Purification,Identification And Functional Properties Of Novel Yellow Pigment Obtained From Cordyceps Militaris

Cordyceps militaris,a precious edible and medicinal mushroom,is rich in nucleosides,cordyceps polysaccharides and water-soluble carotenoids,possessing a variety of medicinal activities such as anti-inflammatory,anti-tumor,anti-aging.The color of C.militaris will change from white to orange when it is exposed to sun light radiation,and produce yellow pigment.In recent years,six kinds of carotenoids have been found from C.militaris fruiting body,and four of them are cordyxanthins with strong water solubility.Compared with the traditional fat-soluble carotenoids,these four natural water-soluble carotenoids are more easily digested and absorbed by human,and theoretically have stronger potential physiological activities.In order to further study the functional activity of yellow pigment from C.militaris,this study used HPLC and HRMS,NMR to identify the structure of yellow pigment.The main results of this paper are as listed follows:（1）A highly efficient method for isolation of cordycepic pigment was established with HPLC,and the preparations of No.4 and No.5 peaks were prepared by Prep-HPLC,named as CMPP-1 and CMPP-2,respectively.The results showed that the purity of these two purified pigments was>95%.（2）Extraction conditions for CMPP-2 was optimized as ethanol concentration 57.68%,extraction temperature 44.40°C,extraction time 52.44 min,and the actual content of CMPP-2 was 2252.23±83.98μg/g,and the extraction series was determined to be 3 grades.（3）Struction of CMPP-2 was identified,and abbreviated as cordycepene.CMPP-2 was detected the maximum absorption wavelength at 278,449 and 477 nm.It’s molecular weight was 236.1158 m/z,and the molecular formula was C₁₄H₁₇N₁O₄.（4）The stability of cordycepene was investigated.The results showed that the order of influence of different light and air on cordycepene was:ultraviolet light>natural light>air.High temperature has obvious influence on the stability of cordycepene.Fe³⁺had protective effect on cordycepene,while K⁺,Na⁺,Ca²⁺had little effect on cordycepene.The order of influence of different p H values on cordycepene stability was:1>3>13>11>5>9>7.The effect of food additives showed that the effect of each additive on the preservation rate increased with the increase of the added concentration.The highest concentration（1.5%）of citric acid,preservative,redox agent had a greater effect on the preservation rate.（5）The antioxidant ability of cordycepene was investigated.The results showed that the DPPH free radical scavenging activity of cordycepene was significantly higher than that of crude extract.When the concentration was increased to 2.0 mg/m L,the scavenging activity of cordycepene was close to that of the control group Vc.Cordycepene（0-50μg/m L）have no cytotoxic effect on HSF cells,and showed a proliferative effect on cells at a concentration of 25 and 50μg/m L for 72 h.Cordycepene（25-50μg/m L）significantly increased the activity of senescent HSF cells after 48 and 72 h,but no difference with the control group without UVB light treatment.At the same time,cordycepene significantly increased the activities of antioxidant enzyme（SOD,GSH-Px and CAT）and decreased MDA levels in a concentration-dependent manner.