Isolation of Pandangolide 1 from Cladosporium oxysporum , An Endophyte of the Terrestrial Plant Alyxia reinwardtii

Pandangolide 1 was isolated from the ethyl acetate ex ract of Cladosporium oxysporum cultures. The fungus was originally obtained from Alyxia reinwardtii. The structure of pandangolide 1 was elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopy and accurate m ss spectrometric data. This is the first report o f the isolation of pandangolide 1 from endophytic C. oxysporum derived from a terrestrial host plant.


Introduction
Endophytic fungi are those that grow intra-or intercellularly within the tissues of higher plants without causing overt symptoms of disease [1].Endophytic fungi are known to produce various bioactive metabolites such as agrochemicals, antibiotics, immunosuppressants, antiparasitics, antioxidants, and compounds with anticancer, antiviral, insecticidal, and antidiabetic activity [2][3][4].
To the best of our knowledge, no report has yet appeared on metabolites of endophytic C. oxysporum.In this work, we report the isolation and structural elucidation of a metabolite from the fungus C. oxysporum growing as an endophyte in A. reinwardtii.

Methods
General experimental procedures.Electrospray ionization mass spectrometry (ESI-MS) was recorded on a UPLC Dionex Ultimate 3000 coupled to a QTOF Bruker Maxis Impact HD.NMR spectra were recorded on a VNMRS400 Varian 400MHz instrument.TLC was carried out on silica gel 60 F 254 TLC plates (Merck) and reversed phase C-18 TLC plates (Merck).Column chromatography used silica gel 60 (Merck) as the stationary phase.Detection was performed by UV light at wavelengths of 254 and 366 nm, and also through derivatization with p-anisaldehyde-H 2 SO 4 reagent.

Plant material and fungal isolation.
A.reinwardtii was collected from Purwodadi Botanical Garden, East Java, Indonesia in April 2003.The plant material was identified by Dr. Irawati at the Research Center for Biology, Indonesian Institute of Sciences, Bogor, Indonesia (voucher no.710/IPH.1.02.If.8/2003).Stems of A. reinwardtii were surface sterilized with 70% ethanol and 75% Clorox (Bayclin TM ).The sterilized stems were cut aseptically and parts of the inner tissues were imprinted onto agar plates containing malt extract agar (MEA) medium with added powdered plant material (15 g/L) and chloramphenicol (0.2 g/L).Pure strains were obtained by repeated inoculation of growing fungi on agar plates with fresh MEA medium (without powdered plant material and chloramphenicol).The isolated fungus (SUB-ALE-A) was identified by Dr. Arnulf Diesel (Heinrich-Heine-Universität Düsseldorf, Germany) as C. oxysporum based on its ITS sequence, as described previously [5,6].

Malt extract broth (MEB) culture of C. oxysporum.
Flasks (500 mL) containing 250 mL of MEB medium were sterilized.A small part of a fungal culture from slant agar medium was transferred under sterile conditions to the MEB medium.The fungus was grown under static conditions at room temperature (approx.30±3 °C) for four weeks.
In the HMBC experiment (Figure2 and Table 1), an important correlation was observed between the oxyme thine proton at δ 4.88 (H-11) and the carbonyl group at δ 174.4 (C-1), which confirmed the position of the lactone moiety.Based on this evidence and on comparison with previously reported NMR data [10,16], 1 was unambiguously identified as pandangolide 1.

Conclusion
Pandangolide 1 was isolated for the first time from the C. oxysporum endophyte of the terrestrial plant A. reinwardtii.

Table 1 . Comparison of NMR Data of 1 with Pandangolide 1
Position δ H , mult.