Screening, Isolation And Identification Of Anti-tumour Compounds From Mycelia Of Selected Entomogenous Fungi

A resazurin cytotoxity assay model with Chinese hamster ovary cells(CHO) was employed to study anti-tumor activity of extracts from mycelium of 40 entomogenous fungi strains deposited in Research Center for Entomogenous Fungi of Anhui Agricultural University collected in various provinces of China.The results showed that mycelia extracts from the strain RCEF0851,RCEF0508,RCEF0514 and RCEF0108 had relative strong activity.At the concentration of 100μg/mL their inhibition rates against CHO cells were more than 70%.Secondary screening revealed that ethylacetate and dichloroform(EAE) mixture extract of RCEF0851 mycelia had the strongest activity.At the concentration of 80μg/ml its inhibition rate against CHO cells was 97.44±1.43%。At the same time the inhibition rate of extracts from the other strains were also more than 67%.So,further investigation is warranted.Considering working effect we just studied the strongest strain RCEF0851 at firstComparison of HPLC analysis with different HPLC columns revealed that a Phenomenex column using Synergi Hydro as solid phase was the best for the EAE extract isolation.The extract was isolated with the column and three pure compounds were obtained.Their purity was confirmed with HPLC method.Activity analysis showed that their inhibition rate against CHO cells were 83.61%,15.38%and 0%respectively at the concentration of 40μg·ml^-1.Compound Hh-1 is a pale yellow powder.HPLC-DAD-HR-ESIMS and ¹H,¹³C, DEPT135,HMQC,COSY and HMBC data revealed that it is Rugulosin.Despite that it is a known anti bacteria compound with special structure discovered at recent years it was discovered for the first time from entomogenous fungi.Its strong activity against CHO cells was also revealed for the first time.For that it showed strong activity and was one of the main components of the extract it could potentially be developed as anti tumor reagent.Compound Hh-2 is a red-orange powder.HPLC-DAD-HR-ESIMS and ¹H,¹³C, DEPT135,HMQC,COSY and HMBC data revealed that it is Skyrin.The molecular formula is C₃₀H₁₈O₁₀.It is a dianthraquinone.Despite that it is a known compound it was discovered from entomogenous fungi for the first time.This compound was reported having anti several tumor cell lines,but our test with CHO cells showed low cytotoxity.Compound Hh-3 is a white powder.MS,one and two dimension NMR analysis showed that it is ergosterol.It had not anti tumor activity.From the structure one can deduce that it has no metabolition relationship with compound Hh-1 and Hh-2.