Investigation on bioactive metabolites produced by an endophytic fungus Trichoderma citrinoviride from the arils of Torreya grandis

Abstract An endophytic fungus Trichoderma citrinoviride capable of producing active substances was isolated from the arils of Torreya grandis. Seven compounds were separated from the ethyl acetate extract of fermentation broth and mycelium by chromatography, respectively identified as trichomerol (1), bisorbicillinolide (2), sohirnone A (3), emodin (4), stigmasterol (5), ergosterol (6), daidzein (7). This study is the first to report of the isolation of the endophytic fungus T. citrinoviride from the arils of T. grandis with complete assignments of 1-7. Compound 1 and 2 exhibited significant antioxidant activity of diphenyl picryl hydrazinyl with IC50 38.92 and 3.91 µg/mL, respectively. Compound 1, 2, 4 and 7 significantly inhibited the growth of Staphylococcus aureus with MIC 0.78; 0.39; 0.20 and 0.20 mg/mL, respectively. Current study indicated the presence of endophytic fungus in the arils of Torreya grandis that could be responsible for the bioactive metabolite synthesis. Graphical Abstract


Introduction
Torreya grandis Fortune ex Lindl. is an evergreen coniferous tree of the Torreya (Taxaceae). The extant Torreya species are restricted to North America and Eastern Asia (Zhang et al. 2020). T. grandis is a rare tree species of economic value in China which mainly produce in Zhejiang and Jiangsu Province. Studies have found that the main structural types of the chemical components in T. grandis are flavonoids, lignans, diterpenoids, fatty acids, volatile oils and amino acids (Tian, Mu, and Zhang 2021). Modern pharmacological studies have found that it has antibacterial, anthelmintic, antitussive, antiviral and antitumor effects. In addition, the extract of T. grandis has been reported to have anti-fatigue and anti-oxidant effects (Duan et al. 2018).
Endophytic fungi are considered as good sources to produce important secondary metabolites with interesting bioactivities (Tanaka et al. 2002). At present, people have gradually found natural compounds similar to the host from endophytes, which not only broadens the resources of medicinal plants, but also promotes the protection and sustainable development of rare and precious species (Rodriguez et al. 2009). In addition, studies shown that secondary metabolites isolated from endophytes also contain biological active ingredients (Chen, Liang, and Zhu 2015). We continue to search for important biologically active compounds from the flora by studying the secondary metabolites produced by an endophytic fungus from the arils of T. grandis. In this study, we reported the isolation of secondary metabolites from an endophytic fungus Trichoderma citrinoviride isolated from the arils of T. grandis (Figure 1).

Results and discussion
The strain identified as Trichoderma citrinoviride was isolated from the arils of T. grandis, which was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan 430072, P. R. China, with the preservation number of CCTCC M 2022156. This is the first report on the isolation of endophytic fungus T. citrinoviride from T. grandis. It was found that the fungus displayed antibacterial activity and antioxidant activity through preliminary pharmacological screening. The ethyl acetate extract obtained from the culture broth and mycelium of T. citrinoviride in Sabouraud medium was subjected to column chromatography on silica gel, Sephadex LH-20, octadecylsilyl silica gel, preparative thin layer chromatography, etc. Seven monomer compounds were separated and purified to obtain. The compounds were investigated by NMR and mass spectrometry. These compounds were determined as trichomerol (1), bisorbicillinolide (2), sohirnone A (3), emodin (4), stigmasterol (5), ergosterol (6), daidzein (7). Compound 1 and 2 exhibited significant antioxidant activity of diphenyl picryl hydrazinyl with IC 50 38.92 and 3.91 mg/mL, respectively. The clearance ability is a positively linear and related to concentration. Compound 1, 2, 4 and 7 significantly inhibited the growth of Staphylococcus aureus with MIC 0.78; 0.39; 0.20 and 0.20 mg/ mL, respectively.
Trichoderma species were known for the production of bioactive products. A variety of Trichoderma were lucratively used as potent biocontrol agents worldwide (Bhardwaj and Kumar 2017). Secondary metabolites of Trichoderma exhibit antifungal and antibacterial properties or can induce resistance to plant pathogens (Mukherjee, Horwitz, and Kenerley 2012). Studies show that Trichoderma could control the growth of Macrophomina phaseolina by disintegration of its DNA (Khan and Javaid 2020;. Three cyclopentenones were isolated from the marine-derived fungus T. viride. Among them, myrothenones A displayed tyrosinase inhibitory activity (Feng et al. 2009). A number of compounds with antifungal activity were isolated from T. harzianum, including 1-hydroxy-3-methylanthraquinone, delta-decanolactone, harzianopyridone and so on (Ahluwalia et al. 2015). Furthermore, a strain of T. citrinoviride was also isolated from marine brown algae, which could produce a new fusicoccane diterpene with antibacterial and antimicroalgal activities (Liang et al. 2016).
Among the compounds we isolated, trichodermol (1) has significant activity and can be used as an anticancer drug. Trichodermol is a derivative of trichodermin. Trichodermin inhibited the activity of peptidyltransferase and affects protein synthesis by binding to the 60S-ribosomal subunit (Wei et al. 1974) . Therefore, it has a strong inhibitory effect on a variety of pathogenic bacteria. Bisorbicillinolide (2) belongs to the category of dimeric sorbicillinoids. This type of compounds has obvious cytotoxic and phytotoxic activity and strong antioxidant activity. A strain of T. citricaria isolated from green algae has the ability to produce sorbitans with potential anti-inflammatory activity (Marra et al. 2019). Mechanism investigation uncovered that some sorbicillinoids could significantly induce cell cycle G2-M phase arresting by increasing the protein levels of p-H3 and cyclin B1 (Guzman-Chavez et al. 2017). Sohirnone A (3) was also isolated from the mangrove endophytic fungus which shows moderate antifungal activity against R. solani, with MIC values of 50 mg/mL (Zhu et al. 2018). Emodin (4) belongs to anthraquinones, and was also isolated from Cascara sagrada. It is a good antibacterial, antitumor and laxative. The antibacterial mechanism of emodin is the comprehensive result of various effects. Emodin could damage bacterial membrane, and inhibit protein synthesis of Staphylococcus aureus (Liu et al. 2015). It could also inhibit activity of succinate dehydrogenase and malate dehydrogenase on oxidative respiratory regulation (Zhou et al. 2011). Stigmasterol (5) and ergosterol (6) are sterols widely found in cell membranes of fungi and are also precursors for the synthesis of hormones and vitamin D. It has the effect of lowering cholesterol and preventing cardiovascular diseases. Daidzein (7) belongs to the flavonoids. Flavonoids have certain efficacies in anti-cancer and anti-tumor, with small side effects and a wide range of sources. It has a variety of antibacterial mechanisms, including the destruction of cell membrane integrity and the increase of cell membrane permeability. In addition, it could inhibit the tricarboxylic acid cycle pathway of Staphylococcus aureus (Ulanowska et al. 2006). Endophytic fungi capable of producing daidzein has also been isolated from the roots of Alnus nepalensis D. Don. The organism exhibited antagonistic activity against Staphylococcus aureus and mouse melanoma cell line B16 (Liu et al. 2009).

Conclusions
This study reported the isolation and identification of seven compounds from the endophytic fungus Trichoderma citrinoviride, which was obtained from the arils of Torreya grandis. Among these compounds, Compound 1 and 2 exhibited significant antioxidant activity. Compound 1, 2, 4 and 7 significantly inhibited the growth of Staphylococcus aureus. The above findings would provide the opportunity to screen for antimicrobial and antioxidant compounds from endophytes.

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

Funding
This work was supported by a key project funded by science and technology department of Zhejiang province under Grant No. 2018C02017, No. 2020C02040, No. 2020C02041 and No. 2020C02055.