Studies On The Regulatory Effect Of Insect Hormones On Liquid Culture Of Cordyceps Militaris And Pharmacological Effects Of The Fermented Mycelia

Regulatory effects of the insect hormones(β-ecdysone and juvenile hormoneⅢ) on the growth and metabolism of Codyceps militaris by liquid fermentation,the protective effects of ethanol extract of the fermented mycelia on hydrocortisone-induced liver-kidney disease in mice and the possible mechanisms were investigated in this study.β-ecdysone(or juvenile hormoneⅢ) was added into the culture medium under different concentrations(Low-dosage group 1.2 mg/l,Mid-dosage group 4 mg/l, High-dosage group 12 mg/l) as one-factor test;β-ecdysone and juvenile hormoneⅢwere simultaneously added into the medium as two-factors test.Mycelial dry weight(MDW), extracellular polysaccharide(EPS,yield and secretory volume),intracellular polysaccharide(IPS,content and yield) and cordycepin(content and yield) were measured, respectively.β-ecdysone obviously had a positive effect on the Cordyceps militaris liquid fermentation.During the whole fermentation process,the MDW maximum of each group was obtained at the 7^th day.Compared with the control group,the yield of IPS was enhanced by 29.2%,when 1.2mg/l ofβ-ecdysone was added.When 4 mg/1 ofβ-ecdysone was provided,the amount of cordycepin per unit of MDW and the total yield of cordycepin were increased by 65.4%and 84.7%,respectively.Adding 12 mg/l ofβ-ecdysone to the culture medium,the MDW,the secretion volume of EPS per unit of MDW and total yield of EPS were increased by 18.5%,181.7%and 63.2%,respectively.The effects ofβ-ecdysone on the yields of MDW,EPS,IPS and cordycepin were also different within the scope of this study,without regard to the concentration ofβ-ecdysone(Cordycepin yield＞EPS yield＞IPS yield＞Mycelial yield).Juvenile hormoneⅢincreased the mycelial yield and cordycepin yield at the 7^th day from 5.1%to 9.7%and 0.1%to 21.1%,however;IPS and EPS yields decreased from 34.5%to 51.6%and 24.9%to 30.8%,respectively.Juvenile hormoneⅢaffected the rhythms of mycelia growth and metabolism of Cordyceps militaris by liquid fermentation. IPS content,EPS yield and cordycepin content of the control group all obtained the maximums at the 7^th day,however;the juvenile hormoneⅢgroups obtained the maximums at the 1^st,3^rd,and 3^rd day,respectively.In addition,juvenile hormoneⅢhad an inhibitory effect on the mycelial growth during the early stage of fermentation.Within the scope of this study,juvenile hormoneⅢdecreased mycelia yield from 52.4 to 59.7%after 1 day,and reached the level of the control group at the 5^th day. With theβ-ecdysone and juvenile hormoneⅢdisposing,mycelial yields were increased at the 7^th day,and the effects were similar to theβ-ecdysone test.On the one hand,compared with the control group,EPS yield was significantly decreased owing to the increasing juvenile hormoneⅢ;on the other hand,compared with the control group and one-factor test,the contents of IPS and cordycepin were increased.Thereinto,the yields of IPS and cordycepin of "juvenile hormoneⅢ:β-ecdysone=1:4" group(juvenile hormoneⅢ1.2 mg/l,β-ecdysone 4.8 mg/l) were increased by 68.4%and 123.2%,respectively.In addition,higher proportion of juvenile hormoneⅢcaused the periods of EPS yields maximum and cordycepin content maximum to occur sooner.HPLC chromatograms of ethanol extract of Cordyceps militaris mycelia between the control group and two-factors group(1:1) at 260 nm and 280 nm showed that the retention times were similar,and it implied thatβ-ecdysone and juvenile hormoneⅢonly affected the yields of the active substance of Cordyceps militaris.Methods of pharmacological test of EECM(ethanol extract of Cordyceps militaris mycelia) were as follows:old male ICR mice were randomly divided into normal,control, EECM groups and the shen-xian-zhuang-shen-jiao-nang group(in kidney test).All groups except the normal control were injected with hydrocortisone(25 mg·kg^-1·d^-1) for 7 days. The EECM groups were treated with EECM at a dose of 0.167,0.334 and 0.668 g·kg^-1·d^-1 for 30 days.At the same time,the normal control group and the model control group were given equivalent amount of 0.85%sodium chloride solution by the same route. Thereafter,MDA,the activities of ATPase,GSH-Px and SOD in the liver and the contents of BUN,Scr,ACTH and Cortisol in the serum were measured.Furthermore,the kidney and hepatic histological changes were also observed by optical microscope.Compared with the model group,the content of MDA in the liver was decreased significantly(P＜0.01);the activities of GSH-Px(P＜0.05),ATPase(P＜0.01) and SOD (P＜0.01) in the liver were markedly improved after preventive treatment with EECM.In addition,the contents of BUN(P＜0.01) and Scr(Mid-dosage group and High-dosage group,P＜0.01;Low-dosage group,P＜0.05) in the serum were obviously decreased;the contents of ACTH and Cortisol in the serum and the avoirdupois of mice were obviously increased(P＜0.01).Furthermore,EECM ameliorated the pathological changes of the liver and kidney and recovered the kidney coefficient that resulted from the kidney pathological edema(Low-dosage group and Mid-dosage group,P＜0.05;High-dosage group,P＜0.01).These results demonstrated thatβ-ecdysone and juvenile hormoneⅢpromoted the yields of MDW,EPS,IPS and cordycepin of Codyceps militaris by liquid fermentation.β-ecdysone showed a more positive role in secondary metabolite(cordycepin) accumulation than in primary metabolite(IPS) production and growth of mycelia.Juvenile hormoneⅢaccelerated the rhythms of mycelial growth and metabolism.The yields of IPS and cordycepin were promoted in a greater degree byβ-ecdysone and juvenile hormoneⅢdisposal.Moreover,the effects ofβ-ecdysone and juvenile hormoneⅢwere dissimilar owing to the different kinds of productions.β-ecdysone and juvenile hormoneⅢnot only showed the synergistic effect but the antagonistic effect each other,and the mentioned effects were related with the proportional diversification of them.Compared with the juvenile hormoneⅢ,effect ofβ-ecdysone was more obvious.In addition,pharmacological test results showed that EECM elevated the oxidation resistance so as to prevent the peroxidative deterioration of hepatic cells,and repaired the cell membrane.Meanwhile, EECM induced the organism to repair the function of Hypothalamic-Pituitary-Adrenal Axis(HPAA),and meliorated the kidney yang deficiency.Furthermore,EECM increased the percolation of kidney globule and repaired the nephric lesions.