Isolation and structure determination of a new indene derivative from endophytic fungus Aspergillus flavipes Y-62

Abstract As part of the search for naturally derived secondary metabolites, a novel indene derivative, compound 1, together with nine known metabolites (2–10) have been purified from an ethyl acetate extract of the plant-associated fungus Aspergillus flavipes Y-62, isolated from Suaeda glauca (Bunge) Bunge which was collected along Zhoushan coast, Zhejiang province, East China. The structure of the new compound 1 was elucidated by extensive use of spectroscopic techniques like 1D, 2D NMR, and HR-TOF-MS, while the known metabolites were established based on both spectral methods as well as by comparison with the previous literature. Compound 1 exhibited antimicrobial activities against the gram-negative pathogen Pseudomonas aeruginosa and Klebsiella pneumoniae with equal MIC values of 32 µg/ml.


General Experimental Procedure
Optical rotation were measured in chloroform on a Perkin-

Isolation and identification
The strain Y-62 was identified as Aspergillus flavipes ( Figure S45), which were isolated from the stems of the plant Suaeda glauca (Bunge) Bunge collected along Zhoushan coast, Zhejiang province, East China, according to the method described by Petrini (Petrini 1986). On the very first step, the plant material was washed out in tap water to remove the waste in the form of dust and debris then stems were cut into pieces of approximately 4-5 mm by a sterilized blade. The pieces were than surface sterilized by 70% ethanol for one minute and dipped in sodium hypochlorite (NaOCl) solution for one to two minutes. The samples were washed with sterile distilled water for one minute and then left to dry. After proper drying, the sample pieces were inoculated in potato dextrose agar medium (PDA) plate and incubated at 28 ±1 0 C for 5 to 7 days.
Finally, the purified colonies were transfered to PDA slant.

Fermentation
The fermentation process of an endophytic strain Y-62 was carried out for extraction and target compound separation into 500 mL flask including 200 mL PDB (Potato dextrose broth) composed of glucose (10.0 g/L) and potato extract (20.0 g/L) for 15 days at 28 0 C under static condition. After 15 days of culture, the mycelium was removed from the whole 40L of culture broth, while remaining filtrate was extracted with an equal volume of ethyl acetate (EtOAc) and subjected to HPLC for analysis.

Large Scale Fermentation
40 L of culture broth were prepared. The whole fermentation broth was filtered. The air-dried mycelia from culture were extracted separately with MeOH (3 × 1L), respectively. The extracts were evaporated in vacuo to afford a gummy residue. The residue was then dissolved in water (250 mL) and extracted with ethyl acetate (3 × 500 mL). Subsequently, the (40 L) filtrate was extracted with (EtOAc) ethyl acetate (2×200 mL) twice and treated in vacuo for dryness. Both extracts were combined and concentrated to afford an organic extract of (10.5 g).
The concentrated extract (10.5 g) was dissolved in methanol with silica gel and treated in vacuo for dryness. The extract was then subjected to silica gel column chromatography (200-300 mesh), using petroleum ether (PE) / EtOAc / MeOH as an eluent mixture flow rate of 3 ml/min (PE / EtOAc = 1:0, 20:1, 15:1, 10:1, 5:1, 2:1, 0:1 and MeOH) to obtain several fractions. As such, six combined fractions were obtained on the basis of TLC examination excluding three more fractions which were combined and subjected again to silica gel column chromatography.
Finally, the main fractions were dissolved in methanol and centrifuged at 12,000 rpm for 10 minutes.

Antimicrobial activity
Microbial activity were carried out using a standardized microdilution method for measuring the