Antioxidative Indenone and Benzophenone Derivatives from the Mangrove-Derived Fungus Cytospora heveae NSHSJ-2

Abstract

Seven new polyketides, including four indenone derivatives, cytoindenones A–C (1, 3–4), 3′-methoxycytoindenone A (2), a benzophenone derivative, cytorhizophin J (6), and a pair of tetralone enantiomers, (±)-4,6-dihydroxy-5-methoxy-α-tetralone (7), together with a known compound (5) were obtained from the endophytic fungus Cytospora heveae NSHSJ-2 isolated from the fresh stem of the mangrove plant Sonneratia caseolaris. Compound 3 represented the first natural indenone monomer substituted by two benzene moieties at C-2 and C-3. Their structures were determined by the analysis of 1D and 2D NMR, as well as mass spectroscopic data, and the absolute configurations of (±)-7 were determined on the basis of the observed specific rotation value compared with those of the tetralone derivatives previously reported. In bioactivity assays, compounds 1, 4–6 showed potent DPPH· scavenging activities, with EC₅₀ values ranging from 9.5 to 16.6 µM, better than the positive control ascorbic acid (21.9 µM); compounds 2–3 also exhibited DPPH· scavenging activities comparable to ascorbic acid.

1. Introduction

Indenones are characterized by a cyclopentenone ring fused with an aromatic benzene ring, providing a rigid bicyclic ring framework which enables the extensive evaluation of structure–activity relationship analysis of target therapeutic molecules [1], and indenone derivatives have been synthesized extensively for drug discovery [2,3,4,5]. The indenone moiety usually exists in natural products as a structural fragment or a small independent molecule [6,7,8,9,10,11], and 2,3-diaryl indenone analogues are rarely reported [12,13,14]. These compounds were considered to be dimers of benzophenone, xanthone, diphenyl ether moieties and indanone moieties, and there was no natural 2,3-diphenyl indenone monomer reported previously. Indenones have multiple bioactivities, including cytotoxicity, DPPH· scavenging activity, anti-inflammatory activity, anti-osteoporosis activity, human DNA dealkylation repair enzyme AlkBH3 inhibitory activity, and PPAR γ agonistic activity [2,3,4,5,8,13,14,15].

Mangrove-associated fungi are known to be an essential source of natural products for the discovery of new drug leads [16,17]. In our continuing search for structurally diverse and biologically active metabolites from mangrove endophytic fungi [18,19,20,21,22], a chemical investigation for new secondary metabolites from mangrove endophytic fungus Cytospora heveae NSHSJ-2, which was isolated from the fresh stem of the mangrove plant Sonneratia caseolaris, led to the isolation and characterization of seven new polyketides (Figure 1), including four new indenone derivatives, cytoindenones A–C (1, 3–4), 3′-methoxycytoindenone A (2), a new benzophenone derivative, cytorhizophin J (6), and a pair of undescribed tetralone enantiomers, (±)-4,6-dihydroxy-5-methoxy-α-tetralone (7), together with a known compound, cytosporaphenones E (5) [23]. Among them, compound 3 represented the first natural indenone monomer substituted by two benzene moieties at C-2 and C-3. Herein, the isolation, structure elucidation, and DPPH· radical scavenging activities of these compounds are described.

2. Results

2.1. Structure Elucidation

Compound 1 was obtained as brown oil. Its molecular formula was assigned as C₁₉H₁₆O₆ on the basis of HRESIMS analysis at m/z 363.08383 [M + Na]⁺ (calcd. For C₁₉H₁₆O₆Na, 363.08391), which was determined to possess 12 degrees of unsaturation. In the ¹H NMR spectrum (

Table 1. ¹H NMR data of 1−4 (J in Hz).

No.	1 a	2 b	3 c	4 c
5	6.65, s	6.54, s	6.63, s	6.58, s
7	6.79, s	6.82, s	6.87, s	6.75, s
10	6.50, d (8.1)	6.57, d (8.3)	6.33, d (8.1)	6.40, d (8.2)
11	7.06, t, (8.1)	7.15, t (8.3)	6.99, t (8.1)	7.02, t (8.2)
12	6.50, d (8.1)	6.57, d (8.3)	6.33, d (8.1)	6.40, d (8.2)
14	2.24, s	2.17, s	2.27, s	2.22, s
1′	2.42, m	2.42, t (7.1)		3.41, s
2′	2.49, m	2.66, t (7.1)	7.30, m
3′			7.15, m	7.09, m
4′		3.58, s	7.13, m	7.07, m
5′			7.15, m	7.02, t (8.2)
6′			7.30, m	7.07, m
7′				7.09, m

^a Data were recorded in Actone-d₆ at 400 MHz for ¹H NMR. ^b Data were recorded in CDCl₃ at 400 MHz for ¹H NMR. ^c Data were recorded in CD₃OD at 600 MHz for ¹H NMR.

), the signals for five olefinic protons (δ_H 7.06, 6.79, 6.65, 6.50 and 6.50), two methylenes (δ_H 2.49 and 2.42) and one methyl (δ_H 2.24) were observed. The ¹³C NMR data (

Table 2. ¹³C NMR data of 1−4.

No.	1 a	2 b	3 c	4 c
1	198.2, C	197.5, C	199.1, C	200.2, C
2	134.4, C	135.2, C	133.6, C	134.8, C
3	152.1, C	148.3, C	154.0, C	154.8, C
3a	127.1, C	123.7, C	127.1, C	127.4, C
4	151.7, C	150.0, C	153.1, C	152.3, C
5	124.5, CH	124.7, CH	125.1, CH	125.0, CH
6	140.7, C	141.5, C	141.9, C	141.1, C
7	116.4, CH	118.2, CH	116.9, CH	116.6, CH
7a	134.3, C	132.5, C	134.4, C	134.6, C
8	110.6, C	108.4, C	112.1, C	111.4, C
9	156.2, C	153.6, C	156.5, C	156.6, C
10	108.0, CH	109.4, CH	107.9, CH	107.8, CH
11	130.7, CH	131.9, CH	130.7, CH	130.8, CH
12	108.0, CH	109.4, CH	107.9, CH	107.8, CH
13	156.2, C	153.6, C	156.5, C	156.6, C
14	21.0, CH3	21.3, CH3	21.2, CH3	21.1, CH3
1′	20.4, CH2	19.6, CH2	133.7, C	30.5, CH2
2′	32.4, CH2	31.0, CH2	129.9, CH	140.9, C
3′	174.3, C	175.1, C	128.5, CH	129.7, CH
4′		52.2, CH3	127.9, CH	128.9, CH
5′			128.5, CH	126.5, CH
6′			129.9, CH	128.9, CH
7′				129.7, CH

^a Data were recorded in Actone-d₆ at 100 MHz for ¹³C NMR. ^b Data were recorded in CDCl₃ at 100 MHz for ¹³C NMR. ^c Data were recorded in CD₃OD at 150 MHz for ¹³C NMR.

) exhibited 19 carbon resonances, including two carbonyls (δ_C 198.2 and 174.3), two aromatic rings (A and C) (δ_C 156.2, 156.2, 151.7, 140.7, 134.3, 130.7, 127.1, 124.5, 116.4, 110.6, 108.0, 108.0), two olefinic carbons for one double bond (δ_C 152.1, 134.4), two methylenes (δ_C 32.4 and 20.4) and one methyl (δ_C 21.0).

The HMBC correlations from H-1′, to C-1, C-2, C-3, from H-14 to C-5, C-6, C-7, from H-5 to C-3a, C-4, and from H-7 to C-1, C-3a suggested the presence of an indenone fragment (rings A and B) (Figure 2). Additionally, the ¹H-¹H COSY correlations of H-10/H-11/H-12, together with the HMBC correlations from H-12 to C-3, C-8 and C-13, and from H-11 to C-13, completed the 2,6-dihydroxybenzoyl fragment (ring C), which connected to the indenone ring at C-3. The structures of ring A, B and C were further confirmed by comparison of ¹H and ¹³C NMR spectra between 1 and 5 [23]. Furthermore, the ¹H-¹H COSY correlation of H-1′/H-2′ and the HMBC correlations from H-1′, H-2′ to C-3′, from H-1′ to C-1, C-2, C-3, and from H-2′, to C-2 indicated the presence of the 2-carboxyethyl group, which was assigned to be connected to the indenone ring at C-2. Thus, the structure of 1 was deduced, named cytoindenone A.

Compound 2 was isolated as brown oil. Its molecular formula was determined as C₂₀H₁₈O₆ (12 degrees of unsaturation) in terms of HREIMS analysis at m/z 377.09985 [M + Na]⁺ (calcd. for C₂₀H₁₈O₆Na, 377.09956). Analysis of the ¹H and ¹³C NMR spectroscopic data of 2 (Table 1 and Table 2) revealed mostly similarities with that of 1, except that the hydroxyl group was substituted with the methoxy group (δ_H 3.58, δ_C 52.2) at C-3′. Combined with the HMBC from H-4′ to C-3′ (Figure 2), the structure of compound 2 was clearly confirmed, named 3′-methoxycytoindenone A.

Compound 3 was acquired as brown oil and had a molecular formula of C₂₂H₁₆O₄, determined by HRESIMS data m/z 367.09424 [M + Na]⁺ (calcd. 367.09408) with 15 degrees of unsaturation. The ¹H NMR spectrum of 3 displayed the signal for ten olefinic protons (δ_H 7.30, 7.30, 7.15, 7.15, 7.13, 6.99, 6.87, 6.63, 6.33 and 6.33) and one methyl (δ_H 2.27). The ¹³C NMR data exhibited one carbonyl (δ_C 199.1), three aromatic rings (δ_C 156.5, 156.5, 153.1, 141.9, 134.4, 133.7, 130.7, 129.9, 129.9, 128.5, 128.5, 127.9, 127.1, 125.1, 116.9, 112.1, 107.9 and 107.9), two olefinic carbons for one double bond (δ_C 154.0, 133.6) and one methyl (δ_C 21.2) (Table 1 and Table 2). According to 1D NMR and 2D NMR data, the rings A, B and C of 2 were similar to that of 1. The obvious difference was the absence of the 2-carboxyethyl group at the C-2 position of compound 1 and the presence of a phenyl group (ring D) at the C-2 position of compound 3. Meanwhile, the ¹H-¹H COSY correlations of H-3′/H-4′/H-5′ failed to be identified because the chemical shifts of H-3′, H-4′ and H-5′ were overlapped; the ¹H-¹H COSY correlations of H-2′/H-3′, the HMBC from H-2′ to C-2, C-5′, and from H-3′ to C-1′ also indicated that ring D was formed and connected to the indenone ring at C-2, and the structure of compound 3 was determined, named cytoindenone B.

Compound 4 was obtained as brown oil. The molecular formula was determined as C₂₃H₁₈O₄ on the basis of HRESIMS data at m/z 381.10980 [M + Na]⁺ (calcd. for C₂₃H₁₈O₄Na, 381.10973), which was thus determined to possess 15 degrees of unsaturation. The ¹H and ¹³C NMR spectroscopic data were listed in Table 1 and Table 2, which suggested that the structure of compound 4 was similar to compound 3, except the presence of methylenes (δ_H 3.41, δ_C 30.5). Similarly, the ¹H-¹H COSY correlations of H-3′/H-4′/H-5′/H-6′/H-7′ failed to be identified because of the overlapping chemical shifts. Combined with the HMBC from H-1′ to C-1, C-2, C-3, C-2′, C-3′, and from H-3′, H-4′ to C-5′ (Figure 2), ring D was formed and C-1′ was connected to the indenone ring and ring D at C-2 and C-2′, and the structure of compound 5 was clearly confirmed, named cytoindenone C.

Compound 6 was isolated as white powder and assigned an HRESIMS ion peak at m/z 395.11005 ([M + Na]⁺, calcd. for C₂₀H₂₀O₇Na, 395.11012), which perfectly matched the molecular formula of C₂₀H₂₀O₇ with 11 degrees of unsaturation. The ¹H NMR spectrum of 6 displayed the signal for five olefinic protons (δ_H 7.25, 7.18, 6.87, 6.27 and 6.27), one methoxyl (δ_H 3.63), three methylenes (δ_H 2.96, 2.32 and 1.85) and one methyl (δ_H 2.36). The ¹³C NMR data revealed 20 carbon resonances, involving two carbonyls (δ_C 204.1 and 202.2), one ester carbonyl (d_C 175.5), two aromatic rings (δ_C 163.2, 163.2, 155.0, 140.8, 137.8, 137.0, 130.0, 121.6, 121.6, 112.7, 108.1, 108.1), one methoxyl (δ_C 52.0), three methylenes (δ_C 38.9, 33.7 and 20.6) and one methyl (δ_C 21.3) (

Table 3. ¹H and ¹³C NMR data for 6.

	6 a
No.	δC, Type	δH Mult (J in Hz)
1	108.1, CH	6.27, d (8.2)
2	137.0, CH	7.18, t (8.2)
3	108.1, CH	6.27, d (8.2)
4	163.2, C
5	112.7, C
6	163.2, C
7	204.1, C
8	130.0, C
9	155.0, C
10	121.6, CH	6.87, s
11	140.8, C
12	121.6, CH	7.25, s
13	137.8, C
14	21.3, CH3	2.36, s
15	202.2, C
16	38.9, CH2	2.96, t (7.5)
17	20.6, CH2	1.85, m
18	33.7, CH2	2.32, t (7.3)
19	175.5, C
20	52.0, CH3	3.63, s

^a Data were recorded in CD₃OD at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR.

). According to 1D NMR and 2D NMR data, the benzophenone moiety of 6 was similar to cytorhizophin C [24]. The only difference between them were that the popionyl group at the C-13 position of cytorhizophin C was replaced by the 5-methoxy-5-oxopentanoyl group of compound 6. The ¹H-¹H COSY correlations of H-16/H-17/H-18, together with the HMBC correlations from H-16 to C-13 and C-15, from H-17 to C-15, and from H-18, H-20 to C-19 indicated that the 5-methoxy-5-oxopentanoyl group was located at C-13. Therefore, the structure of 6 was deduced and named cytorhizophin J.

Compound 7 was acquired as colorless oil. Its molecular formula C₁₁H₁₂O₄ (six degrees of unsaturation) was established on the basis of HREIMS analysis at m/z 209.08093 [M + H]⁺ (calcd. For C₁₁H₁₃O₄, 209.08084). Analysis of the ¹H and ¹³C NMR spectroscopic data of 7 (

Table 4. ¹H and ¹³C NMR data for 7.

	7 a
No.	δC, Type	δH Mult (J in Hz)
1	199.6, C
2	33.0, CH2	2.99, m
		2.43, dt (17.2, 3.6)
3	31.6, CH2	2.26, m
		2.17, m
4	61.7, CH	5.26, t (3.1)
4a	139.9, C
5	146.2, C
6	157.5, C
7	117.8, C	6.92, d (8.6)
8	125.4, CH	7.67, d (8.6)
8a	125.6, C
9	61.9, CH3

^a Data were recorded in CD₃OD at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR.

) revealed mostly similarities to 3,4-dihydro-4β,6-dihydroxy-5-methoxy-2α-methyl-1(2H)-naphthalenone [25]. The main difference between them were the absence of one methine at δ_H 2.98 (1H, m, H-2β) and one methyl at δ_H 1.11 (3H, d, J = 6.8 Hz, 2-Me) in 3,4-dihydro-4β,6-dihydroxy-5-methoxy-2α-methyl-1(2H)-naphthalenone and the presence of one methylene at δ_H 2.99 (1H, m, H_a-2) and 2.43 (1H, dt, J = 17.2, 3.6, H_b-2) in 7, which was confirmed by the ¹H-¹H COSY correlations of H_{a, b}-2/H_{a, b}-3/H-4, and the HMBC correlations (Figure 2) from H_{a, b}-2 to C-1, C-8a. Thus, compound 7 was assigned as shown in Figure 1, and named 4,6-dihydroxy-5-methoxy-α-tetralone. However, chiral HPLC analysis of 7 showed two peaks (t_R 21.3 min and 24.6 min), and subsequent chiral HPLC purification of (±)-7 led to the separation of the two enantiomers (+)-7 and (−)-7. The absolute configurations of (+)-7 and (−)-7 were determined as 4S and 4R by the comparison of the observed specific rotation value [(+)-7: ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ + 31.3, (+)-7: ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ − 31.5)] of compounds (±)-7 with those for (4S)-4,8-dihydroxy-α-tetralone (${\left[\alpha \right]}_{\mathrm{D}}^{27}$ + 24.5), (4S)-5-hydroxy-4-methoxy-α-tetralone (${\left[\alpha \right]}_{\mathrm{D}}^{27}$ + 50.0), (4R)-4,8-dihydroxy-α-tetralone (${\left[\alpha \right]}_{\mathrm{D}}^{27}$ − 26.0) and (4R)-5-hydroxy-4-methoxy-α-tetralone (${\left[\alpha \right]}_{\mathrm{D}}^{27}$ − 50.0) (Figure S39) [26].

2.2. Biological Evaluation

Compounds 1–7 were tested for their DPPH· scavenging activity. As seen in

Table 5. DPPH· scavenging activities of compounds 1–9.

Compound	% Inhibition (100 µM)	EC50 (µM)
1	90.8	11.5 ± 0.1
2	72.5	21.5 ± 1.0
3	69.0	19.7 ± 1.8
4	78.2	16.6 ± 0.4
5	81.0	12.4 ± 0.5
6	87.3	9.5 ± 0.1
(+)-7	12.0	–
(–)-7	4.2	–
ascorbic acid a	91.4	21.9 ± 0.3

^a positive control.

, the results indicated that compounds 1, 4–6 showed significant DPPH· scavenging activities with EC₅₀ values ranging from 9.5 to 16.6 µM, better than the positive control ascorbic acid (21.9 µM) [27,28]; compounds 2–3 also exhibited DPPH· scavenging activities comparable to ascorbic acid.

The antioxidant activities of phenolic compounds were widely investigated and the phenolic content and the side chain functional groups had significant influences on DPPH· scavenging activities [29,30]. Comparing the activities of compounds 1−2, when the carboxyl group at C-3′ was esterified by the methyl group, the antioxidant activity of 2 decreases significantly. Comparing the activities of compounds 2−5, the higher activity of compound 5 was due to the accessibility of the phenolic OH group by DPPH·. The activities of compounds 2−4 were due to the presence of bulky groups at C-2 obstructing DPPH· access to the phenolic OH group. Compound 6 could be regarded as a precursor of compound 2, which formed ring B through C7−C16 aldol-type cyclization. Compound 6 exhibited the strongest antioxidant activity due to the disconnection of ring B and the smallest steric hindrance of phenolic ring C. Compounds (+)-7 and (–)-7 showed no antioxidant activities due to the reduction of the phenolic content.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were performed on an MCP300 (Anton Paar, Shanghai, China). UV data were measured on a Shimadzu UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). The ECD experiment data were conducted with a J-810 spectropolarimeter (JASCO, Tokyo, Japan). IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu, Kyoto, Japan). Melting points were recorded on a Fisher-Johns hot-stage apparatus. The NMR spectra were tested on a Bruker Avance spectrometer (Bruker, Beijing, China) (Compounds 3–4: 600 MHz for ¹H and 150 MHz for ¹³C; compounds 1–2 and 5–7: 400 MHz for ¹H and 100 MHz for ¹³C, respectively). HRESIMS data were conducted on a ThermoFisher LTQ-Orbitrap-LC-MS spectrometer (Palo Alto, CA, USA). Column chromatography (CC) was performed on silica gel (200–300 mesh, Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia, Piscataway, NJ, USA). Semi-preparative HPLC (Ultimate 3000 BioRS, Thermo Scientific, Germany) were conducted using the Chiral INA column (5 μm, 4.6 × 250 mm, Phenomenex, Piscataway, NJ, USA), and the Chiralcel ODH column (5 μm, 4.6 × 250 mm, Daicel, Tokyo, Japan) for chiral separation.

3.2. Fungal Material

The fungal strain NSHSJ-2 used in this study was isolated from the fresh stem of mangrove plant Sonneratia caseolaris, which was collected in June 2020 from the Nansha Mangrove National Nature Reserve in Guangdong Province, China. The strain was identified as Cytospora heveae (compared to no. OQ423127) upon the analysis of ITS sequence data of the rDNA gene. The ITS sequence data obtained from the fungal strain has been submitted to GenBank with accession no. OL780505.1. A voucher strain was deposited in our laboratory.

3.3. Fermentation, Extraction and Isolation

The fungus Cytospora heveae NSHSJ-2 was fermented on solid cultured medium (sixty 1000 mL Erlenmeyer flasks, each containing 50 g of rice and 50 mL of distilled water with 3% sea salt) for 30 days at 25 °C. The cultures were extracted three times with MeOH to yield 22.9 g of residue. Then, the crude extract was eluted by using a gradient elution with petroleum ether/EtOAc from 9:1 to 0:10 (v/v) on silica gel CC to get six fractions (Fr. A–F). Fr. D (297 mg) was subjected to silica gel CC (CH₂Cl₂/MeOH, v/v, 800:1 to 200:1) to obtain three subfractions (Fr. D₁–D₃). Fr. D₂ (9.4 mg) was separated by normal phase HPLC on a chiral column (INA), using hexane/isopropanol (80:20, v/v, flow rate: 1.0 mL/min) as the solvent system, to yield compounds 3 (1.6 mg, t_R 14.0 min) and 4 (4.3 mg, t_R 21.2 min). Fr. D₃ (83.4 mg) was applied to Sephadex LH-20 CC (CH₂Cl₂/MeOH, v/v, 1:1) to yield compound 5 (26 mg). Fr. E (749 mg) was subjected to silica gel CC (CH₂Cl₂/MeOH, v/v, 100:1 to 20:1) to afford four fractions (Fr. E₁−E₄). Fr. E₂ (204 mg) was subjected to silica gel CC (petroleum ether/EtOAc, v/v, 7:3) to yield compounds 2 (46.5 mg). Fr. E₃ (56.4 mg) was subjected to silica gel CC (petroleum ether/EtOAc, v/v, 6:4) to yield compounds 6 (15.4 mg) and (±)-7 (9.4 mg). The chiral HPLC separation of (±)-7 was accomplished over a chiral column (ODH) (column size: 4.6 × 250 mm, 5 μm; flow rate: 1.0 mL/min; solvent: n-hexane-isopropanol = 90:10) to yield (+)-7 (1.4 mg, t_R 21.3 min) and (−)-7 (7.3 mg, t_R 24.6 min). Fr. E₄ (103 mg) was purified by Sephadex LH-20 CC and eluted with MeOH to obtain compound 1 (27.9 mg).

Cytoindenone A (1): brown oil; UV (MeOH) λ_max (log ε): 205 (1.24), 247 (0.53) nm; IR υ_max 3282, 2949, 2835, 1695, 1435, 1276, 1010, 781 cm⁻¹; HRESIMS m/z 363.08383 [M + Na]⁺ (calcd. for C₁₉H₁₆O₆Na, 363.08391); ¹H NMR (400 MHz, Actone-d₆) data and ¹³C NMR (100 MHz, Actone-d₆) data (see Table 1 and Table 2).

3′-methoxycytoindenone A (2): brown oil; UV (MeOH) λ_max (log ε): 204 (0.90), 248 (0.42) nm; IR υ_max 3360, 2954, 2920, 1697, 1622, 1462, 1278, 1012, 783 cm⁻¹; HRESIMS m/z 377.09985 [M + Na]⁺ (calcd. for C₂₀H₁₈O₆Na, 377.09956); ¹H NMR (400 MHz, CDCl₃) data and ¹³C NMR (100 MHz, CDCl₃) data (see Table 1 and Table 2).

Cytoindenone B (3): brown oil; UV (MeOH) λ_max (log ε): 203 (0.32), 272 (0.15) nm; IR υ_max 3365, 2949, 2850, 1689, 1618, 1462, 1280, 1014, 792 cm⁻¹; HRESIMS m/z 367.09424 [M + Na]⁺ (calcd. for C₂₂H₁₆O₄Na, 367.09408); ¹H NMR (600 MHz, CD₃OD) data and ¹³C NMR (150 MHz, CD₃OD) data (see Table 1 and Table 2).

Cytoindenone C (4): brown oil; UV (MeOH) λ_max (log ε): 205 (0.80), 249 (0.35) nm; IR υ_max 3358, 2922, 2852, 1683, 1618, 1462, 1276, 1012, 700 cm⁻¹; HRESIMS m/z 381.10980 [M + Na]⁺ (calcd. for C₂₃H₁₈O₄Na, 381.10973); ¹H NMR (600 MHz, CD₃OD) data and ¹³C NMR (150 MHz, CD₃OD) data (see Table 1 and Table 2).

Cytorhizophin J (6): white powder, mp 190.2−191.6 ◦C; UV (MeOH) λ_max (log ε): 216 (1.43), 270 (0.67) nm; IR υ_max 3342, 2924, 1716, 1627, 1456, 1338, 1226, 1031, 925, cm⁻¹; HRESIMS m/z 395.11005 [M + Na]⁺ (calcd. for C₂₀H₂₀O₇Na, 395.11012); ¹H NMR (400 MHz, CD₃OD) and ¹³C NMR (100 MHz, CD₃OD) data (see Table 3).

(±)-4,6-dihydroxy-5-methoxy-α-tetralone (7): colorless oil; UV (MeOH) λ_max (log ε): 205 (1.63), 230 (1.29), 282 (1.01) nm; IR ν_max 3261, 2943, 2839, 1660, 1578, 1305, 1290, 1190, 1012 cm⁻¹; ¹H NMR (400 MHz, CD₃OD) data and ¹³C NMR (100 MHz, CD₃OD) data (see Table 4); HRESIMS m/z 209.08093 [M + H]⁺ (calcd for C₁₁H₁₃O₄, 209.08084). (+)-7, ${\left[\alpha \right]}_{\mathrm{D}}^{25}$+ 31.3 (c 0.1 MeOH); ECD (c = 0.18 mg/mL, MeOH) λ_max (Δε) 205 (+13.5), 230 (+8.4), 284 (+7.0), 327 (−6.0). (−)-7, ${\left[\alpha \right]}_{\mathrm{D}}^{25}$− 31.5 (c 0.1 MeOH); ECD (c = 0.17 mg/mL, MeOH) λ_max (Δε) 205 (−14.9), 225 (−6.1), 277 (−5.4), 320 (+5.8).

3.4. Biological Assays

The DPPH·radical scavenging activities of compounds 1−7 were determined according to the reported method [14,28]. The DPPH· radical scavenging test was performed in 96-well microplates. Testing materials (compounds 1–7) were added to 100 µL (0.16 mmol/L) DPPH solution in MeOH at a range of 100 µL solutions of different concentrations (6.25−100 µM). Ascorbic acid was prepared as positive control at the same concentrations (Table 5). Absorbance was recorded at λ = 517 nm after 45 min of incubation in the dark. The DPPH·radical scavenging activity was calculated using the formula:

DPPH radical scavenging activity (%) = [(Abs_control − Abs_sample)/Abs_control] × 100

4. Conclusions

In summary, seven new polyketides including four indenone derivatives, cytoindenones A–C (1, 3–4), 3′-methoxycytoindenone A (2), a new benzophenone derivative, cytorhizophin J (6) and a pair of undescribed tetralone enantiomers, (±)-4,6-dihydroxy-5-methoxy-1-tetralone (7), together with a known compound (5), were isolated from the endophytic fungus Cytospora heveae NSHSJ-2. Compound 3 represented the first natural indenone monomer substituted by two benzene moieties at C-2 and C-3. Their structures were confirmed by the analysis of NMR, HR-MS and ECD spectra. All of the compounds were tested for their antioxidative activities. Compounds 1, 4–6 showed potent DPPH· scavenging activities with EC₅₀ values ranging from 9.5 to 16.6 µM, better than the positive control ascorbic acid (21.9 µM); compounds 2–3 also exhibited DPPH· scavenging activities comparable to ascorbic acid.