Study On Enzyme-assisted Extraction Of Hypericin From Hypericum Perforatum L And Its Microwave-assisted Synthesis

In the herbal plant Hypericum perforatum L, hypericin is a low-content naphthodianthrone with anti-tumor and antiviral activities. Since many deficiencies existed in the currently used extraction methods, the extraction rate of hypericin is very low. In this study, cellulases secreted from Aspergillus niger and Trichoderma viride were improved by using of electron and heavy-ion beams, then used as a cell-wall degrading agent for improvement of the extraction rate of this compound and so reduced the waste of Hypericum perforatum L. Furthermore, microwave-assisted synthesis has been introduced in the synthetic route of hypericin to minimise by-products and improve yield. The research works performed were as follows.(1) The fermentation conditions of Aspergillus niger GSICC 60108 and Trichoderma viride GSICC 62010 were optimized. The conditions for Aspergillus niger fermentation were optimized as: culture time 72 h, inoculation rate 8.0%, incubation temperature 28℃, initial pH 4.5 and 1.0% Tween-80 addition. The optimum carbon and nitrogen sources were respectively straw powder and the mixture of ammonium sulfate and wheat bran in the proportion of 1:5. For Trichoderma viride, the optimized incubation temperature and initial pH were 35℃and 5.0, respectively. Other conditions were the same as Aspergillus niger.(2) The high-throughput screening methods for cellulase-producing microorganisms were established, using cellulose-Congo red plate for primary screening and a new microplate-based method for secondary screening. The regression equation for the established secondary screening method was Y=0.208X+ 0.0006, with a linear correlation coefficient of 0.99975 in the concetation range of 0 mg/mL-10 mg/mL. The accuracy, stability and repeatability of the secondary screening method were acceptable for the high-throughput screening. It did not show any significant difference when compared with the international standard method for determination of filter paper activity.(3) Sigle spore suspensions prepared from the parental strains of Aspergillus niger GSICC 60108 and Trichoderma viride GSICC 62010 were irradiated by electron beam. The lethal doses for Aspergillus niger and Trichoderma viride spores were respecitively 1000 Gy and 750 Gy. After the determination of mutation frequencies and filter paper activities of the spore solutions with different irradiation doses, it was found that the highest positive mutation rates (12.0% for Aspergillus niger and 11.0% for Trichoderma viride) and highest filter paper activities were determined with the radiation doses of 750 Gy and 500 Gy, respectively. The R values for two obtained mutants Aspergillus niger EBA 105 and Trichoderma viride EBT 18 were respectively 3.9 and 3.5. For Aspergillus niger EBA 105, the filter paper activity was 152.5 FPU/mL, the endoglucanase activity was 91.4 IU/mL, the exoglucanase activity was 29.6 IU/mL and theβ-glucosidase activity was 153.3 IU/mL. These values were increased by 2.6 times-3.2 times when compared with parent strains. For Trichoderma viride EBT 18, the filter paper, endoglucanase, exoglucanase andβ-glucosidase activities were 186.9 FPU/mL, 108.2 IU/mL, 61.3 IU/mL and 153.3 IU/mL, respectively. The enzyme activities were increased by 2.6 times-3.2 times compared with Trichoderma viride GSICC 62010. The cellulase gene sequence analysis of the mutants showed that the mutations were mainly point mutations.(4) In order to obtain the mutants with higher cellulase activities, Aspergillus niger EBA 105 and Trichoderma viride EBT 18 were irradiated by ¹²C⁶⁺-ion beam once again. The lethal doses for these two mutants were respecitively 150 Gy and 125 Gy. It was found that the highest positive mutation rates (11.0% for Aspergillus niger and 16.0% for Trichoderma viride) and highest filter paper activities were determined with the radiation doses of 80 Gy and 70 Gy, respectively. The R values for two obtained mutants Aspergillus niger CIA 915 and Trichoderma viride CIT 626 were respectively 5.3 and 5.5. For Aspergillus niger CIA 915, the filter paper activity was 431.6 FPU/mL, the endoglucanase activity was 310.8 IU/mL, the exoglucanase activity was 106.2 IU/mL and theβ-glucosidase activity was 95.9 IU/mL. These values were increased by 2.7 times-3.6 times when compared with Aspergillus niger EBA 105. For Trichoderma viride CIT 626, the filter paper, endoglucanase, exoglucanase andβ-glucosidase activities were 535.3 FPU/mL, 336.5 IU/mL, 231.8 IU/mL and 437.6 IU/mL, respectively. The enzyme activities were increased by 2.9 times-3.8 times compared with Trichoderma viride EBT 18. After the sequence analysis of cellulase genes of the mutants, it was demonstrated that the main mutation type was point mutation.(5) The two mutants Aspergillus niger EBA 105 and Trichoderma viride EBT 18 were also irradiated by ²⁰Ne¹⁰⁺-ion beam. The lethal doses were in order of 125 Gy and 100 Gy. It was found that the highest positive mutation rates (9.0% for Aspergillus niger and Trichoderma viride) and highest filter paper activities were obtained with the radiation doses of 90 Gy and 80 Gy, respectively. The two mutants Aspergillus niger NIA 301 and Trichoderma viride NIT 385 were screened with respective R values of 3.6 and 4.2. For Aspergillus niger CIA 915, the filter paper activity was 293.8 FPU/mL, the endoglucanase activity was 176.5 IU/mL, the exoglucanase activity was 61.3 IU/mL and theβ-glucosidase activity was 68.1 IU/mL. These values were increased by 1.9 times-2.1 times when compared with Aspergillus niger EBA 105. For Trichoderma viride CIT 626, the filter paper, endoglucanase, exoglucanase andβ-glucosidase activities were 325.2 FPU/mL, 183.5 IU/mL, 110.2 IU/mL and 275.8 IU/mL, respectively. The enzyme activities were increased by 1.7 times-1.8 times compared with Trichoderma viride EBT 18. The cellulase gene sequence analysis of the mutants demonstrated that point mutation was the main mutation type.(6) After selection of the high-yileld cellulase strains, the two crude enzyme solutions from Aspergillus niger CIA 915 and Trichoderma viride CIT 626 were refined and the mixed proportion of the two solutions were determined as 1:6 to produce a maximum filter paper activity. The optimum extraction conditions were summaried as: pH value 5.0, incubation temperature 40℃, incubation time 6 h, enzyme concentration 150.0 FPU/g and solid-liquid ratio 1:2. The resulted extraction yield of hypericin was up to 166.5μg/g, increased by 46.7% in comparation with ultrasonic extraction method. The degradation effect of cellulose was then validated using a scanning electron microscope before and after the cellulase hydrolysis of the powder of Hypericum perforatum L. It was demonstrated that the microstructure of cellulose has been destructed and the morphology has been changed greatly.(7) In the synthetic route towards hypericin, self-condensation reaction of emodin anthrone was prompted by means of the microwave-assisted organic synthesis. Three influencing factors of this step such as the microwave heating temperature, irradiation time and catalyst were studied and the resulting optimal conditions were determined as follows: 150°C, 1 h and no catalyst used. The yield of this step was increased to 82.2% with microwave assisted synthesis, and the total yield of hypericin synthesis was achieved as 74.3%.In conclusion, the mutants induced by the compound mutagenesis with electron and ¹²C⁶⁺-ion beams had the highest cellulase activities. The cellulase hyperproducing mutants and their enzyme solutions not only improved the extraction of hypericin from Hypericum perforatum L, but also could be used in other industries. Furthermore, in comparison with conventional heating method, it is shown that the use of the microwave method has enhanced the reaction efficiency and yield, reduced side-reactions and made the synthetic procedure more beneficial to low-carbon and environmental protection.