A new bioactive diterpenoid from pestalotiopsis adusta, an endophytic fungus from clerodendrum canescens

Abstract Bioassay-guided fractionation of the culture extract of Pestalotiopsis adusta, an endophytic fungus isolated from the medicinal plant Clerodendrum canescens, led to the isolation of one new, (10S)-12,16-epoxy-17(15→16)-abeo-3,5,8,12,15-abietapentaen-2,7,11,14-tetraone (1), and four known diterpenoids, teuvincenone F (2), uncinatone (3), coleon U (4), coleon U-12-methyl ether (5). These structures were identified by using spectroscopic methods, including UV, MS, 1D and 2D NMR experiments. This is the first report of these compounds being isolated from a Pestalotiopsis species. The cytotoxic activities of the compounds were evaluated, and compounds 1 and 3 demonstrated cytotoxic activities against the HL-60 tumour cell line (IC50 ＜ 20 μM). Graphical abstract

Compound 1 anticancer agent taxol from an endophytic fungal strain of the genus Pestalotiopsis, interest in bioactive compounds from this fungal genus has increased considerably (Xu et al. 2010;Yang et al. 2012). Previous chemical investigations of Pestalotiopsis spp. led to the discovery of various bioactive natural products, such as diterpenoids, flavonoid glycosides, polyketides and terpenoids (Subban et al. 2013;Bharitkar et al. 2015;Nandi et al. 2015;Yang et al. 2015;Yue et al. 2015). as part of our ongoing efforts to isolate and identify bioactive substances from plants and fungi, we noted that Pestalotiopsis adusta, an endophytic fungus from Clerodendrum canescens contained cytotoxic substances. In this report, we describe the isolation, structure elucidation and cytotoxic activities of the five compounds from P. adusta. Our research showed that the endophytic P. adusta is a potentially significant resource of natural products.

Results and discussion
Compound 1 (224, 268, 489 nm) exhibited the presence of a benzoquinone moiety. In the IR spectrum, a carbonyl signal was observed at 1724 cm −1 in addition to the absorption peaks at 1675 and 1617 cm −1 for the p-quinone moiety.
The 13 C NMR and DEPT spectra of 1 showed the presence of 20 carbon atoms: four methyls, one methylene, two methines, four carbonyl groups and nine quaternary carbons. Its 1 H and 13 C NMR spectra were almost identical with those of teuvincenone F (Cuadrado et al. 1992). In fact, the only differences were consistent with the presence in compound 1 of a keto carbonyl at 182.3 ppm in the 13 C NMR spectrum allowed it to be assigned to C-7 and thus conjugated to the p-quinone moiety rather than being an isolated oxo group. Two quinone carbonyls were observed at 179.3 and 173.5 ppm, and the four downfield carbon signals at δ C 129.6, 154.5, 160.9, 129.2 ppm were assignable to olefinic carbon atoms as members of the quinone ring. The 1 H NMR spectrum of 1 showed the presence of two olefinic protons at δ H 6.66 and 6.51, and four methyl singlets at δ H 2.48, 2.02, 2.20 and 1.70, assigned to C-17, C-18, C-19 and C-20, respectively. Coupled resonances at δ H 2.51 and 3.93 were assigned to a methylene at C-1. The presence of four methyl singlets in the 1 H NMR and 20 carbon signals in the 13 C NMR spectrum suggested that 1 possessed an abietane diterpenoid structure. Our assignments were supported by HMBC data that showed correlations from H-17 to C-15, from H-15 to C-12, and C-16. The negative absorption at 305 nm in the CD spectrum showed that the structure had the same abietane absolute configuration as mandarone a (Fan et al. 1999). Therefore, the structure of 1 was elucidated as (10S)-12,16-epoxy-17(15→16)-abeo-3,5,8,12,15-abietapentaen-2,7,11,14-tetraone.
The cytotoxic activities of 1-5 are summarised in Table 1. all showed some degree of cytotoxic activities, and compounds 1 and 3 exhibiting moderate cytotoxic activities (IC 50 values of 12.54 ± 1.18 and 15.66 ± 2.01, respectively) against HL-60 tumour cell line, comparable to with those observed for positive control (IC 50 9.20 ± 1.02 μM).

Identification of C. canescens and fungul isolation
Fresh, healthy stems of C. canescens were collected in September 2012 from the mountain of South Yandang, Zhejiang Province, People's Republic of China. The plant was identified by Dr Chunhui Dai in Zhejiang academy of Traditional Chinese Medicine. Voucher specimens (201206) have been deposited in the Key Laboratory for Genetic Improvement and Quality Control of Medical Plants of Zhejiang Province. The plant stems were washed three times in sterilised distilled water to remove dust and debris and dried on sterile filter paper. The clean material was cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3 ). Sterile conditions were maintained for the isolation of endophytes and all the work was performed in a laminar flow hood to avoid contamination. Surface sterilisation of the samples was achieved with 95% ethanol for 1 min, 10% sodium hypochlorite for 10 min and 70% ethanol for 2 min, and then, the stems were allowed to dry in a sterile environment. The tissues were placed on isolation media (potato dextrose agar; PDa) in Petri dishes supplemented with 200 mg/L chloramphenicol and 200 mg/L streptomycin to suppress bacterial growth, and incubated at room temperature (27 °C) until the outgrowth of endophytes was observed. Single fungal colonies were removed and transferred onto sterile potato dextrose agar (PDa: cooking water from 200 g potatoes/L, added with glucose 40 g/L, and agar 20 g/L) and potato sucrose agar (PSa: potato cooking water (200 g potatoes/L), sucrose 40 g/L, agar-agar 20 g/L) or M2 agar (malt extract 10 g/L, yeast extract 4 g/L, glucose 4 g/L, and agar-agar 15 g/L) and periodically checked for purity. Each isolate was kept in a slant agar tube for future investigations.

Identification of the endophytic fungus
The strain was identified to be P. adusta based on sequence analysis of the ITS region of the ribosomal DNa. The sequence data have been deposited at GenBank (accession number KF011509). a voucher strain has been stored at the Key Laboratory for Genetic Improvement and Quality Control of Medical Plants of Zhejiang Province.

Fermentation, extraction and isolation
The fungus isolated CCa-1 was cultured on slants of PDa at 27 °C for 7 days. agar plugs were cut into small pieces under aseptic conditions and 200 pieces were used to inoculate 100 Erlenmeyer flasks, each containing 100 mL potato dextrose broth (0.4% glucose, 1% malt extract and 0.4% yeast extract); the final pH of the media was adjusted to 6.5 and sterilised by autoclave. 100 flasks of the inoculated media were incubated at room temperature (27 °C) using a rotary shaker for 14 days. Following incubation, the mycelia and solid rice media were extracted three times with EtOac and the filtrate was concentrated in a rotating evaporator under reduced pressure to afford a dark brown gum (30.5 g). a portion of the extract (25.0 g) was fractionated by CC on silica gel to give four fractions (F 1 -F 4 ), eluted with dichloromethane-methanol (CH 2 Cl 2 -MeOH) mixtures of increasing polarity. Fraction F 2 which eluted with

Cytotoxicity assay
The inhibitory effects of the compounds against HL-60 cells were determined using a MTT assay to measure viable cells using cisplatin as a positive control and activity is expressed as percentage inhibition relative to the negative control (He et al. 2012). The dose resulting in 50% inhibition of cell growth (IC 50 ) was calculated by NDST software.

Disclosure statement
No potential conflict of interest was reported by the authors.