An investigation on the bioactive properties of the chemical components and endophytic fungi of Cephalotaxus oliveri Mast.

Abstract

It is widely accepted that endophytic fungi are a rich source of active compounds. Many natural products with diverse bioactivities have been obtained from these fungi, including ones, such as taxol and podophyllotoxin, which were originally thought to be isolated from plants. These results imply that endophytic fungi are a potentially large source of untapped bioactive compounds awaiting exploration. Cephalotaxus oliveri Mast. is a relic, dioecious plant species and endemic to some subtropical forests in China. Its richness in harringtonine (HT) and homoharringtonine (HHT), two phytochemical drugs used to treat leukemia and other cancers is well established. To date, little information is available on the bioactivities of the chemical components and the endophytic fungi of C. oliveri. Hence, this plant seems to be a good candidate to explore the bioactivities of its chemical components and endophytic fungi, to analyze the chemical profile and their antioxidant activities, to isolate and identify the endophytic fungi, and evaluate their bioactivity potential, and to search for HT- and/or HHT- producing strains amongst these fungi, and finally to isolate the pure active compounds from the endophytic strain with the most potent bioactivities. Antioxidant activities of C. oliveri as indicated by the DPPH (2,2-diphenyl -1-picryl hydrazyl), ABTS [2,2'-azinobis (3-ethyl- benzothiazoline-6- sulfonic acid)], superoxide, reducing power coupled with the total phenolic content assays suggest the plant has potent antioxidant potentials. The methanol extract had the most potent activities in DPPH, ABTS and reducing power assays with the highest level of phenolic content among the test extracts. LC-MS/MS and GC-MS analyses resulted in the detection of 22 compounds in the methanol extract, among which are 15 phenolic compounds. The considerable antioxidant potentials and high phenolic content suggest C. oliveri is a rich source of natural phenolic antioxidants. A total of 101 endophytic fungal strains were isolated from C. oliveri using successive surface-sterilization, fragment plating and hyphal tip methods. Nineteen representative strains, identified as either Guignardia (anamorph Phyllosticta) or Colletotrichum species by 18S rRNA gene sequencing, show evident antioxidant, anticancer and antimicrobial activities. The phenolic compounds produced by the endophytes contributed to their antioxidant activities as assessed in DPPH test. Among the endophytic strains, Guignardia sp. L79 was the most antioxidative and cytotoxic. LC-MS/MS and GC-MS analyses indicate the presence of four phenolic compounds including protocatechuic acid, gentisic acid, tyrosol and syringol as secondary metabolites in Guignardia sp. L79. High yields suggest a promising alternative source of the four phenolic compounds can be produced by controlled fungal fermentation. Tyrosol and syringol were isolated from the ethyl acetate extract of Guignardia sp. L79 culture liquid by bioactivity guide fractionation. Syringol displayed more potent antioxidant activities than the commonly used synthetic antioxidant BHT in DPPH, ABTS and reducing power assays, whereas tyrosol exhibited only weak activities in the three assays. Syringol had overt cytotoxic activities to all the three test cancer cell lines, while tyrosol exerted significant inhibitory action to KB and HeLa cells. This is the first report on tyrosol from Guignardia (anamorph Phyllosticta) species and syringol from endophytes as secondary metabolites. However, no trace of HT and HHT was detected in the culture filtrates of all the 101 endophytic strains from C. oliveri using HPLC-DAD analysis.