Research Of Submerged Fermentation And Extract Of Activated Substance Triterpenoids From Antrodia Camphorata

Antrodia camphorata is a well-known special medicinal mushroom that grown only in Taiwan.The recent research indicated that A.camphorata had many kind of pharmacoloty activity,such as hepatoprotective,resisting inflammatory reaction,antioxidant properties,anti-tumor,anti-angiogenic,and so on.While triterpenoids is one of the most important bioactive substances in it.This paper the solid culture and liquid culture were optimized,the antioxidant properties of different extracts from broth and mycelia were studied,the extract condition of triterpenoids were optimized and the triterpenoids were further purified and preliminary identified?The research of solid culture indicated that compared with PDA and SDA culture,MEA culture was more conductive to the growth of A.camphorata,mycelia grow quickly and concentrated.Althouth the macromophology was different,the micromopholoty was almost the same by microscope.The mycelia was thin and dendritic distribution.The spores were mostly ellipsoidal conidia and could be dyed by Alkaline Meilan.Triterpenoids production could reach a relatively high value by two-stage light and temperature train.We evaluate the impact of adding 16 kinds of nutrients include carbon sources,nitrogen sources and growth factors to enhance the formation of triterpenoids in the submerged culture of A.camphorate.Malt extract,bran cooking liquid,glucose,mannitol,beef extract,camphor oil were identified as more significant variables for triterpenoids production by Plackett-Burman design.Single factor experiment and Box-Benhnken design were adopted to determine the optimal medium.The result indicated that when 1.5%malt extract,5.0%bran cooking liquid,3.0%glucose,0.1%mannitol,1.0%beef extract,0.2%camphor oil,while 0.1%MgSO4-7H2O,0.1%KH2PO4 were added,the maximal production of triterpenoids was 6.22 mg/100mL after a 7 d fermentation period,which was almost 8 times as many as the production before optimization.The different extracts were obtained by methanol,ethanol and isopropanol from Antrodia camphorata fermentation broth and mycelia.Their reducing power,scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals(DPPH),hydroxyl radicals,superoxide radicals,hydrogen peroxide and chelating effect on ferrous ions were tested to compare the antioxidant properties of different extracts from broth and mycelia and extracts of different extractants.The result showed that:(1)Considering the extracts of fermentation broth,isopropanol extract had the highest reducing power and the ethanol extract had the highest ferrous iron chelating ability.The highest DPPH scavenging ratio of ethanol extract and isopropanol extract reached 97.88%.The highest hydroxyl radicals scavenging ratio 84.22%was achieved by ethanol extract.These three extracts all had high superoxide radicals scavenging ability and the highest one was isopropanol extract,the scavenging ratio reached 94.89%.The highest hydrogen peroxide scavenging ratio 97.53%was achieved by ethanol extract.(2)Considering the extracts of mycelia,the ethanol extract both had the highest reducing power and ferrous iron chelating ability.The highest DPPH scavenging ratio of ethanol extract reached 89.95%.The hydroxyl radicals scavenging ability of these three extracts all low,the highest one was isopropanol extract,the scavenging ratio was just 37.28%,while the superoxide radicals scavenging ability were generally higher,the highest scavenging ratio 90.05%was achieved by methanol extract.The ethanol extract and isopropanol extract both had the highest hydrogen peroxide scavenging ability,the highest ratio were both 95.06%.Our studies provided a basis for development and utilization of Antrodia camphorata.Single factor experiments and quadratic orthogonal rotation combination design were adopted to determine the optimal condition of solvent,extracting agent,methods of extracting,concentration of solvent,solid-liquid ratio,time,temperature and power.The result indicated that the better extracting agent was methylene chloride,the better solvent was 95%ethanol and the better method of extracting was microwave-assisted extraction.The optimal conditions were achieved by multiple regression analysis:solid-liquid ratio,time,temperature and power were respectively 51.2,9.56 min,63.2?,613.7W and the predict amount of treterpenoids was 7.91mg/g.Combined with actual conditons,the optimization was adjusted to 51,9.5mIn,65? 600W and the amount of treterpenoids reached 7.65mg/g,which was basically consistent with predictive value.Oleanolic acid was used as the standard to study the condition of HPLC.The best mobile phase was methanol:water=90:10.Because of the concentration and condition differences,the relative retention time had slightly change,but almost at 7.50min.The standard curve of Oleanolic acid was Y=6375123.6866x(Y was peak area,x was concentration).HPLC had good reproducibility and the RSD of relabtive retention time and peak area were 0.370%and 5.813%,respectively.The result of isolation and purification showed that the extraction washing method was better than acid and alkaline precipitation method.The best eluent of TLC was petroleum ether:ethyl acetate=2:1.The Rf value of Oleanolic acid was about 0.6 after been chromogeniced by 10%sulphuric acid ethanol,the color was reddish purple.At the correspond station,the sample had similar substance.After purification by column chromatography and preliminary identification,the two sample were confirmed triterpenoids.