A new cyclopentenone derivative from Trichoderma atroviride HH-01

Abstract A new cyclopentenone derivative, atrovinol (1), together with ten known compounds (2-11) were isolated from Trichoderma atroviride HH-01, an endophytic fungus from Illigera rhodantha (Hernandiaceae). Their structures were identified by HRESIMS, 1 D/2D NMR, and electronic circular dichroism (ECD) spectra. Compound 1 exhibited moderate inhibitory activity against Staphylococcus aureus and Bacillus subtilis with MIC values of 8.0 µg/mL and 16.0 µg/mL, respectively. Graphical Abstract


Introduction
Illigera rhodantha Hance belongs to the Hernandiaceae family, which was used as a folk medicine to treat rheumatism and arthritis in ancient China (Li et al. 2011). The medical application and market requirement of I. rhodantha has been relying on the procurement of wild herbs, which resulted in a sharp drop of this wild plant (Hang and Feng 2019 ). Therefore, it is vital to develop a new production process for this high-value medicinal plant.

Structural elucidation
Compound 1 was obtained as a yellowish-brown colloid with an optical rotation value ½a 20 D À4.7 (c 0.3, CH 3 OH). Its molecular formula was deduced to be C 7 H 10 O 4 by HR-ESI-MS at m/z 157.0502 [M-H] -(calcd for C 7 H 9 O 4 , 157.0506). The IR spectrum revealed the presence of hydroxyl groups (3354 cm À1 ), carbonyl function (1664 cm À1 ) and double bond (1452 cm À1 ). The 1 H and 13 C NMR spectra indicated that compound 1 contains seven carbons, including two methylenes (d C 33.7 t, 60.1 t), three methines (d C 79.0 d, 82.2 d, 128.6 d), and two quaternary carbons (d C 177.4 s, 205.2 s). Detailed analysis of the 1 H and 13 C NMR spectroscopic data of 1 revealed that it was very similar to those of trichoderone (You et al. 2010). The main difference between them was that 1 possessed an oxygenated methylene instead of a methyl in trichoderone. In the HMBC spectrum, the correlations from H-6 (d H 2.72 t, J ¼ 6.8 Hz) to C-1 (d C 79.0 d), C-4 (d C 128.6 d) and C-5 (d C 177.4 s), revealed that C-1, C-4 and C-6 (d C 33.7 t) were connected to C-5. Key HMBC correlations from H-4 (d H 6.00 s) to C-3 (d C 205.2 s) and C-2 (d C 82.2 d) indicated that C-3 was directly attached to C-2 and C-4. 1 H-1 H COSY correlation between H-1 (d H 4.50 d, J ¼ 3.0 Hz) and H-2 (d H 4.02 d, J ¼ 2.9 Hz), H-6 and H-7 (d H 3.85 t, J ¼ 6.4 Hz), demonstrated the neighbourhood of C-1 and C-2, and of C-6 and C-7 (d C 60.1 t). The 13 C NMR spectrum showed three hydroxyl groups (d C 82.2 d; 79.0 d; 60.1 t) were placed at C-1, C-2 and C-7, respectively. Therefore, the structure of 1 was solved as shown in Figure S1.

Assessment of antimicrobial activity
The antimicrobial activities of compounds 1-11 were evaluated against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli. The results indicated that 1 has moderate antibacterial activity against S. aureus and B. subtilis with minimum inhibitory concentrations (MICs) of 8.0 mg/mL and 16.0 mg/mL, respectively (kanamycin, 1.0 mg/ mL). Compound 2 existed antibacterial activities against E. coli and B. subtilis with MIC value of 4.0 mg/mL. 3 showed antibacterial activities against E. coli and S. aureus with MIC value of 16.0 mg/mL. The minimal inhibitory concentration (MIC) of 7 against B. subtilis was 32.0 mg/mL. 9 was reported with promising anticancer agent that exhibits high selective toxicity to cancer cells (You et al. 2010). Other compounds showed no antibacterial activity. Comparing 1 with 9, the main difference between them was that 1 possessed an oxygenated methylene instead of a methyl in dihydroterrein. An analysis of the structure-activity relationship suggested that hydroxy group at C-7 and hydroxy oriented at C-1 and C-2 may greatly contribute to the antibacterial activity of atrovinol (1).

General experimental procedures
Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Nicolet Magna-IR 550 spectrometer (Thermo Nicolet Co., Madison, WI, USA) was used for scanning IR spectroscopy in CH 3 OH. NMR spectra were recorded on Bruker Avance 400 MHz spectrometer or Bruker Avance III 600 MHz spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. ESI-MS analyses were recorded with Agilent G3250AA (Agilent, Santa Clara, CA, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, MA, USA). Silica gels (200-300 mesh and 300-400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) were used for column chromatography (CC). Fractions were monitored by TLC and visualised by spraying with 10% H 2 SO 4 in ethanol followed by heating.

Material and fermentation
I. rhodantha (Hernandiaceae) were collected in Zhipu County, Guilin City, in Guangxi Province of China, in April 2019, and identified by professor Yang Chen from Guizhou Medicinal University, PR China. The voucher specimen (2019-HHQT-1) has been deposited in the school of Chemical Science and Technology of Yunnan University. T. atroviride was separated from I. rhodantha. The strain was identified by analysis of the internal transcribed spacer (ITS) region of the rRNA sequence, which was most closely related to Trichoderma sp. (99% GenBank accession no. MG198882.1).
Potato dextrose agar (PDA; 1 L of water, 200 g of potato, 20 g of dextrose, and 15 g of agar, natural pH) slant culture medium was inoculated with the aforementioned fungus and incubated at 28 C for 5 days. After that T. atroviride was added to the fermentation culture medium (80 g potato in each seed bottle) that had been sterilised at 121 C for 30 min before, it was then incubated at 28 C for 30 days.

Antimicrobial activity assay
Antimicrobial activity assays were performed in sterilised 96-well microplates using a microdilution method described previously . The 18-h-old bacterial cultures from B. subtilis (ATCC 6633), E. coli (ATCC 25922) and S. aureus (ATCC 27217) were added to LB broth medium (1 L of water, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl) to reach 1 Â 10 5 colony-forming units/mL. The 24-h-old fungal cultures from C. albicans (ATCC 10231) were added to PDA medium (1 L of water, 200 g of potato, 20 g of dextrose and natural pH)) to reach 1 Â 10 5 colony-forming units/mL. Compounds 1-11 and the positive control were dissolved in dimethyl sulfoxide (DMSO) and their final concentrations ranged from 512 to 1 mg/mL with a 2-fold serial dilution method. The wells containing test strains and diluted compounds were incubated at 37 C (24 h). The wells containing a culture suspension and DMSO were run as negative controls. Kanamycin was introduced as a positive control in the experiments on account of its prominent antimicrobial activity against B. subtilis, E. coli and S. aureus. Nysfungin was selected as positive control of C. albicans. All experiments were repeated two times. The MICs were determined as the lowest concentrations that produce complete growth inhibition of tested microorganisms.

Conclusion
In conclusion, A new cyclopentenone derivative, atrovinol (1) was isolated from Trichoderma atroviride HH-01. The structural determination was accomplished by HRESIMS, EIMS, 1 D/2D NMR, and electronic circular dichroism (ECD) spectra. compound 1 showed moderate antibacterial activity against S. aureus and B. subtilis. An analysis of the structure-activity relationship suggested that hydroxy group at C-7 and hydroxy oriented at C-1 and C-2 may greatly contribute to the antibacterial activity of atrovinol (1).

Acknowledgment
Thanks to Advanced Analysis and Measurement Center of Yunnan University for the sample testing service.

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

Funding
This work was financially supported by a grant from the Natural Science Foundation of the Yunnan Province, grant number 202001BB050042; a Natural Science Foundation of China, grant number 81960760; a Program for Excellent Young Talents in Yunnan University; and an Undergraduates Innovative Experiment Project from Yunnan Province. Ten Thousand Talent Plans for Young Top-notch Talents of Yunnan Province.