| Cordyceps militaris(Vuill.)Fr.is a valuable traditional edible Chinese herbal medicines and important under-forest edible mushroom resources with antioxidant,anti-aging,anti-tumor,immunomodulatory and many other pharmacological effects.Selenium is an essential trace element for human body.Selenium deficiency,which could lead to a variety of human diseases,is now prevalent in the world.In recent years,organic selenium has been widely recognized because it can be easily absorbed by animals and has a high toxicity threshold and bioavailability as well as some other advantages.C.militaris has good selenium-enriched abilities,and Se-polysaccharides it contains has both selenium and polysaccharide biological activities,which is an excellent edible fungus selenium-rich species and can be used as an ideal selenium dietary supplements for research and development.In this study,C.militaris was chosen to be a study object.Based on the optimization of Se-enriched cultivation technique,the accumulation and distribution of selenium in fruiting body and protein were investigated.And then,the extraction,purification,structural characterization,anti-oxidation and in vivo immunoregulatory activity of Se-polysaccharides were also studied which provided the theoretical basis for the further utilization of Se-enriched C.militaris.Below are key research findings:(1)Choosing the yield and selenium content of CM as an indicator,9 factors influencing the cultivation effect were screened by Plackett-Burman and then Box-Behnken experiment design was used to optimize the key factors.The optimum cultivation medium was obtained as follow:glucose 20 g/L,peptone 12 g/L,potassium dihydrogen phosphate 3 g/L,magnesium sulfate 1.5 g/L,triammonium citrate 1 g/L,vitamin B1 50 mg/L,sodium selenite added amount 240 mg/L.The optimum culture conditions were as follows:soaking at room temperature for 26 h,the temperature of germination was 18?,the temperature of induced primordium was 16°C,the temperature of stroma growth was 22°C;under these conditions,the yield of SECM was 175.57 g/kg,and the selenium content in stroma was 197.32 ?g/g.Compared with ordinary cultivation,the yield,polysaccharide and cordycepin content were increased by 9.04%,24.06%and 69.42%respectively,the Selenium content were increased 52.86 times.(2)The selenium accumulation in fuiting body and protein fractions during germination of Se-enriched C.militaris and molecular weights distribution of Se-containing proteins were investigated according to the growth curve.Results showed that the amount of selenium yiled,selenium content,protein selenium content were significantly(p<0.05)increased after germination with 30-240 mg/kg sodium selenite.Under the of same sodium selenite,the selenium yiled was significantly(p<0.05)increased with the growth Se-enriched C.militaris,while the protein selenium content and the ratio of protein selenium to total selenium were decreased significantly(p<0.05).However,the selenium content was decrease firstly and then increase which means the selenium content was significantly(p<0.05)enhanced in the mature period.Selenium can significantly enhance the alkali-soluble protein content(p<0.05),but the content of water-soluble,salt-soluble and alcohol-soluble protein in stroma was significantly decreased(p<0.05)by large selenium added amount(>120 mg/kg).And with the growth of Se-enriched C.militaris,four protein fractionsc ontent gradually decreased.A very non-uniform distribution of Se in Se-containing proteins was observed,and the accumulation of proteins selenium were in the order of alkali-soluble protein>water soluble protein>salt soluble protein>alcohol soluble protein,but water-soluble protein had a greater enrichment capacity of selenium.SDS-PAGE analysis showed that Se was distributed in all the proteins of which molecular weights varied from 11.43 to 92.28 kDa,however,59.98%of Se was observed in the proteins whose molecular weights less than 12.6 kDa that existed a negative relationship in molecular weights.(3)Response surface methodology was used to optimize the hot water extraction(HWE),microwave extraction(ME)and ultrasound/microwave assisted extraction(UME)parameters for crude Se-polysaccharides from Se-enriched C.militaris(CSCMP)based on the single factor experiment.Then,the difference in yield and antioxidant activities of SCMP in vitro were further compared with the three metods.The results indicated that the optimum conditions for UME were as follows:ultrasonication for 26 min,subsequent microwave treatment for 3.2 min,microwave power 350 W,ratio of liquor to material 32 mL/g.Under these conditions,the extraction yield of CSCMP was 5.05%,which was increased by 6.09%and 3.70%compared with ME and HWE.Furthermore,the CSCMP prepared by UME exhibited the strongest antioxidant activity as compared with UE and HWE.These results suggested that ultrasound/microwave assisted extraction was the optimum extraction method for CSCMP.(4)The CMPs(CMP-I,CMP-?,CMP-?)and Se-CMPs(Se-CMP-?,Se-CMP-?,Se-CMP-?)were isolated from CCMP and CSCMP using DEAE-52 cellulose and Sephacryl S-400 column chromatography,which Se-/CMP-? had the highest content in the fuiting body of C.militaris.The results of HPSEC showed that except Se-/CMP-I,Se-/CMP-? and Se-/CMP-?were all polysaccharides homogeneous fraction,and the average molecular weight were consistent.The results of selenium content and uronic acid determination showed that selenium content in three selenium polysaccharides fraction was followed as Se-CMP-?>Se-CMP-?>Se-CMP-?.That is,selenium was more distributed in high molecular weight fractions,and there were no significant difference in the content of uronic acid of Se-/CMP-?,Se-/CMP-? or Se-/CMP-?.The results of monosaccharide composition showed that Se-/CMP-? were mainly composed of mannose,galactose and glucose,and Se-/CMP-? were a homogeneous polysaccharide composed of glucose,and the composition ratio of CMP-? and Se-CMP-?monosaccharide were basically the same.The IR spectrum 1H NMR and 13C NMR analysis suggested that Se-enriched cultivation did not change the main structure of Se-CMP-? and Se-CMP-?,and the selenite groups had been introduced mainly by O-6 substitution at the terminal ?-D-Glcf(1? units.However,in Se-CMP-?,selenium may also substitute the OH at C-6[?6)-?-D-Glcp-(1? or C-2[?4)-?-D-Glcp-(1?]positions that forming the or(?)structure.(5)To further investigate the structure-activity relationship of Se-CMPs,the antioxidant activities of Se-/CMP-? and Se-/CMP-? were evaluated in vitro.The results indicated that Se-CMP-? and Se-CMP-? showed greater total reducing power,OH radical and ABTS+ radical scavenging activities(p<0.01)compared with CMP-? and CMP-?.No significant differences were observed between ·O2-radical scavenging rate of CMP-? and Se-CMP-?,but Se-CMP-? was significantly(p<0.01)higher than that of CMP-?.(6)The in vivo antioxidant activities in D-galactose-induced aging mice of Se-/CMP-?were investigated.Both CMP-? and Se-CMP-? showed potential antioxidant activities in dose-dependent manner via reducing the contents of malonaldehyde(MDA),as well as increasing the activities of total antioxidant capacity(T-AOC),superoxide dismutase(T-SOD)activities,glutathione peroxidase(GSH-Px)activities,and catalase(CAT)activitie.Additionally,the activities of Se-CMP-? was superior to CMP-? which demonstrated that Se-CMP-? may be apply to develop natural antioxidants and anti-aging functional foods.(7)The immunoregulatory activity of CMP-? and Se-CMP-? was evaluated in mice immunosuppressive model established by cyclophosphamide.Both CMP-? and Se-CMP-?protected the body from immunosuppression in a dose-dependent manner.Biological activities related to immunomodulations investigated included increasing weight,enhancing the immune organ indexs,elevating peripheral blood WBC count,accelerating the phagocytotic function of peritoneal macrophage,stimulating the proliferation of splenocytes,balancing the ratio of spleen and peripheral blood lymphocyte subsets,up-regulating the serum immunoglobulin(IgM,Ig A,IgG)and cytokines(TNF-a,IL-6,IL-2,IFN-y)levels,as well as improving liver functions.The degree of modulations were higher in Se-CMP-? than CMP-?,implying that selenium groups the was one of the key bioactive components in Se-CMP-? which are a novel resource for immunostimulant. |
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