Study On Solid-state Fermentation Process And Electron-beam Irradiation Of Hypericum Perforatum L.

In the extraction process of traditional Chinese medicine, it is necessary to overcome the dual resistance of intercellular substance and cell wall when the active components diffuse out of protoplast into extraction medium. In Hypericum perforatum L, the active ingredients have been wrapped in the cell wall which consistuted by cellulose, hemicellulose and lignin, and so this is unfavorable to the extraction of the effective components. Based on the fact that cellulase can promote effective ingredient extraction and separation, the contents of effective components, hyperincin and hyperin, were increased indirectly through the cellulase degradation of coarse fiber in the solid-state fermentation with Aspergillus niger in this study.The purpose of this thesis is to increase the hypericin and hyperin contents by fermentation with Aspergillus niger. First, the high performance liquid chromatography (HPLC) method of hypericin and hyperin were established. And then, the fermentation process was optimized. Finally, the irradiation of Hypericum perforatum L and its leavening by electron-beam was investigated.1. Development of reverse phase HPLC method of hypericin. The optimized HPLC parameters as follows: Separations were achieved on a Agilent HC-C18(5μm, 4.6 mm×250 mm)column at 25℃, and the mobile phase used was 0.30 mol/L ammonium acetate buffer (adjusted the pH to 3.85 with acetic acid), methanol and acetonitrile (10︰40︰50) at a 1 mL/min flow rate . Hypericin was injected 20μL and detected at 588nm. The rentention time was 11 min, and the carlibration equation was Y=3×10~6X-144070(r=0.9999). The linear range and the spiked rescovery were 0.0512μg/mL～10.0μg/mL and 94.55%-101.07% respectively. Other parameters, for example, stability, precision and repeatability etc. were all in conformity with the requirement of"Pharmacopoeia of the People's Republic of China". The established method was more than enough to meet the requirements for the determination of hypericin.2. Development of reverse phase HPLC method of hyperin. The optimized HPLC parameters as follows: Compounds on the column (Hypersil ODS2, 5μm, 4.6 mm×250 mm) were separated with a mobil phase comprising acetonitrile: tetrahydrofuran : 0.1% Phosphoric acid (17︰10︰73). The analyte was detected using the wavelength of 360 nm at 25℃. Under these conditions, hyperin was eluted from the column at 8.6 min. The folw rate and injection volume were 1.0 mL/min and 20μL respectively. The calibration equation [Y = 473282X + 3×10~6 (r=0.9989)] with the linear range of 6.547μg/mL～50.520μg/mL was ploted, and the fortified recovery ranged from 95.03% to 100.32%. Other parameters, were all in conformity with the regulation, and suitable for the detection the hyperin content in whole plant.3. Fermentation of Hypericum perforatum L. by Aspergillus niger. The single-factor test was designed to investigate the effects of seven factors of solid fermentation temperature, initial pH, time, solid-to-liquid ratio, glucose content, inoculation and the volume of surfactant Tween80 on the contents of hypericin and hyperin. And the optimum fermentation process was determined after investigation five factors of initial pH, solid-to-liquid ratio, inoculation and glucose content by L16(45)orthogonal test. The obtained optimum process as follows: 10 g Hypericum perforatum L. was weighed after the irradiated sterilization, added 20 mL Mandels medium (5% glucose content, pH 5.5), and then inoculated with 1 mL seed solution. The fermentation was carried out for 96 h (24 h at 30℃and then 72 h at 25℃). The results showed that the crude fiber content decreased by 16.74%, hypericin increased by 22.79 mg (24.02 %), and an increase of hyperin content of 76.68 mg (19.56%).4. Study of the electron-beam irradiation on the fermentation product. Above all, the effect of variation in irradiation dose on the contents of hypericin and hyperin in Hypericum perforatum L was investigated, and the D10 values were calculated on the basis of the linear relationship between the logarithmic value of surviving microorganism and radiation dose. Furthermore, the microbial growth in the fenmentation product was also evaluated with the irradiation dose of 6.0 kGy. The result showed the samples could be conserved 60 days with the optimum irradiation dose of 6.0 kGy, and all the hygiene parameters meet the standard of"Pharmacopoeia of the People's Republic of China". Finally, by comparing the Hypericum extract 2 and 4, the total amount of hypericin was found to be significantly increased after the irradiation. The amount of hypericin has been increased by 11.08 mg (9.42%). And the contents of hypericin and hyperin could be increased to the maximal extent under combined action of Aspergillus niger fermentation and electron-beam irradiation by comparison of Hypericum extract 1 and 3. The hypericin and hyperin amounts were increased by 33.87 mg (35.71%) and 82.93 mg (21.15%) respectively.For the sake of improving the extraction yield, it is the first time to discrible the fermentation of Hypericum perforatum L with Aspergillus niger and the irradiated decontamination by electron beam. In this thesis, the parameters of solid-state fermentation were optimized and it was also found the best appropriate dose of electron-beam irradiation for significantly increase of the contents of effective components hypericin and hyperin. These studies conducted will provide the new method for the development and application of traditional Chinese veterinary medicine.