Investigation On Active Constituents Of The Cultured Mycelia Of Cordyceps Ophioglossoides And The Molecular Mechanism For Its Anti-tumor Effect

Cordyceps ophioglossoides is a species of family Ophioglossoides and a parasite of certain types of Elaphomyces. It has been used as a rare medicinal fungus for treating metrorrhagia and irregular menstruation. The resources of wild C. ophioglossoides are quite limited and it is nearly impossible to get the crude fungi. There are not many studies on this fungus including the systematic analysis of chemical constituents and the mechanisms of active compounds. The classical Chinese medicine lacked of clear exposition of the material basis of C. ophioglossoides, which hampered the depth development of it. This paper dealt with the investigation of chemical constituents and their bioactivities from the cultured mycelia of C. ophioglossoides. The mechanism of anti-tumor activity was also investigated.1. The chemical constituents of cultured mycelia of C. ophioglossoides was systematic analyzed. Fourteen compounds were established by spectroscopic means, including two sterols, four sesquiterpenes, one anthraquinones, four polyphenols, one phenols and two flavonoid glycosides. Thirteen of them were first reported from this cultured mycelia. The four sesquiterpenes named Cordycepol A-D were new compounds and submitted a patent application (201110111071.5). Cordycepol A-C had an unusual spiro[4.5]decane carbon structure and belonged to acorane sesquiterpenes. Cordycepol D was a derivative of a tumor angiogenesis inhibitor 5-demethoxyfumagillol. Two flavonoid glycosides with five-carbon furanose sugars may be the new natural products by comparing with the known database. Meanwhile, thirteen volatile components including one phenols, one alkene, one sterol, three unsaturated fatty acids and seven long-chain unsaturated fatty acid esters were elucidated from the oil part of the extraction by gas chromatograhpy-mass spectormetry (GC-MS).2. The 50% maximal inhibitory concentration (IC50) of Compound 3 to Human lung carcinoma A549, human hepatic carcinoma HepG2 and human cervical carcinoma HeLa for 24 h was 36.2μg/ml,21.1μg/ml and 40.2μg/ml through the MTT assay in vitro. Compound 3 showed no cytotoxicity to human breast carcinoma MCF-7. The IC50 of Compound 9 to HepG2,Hela,A549 and MCF-7 cells were 39.45μg/ml,66.35μg/ml,37.9μg/ml and 18.9μg/ml. Both compound 3 and 9 have no growth inhibition on normal live cell L-02 (IC50>80μg/ml). What's more, the research on the inhibition of tumor matastasis by a polyphenolic compound daidzein in MDA-MB-231 cells was performed and another polyphenol compound Hispolon from Phellinus linteus was used as a control. Though daidzein had anti-tumor activity, it had no effects on breast tumor matastasis. Hispolon can inhibit MDA-MB-231 cells matastasis by reducing of MMP-9 transcription and expression.3 The mechanism of antitumor efficacy by Cordycepol C was revealed. Nuclear condensation, cleavage of PARP-1, apoptotic bodies, the phosphatidylserine of cells redistributed from the inner to the outer leaflet of the plasma membrane were observed. These typical morphological characteristics of apoptosis indicated that Cordycepol C could induce apoptosis in HepG2 cells. Moreover, Cordycepol C caused G2/M phase growth arrest in HepG2 cells. Cordycepol C could trigger the loss of mitochondrial membrane potential of HepG2 cells in a time-dependent manner, resulting in the translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) from the mitochondria to the nucleus. However, procaspase-3,-8,-9 were not cleaved and a caspase inhibitor, Z-VAD-fmk, did not prevent the apoptosis in HepG2 cells. The expression of Bax protein increased in the whole cell extracts and the mitochondrial translocation of Bax were detected by Western blot. These findings indicated that Cordycepol C induced caspase-independent apoptosis in HepG2 cells through a Bax-mediated mitochondrial pathway that lead to nuclear translocations of AIF and Endo G.Taken together, this study revealed the pharmacodynamic material basis of cultured mycelia of C. ophioglossoides. It's the first time to report the mechanism of Caspase-independent apoptosis through mitochondrial pathway induced by Cordycepol C. This work laid a foundation of further applications of C. ophioglossoides.