Endophytic Fungi From Broussonetia Papyrifera And Their Anti-tumor Secondary Metabolites

Endophytic fungi are those who live inter-and/or intra-cellularly inside thehealthy plant tissues causing asymptomatic infections for all or part of their life cycle.Recent researches have demonstrated that endophytic fungi have abundant species, andtheir secondary metabolites even have various structural types with diverse biologicalactivities, including antitumor, antimicrobial, antiviral and insecticidal. Based on thespecific advantages, endophytic fungi are increasingly attracting attention to becomethe important resources for new biological active materials.The paper mulberry (Broussonetia papyrifera (L.) Vent.) is a deciduous tree inthe family moraceae with rich resources and high medicinal value in China. Its fructushas been recorded as traditional chinese medicine at the earliest in SupplementaryRecords of Famous Physicians, and has the activities of nourishing kidney, clearingliver-fire, improving eyesight and inducing diuresis. Our previous research found thattheir extractive has antitumor activity. Recent researches have demonstrated thatendophytic fungi can produce secondary metabolites with novel structures and uniquefunctions, when they grow under special habitat. In this paper, we selected B.papyrifera, which can grow well under heavy metal stress, to study bioactivity and thetolerance against heavy metal of its endophytes. Then, we focused on isolation andelucidation of the secondary metabolites in two bioactive strains. The main results areas following:1. Study on the endurance against heavy metal of endophytic fungi from B.papeyriferaThe fresh tissues of B. papyrifera were collected from Qixia lead-zinc deposit,Nanjing, Jiangsu province, China. Taking potato dextrose agar mediums, a total of47strains were isolated from paper mulberry. Their leaves have high islation rates(average0.91), which indicated that endophytes from B. papyrifera have richbiodiversity and can be a potential resource bank.To research the tolerance against heavy metal of isolated endophytic fungi, thegrowth status of95endophytic fungi, including48isolates from previous research,were evaluated by exposing to mediums with Zn2+, Pb2+, and Cu2+in differentconcentrations. The results showed that there are five endophytic fungi which can grow well when concentration of zinc ion reaches up to200mmol/L. Those isolatesare Apiospora montagnei, Arthrinium phaeospermum, Aspergillus sp.,Stagonosporopsis cucurbitacearum， Eyronellaea australis。2. Study on bioactivity of endophytic fungi from B. papeyriferaWe screened the anti-inflammatory activity of fermentation products with micemacrophages RAW264.7as vitro inflammation model. The results indicated that71percent of fermentation products of isolated endophytic fungi presentanti-inflammatory activity. But the inhibitory rate in all anti-inflammation activeendophytic fungi is higher than100%when the concentration is100μg/mL. Weassume this result may be related with their cytotoxic activity.The cytotoxic activity of fermentation products was evaluated by the MTTmethod using human hepatocellular carcinoma cell lines (SMMC-7721andQJY7023). The result showed that five endophytic fungi have significantanti-hepatoma activity, including Chaetomium globousm, Fusarium sp., Gibberellaintermedia, Alternaria sp. and an unidentified strain.3. Study on metabolites of anti-tumor activity fungiThe strain Chaetomium globousm and Alternaria sp. with preferable activitywere amplifiedly fermented.36compounds were isolated from the ethyl acetateextract of the two strains by Silica Gel column, Sephadex LH-20, PreparativeTLC and preparative HPLC. Of those compounds, the structures of21compounds were elucidated by the UV, IR, HR-ESI-MS,1H-NMR,13C-NMRand2D-NMR data, including two new componds and one new natural products.11compounds were isolated from Alternaria sp., and the major types of thoseare naphthoquinones, resorcylic lactones, and perylenequinones etc. among those ofaltertoxin IV is a new compound.10compounds were isolated from Chaetomiumglobousm, including steroids, indole alkaloids, and azaphilone, among those of15β-hydroxyl-(22E,24R)-ergosta-3,5,22-trien-7-one is a new compound.Sixteen isolates were evaluated in vitro for their inhibitory ability against thegrowth of human lung adenocarcinoma (A-549), human osteosarcoma (MG-63), andhuman hepatocellular carcinoma (SMMC-7721) cell lines.3,4’,5’-trihydroxy-5-Methoxy-7H-benzo[c]chromen-7-one showed significantcytotoxic activities against A549, MG-63and SMMC-7721cell lines with IC50values of1.47,2.11, and7.34μg/mL. Altersolanol A showed significant inhibition to MG-63and SMMC-7721with IC50values of0.53and2.92μg/mL.In summary, endophytic fungi of Broussonetia papyrifera possess the toleranceagainst heavy metal as same as their host plant, and can produce secondarymetabolites with antitumor activity. Our study therefore underscores that endophyticfungi of Broussonetia papyrifera are promising sources of natural bioactivemetabolites.