Metabolites with Anti-Inflammatory Activity from the Mangrove Endophytic Fungus Diaporthe sp. QYM12

One new diterpenoid, diaporpenoid A (1), two new sesquiterpenoids, diaporpenoids B–C (2,3) and three new α-pyrone derivatives, diaporpyrones A–C (4–6) were isolated from an MeOH extract obtained from cultures of the mangrove endophytic fungus Diaporthe sp. QYM12. Their structures were elucidated by extensive analysis of spectroscopic data. The absolute configurations were determined by electronic circular dichroism (ECD) calculations and a comparison of the specific rotation. Compound 1 had an unusual 5/10/5-fused tricyclic ring system. Compounds 1 and 4 showed potent anti-inflammatory activities by inhibiting the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW264.7 cells with IC50 values of 21.5 and 12.5 μM, respectively.


Introduction
Mangrove endophytic fungi are the second largest ecological group of the marine fungi [1]. The particular environmental conditions of mangroves allow the activation of unique metabolic pathways in endophytic fungi, enabling the production of novel chemical backbones with diverse biological activities, making them a promising source of drug leads [2][3][4][5]. Diaporthe is a ubiquitous fungus commonly isolated from most plant hosts [6]. It is known to produce diverse compounds with antibacterial [7], antifungal [6], cytotoxic [8], antitubercular [9], antiparasitic [10] and anticancer [11] activities. With the aim of seeking new bioactive natural products from marine microorganisms, a mangrove endophytic fungus Diaporthe sp. QYM12, which was isolated from Kandelia candel collected from the South China Sea, was cultured in solid rice medium. As a result, six new metabolites including diaporpenoids A-C (1-3) and diaporpyrones A-C (4-6) together with one known analogue, 4-O-methylgermicidin L (7) [12], were isolated ( Figure 1). Herein, the isolation, structure elucidation and anti-inflammatory activity of all isolated compounds are described.

Results
Compound 1 has a molecular formula of C20H32O6 based on the (+)-HRESIMS (m/z: 391.20900 [M + Na] + ), requiring five indices of hydrogen deficiency. The 1 H NMR data (Table 1) Figure 2) from H3-11 to C-1, C-2 and C-10, and from H3-16 to C-5, C-6 and C-7 implied the existence of a ten-membered ring core structure. Moreover, the correlations from H-3 to C-12, from H3-14 to C-4 and C-12, from H3-19 to C-9 and C-17, and from H-8 to C-17 were consistent with the existence of two five-membered lactones. The NOESY correlations (

Results
Compound 1 has a molecular formula of C 20 Figure 2) from H 3 -11 to C-1, C-2 and C-10, and from H 3 -16 to C-5, C-6 and C-7 implied the existence of a ten-membered ring core structure. Moreover, the correlations from H-3 to C-12, from H 3 -14 to C-4 and C-12, from H 3 -19 to C-9 and C-17, and from H-8 to C-17 were consistent with the existence of two five-membered lactones. The NOESY correlations (   H3-20). Twenty carbon resonances in the 13 C NMR data showed six methyls, two sp 3 methylenes, eight sp 3 methines and four quaternary carbons (two carbonyl carbons). These data suggested that 1 may be a tricyclic diterpenoid.  Figure 2) from H3-11 to C-1, C-2 and C-10, and from H3-16 to C-5, C-6 and C-7 implied the existence of a ten-membered ring core structure. Moreover, the correlations from H-3 to C-12, from H3-14 to C-4 and C-12, from H3-19 to C-9 and C-17, and from H-8 to C-17 were consistent with the existence of two five-membered lactones. The NOESY correlations ( Figure 3         Compound 2 was isolated as a colorless oil and had a molecular formula of C 23 H 32 O 3 via HRESIMS. The NMR data of 2 were similar to those of pughiinin A [13]. It was confirmed that 2 had the same planar structure as pughiinin A by analyzing the COSY and HMBC correlations ( Figure 2). The main difference was the 11E-configuration of the double bond between C-11 and C-12, which was confirmed by the NOESY correlation ( Figure 3) from Hα-10/H 3 -20. The chemical shift at C-20 (δ C 10.6) in 2 further supported the 11E-configuration [14]. The relative configuration of 2 was elucidated by the NOESY correlations from H-13/H 3 -20, H 3 -20/H 3 -17, H 3 -17/H-6β, H-6α/H-15α and H-15α/H-14a. Thus, the structure of 2 was defined as shown in Figure 1    The HRESIMS data of 3 suggested a molecular formula of C 15 H 22 O 4 . The 13 C NMR data ( Table 2) showed 15 carbon resonances, including three methyls, three sp 3 methylenes, five methines (two oxygen-bearing and three olefinic) and four quaternary carbons (one olefinic and one carbonyl). The COSY correlations ( Figure 2) revealed the presence of three spin systems from H-1/H-2/H 2 -3, H-5/H 2 -6/H-7 and H-9/H 2 -10. The HMBC correlations from H 3 -12 to C-3, C-4 and C-5, H 3 -13 to C-7, C-8 and C-9, H 3 -14 to C-10, C-11 and C-15, and H-1 to C-11 and C-15 established the 11-membered ring core structure. The presence of a 4,5-oxirane ring was determined by the chemical shift values of C-4 (δ C 64.6) and C-5 (δ C 60.7). The NOESY correlations ( Figure 3) from Ha-3/H-5, Hb-3/H 3 -12, H 3 -12/H-7, H-7/H-9 and H-9/H 3 -14 indicated the relative configuration as 4R*, 5R*, 7R*, 11R*. The limited quantity did not allow one to define the absolute configuration of 3 through the modified Mosher's method. and five other carbons (one carbonyl carbon and two olefinic carbons). Similar NMR data suggested that the structure of 4 was similar to that of proasperfuranone B [15]. The main difference was that the ketone carbonyl group in proasperfuranone B was reduced to a hydroxyl group in 4. The deduction was confirmed by the HMBC correlations from H-8 to C-6, C-7 and C-9 ( Figure 5). Thus, the planar structure of 4 was established. The calculated ECD spectrum fit the experimental spectrum perfectly well ( Figure 6) at the BVP86/LANL2MB level in methanol; the absolute configuration of C-8 was determined as 8R.

Compound
. Drugs 2021, 19, x FOR PEER REVIEW 5 of 10 BVP86/LANL2MB level in methanol; the absolute configuration of C-8 was determined as 8R.  Compound 5, isolated as a colorless oil, gave a molecular formula of C11H16O4 by HRESIMS data. The 1 H NMR data (Table 3) [17], the absolute configuration of 5 was assigned as 7R.    Compound 5, isolated as a colorless oil, gave a molecular formula of C11H16O4 by HRESIMS data. The 1 H NMR data (Table 3) [17], the absolute configuration of 5 was assigned as 7R.  Compound 5, isolated as a colorless oil, gave a molecular formula of C 11 H 16 O 4 by HRESIMS data. The 1 H NMR data (Table 3) exhibited the presence of three methyl signals at δ H 0.92 (t, J = 7.4 Hz, 3H), 1.91 (s, 3H) and 3.90 (s, 3H), and one olefinic proton at δ H 6.10 (s, 1H). The 13 C NMR data showed 11 carbon resonances assigned to two methyls (δ C 8.5, 11.7), one methoxy (δ C 56.2), two methylenes (δ C 63. 6 [17], the absolute configuration of 5 was assigned as 7R.  (Table 3) of 6 were similar to those of 5, revealing an α-pyrone derivative. Moreover, the planar structure of 6 was established by the spin system of H 3 -10/H-7/H 2 -8/H 2 -9 from 1 H-1 H COSY spectra together with the HMBC correlations ( Figure 5) from H-7 and H 2 -8 to C-6. Meanwhile, the planar structure of 6 was identified as being the same as phomopyronol [18]. Finally, the calculated ECD spectrum and the experimental data ( Figure 7) were well matched, indicating the 7R configuration of 6.
Mar. Drugs 2021, 19, x FOR PEER REVIEW 6 of 10 Compound 6 was obtained as a colorless oil and had a molecular formula of C11H16O4 by HRESIMS. The 1 H and 13 C NMR data (Table 3) of 6 were similar to those of 5, revealing an α-pyrone derivative. Moreover, the planar structure of 6 was established by the spin system of H3-10/H-7/H2-8/H2-9 from 1 H-1 H COSY spectra together with the HMBC correlations ( Figure 5) from H-7 and H2-8 to C-6. Meanwhile, the planar structure of 6 was identified as being the same as phomopyronol [18]. Finally, the calculated ECD spectrum and the experimental data ( Figure 7) were well matched, indicating the 7R configuration of 6. Compound 7 was identified as 4-O-methylgermicidin L (7) [12] by a comparison of the spectroscopic data with the literature.
Nitric oxide (NO) is a key biological signaling molecule regulating the variety of physiological functions [19]. The excessive production of NO could induce tissue damage, and it is essential to find new effective NO inhibitors to treat inflammatory diseases and related disorders. Thus, the anti-inflammatory activity of isolated compounds was evaluated against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. The results (Table 4 and Table S1) showed that 4 exhibited a potent inhibitory activity with an IC50 value of 12.5 μM. Compounds 1-2 showed a moderate activity with IC50 values of 21.5 and 36.8 μM, respectively, when compared to the positive control (L-NMMA, IC50: 15.0 μM). All the tested compounds were nontoxic at the tested concentration.   Compound 7 was identified as 4-O-methylgermicidin L (7) [12] by a comparison of the spectroscopic data with the literature.
Nitric oxide (NO) is a key biological signaling molecule regulating the variety of physiological functions [19]. The excessive production of NO could induce tissue damage, and it is essential to find new effective NO inhibitors to treat inflammatory diseases and related disorders. Thus, the anti-inflammatory activity of isolated compounds was evaluated against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. The results (Table 4 and Table S1) showed that 4 exhibited a potent inhibitory activity with an IC 50 value of 12.5 µM. Compounds 1-2 showed a moderate activity with IC 50 values of 21.5 and 36.8 µM, respectively, when compared to the positive control ( L -NMMA, IC 50 : 15.0 µM). All the tested compounds were nontoxic at the tested concentration.

General Experimental Procedures
Specific rotations were taken on a MCP 300 (Anton Paar) polarimeter at 28 • C. UV spectra were recorded in MeOH using a PERSEE TU-1900 spectrophotometer, and ECD data were measured on a Chirascan CD spectrometer (Applied Photophysics). IR spectra were obtained on a Nicolet Nexus 670 spectrophotometer, in KBr discs. All NMR experiments were performed on a Bruker Avance 500 spectrometer at room temperature. HRESIMS spectra were obtained on a Thermo Fisher Scientific Q-TOF mass spectrometer. Column chromatography (CC) was conducted using silica gel (200-300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Pharmacia). Semipreparative HPLC was carried out using a C18 column (ODS, 250 × 10 mm, 5 µm). Thin-layer chromatography (TLC) was performed on silica gel plates (Qingdao Huang Hai Chemical Group Co., G60, F-254).

Fungal Material, Fermentation and Isolation
The strain QYM12 was isolated from the healthy leaves of Kandelia candel, which were collected in June 2017 from the South China Sea, Dongzhai Harbor Mangrove Nature Reserve Area, Hainan Province, China. Fungal identification was achieved using a molecular biological protocol by DNA amplification and ITS sequence [20]. The sequence was the most similar (99%) to the sequence of Diaporthe sp. (GU066666.1) via BLAST research. The sequence data of the strain has been deposited at GenBank with the accession number MW332459. The fungus was preserved at Sun Yat-Sen University, China. The strain was cultured on PDA medium for four days. Then, the seed culture was prepared by the mycelia of the fungus being inoculated into 500 mL of PDB medium for five days. Thereafter, the seed culture was transferred into solid rice medium (800 × 1000 Erlenmeyer flasks each containing 80 g of raw rice and 70 mL of 0.3% seawater) at 28 • C for 30 days.
Thereafter, the fermented material was extracted with MeOH three times, and organic phases were combined and evaporated under reduced pressure to yield an extract of 25.0 g. Then, the residue was fractionated by silica gel column chromatography with a gradient of petroleum ether/EtOAc from 10:0 to 0:10 to give eight fractions (Fr.1-Fr.8, per 10 mL).

ECD Calculation Methods
The calculation was accomplished according to the method described previously [21]. The conformers of compounds 1, 4 and 6 were first optimized by DFT methods at the B3LYP/6-31G (d) level in the Gaussian 05 program. Then, the theoretical calculation was performed using the time-dependent density functional theory (TD-DFT) at the level of B3LYP/DGDZVP, BVP86/LANL2MB and B3LYP/DGTZVP, respectively.

Anti-Inflammatory Assay
The RAW264.7 cells were purchased from Macrophage Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The method for the assay of the anti-inflammatory activity was conducted according to a previously published paper [20]. The detailed process is described in the Supplementary Materials.

Conclusions
In summary, the strain Diaporthe sp. QYM12, which was isolated from Kandelia candel, Dongzhai Harbor Mangrove Nature Reserve Area, was cultured in solid rice medium, leading to the identification of six new metabolite diaporpenoids A-C (1-3) and diaporpyrones A-C (4-6). Compound 1 was a macrocyclic diterpenoid featuring a rare 5/10/5-fused tricyclic ring system, and compounds 2,3 were macrocyclic sesquiterpenoids possessing a hendecane core. Macrocyclic sesquiterpenoids and diterpenoids are a functionally diverse group of natural products with versatile bioactivities [22]. For instance, junceellolide C showed an anti-HBV activity [23], flaccidenol A displayed a cytotoxic activity [24], antipacid B exhibited an anti-inflammatory activity [25], and euphorbesulins A revealed an antimalarial activity [26]. The anti-inflammatory assay suggested that compound 1 showed a moderate activity with an IC 50 value of 21.5 µM. Compound 4 exhibited a potent inhibitory activity with an IC 50 value of 12.5 µM. Proinflammatory enzymes, including nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), were reported to play key roles in inflammatory processes [27]. Thus, further research is required to clarify the underlying mechanisms of the active compounds. This study has suggested that these macrocyclic sesquiterpenoids and α-pyrone derivatives have the potential to develop lead compounds for anti-inflammatory agents.