Studies Of Natural Pigments From Mycelia Of Several Cordyceps-related Fungi

A series studies were carried out on natural pigments from mycelia of three fungiRCEF4585、 RCEF4337、 RCEF4022isolated from Cordyceps pruinosa Petch、Septafusidium bifusisporum、 Cordyceps martialis Speg., respectively. The studiesincluded HPLC-MS analysis of the crude extraction; isolation of the target pigment;identification of the pigment; stability tests of the pigment.HPLC-MS analysis showed that the natural pigment in mycelia of RCEF4585was amixture. With the help of high resolution MS, the molecular formula of the main pigmentwasidentified as C₂₆H₃₂O₉. UV analysis results revealed that this compound hasultra-violet absorbance at228nm、260nm、413nm、434nm. The main pigment was possiblya new compound according the searching results of Chapman&Hall natural productsdatabase.There was only one pigment found from the methanol extracts of the mycelia ofRCEF3669. Analysis results of HPLC-DAD-MS revealed that the molecular formula of thepigment was C₃₀H₁₈O₁₂, and that the ultra-violet absorbance of the pigment was225nm、266nm、297nm、467nm. The pigment compound was identified as dimeric anthraquinone,according to the Ms and UV data. This pigment was the first time isolated fromentomogenous fungi.HPLC-MS analysis showed that the natural pigments in the methanol extracts frommycelia of RCEF4337were similar to those pigments from RCEF4584. However, onedifferent pigment with molecular formula C₃₀H₁₈O₁₃was found in ethyl acetate extractsfrom RCEF4337. The pigment has ultra-violet absorbance at225nm、266nm、297nm、503nm. According to molecular formula and ultra-violet absorbance, the searching resultsof Chapman&Hall natural products database revealed that this pigment was possibly a newcompound.High-speed counter-current chromatography was used for separation and purificationof natural pigment P-1from the mycelia of RCEF4585. The tow-phase solvent system wasethylacetate-n-butanol-methanol-water （10:3:3:10）. The upper phase was stationary phaseand the lower phase was mobile phase. The flow-rate of the mobile phase was2.0ml/minand the rotation speed was850r/min. The effluent of the column was monitored with a UVdetector at340nm. And Semi-Preparation HPLC was employed to separate and purifynatural pigment, tow pigment compounds P-2and P-4were gained from the mycelia ofRCEF4022and RCEF4337. The purity of the pigment fractions evaluated by high-performance liquid chromatography was more than95%. High resolution MS and UVanalysis results showed that these purified pigments were the same as that of the crudeextracts suggesting a successful separation and purification of the pigment compounds.The three pigment compounds were identified by color development reaction asanthraquinones. Stability of the pigment P-4was studied. The results showed that thepigment could be dissolved in methanol、acetonitrile and ethyl acetate, and heat andoxidizing agent had less effect on the pigment, however, hydrogen peroxide could reducestability of the pigment. Stability tests on metal elements revealed that the tested metal ionshad almost no effect on stability of the pigment, except that iron ion could reduce someabsorbency of the pigment.