Preliminary Study On Liquid Fermentation And Serum Pharmaceutical Chemistry Of Cordyceps Militaris

Cordyceps sinensis as a expensive traditional Chinese medicine and health product,Cordyceps militaris also known as north Cordyceps sinensis.Because of its similar activity and pharmacological effects with the Cordyceps sinensis,is expected to become a new fungal drug source substitute for Cordyceps sinensis.The market demand is huge and scarce wild resources,so its research and development has become a hot issue in recent years.At present,most of the researches focus on the extraction and functional research of active ingredients.There is little research on the formation process of active components during the liquid fermentation of Cordyceps militaris,and the pharmacological basis of active components of Cordyceps militaris is not clear.In this paper,the metabolic changes of the main active components and the liquid fermentation process were studied during liquid fermentation of Cordyceps militaris.On this basis,the fingerprints of Cordyceps militaris,Cordyceps militaris and mycelia of Cordyceps militaris were studied.Analysis the blood transfer components of Cordyceps militaris and its liquid fermentation products and to explore the possible pharmacological basis of the drug.Objective:1.To study the metabolic changes of the main active ingredients during liquid fermentation of Cordyceps militaris,and to determine the best collection time of different active ingredients.2.Determine the best fermentation process by optimized the fermentation conditions of Cordyceps militaris3.To study the correlation between the fingerprints and its serum chromatogram of Cordyceps militaris,mycelia of Cordyceps militaris and the liquid fermentation broth of Cordyceps militaris,and to explore its possible pharmacological basis.Methods:1.The metabolic changes of the main active ingredients during liquid fermentation of Cordyceps militaris: The GIM5.270 of Cordyceps militaris was selected as the index,and the basal medium of Cordyceps militaris was selected.On this basis,the liquid fermentation time of Cordyceps militaris was prolonged.According to the different active ingredients in the fermentation system,the content of the main active ingredients in the mycelium and the fermentation broth was determined by the corresponding analysis method.The total sugar was determined by phenol-method;reducing sugar by DNS method;cordyceps acid using colorimetric method;cordycepin and adenosine using HPLC.And the optimal collection time of various active ingredients was determined,according to the distribution and metabolic changes of the active components of Cordyceps militaris in the fermentation system.2.Study on process of during liquid fermentation of Cordyceps militaris: On the basis of the best collection time,the mycelia and extracellular crude polysaccharide yield as the index,study on the effects of nutrient factors(including carbon source,the amount of carbon source,nitrogen source,nitrogen ratio,the amount of nitrogen source,inorganic salt ratio,the amount of inorganic salt,the amount of VB1)and the effect of culture conditions(temperature,p H,shaker speed and liquid volume)on the yield of active ingredients.Based on this,orthogonal experiment the conditions of culture conditions to optimize the liquid fermentation process,screening the best liquid fermentation process Cordyceps militaris.3.Preliminary study on serum pharmacochemistry of Cordyceps militaris: The HPLC fingerprinting method of Cordyceps militaris was established by screening the elution gradient,detection wavelength,column temperature,mobile phase,elution rate and loading volume.The fingerprints of Cordyceps militaris extract were evaluated by HPLC fingerprints and the method was validated..The fingerprints of artificial mycelia and fermentation broth were established,the product was analyzed for mass ratio.On the basis,the extracts of Cordyceps militaris were injected into the rats,and the administration of serum were obtained.The HPLC chromatograms of the sera were determined by the same chromatographic conditions as the fingerprints of Cordyceps militaris,Fingerprints were compared,analysis the components of blood flow migration.Results:1.The metabolic changes of the main active ingredients during liquid fermentation of Cordyceps militaris: The results showed that the mycelial yield of potato integrated PDA medium was the highest.The results of detection of main active ingredients in mycelia and fermentation broth during liquid fermentation of Cordyceps militaris showed that as the time progressed,the mycelium was more and more,and the fermentation broth became more and more clear.The content of cordyceps polysaccharide and cordyceps acid in the fermentation system increased first and then decreased,the optimal collection time was about 8 days;the content of adenosine was gradually decreased,the best collection time was about 5 days;the content of cordycepin increased gradually,and the best collection time was about 15 days2.Study on process of during liquid fermentation of Cordyceps militaris: The single factor experiment and orthogonal experiment result showed that the best nutrient factors were as follows: 20% potato juice 1 L,glucose 20 g,peptone 16 g,yeast powder 5 g,KH2PO4 The optimum culture conditions were as follows: 21 ?,150 r,PH6.0(natural),liquid volume of 90 m L(300 m L erlenmeyer flask),no light.In this process,the mycelia biomass and crude polysaccharide synthesis can reach 11.22 g / L.3.Preliminary study on serum pharmacochemistry of Cordyceps militaris: The HPLC method for the determination fingerprints of Cordyceps militaris was established as follows:Age La Venusi L ASB C18(5 um,4.6 × 150 mm),the mobile phase was methanol-water,the detection wavelength was 260 nm,the flow rate was 1.0 ml/min,and the column temperature was 25?,injection volume 10 u L,50 min gradient elution.The fingerprints of Cordyceps militaris were established in 10 batches of Cordyceps militaris extract,the similarity was> 0.9,and the fingerprints of mycelium and fermentation broth were established.The levels of adenosine peak and cordycepin were observed in the three products.The content of adenosine in mycelium is higher,the content of cordycepin is higher in the fermentation broth,and there is more common peak between mycelium and Cordyceps militaris.The results of the comparison of HPLC fingerprints and control fingerprints in rats were showed that there were seven migratory components in the serum of the Cordyceps militaris,and five of them were the composition of the stress product,two of them were metabolized;the mycelium in the serum of the six calibration components in the blank serum did not appear,are the prototype components;fermentation broth of serum calibration of seven migratory components in the blank serum did not appear,of which five for the prototype ingredients,two for the metabolic or stress product components.Two prototype components which in the three types of product extracts were identified as adenosine and cordycepin.Conclusion:In this paper,we selected the Cordyceps militaris species and studied the metabolic changes of the main active ingredients during liquid fermentation of Cordyceps militaris.The optimum collection time of cordyceps polysaccharides,cordycep acid,adenosine and cordycepin were determined respectively.Based on this,The mycelia and crude polysaccharide yield as the comprehensive index,the optimum fermentation process of Cordyceps militaris was determined by single factor and orthogonal experiment.At the same time,the fingerprints of Cordyceps militaris,Cordyceps militaris and mycelia of Cordyceps militaris were studied,Then,study on the correlation between fingerprints and liquid chromatograms of Cordyceps militaris and self-cultured mycelia and fermentation broth.Found that nucleoside cordycepin and adenosine are in the prototype ingredients into the blood,in addition to the administration of some of the components of the metabolites or stress products is not the original liquid,may play a pharmacological basis for the efficacy.The experimental results will provide a basis for the industrial production of Cordyceps militaris,but also provide a reference for revealing the effective components of Cordyceps militaris and its related products.