Two New Diphenylketones and a New Xanthone from Talaromyces islandicus EN-501, an Endophytic Fungus Derived from the Marine Red Alga Laurencia okamurai

Abstract

Two new diphenylketones (1 and 2), a new xanthone (3), and a known xanthone analogue (4) were isolated and identified from Talaromyces islandicus EN-501, an endophytic fungus obtained from the fresh collected marine red alga Laurencia okamurai. Their structures were elucidated on the basis of NMR spectroscopic and X-ray crystallographic analysis. The joint isolation of benzophenones and xanthones from the same fungal strain supports the biogenesis of xanthones via a benzophenone intermediate. It is worth mentioning that xanthones 3 and 4 have a methyl group at C-6 and C-2, respectively, which is uncommon compared with typical xanthones usually having a methyl group at C-8. Compounds 1–4 exhibited potent antioxidative activities against DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) radicals with IC₅₀ values ranging from 0.58 to 6.92 μg/mL, which are stronger than that of the positive controls BHT (butylated hydroxytoluene) and ascorbic acid. Compounds 1, 3, and 4 also showed inhibitory activities against several pathogenic bacteria.

1. Introduction

Marine-derived fungi have been evidenced as a prolific source for the discovery of pharmacologically-active natural products [1,2]. As part of our ongoing research toward the discovery of secondary metabolites from marine-derived fungi [3,4,5,6,7,8], an endophytic fungal strain, Talaromyces islandicus EN-501, was selected for chemical investigation. Recently, several new natural products were isolated from plant-derived endophytic species of the genus Talaromyces [9,10,11]. The fungal strain used in the present work was isolated from the inner tissue of the fresh collected marine red alga Laurencia okamurai. As a result, two new diphenylketones (1 and 2), a new xanthone derivative (3), and a known xanthone analogue (4) (Figure 1), were isolated and identified. It should be mentioned that compound 4 was listed as a substance tested for antitermitic activity [12], but no spectroscopic data or source information were reported. The structure elucidation and fully assigned NMR data of 4 are first described herein. The antioxidative activities against DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) radicals, as well as antibacterial activity against six pathogenic strains, were evaluated. This paper describes the isolation, characterization, and bioactivity of compounds 1–4.

2. Results and Discussion

2.1. Structure Elucidation of the New Compounds

Compound 1 was obtained as yellowish crystals with the molecular formula C₁₄H₁₂O₅ as established by HRESIMS (high resolution electrospray ionization mass spectroscopy, Figure S1) data, indicating nine degrees of unsaturation. The ¹H NMR spectrum (Figure S2) showed signals for a 1,2,3-trisubstituted phenyl group at Δ_H 6.98 (d, J = 7.7 Hz, H-4), 6.77 (t, J = 7.7, Hz, H-5), and 6.71 (d, J = 7.6 Hz, H-6), and a 1,2,3,5-tetrasubstituted phenyl unit at Δ_H 6.91 (br s, H-4′) and 6.62 (br s, H-6′) (

Table 1. ¹H and ¹³C NMR data of compounds 1 and 2 (Δ in ppm, J in Hz).

Position	1 (Measured in DMSO-d6)		2 (Measured in CDCl3)
	ΔC	ΔH	ΔC	ΔH
1	126.1, C		122.1, C
2	143.8, C		148.5, C
3	145.8, C		148.2, C
4	117.7, CH	6.98, d (7.7)	123.1, CH	7.11, d (7.0)
5	119.2, CH	6.77, t (7.7)	118.9, CH	6.89, t (7.2)
6	119.0, CH	6.71, d (7.6)	115.2, CH	7.04, d (7.0)
7	202.5, C		201.7, C
1′	120.2, C		119.0, C
2′	152.3, C		155.0, C
3′	127.1, C		129.0, C
4′	125.3, CH	6.91, br s	125.6, CH	6.94, br s
5′	148.7, C		146.5, C
6′	114.8, CH	6.62, br s	115.5, CH	6.84, br s
7′	15.4, CH3	2.18, s	15.8, CH3	2.27, s
2-OH				9.00, s
3-OH/OMe		9.47, s	56.3, CH3	3.93, s
2′-OH		11.42, s		10.95, s
5′-OH		9.02, s		4.57, s

). The ¹³C NMR spectrum (Figure S3) displayed 14 resonances, which were classified by DEPT (distortionless enhancement by polarization transfer, Figure S3) experiments as one methyl, five aromatic methines, and eight non-protonated (including one ketone and seven aromatic) carbons (Table 1). The ¹H and ¹³C NMR spectroscopic data (Figures S2–S6) are evocative of a benzophenone scaffold [13]. In the HMBC spectrum (Figure S6), the correlations from the proton of 2′-OH (Δ_H 11.42) to C-1′, C-2′, and C-3′ located the OH group at C-2′, while the correlations from H-7′ (Δ_H 2.18) to C-2′, C-3′, and C-4′ placed the methyl group at C-3′, and at last, the correlations from the OH proton at Δ_H 9.02 to C-4′, C-5′, and C-6′ determined this OH group at C-5′. As for the other nucleus, HMBC correlations from H-6 to C-2 and C-7, from H-5 to C-1 and C-3, and from H-4 to C-2, as well as the chemical shifts of C-2 and C-3 at Δ_C 143.8 and 145.8 determined the locations of two other phenolic OH groups at C-2 and C-3 (Figure 2). Thus, compound 1 was characterized as 2,2′,3,5′-tetrahydroxy-3′-methylbenzophenone, and the structure was confirmed by the single-crystal X-ray diffraction analysis (Figure 3).

Compound 2 was obtained as a yellowish solid with the molecular formula C₁₅H₁₄O₅, as established by HRESIMS data (Figure S7), indicating nine degrees of unsaturation. The 1D and 2D NMR spectra data of 2 (Table 1, Figures S8–S12) are reminiscent of those of 1, only differing in the additional resonances from a methoxy group at Δ_H 3.93 and Δ_C 56.3 in the NMR spectra of 2. In the HMBC spectrum, correlation from the protons of the OMe group to C-3 assigned the methoxy group at C-3 (Figure 2). Therefore, the structure of compound 2 was identified as 2,2′,5′-trihydroxy-3-methoxy-3′-methylbenzophenone.

Compound 3 was also obtained as a yellowish solid with the molecular formula C₁₄H₁₀O₅, as established by HRESIMS data (Figure S13), indicating ten degrees of unsaturation. Extensive analysis of the ¹H and ¹³C NMR data (

Table 2. ¹H and ¹³C NMR data of compounds 3 and 4 (Δ in ppm, J in Hz).

Position	3 (Measured in DMSO-d6)		4 (Measured in DMSO-d6)
	ΔC	ΔH	ΔC	ΔH
1	152.0, C		149.7, C
2	110.6, CH	7.42, d (7.2)	117.6, C
3	124.5, CH	7.24, d (7.2)	124.6, CH	7.21, s
4	137.9, C		137.0, C
4a	146.2, C		141.0, C
5	123.8, CH	7.20, s	147.6, C
6	117.1, C		120.8, CH	7.32, d (7.6)
7	149.1, C		124.3, CH	7.27, t (7.8)
8	120.9, CH	7.26, s	113.8, CH	7.54, d (7.7)
8a	120.5, C		120.6, C
9	182.8, C		182.3, C
9a	107.7, C		107.8, C
10a	141.2, C		145.1, C
2-CH3			14.4, CH3	2.17, s
6-CH3	14.5, CH3	2.19, s
1-OH		12.04, s		12.06, s

, Figures S14 and S15) as well as the COSY and HSQC (heteronculear single quantum coherence) spectra (Figures S16 and S17) disclosed that 3 was a xanthone analogue similar to 1,4,7-trihydroxy-xanthone [14]. The main differences are that resonance for one of the five aromatic protons in the ¹H NMR spectrum of 1,4,7-trihydroxy-xanthone disappeared in that of 3, and signal for an additional methyl group was observed at Δ_H 2.19 and Δ_C 14.5 (CH₃-6) in the NMR spectra of 3. Moreover, the resonance of C-6—compared to that of the known xanthone—moved to the shielded field in the ¹³C NMR spectrum of 3. These spectroscopic features suggested that compound 3 was 1,4,7-trihydroxy-6-methylxanthone. The COSY and HMBC correlations (Figure 2, Figures S16 and S18) supported the above deduction.

Compound 4 was obtained as a yellowish solid with the molecular formula C₁₄H₁₀O₅, the same as that of 3, as established by HRESIMS data (Figure S19). The ¹H and ¹³C NMR data of 4 (Table 2, Figures S20 and S21) as well as the COSY and HSQC spectra (Figures S22 and S23) showed a close relationship to that of 3. The methylation of C-2 and hydroxylation of C-5 in 4 were supported by the HMBC correlations from CH₃-2 to C-1, C-2, and C-3 and from H-7 to C-5, respectively (Figure 2 and Figure S24). Thus, compound 4 was characterized as 1,4,5-trihydroxy-2-methylxanthone. This compound was listed as a substance tested for antitermitic activity [12], but no spectroscopic data or source information were reported. So, its structure elucidation and fully assigned NMR data are provided here.

The biogenesis for most xanthones is known to proceed via a benzophenone intermediate in lichens and fungi [15,16]. The joint isolation of benzophenones and xanthones from T. islandicus EN-501 indicates that benzophenone (1) might serve as an intermediate to afford xanthone (4) upon cyclization. Compound 4 is an atypical xanthone with a methyl group at C-2, which is different from most other xanthones that usually possess a methyl group either at C-3/C-6 or at C-1/C-8 [15,16]. Atypical xanthones with a methyl at C-2/C-7 have been described previously [17,18], which might be derived from a site-selective methylation step involving discrete enzymes [17].

2.2. Biological Activities of the Isolated Compounds

In the antioxidative assay, compounds 1–4 exhibited comparable DPPH radical scavenging activity, with IC₅₀ values ranging from 1.23 to 6.92 μg/mL (

Table 3. Antioxidant activity of compounds 1–4 against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate (ABTS) (IC₅₀, μg/mL).

Samples	1	2	3	4	BHT a	Ascorbic Acid
DPPH	1.26	1.33	6.92	1.23	16.27
ABTS	0.69	0.58	2.35	1.27		3.01

^a BHT: butylated hydroxytoluene.

), stronger than that of BHT (butylated hydroxytoluene), a well-known antioxidant (IC₅₀ = 16.27 μg/mL). In addition, compounds 1–4 showed potent ABTS radical scavenging activity with IC₅₀ values ranging from 0.58 to 2.35 μg/mL (Table 3), which are stronger than that of ascorbic acid (IC₅₀ = 3.01 μg/mL). Compounds 1–4 were also evaluated for their antibacterial activities. While compounds 1, 3, and 4 showed potent activities against three human pathogens (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) and three aquatic bacteria (Vibrio alginolyticus, V. harveyi, and V. parahaemolyticus), with minimum inhibitory concentration (MIC) values ranging from 4 to 32 μg/mL (

Table 4. Antibacterial activity of compounds 1–4 (minimum inhibitory concentration, MIC, μg/mL) ^a.

Samples	1	2	3	4	Chloramphenicol
EC	4	>64	32	4	1
PA	4	>64	32	4	4
SA	8	>64	>64	8	2
VA	4	>64	32	4	0.5
VH	8	>64	32	8	2
VP	4	>64	32	4	2

^a EC: E. coli; PA: P. aeruginosa; SA: S. aureus; VA: V. alginolyticus; VH: V. harveyi; VP: V. parahaemolyticus.

), compound 2 showed weak activity against the tested bacteria (IC₅₀ > 64 μg/mL), suggesting that methoxylation at C-3 weakened the antibacterial activities (1 vs. 2).

3. Experimental Section

3.1. General

Melting points were determined with an SGW X-4 micro-melting-point apparatus. UV spectra were measured using a Lengguang Gold S54 spectrophotometer (Shanghai Lengguang Technology Co.Ltd., Shanghai, China). NMR spectra were acquired using a Bruker Avance 500 spectrometer (Bruker Biospin Group, Karlsruhe, Germany). Mass spectra were measured on a VG Autospec 3000 mass spectrometer (VG Instruments, London, UK) or an API QSTAR Pulsar 1 mass spectrometer (Applied Biosystems, Foster City, CA, USA). HPLC analysis was carried out on a Dionex HPLC system (P680 HPLC pump, UVD 340U UV-visible detector) using a C₁₈ column (5 μm, 8.0 mm i.d. × 250 mm). Column chromatography (CC) was performed with Si gel (200–300 mesh, Qingdao Haiyang Chemical Factory, Qingdao, China), Lobar LiChroprep RP-18 (40–60 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 (18–110 μm, Merck).

3.2. Fungal Material

The fungal endophyte, Talaromyces islandicus EN-501, was isolated following surface sterilization from the marine red alga Laurencia okamurai collected from the coast of Qingdao, China, by using the procedures provided in our earlier report [19]. The isolate was identified by analysis of its morphological characteristics and ITS (internal transcribed spacer) gene sequence, which has been submitted to GenBank (accession number KU885935). A BLAST search result indicated that the sequence is almost same (99%) to the sequence of Talaromyces islandicus CBS 117284 (compared with KF984882.1). The strain was preserved in the Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

3.3. Fermentation

The fermentation was carried out statically in solid rice medium in 1 L Erlenmeyer flasks (each containing 70 g rice, 0.1 g corn flour, 0.3 g peptone, 0.1 g sodium glutamate, and 100 mL naturally sourced and filtered seawater, which was obtained from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS, pH 6.5–7.0) for 30 days at room temperature.

3.4. Extraction and Isolation

The fermented whole culture (90 flasks) was extracted with EtOAc to afford an organic extract (80 g), which was partitioned by Si gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity, from petroleum ether (PE) to methanol (MeOH) to yield nine fractions (Frs. 1–9) based on thin layer chromatography (TLC) and HPLC analysis. Fr.4 (5.5 g), eluted with PE–EtOAc (2:1), was further chromatographed over reversed-phase C₁₈ eluting with a MeOH–H₂O gradient (from 1:9 to 1:0, v/v) to afford seven subfractions (Fr.4.1–Fr.4.7). Fr.4.4 (0.5 g) was purified by CC on Si gel (CHCl₃–MeOH, from 100:1 to 20:1, v/v) and then on Sephadex LH-20 (MeOH) to yield compound 1 (120.0 mg), while Fr.4.5 (0.4 g) was purified by CC on Si gel (CHCl₃–MeOH, from 80:1 to 20:1, v/v) and then on Sephadex LH-20 (MeOH) to yield compound 2 (5.3 mg). Fr.4.6 (0.6 g) was purified by CC on Si gel (CHCl₃–acetone, from 40:1 to 3:1, v/v) and Sephadex LH-20 (MeOH), and then purified by prep. TLC (plate: 20 × 20 cm, developing solvents: PE/EtOAc, 2:1) to yield compounds 3 (8.0 mg) and 4 (10.2 mg).

2,2′,3,5-Tetrahydroxy-3′-methylbenzophenone (1): Yellowish crystals; mp 174–176 °C; UV (MeOH) λ_max (log ε) 203 (4.56), 208 (4.58), 221 (4.75), 268 (4.56), 376 (4.16) nm; ¹H and ¹³C NMR data, see Table 1. HRESIMS m/z 261.0755 ([M + H]⁺) (calcd. for C₁₄H₁₃O₅, 261.0757, Δ 0.2 ppm).

2,2′,5′-Trihydroxy-3-methoxy-3′-methylbenzophenone (2): Yellowish solid; UV (MeOH) λ_max (log ε) 203 (4.25), 209 (4.33), 226 (4.42), 266 (4.26), 383 (3.82) nm; ¹H and ¹³C NMR data, see Table 1. HRESIMS m/z 275.0912 ([M + H]⁺) (calcd. for C₁₅H₁₅O₅, 275.0914, Δ 0.2 ppm).

1,4,7-Trihydroxy-6-methylxanthone (3): Yellowish solid; UV (MeOH) λ_max (log ε) 203 (4.92), 213 (4.99), 248 (4.96), 259 (4.92), 314 (4.59) nm; ¹H and ¹³C NMR data, see Table 2. HRESIMS m/z 259.0599 ([M + H]⁺) (calcd. for C₁₄H₁₁O₅, 259.0601, Δ 0.2 ppm).

1,4,5-Trihydroxy-2-methylxanthone (4): Yellowish solid; UV (MeOH) λ_max (log ε) 205 (4.86), 212 (4.96), 248 (4.99), 263 (4.97), 317 (4.52) nm; ¹H and ¹³C NMR data, see Table 2. HRESIMS m/z 259.0598 ([M + H]⁺) (calcd. for C₁₄H₁₁O₅, 259.0601, Δ 0.3 ppm).

3.5. X-ray Crystallographic Analysis of Compound 1 [20]

The crystallographic data were collected on a Bruker Smart-1000 CCD diffractometer equipped with a graphite-monochromatic Mo-Kα radiation (λ = 0.71073 Å) at 298(2) K. The data were corrected for absorption by using the program SADABS [21]. The structure was solved by direct methods with the SHELXTL software package [22]. All non-hydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by full-matrix least-squares techniques [23].

Crystal data for compound 1: C₁₄H₁₂O₅, F.W. = 260.24, orthorhombic space group Pna2(1), unit cell dimensions a = 8.1951(7) Å, b = 17.9481(15) Å, c = 8.1962(8) Å, V = 1205.55(19) ų, α = γ = β = 90°, Z = 4, d_calcd = 1.434 mg/m³, crystal dimensions 0.42 × 0.40 × 0.37 mm, μ = 0.110 mm⁻¹, F(000) = 544. The 5770 measurements yielded 2070 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R₁ = 0.0383 and wR₂ = 0.0789 (I > 2σ(I)).

3.6. Antioxidant Assay

Evaluation of pure compounds for antioxidative activity against DPPH and ABTS free radicals was carried out by the method described previously [8]. BHT and ascorbic acid were used as positive controls against DPPH and ABTS free radicals, respectively.

3.7. Antimicrobial Assay

Antimicrobial evaluation against three human pathogens (E. coli, P. aeruginosa, S. aureus) and three aquatic bacteria (V. alginolyticus, V. harveyi, and V. parahaemolyticus) was carried out by the microplate assay [24]. Chloramphenicol was used as positive control.

4. Conclusions

In summary, two new diphenylketones (1 and 2), a new xanthone (3), and a known xanthone analogue (4) were isolated and identified from the marine algal-derived endophytic fungus T. islandicus EN-501. Each of these compounds exhibited potent antioxidative activities against DPPH and ABTS radicals, and compounds 1, 3, and 4 showed strong antibacterial activities.