Lasiodiplodins from mangrove endophytic fungus Lasiodiplodia sp. 318#

Four new lasiodiplodins (1–4), together with three known analogues, have been isolated from a mangrove endophytic fungus, Lasiodiplodia sp. 318#. Their structures were elucidated by spectroscopic techniques. Cytotoxic activities of compounds 1–7 were evaluated in vitro against human cancer lines THP1, MDA-MB-435, A549, HepG2 and HCT-116. Compound 4 exhibited moderate cytotoxic activities.


Introduction
Mangrove forests are inhabited by large number of fungal communities due to uniqueecosystem and are biodiversity hotspots for marinefungi (Carol et al. 2007). Mangrove fungi constitutethe secondl argest part of marine-derived fungi (Sridhar 2004). Mangrove fungi not only play an important role in the nutritive cycle, but alsop ossess immense biotechnological potentials (Hrudayanath et al. 2013). Becauseo ft he uniquea nd extremee nvironmental conditions, including extensives alinity, moisture,h igh temperature, tidal activities and high microbial competition, mangrove endophytic fungi are believed to contribute to mangrove adaptation to the extremeenvironment (Abdessamad et al. 2013). In addition, these fungi are also arich source of natural products.M any novel bioactive metabolites from mangrove fungi are used in pharmaceutical industry as lead compounds of antiviral, anticancer, antibiotic and immunosuppressive drugs and so on .
In the course of continuing search for novel bioactive natural compounds, ourattention has focused on af ungals train, Lasiodiplodia sp. 318#, which was collectedf rom Excoecaria agallocha of Mangrove National Nature Reserve in Gaoqiao, Zhanjiang city, Guangdong Province, China. Lasiodiplodins have been reportedasfungalmetabolites in 1971and have also been found in plants (Aldridgee ta l. 1971; Lee et al. 1982). This class of compound showed cytotoxicity against the P388 murine leukaemia cell line (Sadia et al. 2014). In this paper, four new lasiodiplodins 1 -4 ,t ogether with three known lasiodiplodinsw ere isolated from Lasiodiplodia sp. 318# (Figure1)a nd the cytotoxic activities were evaluated in vitro.

Results andd iscussion
The fungus Lasiodiplodia sp. 318# was fermentedusing rice solid medium. The EtOAc extract of the cultureo f Lasiodiplodia sp. 318# was fractionated and purified by SiO 2 column chromatography (CC), Sephadex LH-20 and HPLC to obtain compounds 1 -7 (Figure 1). Known compound 5 was assigned as lasiodiplodin by comparison with spectral data and singlecrystal X-ray evidence reported in the literature (Lee et al. 1982) ( Figure S1). Compounds 6 -7 were also identified by comparison of their spectroscopicdata with those of literature (Matsuura et al. 1998).
Compound 3 was isolated as an orangered solid, and itsmolecular formula was determined to be C 17 H 22 O 5 with 7degrees of unsaturation, alsofitted the lasiodiplodin structural class. The gross structure of compound 3 was identified by comparison of its NMR data to thoseo f compound 5 .The 1 HNMR and 13 CNMR data of 3 resembled closely to those of lasiodiplodin ( 5 ), the main differencesbeing in the 1 HNMR signals of benzene protons ( d H 6.20, 6.21 for 5 and d H 5.85 for 3 )and the downfield shifts observed for C-12 ( d C 180.9) and C-13 ( d C 178.0) in the 13 CN MR spectrum of 3 ,i ndicated that two aromatic carbons in 5 were replacedb yt wo ketone carbonyl carbons in 3 .T he location of the ketone carbonyl carbons was also demonstrated by the HMBC correlations from H-10 to C-11, C-12, and H-14 to C-12, C-13. The 12-membered lactones moiety in 3 was confirmed by the 1 H-1 HCOSY correlations from H-17 to H-10 and the HMBC correlations from H-17 to C-4, H-4 to C-5, H-5 to C-6, H-6 to C-7, H-9 to C-8, which was the samet o 5 ( Figure S19). Therefore, the structure of 3 was established. The absoluteconfiguration of 3 was tentativelyassigned as 3R on the basis of optical rotation values for 3 and 5 .

Cytotoxicity assay
Cytotoxic activity was tested by MTS assay ( Bliss 1935). The cytotoxicity activities of all the compounds were evaluated. Four humanc ancer celll ines MDA-MB-435, HepG2, HCT-116, A549 and human leukaemia THP1 cell line were used in the cytotoxicity bioassay. Epirubicin was used as positive control.

Conclusion
In summary, aseries of lasiodiplodins have been isolated from amangrove endophytic fungus by culturing the fungus in rice medium. The cytotoxicity activities of all the compounds were tested against THP1, MDA-MB-435, A549, HepG2 and HCT-116. Compound 4 showed moderate cytotoxica ctivities, which indicated that the open-ring structure contributest oc ytotoxicity activities.