Endophytes are endosymbiotic microorganisms, which grow in the internal tissues of living plants without causing apparent harm to the host [1]. Though the exact underlying mechanisms of the relationship of endophytes with the host plant are still under study, it is evident that endophytes have mutualistic symbiotic correlations with their host plant [2]. There have been reports that secondary metabolites produced from endophytes may protect the host plants by exhibiting antifungal, antibacterial, antiviral, plant growth, and regulatory activities [3]. In addition, endophytes play various indispensable functions for plant development, stress tolerance, and adaptation in nature [4]. Furthermore, due to their rich biodiversity, endophytic fungi have been considered as potential sources for the new drug candidates, which possess chemically novel structures [5].

During our ongoing effort to investigate the novel bioactive substances from endophytic fungi, Phoma sp. PF2 was isolated from the leaves of Artemisia princeps Pamp. (Asteraceae). Phoma sp., a genus of coelomycetous fungi, has been reported to produce various bioactive secondary metabolites, such as cytotoxic α-pyrones (phomones C-F) [6], thiodiketopiperazine (phomazine B) [7], antiangiogenic polyketide (phomaketide A) [8], anti-inflammatory polyketide (phomaketide C) [8], and antibacterial polyketide (barceloneic acid C) [9]. A. princeps, a perennial herb native to Korea, China, and Japan, has been traditionally used to treat inflammation, diarrhea, bacterial infection, and circular disorders [10]. Recent studies have found that the extract of A. princeps and its constituents show gastroprotective [11], neuroprotective [12], anti-oxidant [10], anti-inflammatory [13], anti-obesity [14], and anti-diabetic activities [14].

In this study, chemical investigation of the cultures of an endophytic fungus Phoma sp. PF2 derived from A. princeps resulted in the isolation and identification of eight compounds (1−8), including two new isochromanone derivatives (1 and 2) (Fig. 1). Since A. princeps is well known to have anti-inflammatory effect, the anti-inflammatory activities of the isolated compounds (1−8) were evaluated by measuring the inhibitory activity of nitric oxide (NO) production levels in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells.

Fig. 1
figure 1

Chemical structures of 1–8 isolated from Phoma sp. PF2 fungi derived from Artemisia princeps

Compound 1 was obtained as an amorphous white powder and its molecular formula was deduced as C13H16O5 from the negative mode HRESIMS ion peak at m/z 251.0914 [M−H] (calcd for C13H15O5, 251.0919), indicating six degrees of unsaturation. The 1H NMR spectrum of 1 showed the signals of three methyls [δH 1.22 (3 H, d, J = 7.1 Hz), 1.77 (3 H, s), and 2.09 (3 H, s)], a methoxy group [δH 3.86 (3 H, s)], a methine [δH 3.25 (1 H, q, J = 7.1 Hz)], and an aromatic proton [δH 6.38 (1 H, s)], indicating the presence of a pentasubstituted aromatic ring (Table 1). The 13C NMR spectrum exhibited 13 carbon resonances, which comprised four methyls (δC 10.0, 17.2, 25.8, and 55.7), two methines (δC 38.7 and 97.4), and seven quaternary carbons (δC 99.0, 104.2, 115.5, 142.1, 163.0, 164.6, and 169.2). The HMBC spectrum correlation signals from H-4 (δH 3.25) to C-3 (δC 104.2), C-4a (δC 142.1), and C-8a (δC 99.0), and from H-7 (δH 6.38) to C-5 (δC 115.5), C-6 (δC 164.6), C-8 (δC 163.0), and C-8a revealed the presence of an 1-isochromanone skeleton in the structure (Fig. 2a). In addition, the correlations from H3-9 (δH 1.77) to C-3, from H3-10 (δH 1.22) to C-3, C-4 (δC 38.7), and C-4a, from H3-11 (δH 2.09) to C-4a, C-5, and C-6, and from H3-12 (δH 3.86) to C-6 in the HMBC spectrum established the position of three methyls and a methoxy group. Since 1 had stereogenic centers at C-3 and C-4, the stereochemistry of this compound should be established. The strong ROESY correlations between H3-9 (δH 1.77) and H3-10 (δH 1.22) deduced that the two methyls of C-9 and C-10 were in the same face of the molecule. Therefore, the methyl group at C-10 and the hydroxyl group at C-9 were found to be in “anti” orientation. The absolute configuration at C-3 and C-4 of 1 was determined by the measurement of CD spectrum, as well as ROESY data. It displayed positive Cotton effects at 238 and 270 nm, establishing the absolute configuration of C-4 to be in S (Fig. 3a) [15]. Accordingly, the absolute stereochemistry of C-3 was also concluded to be in S based on the relative stereochemistry obtained by ROESY data [15]. Based on these results, compound 1 was assigned as (3S,4S)-3,8-dihydroxy-6-methoxy-3,4,5-trimethylisochroman-1-one.

Table 1 1H (300 MHz) and 13C (75 MHz) NMR spectral data of 1 and 2 in CDCl3 (δ in ppm, J values in parentheses)
Fig. 2
figure 2

Key HMBC correlations of 1 (a) and 2 (b)

Fig. 3
figure 3

a Experimental CD spectrum of compound 1. b Comparison of the experimental CD spectrum (solid) and the calculated ECD spectra (dashed) of compound 2

Compound 2 was isolated as an amorphous white powder and the molecular formula, C14H18O5, was established by a HRESIMS peak in the negative ion mode at m/z 265.1074 [M−H] (calcd for C14H17O5, 265.1076). The 1H NMR spectrum of 2 was similar to those of 1, except for the presence of two methoxy groups [δH 3.77 (H3-13) and 3.82 (H3-12)] and the downfield shifted appearance of a quartet methine signal [δH 4.03 (1 H, q, J = 7.2 Hz, H-4)] (Table 1). The 13C NMR spectrum of 2 showed 14 carbon signals including overlapped quaternary carbons at δC 162.9 (C-6 and C-8). Compared to that of compound 1, it showed an additional methoxy (δC 50.9, C-13) group and a ketone (δC 207.6, C-3) group, respectively, instead of a hemiacetal carbon at δC 104.2 (C-3) shown in 1. Considering 6 degrees of unsaturation of this molecule, two carbonyl carbons were deduced to be present in 2, suggesting the opening of the lactone ring moiety of an 1-isochromanone skeleton. The HMBC correlation from H3-13 (δH 3.99, s) to C-1 (δC 170.8) indicated that the additional methoxy group was attached to C-1, which supported the above assumption. Moreover, the HMBC correlation signals from H3-9 (δH 1.93, s) to C-3 (δC 207.6) and C-4 (δC 51.1) and from H3-10 [δH 1.33, d (J = 6.8 Hz)] to C-3, C-4, and C-4a (δC 142.5) revealed the presence of an acetyl group linked to the methyl-bearing methine carbon C-4. (Fig. 2b). The absolute configuration of C-4 was determined by comparing the experimental CD spectrum with the calculated ECD spectrum. The experimental CD spectrum of 2 displayed a negative Cotton effect at 226 nm and positive Cotton effects at 260 and 283 nm, which was more consistent with those in the calculated ECD spectrum of 4S-form than that of 4R-form of 2 (Fig. 3b). Therefore, the stereochemistry of the chiral carbon C-4 in 2 was established to be S, which agreed with the absolute stereochemistry of C-4 in compound 1. From these data, the structure of compound 2 was concluded as methyl (S)-8-hydroxy-6-methoxy-5-methyl-4a-(3-oxobutan-2-yl)benzoate.

The six known compounds were assigned as (4S)-clearanol C (3) [15], (3R,4S)-3,8-dihydroxy-3-hydroxymethyl-6-methoxy-4,5-dimethylisochroman-1-one (4) [15], polygonolide (5) [16], (7S,8S)-8-ethyl-7-hydroxy-2,3,8-trimethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (6) [17], (7R,8S)-8-ethyl-7-hydroxy-2,3,8-trimethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (7) [17], and leptosphaerone (=(7S,8S)-7-ethyl-7,8-dihydro-8-hydroxy-3,4,7-trimethyl-6H-2-benzopyran-6-one) (8) [18] by comparing their spectroscopic data with previously reported literature values (Fig. 1). To the best of our knowledge, the known compounds 36 and 8 were isolated from Phoma sp. for the first time.

Several previous researches have reported the anti-inflammatory activities of isochromanone-type compounds. In particular, 8-dihydroxy-3-methyl-isochromanone was found to inhibit LPS-induced vascular inflammation in human umbilical vein endothelial cells through inhibition of mitogen-activated protein kinase and nuclear transcription factor-kappa B (NF-κB) signaling pathway activation [19]. In addition, 1-phenyl-6,7-dihydroxy-isochroman was reported to attenuate LPS-stimulated inflammatory response through downregulation of inducible isoforms of nitric oxide synthase and cyclooxygenase (COX)-2, as well as of NF-κB in primary cultured rat microglial cells [20]. Therefore, to investigate the anti-inflammatory activities of the isochromanones, the compounds (18) isolated from the endophytic fungus in this study were subjected to the inhibition assay on NO production in LPS-induced RAW264.7 macrophage cells (S17). Most isolates except for 6 did not show the cell cytotoxicity at 50 μM on RAW264.7 cells in preliminary MTT assay. All the isolated compounds exhibited moderate inhibitory activities on NO production in LPS-stimulated RAW264.7 cells.

In summary, eight secondary metabolites (18), including two new isochromanone derivatives, (3 S,4 S)-3,8-dihydroxy-6-methoxy-3,4,5-trimethylisochroman-1-one (1) and methyl 6-hydroxy-4-methoxy-3-methyl-2-(3-oxobutan-2-yl)benzoate (2), were isolated from the ethyl acetate extract of the cultures of an endophytic fungus Phoma sp. (PF2) derived from A. princeps. Among the isolates, 1 and 4 showed inhibitory activities on NO production against LPS-activated RAW264.7 mouse macrophage cells without cell cytotoxicities. These results provide further information about the chemical characteristics of the endophytic fungus Phoma sp., as well as its potential use as a natural source for anti-inflammatory agents.