Bioactive compounds from an endophytic fungi Nigrospora aurantiaca

Many groups of fungi live as an endophyte in plants. Both published and undiscovered bioactive compounds can be found in endophytic fungi. Various biological activities of bioactive compounds from endophytic fungi had been reported, including anti-inflammatory and anticancerous effects. The chemical investigation of biologically active compounds from endophytic fungi Melaleuca leucadendra Linn. have not yet been stated. One new compound, namely nigaurdiol (1), along with five known compounds, xyloketal K (2), bostrycin (3), deoxybostrycin (4), xylanthraquinone (5), and ergosterol (6), were isolated from the Melaleuca leucadendra Linn. associated fungal strain Nigrospora aurantiaca#TMU062. Their chemical structures were elucidated by spectroscopic data and compared with literature. All isolated compounds were evaluated for inhibitory effect of NO production in LPS-activated microglial BV-2 cells. Compound 6 exhibited considerable inhibitory effect on NO production with IC50 values of 7.2 ± 1.4 µM and the survival rate of the cells was 90.8 ± 6.7% at the concentration of 10 µM.


Background
Endophytes are defined as microorganisms that spend at least parts of their life cycle inhabiting in its host plants without causing apparent harm to the host (Hardoim et al. 2015). Endophytic fungi is one of the potential resources for obtaining bioactive compounds because of its complex interaction with their host plants or other microorganisms within the host plants. Previous studies showed that many bioactive compounds produced by endophytic fungi exhibit antioxidant, anticancer, antiinflammatory, antimicrobial, and other biological activities (Kumari et al. 2018;Ukwatta et al. 2020). Some of the medicinal plants have been found to rear a number of highly diversified endophytic fungi, which could even produce the same compounds as their host plants. For instance, ginkgolide B can be produced by both Fusarium oxysporum and its host plant Ginkgo biloba (Cui et al. 2012). Thus, many of the folk medicinal plants were chosen to screen the associated fungal strains with significant biological activities in the recent past.
Melaleuca leucadendra Linn. of the Myrtaceae family is distributed across Australia and Southeast Asia countries like Indonesia (Pujiarti et al. 2011). The leaves of this family are known to contain a high concentration of terpenes with varied quality and quantity (Keszei et al. 2008). As a folk medicine, M. leucadendra Linn. was reported to exhibit antioxidant, antiproliferative, and anticancer activities (Rini et al. 2012;Monzote et al. 2020

Fungal material
The fungal strain Nigrospora aurantiaca was isolated from a healthy leaf of Melaleuca leucadendra linn collected in the yard of National Taiwan University and was identified by sequencing the internal transcribed spacer regions of the rDNA (ITS). A BLAST search of the sequence led to the best match of Nigrospora aurantiaca. Mycelium Nigrospora aurantiaca # TMU062 was inoculated into two different media-liquid medium and solid medium. Inoculation in liquid medium was done in 5 L serum bottles, each containing 50 g of malt extract (Becton, Dickinson and Company, Sparks, USA) and 3.5 L of deionized water. The fermentation was conducted with aeration at 25-30 °C for 14 days. As for solid medium, 250 mL flasks were used-each containing 20 g of barley and 0.2 g of potato dextrose agar (Becton, Dickinson and Company, Sparks, USA). After adding 15 mL of deionized water, they were fermented for 30 days at 27-30 °C.

Extraction and isolation
The fermented broth (9.5 L) was filtered and partitioned five times with equal volumes of EtOAc and subsequently concentrated in vacuum to obtain crude extract (5.8 g). The crude extract was re-dissolved in 50 mL MeOH to obtain MeOH layer and sediment (2.3 g). Then, the sediment was dissolved in 10 mL DMSO and purified by HPLC semipreparative reversed-phase column  Data are as the mean ± SD (n = 3) * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the stimulation (V); ### p < 0.001 compared with the resting (R)

Microglial culture
The murine BV-2 microglial cell line cultured followed the procedure of our previous reports (Hsiao et al. 2020). In summary, BV-2 cells were cultured with DMEM containing Fetal Bovine Serum (FBS), streptomycin/penicillin, Lglutamine and HEPES at 37 °C, humidified 5% CO 2 . Prior to the study, BV-2 cells were cultured in FBS media (5%), pretreated with carrier media or various concentrations of compounds for 15 min, and eventually collected after 24 h of stimulation with LPS (150 ng/mL).

Cell viability assays
As in our previous report, cellular viability was assessed using MTT test where BV-2 cells, along with other compounds were treated for 24 h (Hsiao et al. 2020).

Detection of nitric oxide production
The level of nitric oxide (NO) metabolites from the production of activated BV-2 cells was measured with reference to the Griess method (Wang et al. 2018).