Sesquiterpenes produced by Pestalotiopsis microspora HF 12440 isolated from Artocarpus heterophyllus

Abstract A drimane-type sesquiterpene, (+)-dendocarbin L (1) together with two bisabolane-type sesquiterpenes, (+)-sydonic acid (2) and (+)-sydowic acid (3) were isolated from the mycelium of Pestalotiopsis microspora HF 12440, an endophytic fungus from the stem of Artocarpus heterophyllus. The structures of all compounds were elucidated using spectroscopic methods and by comparison with the literature. Compound 1 was isolated from the fungi for the first time, compounds 2 and 3 were firstly obtained from this endophytic fungus. Compound 3 showed cytotoxicity (IC50 2.56 μg/mL) against murine leukemia P-388 cells. Graphical Abstract


Introduction
Artocarpus, together with Morus and Ficus, are the important plant genus in family Moraceae. Traditionally, Artocarpus have been used as medicines, such as to treat inflammation, malaria, fever and diarrhea (Jagtap and Bapat 2010). Chemical investigation of Artocarpus in our laboratory indicated that the main secondary metabolites from Artocarpus are prenylated phenolic compounds such as flavonoids, stilbenes and 2-arylbenzofuranes with important bioactivities, including cytotoxicity Hakim et al. 2002;Syah et al. 2006). In a continuing project on the bioactive compounds from Artocarpus, we have explored endophytic fungi isolated from this genus plant. Endophyte can be described as microbes that live reside in various tissues of plants (Kaul et al. 2012;Jia et al. 2016;Deng and Cao 2017). Endophytic fungi can produce many secondary metabolites with the variety of chemical structures and bioactivities. A fungus, P. microspora HF 12440, was isolated from the stem of A. heterophyllus. Pestalotiopsis has been reported to produce terpenoids, a-pyrones, steroids, phenolic derivates and coumarins (Xu et al. 2010;Yang et al. 2012;Xu et al. 2016;Zhou et al. 2017;Li et al. 2018). Herein, we report the isolation, characterization and bioactivity of three sesquiterpenes, (þ)-dendocarbin L (1), (þ)-sydonic acid (2) and (þ)-sydowic acid (3) from the mycelial P. microspora HF 12440 isolated from the stem of A. heterophyllus. All sesquiterpenoids were assayed for their cytotoxic activity against murine leukemia P-388 cells. Compound 3 demonstrated cytotoxic activity with IC 50 2.56 lg/mL. In fact, no report on phytochemical constituents from Pestalotiopsis living in A. heterophyllus has been found.

Results and discussion
The fresh stem of A. heterophyllus was sterilized with ethanol 70%, NaOCl 3.5% and aquabidest and then inoculated on Potato Dextrose Agar (PDA) media. A single strain of fungus, P. microspora HF 12440, was identified by morphological assessment and internal transcribed spacer (ITS) sequence analysis in Indonesian Institute of Science, West Java. The fungus was transferred onto Potato Dextrose Broth (PDB) media. After 14 days of cultivation, the filtrate was separated with Buchner funnel and the mycelium was extracted with methanol. Separation of methanol extract with vacuum liquid chromatography, radial chromatography and column chromatography yielded compounds 1, 2 and 3 ( Figure 1).
All structures of isolated compounds were determined by means of HR/ESI/TOF-MS, extensive NMR (1D and 2D) spectroscopic studies and by comparison with previously published data. Compounds 1, 2 and 3 were identified as (þ)-dendocarbin L (Sultana et al. 2011), (þ)-sydonic acid (Kudo et al. 2009) and (þ)-sydowic acid (Serra 2000), respectively. To our knowledge, compound 1 has never been previously reported from endophytic fungi. Additionally, isolation of compounds 2 and 3 from Pestalotiopsis has been investigated for the first time in this study.
Cytotoxicity of pure compounds was assessed in murine leukemia P-388 cells. The results showed that (þ)-sydowic acid (3) was most potential with IC 50 2.56 lg/mL, whereas compounds 1 and 2 were inactive with IC 50 18.78; 20.30 lg/mL, respectively.

Conclusion
Phytochemical evaluation on the mycelium of P. microspora led to the isolation and identification of three sesquiterpenes, (þ)-dendocarbin L (1), (þ)-sydonic acid (2) and (þ)-sydowic acid (3). Significant cytotoxic activity of compound 3 against murine leukemia P-388 cells was observed with IC 50 2.56 lg/mL. Interestingly, there is no report on compound 1 isolated from endophytic fungi. In addition, compounds 2 and 3 were present in extracts from Pestalotiopsis for the first time.