than that in Rehmannia glutinosa. But acteoside in R．piasezkii and R．solanifolia arehigher. Even though, the relatives have little possibility to take the place of R. glutinosa inmedicine. Cross results showed that the species were all entomophilous pollination plants.R．solanifolia was infertility species and its origination and classification position wasdiscussed. Reciprocal crosses between the species showed that the cross between R．henryiand R．piasezkii could give germinative seeds. The other cross pairs gave few germinativeseeds.In order to make the best use of germplasm resource of R. glutinosa and to breed newmedical or food materials adapting to local climate, breeding was conducted with naturalcross seeds of varieties and special materials.21materials were selected for medical use byfresh rehmannia fingerprint method and on synthesis value of major components in PCA.10materials were chosen for food use on relative content of total iridoid glycoside which wasdetermined by colorimetry with paradime thylaminobenzaldehyde to present color.