Introduction

Although the advent of combinatorial chemistry has shifted the research focus away from natural products, fungal endophytes continue to be a new and feasible source for novel drugs (Suryanarayanan et al. 2009). Endophytic fungi, microorganisms occuring within the living tissues of plants without causing visible disease symptoms at a particular time (Rodriguez et al. 2009), have been found in all plant species examined to date (Arnold et al. 2000). They have a capacity to produce an array of metabolites of varied structural groups such as terpenoids, steroids, xanthones, chinones, phenols, isocoumarins, benzopyranones, tetralones, cytochalasines, and enniatines (Schulz et al. 2002) possessing a variety of biological activity include antibacterial, antiviral, antifungal, and anticancer activities (Gunatilaka 2006). However, only few of them have been cultivated and screened for drug use.

Taxus chinensis var. mairei (Lemée et Lévl) Cheng et L.K. Fu, one Taxus species endemic to China, is ubiquitous to Yangtze River southward in China. Yew trees are a rich source of biological active diterpenoids belonging to the unique structure class of taxanes (Baloglu and Kingston 1999; Parmar et al. 1999) such as taxol (paclitaxel), which is one of the most important anticancer drugs currently on the market for the treatment of ovarian and breast cancers and shows promise for the treatment of neck, lung, gastrointestinal and bladder cancers (Li et al. 2008). In spite of taxol-producing endophytic fungi having generated worldwide interest (Zhang et al. 2009a, b; Raja et al. 2008; Kumaran et al. 2010), there are a few studies on other bioactive compounds produced by endophytes harbored in Taxus species (Li et al. 2009).

In the course of our continuous search for novel bioactive secondary metabolites from endophyte cultures (Xu et al. 2009; You et al. 2009; Sun et al. 2011), we isolated a fungus Perenniporia tephropora Z41with high anti-Pyricularia oryzae activity from the bark of T. chinensis var. mairei collected in Jingning, Zhejiang Province, People’s Republic of China. Through bioassay-oriented fractionation, a new sesquiterpenoid, namely perenniporin A (1), together with three known compounds, ergosterol (2), rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]furan-5-ol (3), and albicanol (4), were isolated from the culture broth of P. tephropora Z41. We report herein the details of the isolation and identification of endophytes and compounds and the evaluation for cytotoxic activity of those isolated compounds.

Materials and methods

Isolation and identification of the endophytic fungus

Healthy bark of T. chinensis var. mairei was collected in Jingning, Zhejiang Province, PR China. Samples were immediately placed in plastic bags and taken to the laboratory store at 4 °C for isolation of endophytic fungi within 48 h of collection. The samples were cleaned in tap water and then cut into 1 × 1 cm pieces before surface-sterilization. Bark pieces were consecutive immersion in 75 % ethanol for 1 min, 3 min in 2.5 % sodium hypochlorite, and 30 s in 75 % ethanol. The surface sterilized bark was cut into 0.5 × 0.5 cm pieces, and the tissues were placed on potato dextrose agar (PDA) media containing 50 mg/L penicillin to inhibit bacterial contamination and incubated at room temperature for 7–14 days. The hyphal tip was removed and placed on PDA media, incubated at 26 °C, and replated until a pure culture was obtained.

The isolated endophytic fungus Z41 was identified according to its 5.8S gene and ITS (ITS1 and ITS2 regions) sequence by using the universal primers ITS5 and ITS4 following the protocol of Guo et al. (2003). The 5.8S gene and ITS regions sequence was compared by Blast search with reference sequences at the GenBank and aligned with CLUSTAL X software (Thompson et al. 1997) using 1,000 bootstrap replicates. The phylogenetic tree was performed using the neighbor-joining method (Saitou and Nei 1987). Identification at species taxonomic levels was based on ≥97 % ITS similarity (Higgins et al. 2007). The sequence was submitted to GenBank (accession No.JN198488). The fungal strain Z41 was deposited in the China General Microbiological Culture Collection Center (CGMCC) as CGMCC 3.14976.

Fermentation and compounds isolation

The endophytic fungus Z41 initially grown on a PDA medium in Petri dishes was transferred into a shake flask culture by punching out 5 mm of the agar plate culture with a self-designed cutter (Wang et al. 2006). The shake flask culture was carried out (250 mL) containing 100 ml potato dextrose liquid medium at 26 °C with 180 rpm for 7 days and then enriched by transferring into 5 L Erlenmeyer flasks. About 60 L of fermentation liquid was obtained after incubation in the same condition for 10 days. The mycelial pellets were harvested by filtration and then thoroughly crushed in a mortar. The fermentation broths and ground mycelia were subjected to ultrasound-assisted extraction three times with equal volume of ethyl acetate at room temperature for 1 h. All extracts were combined and condensed in a rotating evaporator under reduced pressure. The obtained crude extract (5.62 g) was chromatographed on a silica gel column eluting with a step gradient of petroleum ether- EtOAc (20:1, 10:1, 5:1, 1:1, 1:2, each 1.5 L, v/v) to give eight fractions (Fr1–Fr8). Fr3 was recrystallized to give compound 2 (18 mg). Fr4 (305 mg) was separated over silica gel with petroleum ether–EtOAc (5:1, 3:1, 1:1, each 0.5 L, v/v) to give two subfractions (subFr1 and subFr2). SubFr1 (45 mg) was separated using preparative TLC (petroleum ether–EtOAc, 3:1, v/v) to obtain compound 3 (12 mg). SubFr2 (85 mg) was subjected on Sephadex LH-20 (CH2Cl2—MeOH, 1:1; 500 ml, v/v) to yield compound 4 (8 mg). Fr5 (325 mg) was subjected to silica gel with petroleum ether–EtOAc (3:1, 1:1, each 0.5 L, v/v) and Sephadex LH-20 (MeOH—H2O, 1:1; 500 ml, v/v) to give compound 1 (102 mg).

Chemical analysis

NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer with TMS as an internal standard. ESIMS were measured on an Agilent LC/MSD Trap XCT mass spectrometer, whereas HRESIMS were measured using a Q-TOF micro mass spectrometer (Waters, USA). IR spectra were recorded on a Bruker Vector 22 spectrometer with KBr pellets. Optical rotations were acquired with a Perkin-Elmer 341 polarimeter. CD data were obtained on a Jasco 810 spectrometer. Materials for column chromatography were silica gel (100–200 mesh; Huiyou Silical Gel Development Co. Ltd. Yantai, China), silica gel H (10–40 μm; Yantai), Sephadex LH-20 (40–70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-GEL ODS-A (50 μm; YMC, Milford, MA). HSGF254 silica gel TLC plates (Yantai) were used for analytical TLC.

Determination of the absolute configuration of C-10 in compound 1

According to the published literature (Górecki et al. 2007; Di Bari et al. 2001), a mixture of compound 1 (1.1 mg) and Mo2(OAc)4 (1.2 mg) was prepared for CD measurement. The mixture was kept for 30 min to form a stable chiral metal complex, the CD spectrum of which was then recorded. The observed sign of the diagnostic ICD (induced CD spectrum) curve at around 305 nm was correlated with the absolute configuration of C-10 in compound 1 (Górecki et al. 2007; Di Bari et al. 2001).

P. oryzae bioassay

The anti-P. oryzae activity was evaluated following the method of Xu (Xu et al. 2009). In brief, after the spore suspension of P. oryzae P-2b (4 × 104/mL) was added in a 96-well microplate, 50 μL of sample was placed in each well and incubated at 27 °C for 15 h. The spore growth and mycelium shape were observed and compared with Ketoconazole to determine the minimum inhibited concentration (MIC). Each test was done in three replicates and interpretation was based on average value of results.

Cytotoxic assay

The MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide] colorimetric assay, against HeLa, SMMC-7721 and Panc-1 (available in Chinese Academy of Sciences), was performed as described in the literature (Wang et al. 2011). Briefly, the cells were cultured in 10% FBS DMEM medium. Test samples were prepared at six concentrations (0.001, 0.01, 0.1, 1.0, 10.0, and 100.0 μg/mL). After these cell lines were seeded in a 96-well microplate for 12 h, 10 μL of sample was placed in each well and incubated at 37 °C for 48 h, and then 20 μL MTT (5 mg/mL; Sigma, NY) was added for 4 h. After removing the medium and putting 150 μL dimethyl sulfoxide (DMSO) into each well, the plate was shaken for 10 min to dissolve blue formazan crystals and the absorbance was read at a wavelength of 570 nm on a microplate reader (Labsystems, WellscanMR-2).

The cytotoxicity of tested samples against tumor cells was expressed as IC50 values, which were calculated by LOGIT method. Results are representative of three individual experiments.

Results

Identification of the endophytic fungus

The phylogenetic tree (Fig. 1) inferred from the 5.8S gene and ITS regions sequences indicated that the endophytic fungus Z41 was classified into the clade including P. tephropora AB462323, Perenniporia corticola HQ654093, Perenniporia tenuis HQ848474, Perenniporia maackiae HQ654102. The endophytic fungus Z41 closely related to P. tephropora AB462323 with the ITS sequence similarity of 99.3 % and was therefore further identified as P. tephropora.

Fig. 1
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Phylogenetic tree of the endophytic fungus Z41 based on 5.8S and ITS regions sequences. Bootstrap values above 50 % (1,000 replicates) are shown at branches. Hydnopolyporus fimbriatus is used as an outgroup

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Structural determination of the compounds

Compound 1, trivially named perenniporin A, was obtained as yellowish powder; [α]\( _{\text{D}}^{{20}} \) −38.9° (c 0.175, MeOH); UV (MeOH) λ max 289 nm; IR (KBr) v max 3,345, 2,940, 1,672, 1,641, 1,431, 1,383, 1,271, and 685. Its molecular formula was determined to be C15H21O5 by positive HRESIMS [M+Na]+ at m/z 303.1214 (calcd 303.1216), indicating six degrees of unsaturation, which was supported by its NMR data (Fig. 2). The IR spectrum exhibited an OH absorption band at 3,345 cm−1.

Fig. 2
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Supporting Information of compound 1. a 1H-NMR of compound 1; b 13C-NMR of compound 1; c DEPT of compound 1; d HSQC of compound 1; e 1H–1HCOSY of compound 1; f HMBC of compound 1; g ROESY of compound 1

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The 1H NMR spectrum of 1 indicated signals due to two tertiary methyl groups (δ H 1.14 and 1.18), three methylene groups (δ H 2.53 and 2.23, δ H 2.39 and 2.26, and δ H 2.03 and 1.62), one oxygenated methine proton (δ H 4.39, s), three olefinic protons at δ H 6.28, 7.24, and 7.02 assigned to H-8, H-14, and H-2, respectively, and an one-proton multiplet at δ H 2.71 assigned to H-6. The 13C NMR and DEPT (Table 1) analysis revealed 15 carbon signals, including two methyls, three methylenes, five methines, and five quaternary carbons, of which the typical signals were one oxygenated quaternary olefinic carbon (δ C 155.4, C-9), one oxygenated olefinic methine carbon (δ C 136.8, C-14), one quaternary olefinic carbon (δ C 130.1, C-7) and one olefinic methine carbon (δ C 107.1, C-8), which undoubtedly constitute a 2,4-disubstituted furan ring. Furthermore, HMBC correlations observed between H-2/H-4 and C-2/C-4, and between H-1/H-5 and C-1/C-5, strongly supported a 1, 4-disubstituted cyclohexane structure moiety in compound 1. The 1H–1H COSY spectrum revealed a group of five protons connected in the order 7.02 (δ H, m, H-2) to 2.53 (δ H, m, H-1) to 2.71 (δ H, m, H-6) to 1.62 (δ H, m, H-5) to 2.26 (δ H, m, H-4), indicating the existence of fragment –CH–CH2–CH–CH2–CH2–, from C-2 through C-1 to C-6, C-5, and C-4 (Fig. 3). In addition, HMBC cross-peaks from H-4 and H-5 to C-3 (δ C 130.0) show that the C-2/C-3 double bond is part of the cyclohexene ring, which is conjugated with a carboxy group bond to C-3, evidenced by the HMBC correlation from H-2 (δ H 7.02, m) to C-15 (δ C 169.4). A HMBC correlation from H-6 (δ H 2.73, m) to C-7 (δ C 130.1), as well as HMBC correlations from H-1 and H-5 to C-7 and from H-8 (δ H 6.28, s) to C-6 (δ C 29.6), indicated the suggested linkage of the furan ring and the cyclohexane ring from C-7 to C-6 in compound 1. H-14 (δ H 7.22, s) also gives a HMBC correlation over the furan oxygen to C-9 (δ C 155.4), to which a 2-methyl-1,2-propylene glycol moiety is attached based on detailed inspection of the HMBC correlations (Fig. 3). Key ROESY correlations were observed between H-6 and H-2, H-8, and H-10, indicating a β- orientation for both H-6 and H-10. Accordingly, the structure of compound 1 was therefore deduced to be shown in Fig. 4. And the absolute configuration of C-10 in 1 was further assigned using the Snatzke′s method (Górecki et al. 2007; Di Bari et al. 2001). Metal complex of compound 1 in DMSO gave a significant induced CD spectrum (ICD) (Fig. 5), in which the negative Cotton effect observed at 305 nm permitted the assignment of a 10R-configuration for 1. Compound 1 is a new member of the bisabolane sesquiterpenoid metabolites, and to our knowledge, this is the first reported isolation of bisabolane sesquiterpenoid derivative from the genus Perenniporia.

Table 1 1H-NMR (600 MHz), 13C-NMR (150 MHz), and DEPT spectroscopic data (δ in methanol—d 4) for 1
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Fig. 3
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Key HMBC (arrows) correlations and 1H–1H COSY (solid lines) of compound 1

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Fig. 4
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Structures of compounds 1–4

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Fig. 5
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a Conformations of the Mo 4+2 complex of 1. b ICD spectrum of Mo 4+2 complex of 1 in DMSO

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Based on the MS and NMR data, compounds 2–4 were identified as already known compounds: compound 2, ergosterol (Mooney et al. 2007); compound 3, rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]furan-5-ol (Hashimoto et al. 2003); and compound 4, albicanol (Ito et al. 2000). Compound 2, ergosterol, the principal sterol in fungi cell membrane, is abundant in edible mushrooms, yeast lees of wines, and Chinese medicinal mushrooms, such as Ganoderma lucidum and Cordyceps sinensis (Kuo et al. 2011), and is also known to be an important pharmaceutical intermediate and the precursor of vitamin D2 and cortisone (Jasinghe et al. 2005). In addition, the antitumor activity of ergosterol has been reported previously (Yasukawa et al. 1994; Kwon et al. 2002; Ding et al. 2009). Compound 3, a cadinane-type sesquiterpene possessing a cyclic ether linkage, was previously isolated from the liverwort Ptychanthus striatus (Hashimoto et al. 2003) and it was isolated from Perenniporia species for the first time. Compound 4, albicanol, a representative drimane sesquiterpene, was reported to be a pungent principle and antifeedant agent against fish and worm, which also exhibited strong inhibitory effects on the EBV-EA activation and significantly suppressed an in vivo two-stage carcinogenesis on mouse skin (Ito et al. 2000). This is the first report of its presence as a metabolite in Perenniporia species.

Anti-P. oryzae activity

The EtOAc extract of P. tephropora Z41 (EPT) displayed strong inhibitiory activity against P. oryzae (MIC = 7.8 μg/mL). Table 2 showed that compound 2 has the most active anti-P. oryzae activity (MIC = 7.8 μg/mL) among the tested pure compounds. The MIC values of compound 1, 3 and 4 were 15.625, 15.625, and 33.75 μg/mL, respectively.

Table 2 Anti-Pyricularia oryzae activity of EPT and compounds 1–4
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Cytotoxic activity

The EtOAc extract of P. tephropora Z41 (EPT) showed significant cytotoxic activities against HeLa, SMMC-7721 and Panc-1 cell lines with IC50 values of 2.83, 15.60, and 12.58 μg/mL. Compound 2 was the most cytotoxic constituent, especially against the HeLa cell line with IC50 value of 1.16 μg/mL. In contrast, compounds 1, 3, and 4 exhibited moderate cytotoxicity with IC50 values ranging from 6 to 58 μg/mL (Table 3).

Table 3 Antitumor activity of EPT and compounds 1–4 (IC50, in micrograms per milliliter)
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Discussion

Metabolites product by endophytes could be influenced by the chemistry of their host plants (Huang et al. 2008; Mucciarelli et al. 2007; Aly et al. 2010). During the long period of co-evolution, some endophytes have the ability to produce similar or identical biological active compounds as their host plants, such as paclitaxel, podophyllotoxin, camptothecine, vinblastine, hypericin, and diosgenin (Zhao et al. 2011). We have previously investigated endophytic fungi isolated from bark, branches and leaves of T. chinensis var. mairei collected from Jiangxi, Zhejiang and Chongqing in China (data not shown). In order to searching for similar antitumor compounds as the yew trees, the EtOAc crude extracts from the fermentation broths and ground mycelia of isolated 145 endophytic fungal taxa were first tested through P. oryzae bioassay, which is a good primary model for screening of antifungi and antitumor strains (Gunji et al. 1983). As a result, the crude extract of P. tephropora displayed the strongest inhibitiory activity against P. oryzae (minimum morphological deformation concentration = 7.8 μg/ml), which is therefore selected for the present study on its cytotoxic metabolites.

Basidiomyceteous endophytes have been infrequently isolated from plant hosts (Pinruan et al. 2010; Rungjindamai et al. 2008). P. tephropora, belonging to Basidiomycetes, has not been isolated from the other Taxus species yet. Although members of the genus Perenniporia are mostly isolated as wood-inhabiting fungi, some of its species were encountered as endophytes as well, e.g., Perenniporia sp. (Evans et al. 2003; Pinruan et al. 2010). Basidiomycetes produce a large number of secondary metabolites which showed antibacterial, antifungal, antiviral, cytotoxic, and hallucinogenic activity (Suay et al. 2000). However, to our knowledge, there have been few bioactive metabolites, except laccase (Ben Younes et al. 2007), reported from P. tephropora.

In this study, we found that the endophytic fungus P. tephropora Z41 produced different varieties of metabolite classes that were not yet reported from Perenniporia species and that showed potent anti-P. oryzae and cytotoxic activities. Compound 1 was a new member of the bisabolane sesquiterpenoid metabolites with moderate cytotoxic activity, and it represents the first isolation of bisabolane sesquiterpenoid derivative from the genus Perenniporia. Compound 2 was the principal sterol in fungi cell membrane and we found it was the most cytotoxic constituent against the tested three cell lines in our study. Compound 3 was a cadinane-type sesquiterpene and compound 4 was a drimane-type one. Both of them were isolated from Perenniporia for the first time. Compound 4 was previously reported to show significant antitumor promoting activity both in vitro and in vivo. In the present work, it also demonstrated moderate cytotoxic activity against the tested cancer cell lines. Our results indicate that ergosterol 2 and sesquiterpenoids 1, 3, and 4 could be valuable candidates as potent tumor inhibitors and be beneficial in the therapy of cancer diseases. Our study also underscores that endophyte P. tephropora is a promising sources of natural bioactive and novel sesquiterpenoids metabolites, though without diterpenoids like taxols.