Identification of Massoia Lactone and its Hydroxy-derivative from Kabatiella caulivora, an Endophyte of the Terrestrial Plant Alyxia reinwardtii

Massoia lactone (1) and its derivative, 3,5-dihydroxydecanoic acid--lactone (2), were identified from an ethyl acetate extract of the endophytic fungus Kabatiella caulivora, isolated from its host Alyxia reinwardtii. The compounds were identified by GC-MS. To the best of our knowledge, this is the first report of the identification of massoia lactone and its derivative from the endophytic K. caulivora fungus.


Introduction
Endophytic fungi are widely recognized as prolific sources of secondary metabolites that might be useful for the development of new pharmaceutical agents. Many novel bioactive products, such as antibiotics and anticancer and antidiabetic agents, have been isolated from these fungi [1].
Some endophytic fungi, such as Lecythophora sp., Hypocrea cf. koningii, Kabatiella caulivora strains A and B, Cladosporium oxysporum, and Aspergillus penicilloides, have been isolated in our laboratory from Alyxia reinwardtii [2], a slow-growing member of the Apocynaceae found in tropical climates. A. reinwardtii has been used in Indonesian traditional medicine (jamu), but the species is nearly extinct at present [3].

Materials and Methods
Plant material and isolation of the endophytic fungus. K. caulivora strains A and B were isolated from stems of A. reinwardtii (collected from the Purwodadi Botanical Garden, East Java, Indonesia). The sterilized stems were cut aseptically and the inner parts of the tissues were imprinted onto agar malt extract agar (MEA) medium containing powdered plant material (15 g/L) and chloramphenicol (0.2 g/L). Pure strains were obtained by repeated inoculation of the growing fungi on agar plates with fresh MEA medium. The endophyte was identified by Dr. Arnulf Diesel, Institut fuer Pharmazeutische Biologi Universität Düsseldorf, Germany, as described by Sugijanto [2].

Cultivation of the endophytic fungus.
A total of 523 culture bottles (40 mL) containing liquid malt extract medium was used for cultivation of K. caulivora strain A. The fungus was cultivated for 28 days at room temperature (30 ± 3 o C) at an initial pH of 5.75 ±0.05.
Extraction and fractionation. The culture broth and mycelia (20 L) were extracted with ethyl acetate (EtOAc, 73 L). The EtOAc layers were collected, combined, and concentrated under vacuum at 35°C to yield a blackish brown concentrated extract (5.34 g). This extract subjected to column chromatography on 200 g silica gel 60 (62.5  3.5 cm) as the stationary phase and eluted with mixtures of n-hexane, EtOAc, and methanol (MeOH) in increasing polarity, which yielded 17 fractions. Fraction 9 (157.3 mg) showed antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans using the disk diffusion method [11], so fraction 9 was further purified by column chromatography on Sephadex LH 20 (16 g) with MeOH as the mobile phase. This yielded a semi-purified oily fraction a (20.1 mg) that had a specific aroma of massoia bark oil. This fraction a was dissolved in 5 mL MeOH.

Gas Chromatography-Mass Spectrometry (GC-MS).
The GC-MS analysis of fraction a was performed using an Agilent GC 6890N and Mass Selective Detector (MSD) 5973, equipped with an HP-5 column (30 m  0.250 mm  0.25 µm i.d.), and completed using the Wiley 7n.1 data base (2004). The inlet temperature was set at 250°C, the split ratio was 50:1, the flow rate was 1 mL/min (He), and the oven temperature was programmed at 100-250°C, at 5°C/min. The electron impact ionization (EI-MS) source temperature was set at 230°C, the transfer line temperature from the oven to the detector was 280°C, and the energy of ionization was set at 70 eV.

Method of Identification.
The EI MS spectra of peaks 1 and 2 were compared to the Wiley database, the online data bases of the National Institute of Standards and Technology (NIST) [12], the Spectral Database for Organic Compounds (SDBS) [13], and published reports [7,9,[14][15][16], according to the method recommended by the Commission Decision 2002/657/EC [17].

Results and Discussion
The total ion chromatogram (TIC) of fraction a exhibited two main peaks at retention times (Rt) of 9.8 and 15.0 min (see Figure 2). The EI-MS spectrum of peak 1 (see Figure 3) showed a very small molecular ion at m/z 168, with other peaks observed at m/z 97 (base peak), 69, 68, and 41. The Wiley database predicted that peak 1 was massoia lactone (1), with the highest quality score (90%). The EI-MS of the peak 1 was identical to the EI-MS spectra of massoia lactone of published by NIST [12], SDBS [13], previous publications [7,[14][15][16], and the main compound of massoia bark oil (1) [unpublished work]. According to the Commission Decision 2002/657/EC, the identity of a compound can be confirmed if its MS spectrum shows at least 4 identical fragments when compared to the standard MS [17]. All these data confirmed unambiguously that peak 1 was massoia lactone or 2deceno--lactone (1).
The EI-MS spectrum of peak 2 ( Figure 4) showed a similarity with compound (2) according to the Wiley database (highest quality score 90%). The EI-MS of peak 2 showed peaks at m/z 168, 115 (base peak), 97, 73, and 43, which was identical to the EI-MS of 2 in previous publications [9,15]. The presence of a hydroxyl group was confirmed by intense peaks of m/z at 97 and 115. The absence of the molecular ion at m/z 186 was due to a loss of H 2 O from the molecular ion and a fragment at m/z 168 was produced [15]. These data confirmed [17] that peak 2 was 3,5-dihydroxydecanoic acid--lactone (2), a hydroxyl derivative of 1.
Previous publications [7,9] indicated that the genus Kabatiella or Aureobasidium could produce compound 1 and 2, and this present work reported the identification of both compounds from K. caulivora cultures. Consequently, compounds 1 and 2 might be specific to this genus. Further work is needed to confirm this hypothesis.
Compound 1 was isolated from other fungi, including Fusarium solani and Trichoderma viride [18], while compounds 1 and 2 were also identified from the fungus Cephalosporium recifei [15]. Compound 1 was isolated from plants (i.e., sugar cane molasses, coconut and massoia bark-oil, and Solanum muricatum [16,19,20]). This shows that compound 1 is present in both fungi and plants. Compound 1 has reported activity against the growth of Candida sp. [7,21], while the antibacterial activity of compound 1 against some microorganisms was reported by Simionatto et al. [22]. This activity could explain the activity of fraction a against C. albicans, S. aureus, and E. coli. Compounds 1 and 2 could be present in fungi and plants to function as a defense system against microorganisms.

Conclusion
Massoia lactone (1) and 3,5-dihydroxydecanoic acid-lactone (2) were identified for the first time in the endophytic fungus K. caulivora isolated from A. reinwardtii. Compounds 1 and 2 appear to be specific to the genus Kabatiella.