Study On Transformation Of Ginsenoside Re Into Rh₁ By Microorganism

Ginsenosides are the main active components of ginseng,which have the physiological functions of improving memory,promoting blood circulation,anti-tumour and advance the immunity of human body.The content of rare ginsenoside in ginseng is very minor,but the pharmacological function is stronger than the constant component,especially in the anti-bacterial,anti-inflammatory and anti-tumor aspects.In this paper,the preparation of rare ginsenoside Rh₁ was studied by using microbial transformation with constant ginsenoside Re as raw material.Several strains of fungi were isolated and purified from the planting soil of ginseng in Ansan,Liaoning and Fusong,Jilin Province.After screening and re-screening,a highly effective strain,which transformed Re into rare ginsenoside Rh₁,was isolated and purified.The transformation efficiency of the bacteria served as the experimental material and was assessed by conversion rate.The single factor experiment was carried out on the aspect of time,temperature and pH of microbial transformation.The single factor experiment was used to find the optimum reaction conditions of each effect factor.The microbial transformation conditions were optimized by orthogonal test.The optimal conditions for microbial transformation were as follows: peptone 0.15 g,magnesium sulfate 0.06 g,sodium nitrate 0.3 g,dipotassium hydrogen phosphate 0.15 g,culture temperature 34 ?,pH 4.5,substrate concentration in 80 mL fermentation broth 0.6 g / L,and the conversion rate was 23.14%.In order to improve the conversion rate of rare ginsenoside Rh₁,UV mutation experiment was carried out to obtain the high-efficiency strain.The lethal curve is obtained.The best ultraviolet irradiation time was 2.0 min.A number of independent colonies were obtained by UV mutation.Four of them were found to have higher transformation efficiency.Among them,the transformation efficiency of one strain was 54.06%,which was 2.33 times of the original strain.The result of ITS sequencing showed that the strain belonged to the same branch of Aspergillus niger?KT852982.1?.The obtained fermentation broth was extracted with water saturated n-butanol solution and concentrated by evaporation method.The reaction product was purified by using UItimate XB-C18?15 mm × 200 mm,20-40 ?m?semi-preparative column with acetonitrile: water = 20:80 as the mobile phase at a flow rate of 2.5 mL / min.The purity of Rh₁ reached 81.98 % and the yield was 71.83%.In order to investigate the process of Rh₁ transformation and the detection ofginsenosides,a quantitative method for the simultaneous determination of ginsenoside Rg1,Re,Rf,Rg2 and Rh₁ was established.UG80-CAPCELL PAK NH2?4.6 mm ID ×250 mm,5 ?m?serve as the stationary phase and the mobile phase consisted of cetonitrile: water = 6: 94?v / v?.The UV detection wavelength was 203 nm,flow rate was 0.80 mL / min,25?,the samples were well separated.