A new helvolic acid derivative from an endophytic Fusarium sp. of Ficus carica

Abstract A new helvolic acid derivative named helvolic acid methyl ester (1), together with two known helvolic acid compounds, helvolic acid (2) and hydrohelvolic acid (3), were isolated from the fermentation of endophytic fungus Fusarium sp. in Ficus carica leaves. Their structures were elucidated and identified by spectroscopic methods. Compounds 1–3 showed potent antifungal and antibacterial activities.


Introduction
Endophytic fungi of medicinal plant have been recognised as a rich source of potent bioactive natural products (Strobel 2003;Leslie Gunatilaka 2006). An increasing number of bioactive natural products from endophytic fungi of medicinal plants were reported in recent years (Arnold 2007;Rodriguez et al. 2009;Chon 2012;Liu et al. 2011;Luo et al. 2013;Ortega et al. 2013). Ficus carica is not only an edible plant but also a traditional medicinal plant, which is often used in drugs, such as hypotensive (Zhong & Xu 2000), anti-inflammatory (Zhao et al. 2001), hypolipidemic  and hyperglycemic . Fusarium sp. was isolated from F. carica leaves; some bioactive substances have been obtained from Fusarium sp. (Bashyal & Leslie Gunatilaka 2010;Ruan et al. 2014;Dame et al. 2016). Thus, it attracted us to find novel structures and potent bioactive products from endophytic fungus Fusarium sp. of F. carica. In the course of our ongoing research, a new natural product named helvolic acid methyl ester (1), together with two known compounds helvolic acid (2) and hydrohelvolic acid (3), were obtained. This article described the isolation and structural elucidation as well as antimicrobial activities evaluation of three compounds.

Fungus identification
The strain of FL10 was identified as Fusarium sp. (GenBank under accession no. JX119038) based on DnA amplification and the internal transcribed spacer (ITS) sequence.

Biological activities
The result of antimicrobial activity indicated that compounds 1-3 exhibited potent antifungal and antibacterial activities. These results compared with known antibiotics as standards are shown in Table 1.
It was reported that helvolic acid exhibited antibacterial activities mainly against Grampositive bacteria (Qin et al. 2009;Ratnaweera et al. 2014). Previous studies have also reported helvolic acid has been isolated from Xylaria sp., Aspergillus fumigatus and a few other fungal species (Zhang et al. 2008;Ratnaweera et al. 2014). Yet, this is the first study to report the antifungal helvolic acid from an endophytic Fusarium sp.

General
The 1 H nMR and 13 C nMR spectra were recorded on Bruker ADVAnCE 400 MHz spectrometer, with TMS as an internal standard. HR-ESI-MS were obtained in the positive ion mode with Bruker maXis 4G uHR-TOF. Column chromatography was performed with silica gel ( (Huang et al. 2006).

Antimicrobial assay
The antimicrobial evaluation in vitro against two Gram-positive bacteria (S. aureus and B. subtilis), two Gram-negative bacteria (E. coli and P. aeruginosa) and five plant pathogenic fungi (B. cinerea, C. gloeosporioides, F. oxysporum, F. Graminearum and P. capsici) was carried out by the continuous dilution method in the 96-well plates (Giovanni et al. 2008). Solutions of all compounds were the prepared series of concentration from 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, to 0.98 μg/mL in DMSO. Each well containing 100 μL of the solution of sample was inoculated with 100 μL of bacteria suspension containing 10 6 CFu/mL, and plates were incubated 24 h at 37 °C for bacteria and 48 h at 28 °C for fungi. The tested strains were incubated in the liquid mediums, lysogeny broth medium for bacteria and Sabouraud dextrose broth for fungi. The result of assay was recorded by the values of minimal inhibition concentration. The MIC was defined as the lowest concentration that produces complete growth inhibition of the tested. Streptomycin sulphate and penicillin were positive control against bacteria, carbendazim as positive control for the antifungal assay.

Conclusion
In conclusion, a new natural product helvolic acid methyl ester (1), together with helvolic acid (2) and hydrohelvolic acid (3), were first isolated from endophytic fungus Fusarium sp. in F. carica leaves. They exhibited potent antifungal and antibacterial activities. Thus, compounds 1-3 can be as the natural fungicide for plant pathogens and a source of antibacterial drug.

Disclosure statement
no potential conflict of interest was reported by the authors.