Study On Fermentation And Degradation Technology Of Alkaloids Of Epilobium

Radix Aconiti,which comes from the buttercup plant Ocimum sanctum,is pungent and poisonous,with a hot nature,and has the effect of returning Yang and dispelling cold and wind-dampness,and is widely used in Chinese medicine prescriptions.The traditional concoction method of Radix Aconiti is mainly steaming,boiling,frying,simmering,etc.The process of concoction of Radix Aconiti relies mainly on the experience of the operator for subjective control,resulting in unstable efficacy of the product.The main mechanism of traditional concoction is that the more toxic bis-ester alkaloids degrade into less toxic mono-ester alkaloids under the action of heat,and the mono-ester alkaloids are the main active ingredients of Fructus saposhnikii;the mono-ester alkaloids can continue to be hydrolyzed into basically non-toxic aminol alkaloids under heat conditions,so the efficacy of Fructus saposhnikii concocted at high temperature is also severely weakened although the toxicity is obviously reduced.Therefore,in this paper,the process of preparing biotransformed epilobium by solid fermentation method and liquid fermentation method was investigated with raw epilobium as the research object,and the toxicological properties of the biotransformed epilobium obtained from solid fermentation were evaluated by selecting medicinal and food fungi as the fermentation strains.The main conclusions are as follows.1.Screening of the most suitable strains of biotransformed epilobium by solid fermentationEleven species of medicinal and dietary fungi were used as strains,respectively,and inoculated onto the solid medium of raw epiphyllum,and their growth was observed regularly.After the strains grew all over the medium,they were recovered and dried at low temperature to obtain biotransformed epiphyllum,and the biotransformed epiphyllum produced from the above experiments was determined by HPLC method for its alkaloid content.Combined with the growth,substrate conversion rate and alkaloid content,the strain used for the preparation of biotransformed epilobium by solid fermentation was finally determined as: Schizophyllum commne.2?Preparation of solid fermentation biotransformed epilobiumThe fermentation temperature was 25 ?,the water content of the medium was 45%,and the incubation time was 17 d.The content of the toxic substance bis-ester type alkaloids in the biotransformed epilobium was 0.0019%,which was much lower than the2020 version of the national pharmacopoeia standard of 0.020%;the content of the active ingredient mono-ester type alkaloids increased to 0.0352%,much higher than the 2020 version of the national pharmacopoeia standard of 0.010%.According to the above process scale-up,300 g of raw epimedium was fermented in a solid fermenter with a volume of 5 L.After drying,246 g of biotransformed epimedium was obtained with a content of 0.0029% of bis-ester type alkaloids and an increased content of mono-ester type alkaloids to 0.0329%.The biotransformed epiphyllum,raw epiphyllum and commercially purchased medicinal epiphyllum obtained by fermentation in test tubes under optimal conditions after orthogonal tests were studied in comparison by acute toxicity experiments in animals.The results showed that both the raw and medicinal prepared epiphyllum caused prostration and weight loss in animals,while the raw epiphyllum caused death in animals.No such toxicity symptoms were found in the biotransformed epiphyllum group,and no significant differences were observed in behavioral observations and body weight compared with the normal control group,indicating that the toxicity of biotransformed epiphyllum was significantly reduced.3.Preparation of liquid fermented biotransformed epiphyllumThe strains of Schizophyllum commne and Hericium erinaceus,which grew well on the solid medium of epiphyllum,were inoculated on the liquid medium to complete the preparation of the seed solution,and the seed solution was transferred to the fermentation medium of epiphyllum prepared from epiphyllum and water,and fermented in 300 mL triangular flasks at The alkaloid content of biotransformed epilobium was determined by HPLC method after incubation at 25°C and 150 r/min for 10 days.Based on the growth of the strain on the liquid epiphytic medium and the measured alkaloid content,the final decision was made to use Schizosaccharomyces pombe as the liquid fermentation strain.The conditions for the liquid fermentation of raw epiphytes such as: inoculum,substrate concentration,and fermentation time were determined through single-factor experiments;based on this,the best conditions for the fermentation of raw epiphytes by Schizosaccharomyces pombe were optimized by using the response surface method with the concentration of monoester type alkaloids in the fermentation broth as the index: the substrate concentration of fermentation broth epiphytes was 7.20 g/100 mL,the inoculum of seed solution was 5.20 mL/100 mL,and the fermentation time was 9.5 days.The fermentation broth of biotransformed epilobium prepared under these conditions was crushed and broken by colloid mill and homogenizer,and then made into biotransformed epilobium by spray powder dryer.The content of monoester type alkaloids in biotransformed epilobium was 0.0113%,which was slightly higher than the 2020 version of the national pharmacopoeia standard of 0.010%.The content of bidentate alkaloids was lower than the minimum detection value,i.e.the total amount was lower than 0.0054%,which was lower than the national pharmacopoeia standard of 0.020% in the 2020 version.