New lasiodiplodins from mangrove endophytic fungus Lasiodiplodia sp. 318#

Abstract Two new lasiodiplodins (1–2) together with three known analogues, were isolated from a mangrove endophytic fungus, Lasiodiplodia sp. 318#. Their structures were established by spectroscopic techniques (1D- and 2D-NMR, HR-ESI-MS, etc.), and electronic circular dichroism. Cytotoxic activities of compounds 1–5 were evaluated in vitro. Compound 4 was the most potent, with IC50 values of 5.29 μM against MMQ, 13.05 μM against GH3. Preliminary structural-activity analysis indicated that the functional group (resorcinol-3-OH) contributed greatly to the binding of Lasiodiplodins to the cytotoxic activities.


Introduction
Lasiodiplodin consists of a resorcinol moiety fused to a 14/12-member macrocyclic lactone ring, which was first isolated from Lasiodiplodia theobromae and includes a trans double bond, a ketone and a methyl branch (aldridge et al. 1971;El-Elimat et al. 2014). The structure is clearly flexible enough to allow lasiodiplodins to adopt a conformation able to bind to the activity. Lasiodiplodin analogues show a wide range of biological activities, such as potato micro-tuber-inducing in vitro (Li et al. 2005), cytotoxicity (Buayairaksa et al. 2011), mineralocorticoid receptor antagonist (Jiang et al. 2011), anti-microbial activity (Yang et al. 2006a), plant-growth inhibitor (Veiga et al. 2007), ɑ-glucosidase inhibitory activity (Chen et al. 2015) and so on. In previous research, the activity of 12,14-dibromo-lasiodiplodin was seven times stronger than that of the parent compound (Yang et al. 2006b).
In our continued search for natural products from mangrove endophytic fungi on the bioactive principles, our attention has focused on the plant of Excoecaria agallocha, which widely grow in mangrove swamps. E. agallocha belongs to the genus Excoecaria of the family Euphorbiaceae, which was used to treat ulcers, leprosy, some sores and stings from marine creatures (Subhan et al. 2008). This plant is well-protected by chemical defences, and can cause severe human allergies. It can produce secondary metabolites including diterpenoids, triterpenoids and flavonoids, especially in the milky latex (Konishi et al. 1998;Konoshima et al. 2001;Zou et al. 2006;Subhan et al. 2008). under the distinctive environment, the endophytic fungi can produce many bioactive molecules to compete the living space. The Lasiodiplodia sp. 318 # , was collected from E. agallocha in Mangrove National Nature Reserve in Gaoqiao, Zhanjiang city, Guangdong Province, China. In previous study, some new lasiodiplodins had been isolated from this fungus, and tested cytotoxic activity against human cancer lines THP1, MDa-MB-435, a549, HepG2 and HCT-116, which show moderate cytotoxic activity .
In this paper, two new lasiodiplodins 1-2, together with three known lasiodiplodins were isolated from Lasiodiplodia sp. 318 # and their structures were established by spectroscopic techniques and electronic circular dichroism (ECD). Their cytotoxic activities were evaluated in vitro.
The absolute configuration of 1 was assigned by application of ECD. From the Supplementary Figure S2, compound 1 which showed a negative cotton effect at 247 nm, which deduced that, the absolute configuration at C-15 belonged to R.
among the lasiodiplodins, finding the relationship between biological activity and structure, play an important role in exploiting biological function serves in the producing organisms. Cytotoxic activities of seven lasiodiplodins which has been reported were evaluated in vitro against human cancer lines MDa-MB-435, a549, HepG2, HCT-116 and human leukaemia THP1 . The compound ethyl-2,4-dihydroxy-6-(8′-hydroxynonyl)-benzoate, one opening ring structure with hydroxylation of position C-3 of lasiodiplodins, exhibited moderate cytotoxic activities while other compounds showed no notable cytotoxic activities which indicated that the hydroxylation of C-3 or the open-ring structure increased flexibility in the macrocyclic lactone ring and might contribute to cytotoxic activities. In this paper, further studies aimed at discovering the potential activities of these lasiodiplodins, rat prolactinoma cell line MMQ and rat pituitary adenoma GH3 cell line were used. Compounds 1-5 showed different inhibitory effect on MMQ and GH3 cell lines (Supplementary Figure  S3). Compounds 2-5 showed more potent than compound 1, especially compound 4 was the most potent, with IC 50 values of 5.29 μM against MMQ and 13.05 μM against GH3, respectively. Compounds 2-5, all with a hydroxyl group at C-3 position, exhibited cytotoxic activities. and compound 1 with a methoxy moiety at C-3 position was inactive. This demonstrated the importance of the hydroxyl group at C-3 position, which was also supported by our previous studies . Comparing the compounds 2 and 4, carbonylation of position C-15 in compound 2 reduced the cytotoxic activities. In addition, the IC 50 values of compounds 2-4 against normal cells (RPC) higher than the cancer cells (MMQ, GH3), which indicated that compounds 2-4 have some selective between normal cells and cancer cells.

Conclusion
Finally, two new lasiodiplodins together with three known analogues have been isolated from a mangrove endophytic fungus by culturing the fungus in rice medium. The cytotoxic activities of the compound 1-5 were tested. Compound 4 was the most potent, with IC 50 values of 5.29 μM against MMQ, 13.05 μM against GH3, which indicated that the functional group (resorcinol-3-OH) contributed greatly to the binding of Lasiodiplodins to the cytotoxic activities.

Supplementary material
all the spectra of compounds 1-4 and cytotoxic assay are available in the supplementary file.

Author contributions
Jing Li and Lan Liu conceived and designed the experiments, Jiguo Huang, Jiayi Xu, Dilfaraz Khan, Shah Iram niaz, Yongcheng Lin performed the experiments and test, Zhen Wang and Yonghong Zhu contributed the activities assay. Jiguo Huang, Dilfaraz Khan, Jing Li and Lan Liu analysed the data and composed the manuscript.