Daldiniaeschsone A, a Rare Tricyclic Polyketide Having a Chromone Unit Fused to a δ-Lactone and Its Symmetrical Biphenyl Dimer, Daldiniaeschsone B, from an Endophytic Fungus Daldinia eschscholtzii SDBR-CMUNKC745

Daldiniaeschsone A (1), a rare tricyclic polyketide having a chromone unit fused to a δ-lactone and its symmetrical 6,6′-biphenyl dimer, daldiniaeschsone B (2), together with three known compounds (3−5), were isolated from a plant-derived endophytic fungus, Daldinia eschscholtzii SDBR-CMUNKC745. Their structures were elucidated by extensive 1D and 2D NMR spectroscopic data and HRESIMS. All compounds showed α-glucosidase inhibitory activity with IC50 values ranging from 0.16−0.85 mM and compound 1 was the best α-glucosidase inhibitory activity (IC50 = 0.16 mM).


Introduction
Natural products from fungi have been observed to inhibit or kill a wide variety of harmful microorganisms, including, but not limited to, phytopathogens and bacteria, fungi, viruses, and protozoans that affect humans and animals [1][2][3]. Similar to plants, many compounds isolated from microorganisms have become well-known drugs such as penicillin (Penicillium notatum), vancomycin (Amycolatopsis orientalis), and erythromycin (Saccharopolyspora erythraea) [4]. Due to the short period to scale up and the diversity production of secondary metabolites, fungi have become the new resource for natural products. The genus Daldinia belonging to the Xylariaceae is a genus of fungi that produces polyketide compounds with diverse structures and impressive biological activities, including immunosuppressive, cytotoxic, antiangiogenesis, anti-inflammatory, and antibacterial

General Experimental Procedures
The optical rotations were measured with a Jasco P-1010 polarimeter. The UV spectra were recorded with a PerkinElmer UV-Vis spectrophotometer. Infrared (IR) spectra were recorded using a PerkinElmer Frontier Optica FT-IR spectrometer. The 1D and 2D NMR spectra recorded using a 500 MHz Bruker AV-500 spectrometer. Chemical shifts are reported in parts per million (δ) and coupling constants (J) are expressed in hertz. The HRESIMS were carried out on a QTOF 6540 UHD mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Chiral HPLC was performed on a CHIRALCEL OD-H column of 4.6φ-250 mm and attached to Agilent Technologies 1260 Infinity II. Silica gel G60 (60-200 µm, SiliCycle ® Inc., Québec, QC G1P 4S6, Canada) was used to perform column chromatography (CC). Precoated plates of silica gel (60F254, Merck, Kenilworth, NJ, USA) were used for analytical purposes.

Fungal Material
The fungus strain SDBR-CMUNKC745 was isolated from Cinnamomum bejolghota, collected from Chiang Mai, Thailand, in October 2015. This strain was deposited in the Culture Collection of Sustainable Development of Biological Resources (SDBR) Laboratory, Faculty of Science, Chiang Mai University, Thailand. Fungal colonies on potato dextrose agar grew to 80−85 mm at 30 • C in darkness after one week. Colonies were white to pale grey, and the reverse side was whitish-grey and black in the centre ( Figure 1A). Sporulation was observed within three weeks. Conidiophores were 0.8-2 µm long × 0.7-1.5 µm wide, hyaline, mononematous or synnematous, nodulisporium-like branching pattern with dichotomous or trichotomous branched ( Figure 1B). Conidiogeneous cells were 2.8-3.1 × 2.5-2.9 µm, cylindrical, hyaline, and smooth. Conidia were 4.8-6.4 × 2-3.8 µm, holoblastic, hyaline, ellipsoid to obovoid, aseptate, and smooth to finely roughened with flattened base ( Figure 1C). The internal transcribed spacer (ITS) and large subunit (LSU) region of ribosomal DNA, RNA polymerase II second largest subunit (RPB2), and βtubulin (TUB) genes of this strain were deposited in GenBank under the accession number MW1937612 MW193761, MW202236, and MW202237, respectively. The phylogenetic tree from the combined data set of ITS, LSU, RBP2, and TUB indicated that strain SDBR-CMUNKC745 clustered with D. eschscholtzii with 100% bootstrap and 1.0 posterior probability supports ( Figure 2). Moreover, each gene sequence of this strain revealed a similarity greater than 99% to the D. eschscholtzii MUCL 45435.

Fermentation, Extraction, and Isolation of Secondary Metabolites
Daldinia eschscholtzii SDBR-CMUNKC745 from stock culture was cultivated on potato dextrose agar (Difco™, Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) at 30 °C for five days. Then, five fungal mycelial plugs (5 mm in diameter) were inoculated into 500 mL Erlenmeyer flask containing 150 mL of Czapek Dox broth (CDB; 30 g/L sucrose, 2 g/L NaNO2, 1 g/L K2HPO4, 0.5 g/L KCl, 0.5 g/L MgSO4, and 0.01 g/L Fe2SO4, pH 8). Cultivation was performed in the dark at 30 °C with shaking at 100 rpm on a reciprocal shaker. After 10 days of incubation, the cultures were filtrated by Whatman's No. 1 paper to harvest the supernatant. After that, the culture filtrate of endophytic fungus was extracted twice with an equal volume of EtOAc (1:1 v/v). The upper solvent phase was collected and evaporated using a rotary evaporator. The crude extract was stored at −20 o C for further experiments.

α-Glucosidase Inhibitory Activity
A colorimetric α-glucosidase assay was carried out by the previously described procedure [18]. Briefly, the sample solution was dissolved in DMSO after the solutions were diluted with a phosphate buffer (100 mM KH2PO4, pH 6.9) become DMSO (10%, v/v) con-

Fermentation, Extraction, and Isolation of Secondary Metabolites
Daldinia eschscholtzii SDBR-CMUNKC745 from stock culture was cultivated on potato dextrose agar (Difco™, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 30 • C for five days. Then, five fungal mycelial plugs (5 mm in diameter) were inoculated into 500 mL Erlenmeyer flask containing 150 mL of Czapek Dox broth (CDB; 30 g/L sucrose, 2 g/L NaNO 2 , 1 g/L K 2 HPO 4 , 0.5 g/L KCl, 0.5 g/L MgSO 4 , and 0.01 g/L Fe 2 SO 4 , pH 8). Cultivation was performed in the dark at 30 • C with shaking at 100 rpm on a reciprocal shaker. After 10 days of incubation, the cultures were filtrated by Whatman's No. 1 paper to harvest the supernatant. After that, the culture filtrate of endophytic fungus was extracted twice with an equal volume of EtOAc (1:1 v/v). The upper solvent phase was collected and evaporated using a rotary evaporator. The crude extract was stored at −20 • C for further experiments.
After removing the solvent under reduced pressure, the culture medium was extracted with EtOAc to provide a crude extract (4.4 g), which was subjected to silica gel CC (

α-Glucosidase Inhibitory Activity
A colorimetric α-glucosidase assay was carried out by the previously described procedure [18]. Briefly, the sample solution was dissolved in DMSO after the solutions were diluted with a phosphate buffer (100 mM KH 2 PO 4 , pH 6.9) become DMSO (10%, v/v) concentration. The substrate, p-nitrophenyl α-D-glucoside (Sigma, St. Louis, USA, CAS No. N1377), (c 1.5 mM), was prepared by dissolving with phosphate buffer. The tested samples (50 uL) were combined with 100 µL of the α-glucosidase enzyme solution (0.35 U/mL, Sigma, St. Louis, MO, USA, CAS. No. G5003), then the mixture was preincubated for 10 min at 37 • C. The enzymatic reaction was started by applying substrate (100 µL, c 1.5 mM) into the mixture and incubated at 37 • C for 20 min. The reaction was subsequently terminated by the addition of Na 2 CO 3 (1 mL, c 1 M). The absorption was immediately measured at 405 nm by determining the quantity of p-nitrophenol released from the substrate. Acarbose was used as a positive control with an IC 50 value of 0.08 mM.

Nitric Oxide (NO) Production Inhibitory Activity
The NO inhibitory activity was performed using the same procedure as previously reported [19]. RAW 264.7 (American Type Culture Collection, Manassas, VA, USA) were seeded at 4 × 10 4 cells/well in 96 well plates suspended in 100 µL DMEM supplemented with 10% FBS and incubated at 37 • C 5% CO 2 overnight. Cells were incubated with 1 µg/mL LPS for 1 h and treated with various concentrations of sample compounds and vehicle control (DMSO) for 24 h. After 24 h, the NO production inhibitory was determined in the culture supernatant using the Griess reaction by adding 100 µL of Griess reagent in a 96 well plate for 10 min. The determination of nitric oxide was measured at 570 nm with Biochrom EZ Read 400 ELISA microplate reader (Biochrom Ltd., Cambridge, UK). Additionally, the data were presented as IC 50 which was calculated with GraphPad Prism 6.0 software. Indomethacin was used as a positive control of NO production inhibitor with an IC 50 value of 19.61 µM.

Cytotoxicity Assay (MTT Assay)
Cell viability studies were evaluated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay [20]. In brief, RAW 264.7 cells (5 × 10 4 cells/well) were cultured and incubated in 96-well plates for 24 h. The samples were prepared in DMSO at different concentrations (0.01-1000 g/mL). The cells were then treated with samples in DMEM medium (10% FBS, 100 µg/L streptomycin, and 100 IU/mL penicillin at 37 • C in a 5% CO 2 ) for 24 h. The medium was then removed and each well was filled with fresh DMEM containing 0.5 mg/mL MTT solution for 4 h at 37 • C in a 5% CO 2 . After that, the medium was removed, and the formazan precipitate was dissolved in DMSO [21]. The absorbance was measured at 550 nm on a microplate reader. All processes were performed in triplicate.

α-Glucosidase and NO Production Inhibitory Activities
The crude extract and all isolated compounds were evaluated for their α-glucosidase and NO production inhibitory activities ( Table 2). The crude extract showed α-glucosidase inhibitory activity with an IC 50 = 5.69 µg/mL, while compounds 1-5 also showed αglucosidase inhibitory activity with IC 50 values ranging from 0.16 to 0.85 mM. For the NO production inhibitory activity, the crude extract also showed efficient inhibition with an IC 50 value of 19.85 µg/mL. Unfortunately, compounds 1 and 2 were inactive, without toxicity, against RAW 264.7 macrophage cells at 50 µg/mL.
new polyketide biphenyl derivatives, phomalevones A-C, isolated from an u Phoma-like fungus. Daldiniaeschsone B displayed a strong negative CE at 2 weak positive CEs at 273 and 317 nm (Supplementary Materials, Figure S21 similar to data reported for phomalevones A-C which possess P-helicity. Thus ity of the 6,6′-axis of daldiniaeschsone B was assigned as the aS-configuration

Putative Biosynthesis Pathway of Two Novel Compounds
The biosynthetic pathway of monomeric and dimeric polyketide meta been investigated [27]. The putative biosynthetic pathway of compounds shown in Figure 5.  Daldiniaeschsone B (2), [α] 21 D −3.9 (c 1, MeOH), was isolated as a yellow amorphous solid, and the molecular formula C 32 H 30 O 14 was determined based on the HRESITOFMS data (m/z 661.1504 [M + Na] + ) and 13 C NMR spectroscopic data. The UV and IR spectra were almost identical to those of 1 (Supplementary Materials, Figures S17 and S18). Analysis of 1D and 2D NMR spectroscopic data (Table 1) indicated that 2 shared the same core chromone-lactone structure to 1. However, the molecular formula C 32 H 30 O 14 of 2, along with the observation of only 16 carbon resonances, indicated that this compound was a symmetrical dimer. The 1 H and 13 C NMR spectroscopic data of compound 2 were similar to those of 1 except compound 2 displayed 1 H NMR resonances for an AB aromatic proton system [δ H /δ C 7.52 (1H, d, J = 8.5 Hz, H-7/7 )/δ C 141.2 and 6.63 (1H, d, J = 8.5 Hz, H-8/8 )/δ C 107.4] instead of an ABC spin system as in 1. The HMBC correlations of H-7 with C-5 (δ C 159.2), C-6/C-6 (δ C 117.7), and C-8a (δ C 158.4) and the lack of resonances for H-6/H-6 in 1 H NMR spectral data revealed the C-6/C-6 carbon-carbon linkage between the two chromone moieties. A detailed assignment of the protons and carbons of 2 is shown in Table 1. Chiral HPLC analysis indicated a scalemic mixture with an ee of 86.2% (Supplementary Materials, Figure S20). Previously, several biphenyl core units have been synthesised by Bringmann et al. [25] and CD spectra have identified their axial/rotameric configuration (aS or aR isomers). The aS isomers of the biphenyls showed Cotton effects (CEs) similar to daldiniaeschsone B, whereas the aR isomers showed opposite signs. Shim et al. [26] also reported experimental and calculated CD spectra established aS-configuration of three new polyketide biphenyl derivatives, phomalevones A-C, isolated from an unidentified Phoma-like fungus. Daldiniaeschsone B displayed a strong negative CE at 225 nm and weak positive CEs at 273 and 317 nm (Supplementary Materials, Figure S21). This was similar to data reported for phomalevones A-C which possess P-helicity. Thus, the chirality of the 6,6 -axis of daldiniaeschsone B was assigned as the aS-configuration.

Putative Biosynthesis Pathway of Two Novel Compounds
The biosynthetic pathway of monomeric and dimeric polyketide metabolites has been investigated [27]. The putative biosynthetic pathway of compounds 1 and 2 is shown in Figure 5. The multistep condensations of C 16 -octaketide would produce xanthone intermediate i (ravenelin) [27][28][29]

α-Glucosidase and NO Production Inhibitory Activities
The broth extract of D. eschscholtzii was evaluated for its α-glucosidase inhibitory activity at the concentrations of 100, 150, and 200 µg/mL. At the highest concentration (200 µg/mL), the broth extract showed the highest inhibition of α-glucosidase with the % inhibition of 99.4% and its IC50 value was 5.69 µg/mL. However, at the concentrations of 100 and 150 µg/mL, the broth extract had the % inhibition less than 70%, indicating that at these concentrations (100 and 150 µg/mL), the broth extract was inactive. In the case of the pure compounds, compounds 1-5 showed α-glucosidase inhibitory activity with IC50 values of 0.16, 0.23, 0.85, 0.55, and 0.76 mM, respectively, while acarbose, the positive control had an IC50 = 0.08 mM. The broth extract and the most active compounds (1 and 2) were also evaluated for their cytotoxicities against mammalian cells, RAW 264.7 cells, using the MTT assay at the concentrations of 6.25, 12.5, 25, and 50 µg/mL. At the concentration of 50 µg/mL, the broth extract, and compounds 1 and 2 showed cell viability over 75%, indicating that all testing samples had no cytotoxicity at this concentration. The other concentrations (6.25-25 µg/mL) were cytotoxic. Notably, this is the first publication on the αglucosidase inhibitory activity of these compounds. For the NO production inhibitory activity, the broth extract showed weak activity with an IC50 value of 19.85 µg/Ml, whereas compounds 1 and 2 were inactive.

Conclusions
Previous chemical constituent investigations of D. eschscholtzii, using different fungal collections and different conditions of fermentation yielded different compounds. Various types of compounds have been isolated and identified, but polyketides are found as the

α-Glucosidase and NO Production Inhibitory Activities
The broth extract of D. eschscholtzii was evaluated for its α-glucosidase inhibitory activity at the concentrations of 100, 150, and 200 µg/mL. At the highest concentration (200 µg/mL), the broth extract showed the highest inhibition of α-glucosidase with the % inhibition of 99.4% and its IC 50 value was 5.69 µg/mL. However, at the concentrations of 100 and 150 µg/mL, the broth extract had the % inhibition less than 70%, indicating that at these concentrations (100 and 150 µg/mL), the broth extract was inactive. In the case of the pure compounds, compounds 1-5 showed α-glucosidase inhibitory activity with IC 50 values of 0.16, 0.23, 0.85, 0.55, and 0.76 mM, respectively, while acarbose, the positive control had an IC 50 = 0.08 mM. The broth extract and the most active compounds (1 and 2) were also evaluated for their cytotoxicities against mammalian cells, RAW 264.7 cells, using the MTT assay at the concentrations of 6.25, 12.5, 25, and 50 µg/mL. At the concentration of 50 µg/mL, the broth extract, and compounds 1 and 2 showed cell viability over 75%, indicating that all testing samples had no cytotoxicity at this concentration. The other concentrations (6.25-25 µg/mL) were cytotoxic. Notably, this is the first publication on the α-glucosidase inhibitory activity of these compounds. For the NO production inhibitory activity, the broth extract showed weak activity with an IC 50 value of 19.85 µg/Ml, whereas compounds 1 and 2 were inactive.

Conclusions
Previous chemical constituent investigations of D. eschscholtzii, using different fungal collections and different conditions of fermentation yielded different compounds. Various types of compounds have been isolated and identified, but polyketides are found as the major compounds. To the best of our knowledge, two new polyketides (chromones) were isolated and identified for the first time in D. eschscholtzii isolated from the cinnamon plant (Cinnamomum bejolghota) collected from Chiang Mai Province, Thailand. Compounds 1 and 2 are rare tricyclic chromones found in nature, having the chromone unit fused to δ-lactone. Compound 1 was a monomer, whereas compound 2 was a symmetrical dimer with a C-6/C-6 carbon-carbon linkage between the two chromone moieties. The compounds 4 and 5 were discovered from the Daldinia genus for the first time. Many chromones isolated from D. eschscholtzii have shown good inhibitory activities against α-glucosidase and nitric oxide. Unfortunately, in this study, all of the chromone derivatives showed weak α-glucosidase inhibitory, and compounds 1 and 2 showed no NO production inhibitory activities.