Drimane Sesquiterpenoids and Isochromone Derivative from the Endophytic Fungus Pestalotiopsis sp. M-23

Three new drimane sesquiterpenoids (1–3) together with the known 2α-hydroxyisodrimeninol (4), and a new isochromone derivative (5), were obtained from the solid cultures of fungal strain Pestalotiopsis sp. M-23, an endophytic fungus isolated from the leaves of Leucosceptrum canum (Labiatae). Their structures were determined by comprehensive 1D and 2D NMR, and MS analyses. The metabolites were evaluated for their antibacterial activities, and compound 3 showed weak inhibitory activity against Bacillus subtilis.


Introduction
Fungal endophytes are ubiquitous fungi that inhabit healthy plant tissues without causing apparent symptoms of disease, and their colonizations have been found in almost all kinds of plants, from algae to vascular plants [1]. Endophytic fungi have gained prominence as important sources of a variety of new biologically active natural products [2]. For example, the famous anticancer drug paclitaxel can be produced by the endophytic fungus Taxomyces andreance from Pacific yew [3]. An endophytic fungus Thielavia subthermophila [4] from the medicinal herb Hypericum perforatum has been reported to produce the antidepressant napthodianthrone derivative hypericin [5].
The fungal genus Pestalotiopsis, representing one of the largest biomasses of any plant-associated endophytic fungus in the world, produces a large variety of secondary metabolites, including terpenoids, alkaloids, chromone derivatives and phenolics [6], with cytotoxic [7], antibacterial and antifungal activities [8,9]. In this study, an endophytic fungal strain, Pestalotiopsis sp. M-23, was isolated from the leaves of Leucosceptrum canum, a woody Labiatae (=Lamiaceae) plant with unique dark-brown colored nectar that has been proved to be caused by a novel bird attractant proline-benzoquinone, and with a unique class of sesterterpenoids named leucosceptroids that have been shown to function as defense against attack by insect herbivores and pathogens [10][11][12][13][14]. In this study, a phytochemical investigation on the solid culture of the endophytic fungus Pestalotiopsis sp. M-23 was carried out, which led to the isolation and identification of five compounds (1)(2)(3)(4)(5), including three new drimane sesquiterpenoids (1-3) and a new isochromone derivative (5). In addition, the antibacterial activities of these compounds were also evaluated.

Results and Discussion
More than one hundred endophytic fungal strains were isolated from the leaves of L. canum. Among them, M-23 was identified as Pestalotiopsis sp. using molecular biological techniques [15]. To carry out phytochemical investigation, the mycelia and solid culture media of Pestalotiopsis sp. M-23 were extracted with acetone, and the crude extract was fractionated by column chromatography on silica gel. Further purification was performed by repeated normal-phase, Sephadex LH-20, ODS column chromatographies, and reverse-phase semi-preparative HPLC to yield five compounds (1)(2)(3)(4)(5).
Compound 1 was isolated as colorless oil. Its molecular formula was established as C 15 7), and three quaternary carbons (including an oxygen-bearing carbon at d C 79.0). These data suggested that 1 was a drimane sesquiterpenoid [16,17]. Comparing the NMR spectra (Table 1) of 1 with those of 2a-hydroxyisodrimeninol (4) [17,18], a drimane sesquiterpenoid also isolated from this fungus, revealed that the two compounds were similar. Compound 1 showed the typical hemiacetal and oxymethine resonances as those in 4. The major difference between these two compounds was that the tri-substituted double bond in 4 was replaced by a di-substituted double bond located between C-6 and C-7 in 1, which was confirmed by the 1 3.70) revealed that these protons were b-oriented, and the ROESY correlation between H-5 (d H 1.93) and H-9 (d H 2.07) suggested their a-oriented configuration (Fig. 2). Consequently, the structure of 1 was determined as 2a,8adihydroxy-6,7-en-isodrimeninol ( Fig. 1).
Compound 2 was obtained as colorless oil. It gave a molecular formula of C 15 H 24 O 4 according to 13 C NMR spectroscopic and HR-EI-MS data (m/z 268.1677 [M] ? ), with 4°of unsaturation. The 1 H and 13 C NMR spectroscopic data (Table 1) were similar to those of 4, suggesting that 2 was also a drimane sesquiterpenoid with a hemiacetal moiety and a hydroxyl group located at C-11 and C-2, respectively. Major difference between these two compounds was that the resonances of double bond between C-7 and C-8 in 4 were missing, instead an oxymethine at d C 59.6 and an oxygen-bearing quaternary carbon at d C 65.2 appeared in 2. The HMBC correlations from H 2 -12 (d H 3.75 and 4.00) to C-7 (d C 59.6) and C-8 (d C 65.2), and from H-7 (d H 3.38) to C-6 (d C 23.7) in 2, suggested the presence of an epoxide group between C-7 and C-8 in 2. The ROESY spectrum revealed that the relative configuration of 2 was similar to that of 4, and the ROESY correlation of H-7 (d H 3.38) with H-12 (d H 3.75) indicated that H-7 and H-12 were at b-position, and thus the epoxide ring occupied a-position (Fig. 2). Therefore, the structure of 2 was concluded as 2ahydroxy-7a,8a-epoxy-isodrimeninol (Fig. 1).
Compound 3 exhibited an [M ? Na] ? ion peak at m/z 273.1462 in the HR-ESI-MS, corresponding to the molecular formula C 15 H 22 O 3 with 5°of unsaturation, suggesting that 3 was also a sesquiterpenoid. Comparing its 1D NMR spectra data ( Table 1) with those of dendocarbin B [17] indicated that 3 had the similar drimane skeleton with an ester carbonyl group located at C-11 and a double bond between C-7 and C-8, which were further confirmed by analyzing the HMBC and HSQC spectra of 3. The oxymethine at d C 75.5 in 3 obviously appeared at lower field than the one in dendocarbin B (d C 63.6) [17], indicating that the position of the hydroxyl group shifted in 3. In the HMBC spectrum of 3, the correlations from Me-13 (d H 0.93) and Me-14 (d H 0.96) to the oxymethine carbon at d C 75.5 indicated that the hydroxyl group was assignable to C-3. The coupling pattern of H-3 (d H 3.44, br s) indicated an a-oriented configuration of 3-OH [19] (Fig. 2), which was supported by the ROESY cross-peak between H-3 and Me-13, and by the upfield shifted C-14 due to a c-gauch effect from 3a-OH. Thus, the structure of  3 was established as 11-dehydro-3a-hydroxyisodrimeninol (Fig. 1). Compound 5 was obtained as colorless oil and has a molecular formula of C 12 H 14 O 6 as determined from HR-ESI-MS (m/z 249.1105 [M ? Na] ? ), with 6°of unsaturation. In the 1 H NMR spectrum (Table 2), a secondary methyl resonance at d H 1.57 (d, J = 6.7 Hz, 3H) and an oxymethine group at d H 5.46 (q, J = 6.7 Hz, 1H) were evident, indicating the existence of an AB 3 system in 5. A singlet at d H 5.17 (2H) and a pair of AB doublets at d H 4.35 (d, J = 11.3 Hz, 1H) and 4.10 (d, J = 11.3 Hz, 1H) were clearly shown, revealing the presence of two oxygenated methylene groups. In the low-field region, two ortho-coupled aromatic doublets at d H 7.65 (d, J = 7.6 Hz) and 8.19 (d, J = 7.6 Hz) were observed, indicative of a 1,2,3,4-tetra-substituted phenyl ring. The 13 C NMR and DEPT spectra ( Table 2) (Fig. 2). Therefore, the structure of 5 was determined as 4,10-dihydroxy-gamahorin (Fig. 1).
A known drimane sesquiterpenoid was also isolated and identified as 2a-hydroxyisodrimeninol (4) [17,18], by comparing its NMR data with those previously reported in the literature.
Dramine sesquiterpenoids have been shown to possess extensive biologically activities, such as antifeedant, antiinflammatory, cytotoxic, antioxidant and a-amylase inhibitory activities [21][22][23]. Antibacterial activity of 1-5 against Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus were evaluated in this study. Compound 3 showed weak inhibitory effect on B. subtilis with IC 50 value of 280.27 lM. However, none of these compounds showed obvious activity against S. aureus and M. luteus. Despite, our results indicated that Pestalotiopsis sp. M-23 as a prolific resource of characteristic dramine sesquiterpenoids, is an interesting endophyte worthy of further indepth investigation.

General Experimental Procedures
Column chromatographies were performed on 200-300 mesh silica gel (Qingdao Marine Chemical Factory, P. R. China), Sephadex LH-20 (25-100 lm, GE Healthcare), and ODS (75 lm, YMC gel). Optical rotations were measured on a Horiba-SEAP-300 spectropolarimeter. UV spectral data were obtained on a Shimadzu-210A doublebeam spectrophotometer. IR spectra were recorded on a Bruker-Tensor-27 spectrometer with KBr pellets. NMR experiments were carried out on either a Bruker AM-400 or an Avance-600 spectrometer with TMS as internal standard. ESI-MS and HR-ESI-MS were recorded on an API-QSTAR-TOF. EI-MS and HR-EI-MS were recorded on an Autospec Premier P776 mass spectrometer. Semi-preparative HPLC analyses were performed on an Agilent 1200 series instrument equipped with a quaternary pump, a vacuum degasser, an autosampler, a thermostated column compartment and a diode array detector.

Fungal Strain Isolation and Identification
Healthy, asymptomatic leaves of L. canum were harvested at Kunming Botanical Garden, P. R. China, in March 2014, and disinfected with 75 % EtOH for 10 s, and then rinsed with sterile distilled water for 5 times. The surface disinfected leaves were aseptically cut into 2 cm 9 2 cm pieces, and cultivated in potato dextrose agar (PDA) plates containing 30 lg/mL streptomycin to inhibit the bacterial growth at 28°C. A pure strain coded as M-23 was obtained by transferring monosporic isolates to fresh PDA gradually. M-23 was incubated in potato dextrose broth (PDB) at 28°C in 100 mL Erlenmeyer flask for 3 days and shaking at 220 rpm.
Genomic DNA of M-23 was extracted from fungal mycelia (20 mg) grown in PBD using fungal DNA isolation mini kit (Sangon, Shanghai, China). The PCR reaction was performed using isolated genomic DNA as template, ITS5/ITS4 as primer pairs. The amplified DNA fragment was purified and sequenced using the same primer pairs by BGI Inc. The obtained DNA sequence data were searched for similar sequences in GenBank.
M-23 strain, whose ITS sequence data were found to be identical to those of Pestalotiopsis clavata MFLUCC 12-0268 (NR120182), P. neglecta Q13DW (EF055210) and P. lespedezae SY16E (EF055205) using BLAST search in NCBI, was finally identified as Pestalotiopsis sp.. The obtained sequence data were submitted to and deposited at GenBank (Accession No. KT372852).

Antibacterial Tests
Antibacterial activity of 1-5 against Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus were evaluated using broth dilution method [24] with modification. Briefly, test compound was dissolved in proper solvent to obtain the highest concentration of 10.24 mg/mL. Serial dilutions of mother solution were performed with final concentration ranging from 512, 256, 128, 64, 32, 16 and 0 lg/mL. Ampicillin was used as positive control. All assays were performed in triplicate. The results were expressed as the minimum concentration inhibiting 50 % of bacterial growth (IC 50 ) [25]. The IC 50 values were calculated after 24 h of growth at 37°C.