Research On Biotransformation Synthesis And Bioactivities Of Cordyceps Polysaccharide Germanium

Many specices in the Coryceps genus have been used to treat diseases in Traditional Chinese Medicine(TCM).Examples include Cordyceps militaris(L.ex Fr.)Link and C.sinensis.Although C.sinensis is widely used by medicine industry,C.militaris(known as the North Cordyceps,Cordyceps,Cordyceps militaris),is another important medicinal species that has been shown to have equivalent pharmaceutical values as C.sinensis.C.militaris is being developed to a substitute of C.sinensis.C.militaris produces many medicinally active compounds.One of the well-known metabolites is polysaccharide.Numerous basic and clinic researches have demonstrated multiple functions of Cordyceps polysaccharide.The uptake of Cordyceps polysaccharides can enhance body's immunity to prevent against diseases.Consumption of Cordyceps products can improve high blood pressure and reduce heart dieases.Cordyceps polysaccharide germanium(CPG)is one of active compounds produced in C.militaris.CPG results from the reaction of polysaccharides and inorganic germanium in the body of mushroom.The formation of CPG was in C.militaris was investigated.In my expeirments,both solid-agar medium and liquid suspension culture of C.militaris were developed.Germanium dioxide(Ge02)was added to culture media to reach different final concentrations.Analytic assay showed that Ge was absorbed into fruiting body and mycelium.As a result,CPG was extracted from cultured materials,indicating that Ge reacted with endogenous polysaccharide.Regulation of CPG formation was investigated in this study.Experiments included synthesis,separation and purification,structural characterization and bioactivities.Meanwhile,effects of blue light on synthesis of CPG and other main active ingredients of C.militaris were investigated.Effects of both Ge and Se on the formation main active ingredients in sporophore and mycelium of C.militaris were investigated.Results and conclusions are summarized below.1.Effects of Ge concentrations on formation of CPGConcentrations that were tested included 100,150,200,250 and 300 mg/L added into agar-solidified media.Student's test(P<0.05)was completed to evaluate significance in all treatments.Different impacts of concentrations on biomass and CPG formation were observed in our experiments.In solid media,the concentrations at 200mg/L led to the maximum the sporophore biomass,at 300mg/L resulted in the maximum production of CPG in sporophore,and at 250mg/L allowed the maximum conversion of Ge to CPG.In liquid culture mediua,the concentration of Ge at 250mg/L enhanced the maximum biomass of mycelium,at 150mg/L enhanced of the maximum production of CPG in mycelium,and at 200 mg/L resulted in the maximum conversion in mycelium.Quantification of biomass and concentrations of CPG are summarized here.In solid medium,at 250mg/L of Ge,the c ontent of OG in sporophore is 489.86?g/g;at 300mg/L of Ge,the content of PG in sporophore is 216.76?g/g;at 300mg/L of Ge,the conversion ratio of PG in OG of sporophore is 51.9%.In liquid media,at 250mg/L of Ge,the content of OG in mycelium is 584.76?g/g;at 150mg/L,the content of PG in mycelium is 107.29?g/g;at 200 mg/L,the converstion ratio of PG to OG in mycelium is 34.2%.2.Separation,purification,and characterization of CPG and as well as other composition of polysaccharidesDifferent extraction conditions were tested in my experiments.Ethanol:water(70:30)was optimized to result in the maximum extraction of CPG from sporophore powder in 2 hrs.using.In addition,decolorization processing with 2%of activated carbon enhanced extraction efficiency.Different CPG products were extracted from sporophore and in mycelium.Those CPGs are composed of arabia sugar,xylose,galactose and glucose in two materials.The mass ratio of the four monosacchride in sporophore PG was 1:2.7:91.5:7.1,and in mycelium PG was 1:1.1:39.7:3.5.The average molecular weight CPGs are:Mn=9.3733×104g/mol,Mw=4.9465×105g/mol,Mz=1.4283×106g/mol.3.Effects of blue light on Ge concentrations and main compositions in C.militarisIn my experiments,I observed that blue light could regulate Ge concentrations in fruiting body and mycelium of C.militaris and promote differentiation and growth of fruiting body.After one cycle of culture on solid medium,the biomass of fruiting body was increased 26.5%compared to control(with white light).After 36 days of inoculation,nearly 50%of added Ge was converted into CPG in both fruiting body and mycelium.The dynamics of Ge concentrations are summarized in the following.At day 36 and 52 after inoculation on solid medium,the contents of CPG was 5.91%and 6.42%(dry weight)in fruiting body in the blue light condition,3.078-and 3.344-fold higher than in the white light conditions,respectively.At day 5 and 8 after inoculation in liquid medium,the contents of CPG were 5.92%and 6.88%(dry weight)in mycelium in the blue light condition,2.5 and 2.9-fold higher than in the dark condition.The content of cordycepin in fruiting body was 1.44-fold higher in blue light condition than in white light condition.The content of cordycepin in mycelium was 1.297-fold higher in the blue light condition than in the dark condition.Meanwhile,units of CPG was changed in the blue light.GPGs are composed of rhamnose,arabia sugar,xylose,galactose and glucose in fruiting body.Of them,rahmnose was not observed in the white condition.By contrast,this phenomenon was not observed in mycelium culture.4.Effects of Ge on main metabolite composition in fruiting body and mycelium.Contents of cordycepin,adenosine and cordyceps polysaccharide were impacted by different concentrations of Ge.In general,low concentrations of Ge enhanced the production of these three metabolites,while high concentrations decreased their productions.Ge at 250-300 mg/L could increase production of these metabolites in fruiting body.The concentration at 200 mg/L increased the production of these metabolites in cultured mycelium.In addition,addition of both Ge and Se into media could increase production of these metabolites.Particularly,the production of cordycepin was significantly increased.Compared to control expeirments,the production of cordycepin was increased 2.89 fold.5.Investigation of toxicity and safety of Cordyceps polysaccharide germaniumThe Potential toxicity of CGP was tested using cordyceps mice and rats.The resulting data showed that CPGs in a consumption concenratin range did not have toxic effects mice and rats.Anatomic observation showed no abnormal cells and tissues occurred in tested animals,indicating that CPGs are safe products.In addition,Ames test,micronucleus test of bone marrow cell and aberration of sperm test were completed to evaluate potential long-term toxic effects of CPGs on rats.All results showed that CPGs did not have poisoning effects in these tissues.Meanwhile,mutagenic tests were negative(P>0.05).Therefore,CPGs are safe.6.Study on anti-aging benefits of Cordyceps polysaccharide germaniumCPGs were investigated to understand their effects of MAO activity in liver and brain of rats.The resulting data showed that CPGs could significantly influence MAO activity in the liver of male rats and in the brain of female rats(P<0.05).The MAO activity in liver of male rats was increased as the increase of CPG intake.After 26 weeks of feeding with a high dose of CPGs,compared to control animals,the MAO activity in the liver in male rat was increased by 2.56 times.By contrast,the MAO activity in the brain female rat fed with a low dose was decreased by 36.9%.This indicates that a high dose of CPGs has anti-aging effect.Effect of CPGs on lifespan was studied using Drosophila melanogaster.Low doses of CPGs were found to effectively improve life expectancy,extend maximum life span and increase half of the time of death of male and female(P<0.05)P<0.01).By contrast,high doses did not obviously impact life expectancy,maximum life span and half of the time of death of Drosophila melanogaster.In these experiments,CPGs were observed to have stronger effects of on life expectancy,maximum life span and half death time of female than on those of male animals.In one group experiment with 1%CPGs,the average life expectancy,maximum life span and half death time of male animals was increased 25.7%,11.5%,and 22.93%compared with control groups.In one group experiment with 1%CPGs,the average life expectancy,maximum life span and half death time of female animals was increased 44.12%,40.81%,and by 46.19%compared to control ones.In conclusion,CPGs have more effects on lifespan of female D.melanogaster than on lifespan of male D melanogaster.In conclusion,my dissertation has the following innovative results:1.The formation of CPGS was firstly and systematically studied in solid and liquid cultures of C.militaris.Results show differentiation of GPGs formation in sporophore and mycelium.This result will be useful for further investigations of CPGs synthesis.2.Monosaccharides in CPGs are characterized in cultured C.Militaris.3.The concentrations of Ge and blue light can dramatically affect main active components in C militaris..This result provides data to use optimized Ge concentrations and blue light to icnrese the production of CPGs.4.The toxicity of CPGs were firstly and systematically determined to be non-toxic to rat and mice.This result will provide important data for further development of CPGs as new antitumor drugs.5.The effects of cordyceps polysaccharide germanium on MAO activity in the liver and the brain of rats were firstly studied.The resulting data show that CPGs have an anti-aging function.