| Rare ginsenosides are a type of secondary ginsenosides whichglycosyl ligands have been reduced, usually having more bioactivities,such as stronger antitumor activity. But the content of the rareginsenosides in natural ginseng or American ginseng is extremly little, oreven doesnt exist. There are three way to produce the rare ginsenosides:Alkaline hydrolysis, acid hydrolysis and microbial transformation.Microbial transformation of ginsenosides have become the maindirection of the research, because of its advantages, such as exellentspecificity, less isomerization derivatives and higher yield. The purpose ofthe research is to study the strain GsBt3which can transform theginsenosides of American ginseng into rare ginsenosides.According to the result of the investigation of the developingsolvent of TLC, we choose the best chromatographic conditions toseparate the ginsenosides of Americanginseng(chloroform:methanol:H2O=40:10:1, v/v/v). There are fiveproducts of transformation of American ginseng total saponins:ginsenoside Rg1, Rh1, Rd, Rg3and C-K. HPLC analysis was performedon a Welchrom C18column(4.6mm×250mm,5μm); The mobile phasewas acetonitrile(A)-water(B) with the gradient elution (0-20min,25%A'40%A;20-33min,40%A'46%A;33-37min,46%A;37-45min,46%A'50%A;45-62min,50%A'70%A;62-65min,70%A'90%A) at a flow rate of1mL/min; the temperature of column was maintained at30℃, and thedetecting wavelength was set at203nm. The linear range were0.740~7.400μg (r=0.9999) for ginsenoside Rg1,0.536~5.360μg(r=0.9999)for ginsenoside Rh1,1.128~11.280μg (r=0.9999) for ginsenoside Rd,0.308~3.080μg (r=0.9999) for ginsenoside Rg3,0.376~3.760μg (r=0.9999)for ginsenoside C-K. The average recoveries(n=6) of five componentswere97.12%with RSD of1.22%,98.50%with RSD of0.87%,98.00%with RSD of1.06%,98.12%with RSD of1.66%and96.65%with RSD of1.48%.This method is proved simple, sensitive, accurate and reliable. Theanalytic result of conversion product of American ginseng totalsaponins show that, the average content of ginsenoside Rg1, Rh1, Rd,Rg3and C-K are10.47%、7.26%、19.51%、4.20%and5.78%.To transform the American ginseng total saponins into rareginsenosides, we used microbial transformation and alkaline hydrolysismethods. To separate the products with silica gel columnchromatography and got three high purity samples in microbialtransformation method: the conversion product AⅠ,BⅠ,CⅠ. And wealso got four high purity samples in alkaline hydrolysis: the conversionproduct AⅡ,BⅡ,CⅡ,DⅡ. Compare the result of the detection of TLCand HPLC with reference substance. The result confirmed that theconversion product A Ⅰ, A Ⅱ are ginsenoside Rg3, the conversionproduct BⅠ, BⅡare ginsenoside C-K, the conversion product CⅠ, DⅡare ginsenoside Rh1. And we confirmed that the conversion product CⅡis ginsenoside F1by the analysis of MS and NMR data.In order to improve transformation rates of strain GsBt3, we havedone some researches in the carbon source type, adding amount ofcarbon sources, culture medium pH, the volume of culture medium andfermentation time. And we got the best conditions is:1%carbonsources,100mL/500mL, pH=5,28℃,220r/min,5days fermentation.Morphological analysis experiments show that the colony of strainGsBt3is white. After48hours, the top of the colony produce brownspore and the colonies gradually turn into dark brown. There is noexudate and the contrary side of the colony is light yellow. Physiologicaland biochemical experiments show that strain GsBt3has cellulase,protease, amylase enzyme and β-glucosidase. It is negative in the curdenzyme and nitrate reduction. Compare the sequence of strain GsBt3with sequence in Gene Bank, the appraisal results show that GsBt3strain is one of Aspergillus aculeatus. |
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