Antioxidant Aryl-Substituted Phthalan Derivatives Produced by Endophytic Fungus Cytospora rhizophorae

Introduction

The free radicals and reactive oxygen species (ROS) were highly reactive intermediates widely existing in human body, which can react with human biomolecules including lipids, proteins, DNA, etc, thus causing seriously detrimental health effects, such as neurodegenerative diseases, atherosclerosis, liver cirrhosis, cataracts, diabetes, and cancer (Kang et al., 2007; López-Alarcónand Denicola, 2013). With the aim to clear up the oxidative stress resulting by excess amounts of ROS, numerous remarkable results have been reported in the past decades (Cerutti, 1985; Halliwell, 1987; Breimer, 1990; Ding et al., 1999; Grisham et al., 2000; Aitken et al., 2012; Russell and Cotter, 2015; El-Hawary et al., 2019; Kusio et al., 2020). Among them, antioxidant was respected as one of the most efficient therapeutic strategies against human diseases related to oxidative damage by ROS (Beckman et al., 1992; Taniguchi et al., 1993).

In the repertoire of pharmaceutical antioxidant discovery and achievements, natural products exampled by astaxanthin, vitamins, as well as carotenoids have played extremely significant roles (Quiñones, et al., 2012). Additionally, many attentions have been continuously paid to the discovery of natural antioxidants. Consequently, more and more nature-originated antioxidants were emerged and widely used in functional foods, pharmaceutical drugs, and industrial cosmetics (Mussard et al., 2019; Wen et al., 2017). Polyphenols represent a characteristic family of natural-based organic compounds with strong antioxidant activities (Dao et al., 2020; Bodoira and Maestri, 2020). Phthalans featured by a core isobenzofuran skeleton were a typical class of phenols, which have dramatically attracted many medicinal scientists attributable to their affluent structure diversities, novel architecture complexities, and significant pharmaceutical activities in recent years (Naito and Kaneko, 1969; Strobel et al., 2002; Harper et al., 2003; Kapoor et al., 2003; Fotso et al., 2008). Especially, the 1-phenyl-phthalan moiety is frequently encountered in numerous natural products and commercially available drugs or drug lead compounds. Their fascinating biological activities and novel structural features rendered them appealing targets for the natural product and pharmaceutical communities.

As a part of our continuing program to discover structurally unique natural products with significantly biological potentials from the endophytic fungi (Liu et al., 2017; Liu et al., 2019a; Liu H.-X. et al., 2019; Chen et al., 2019), an endophytic fungus, Cytospora rhizophorae A761, was obtained from the stem of Gynochthodes officinalis (F.C.How) Razafim. and B. Bremer (basionym: Morinda officinalis). The chemical investigation on the liquid culture of C. rhizophorae has resulted in the successful purification of six novel polyphenolic natural products cytorhizophins D-I (1-6) as well as three known derivatives cytorhizophin C (7) (Liu et al., 2019c), pestacin (8) (Harper et al., 2003) and rhizophol B (9) (Liu et al., 2019c) (Figure 1). Cytorhizophins D-E (1-2) and F-G (3-4) were two pairs of diastereoisomers, all of them featured a 1-phenyl-1,3-dihydroisobenzofuran scaffold with a highly oxygenated isopentenyl unit. Cytorhizophins H-I (5-6) represent the first examples of phthalide family with a fascinating 6/6/6/5 tetracyclic ring system fusing as unprecedented furo [4,3,2-kl]xanthen-2 (10bH)-one skeleton. Herein, the details of the extraction, purification, structure elucidation, and antioxidant activity of cytorhizophins D-I were described.

FIGURE 1

FIGURE 1. Structures of compounds 1-9.

Materials and Methods

General Experimental Procedures

The general experimental procedures were described in supporting information.

Fungal Material

The information of fungal material used in this study were identical to that of the previous descriptions (Liu et al., 2019a).

Extraction and Isolation

The strain Cytospora rhizophorae A761 was kept for 7 days at 28°C and 120 r/m on a rotary shaker in 150 flasks (1,000 ml) containing 500 ml of potato dextrose broth (potato 20%, glucose 2%, K₂HPO₄ 0.3%, MgSO₄•7H₂O 0.15%, vitamin B 10 mg/L). The fermented broth (75 L) was filtered through cheesecloth to give the broth and mycelia. The fermented broth were subjected to macroporous resin D101 column with ethanol as eluent. The EtOH fraction was concentrated under a vacuum to yield a dark brown gum (37 g). The crude extract was subjected to reversed-phase silica gel C₁₈ using step gradient elution with MeOH/H₂O, 60%→100% to afford six fractions (Fr.): Fr.1-Fr.6.

Then, Fr. 2 (8.92 g) was separated by silica gel flash CC (n-hexane/EtOAc, 20:1→1:1, v/v) to give nine subfractions (Fr.2-1 to Fr.2-9). Fr.2-5 (166.2 mg) was subjected to CC on Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) to give four sub-fractions (Fr. 2-5-1 to Fr. 2-5-4). Fr. 2-5-4 was further purified by silica gel flash column chromatography (n-hexane/EtOAc, 10:1→1:1, v/v) to give 8 (9.0 mg). Fr.2-7 (3.07 g) was subjected to CC on Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) to give ten sub-fractions (Fr. 2-7-1 to Fr. 2-7-10). Fr. 2-7-5 was purified by silica gel flash column chromatography and further purified by semipreparative HPLC (MeOH/H₂O, 60:40, v/v, 3 ml/min) to give 9 (5.0 mg). Fr. 2-7-6 was divided into five sub-fractions (Fr.2-7-6-1 to Fr. 2-7-6-5) by silica gel flash column chromatography (n-hexane/EtOAc, 10:1→1:1, v/v). Fr. 2-7-6-5 was further separated by semipreparative HPLC (MeOH/H₂O, 73:27, v/v, 3 ml/min) to give four sub-fractions (Fr. 2-7-6-5-1 to Fr. 2-7-6-5-4). Fr. 2-7-6-5-2 (10 mg, t_R = 8.3 min) was purified by semipreparative HPLC equipped with a Chiralpak IC column (n-hexane 95%/isopropyl alcohol, 7:3, 3 ml/min) to obtain 3 (5.0 mg, t_R = 8.6 min) and 4 (2.5 mg, t_R = 9.0 min).

Fr. Three was further purified by CC over reversed-phase silica gel C₁₈ (MeOH/H₂O, 20%→100%) to give five subfractions (Fr.3-1 to Fr.3-5). Fr.3-2 (2.0 g) was divided into seven sub-fractions (Fr. 3-2-1 to Fr. 3-2-7) by Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v). Fr. 3-2-1 was further purified by repeated silica gel and semi-preparative HPLC (ACN/H₂O, 50:50, v/v, 3 ml/min) to obtain compound 7 (2.0 mg, t_R = 12.0 min). Fr. 3-2-2 was subjected by silica gel CC (n-hexane/EtOAc, 5:1→1:2, v/v) to yield four sub-fractions (Fr.3-2-2-1 to Fr. 3-2-2-4). Fr. 3-2-2-1 was purified by semipreparative HPLC (MeOH/H₂O, 60:40, v/v, 3 ml/min) to give a mixture (10 mg, t_R = 8.9 min). The mixture was further separated by HPLC (Chiralpak IC column, n-hexane 95%/isopropyl alcohol, 4:1, 3 ml/min) to obtain 2 (4.0 mg, t_R = 20.8 min) and 1 (4.0 mg, t_R = 25.1 min).

Fr.3-4 (1.3 g) was separated by silica gel flash CC (n-hexane/EtOAc, 5:1→1:5, v/v) to yield twelve sub-fractions (Fr.3-3-1 to Fr. 3-3-12). Compound 5 (3.0 mg, t_R = 14.0 min) was obtained from Fr. 3-3-10 by semipreparative HPLC (MeOH/H₂O, 60:40, v/v, 3 ml/min). Fr.3-4 (2.8 g) was separated by Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) to give seven sub-fractions (Fr. 3-4-1 to Fr. 3-4-7). Fr. 3-4-4 was divided into four sub-fractions (Fr.3-4-4-1 to Fr. 3-4-4-4) by silica gel flash CC (n-hexane/EtOAc, 2:1→1:2, v/v). Fr. 3-4-4-2 was purified by HPLC (ACN/H₂O, 55:45, v/v, 2 ml/min) to yield compound 6 (4.0 mg, t_R = 18.7 min).

Cytorhizophin D (1): yellow powder [α]²⁵_D = +34.0 (c 0.12, MeOH); CD (MeOH, 0.4 mg/ml): 206 (−5.3), 214 (+40.5), 230 (+8.6), 247 (−18.2), 260 (+1.9), 288 (−2.3), 306 (−1.7) nm; UV (MeOH) λ_max (log ε) 213 (5.35), 311 (4.23) nm; IR ν_max 3,230, 2,927, 1716, 1,616, 1,472, 1,015, 887, 794 cm⁻¹. For ¹H and ¹³C NMR, see Table 1; HRESIMS: m/z 373.1285 [M + H]⁺ (calcd for C₂₀H₂₁O₇, 373.1282).

TABLE 1

TABLE 1. ¹H (600 MHz) and¹³C (150 MHz) NMR data of 1 and 2 in CD₃COCD₃.

Cytorhizophin E (2): yellow powder [α]²⁵_D = −22.3 (c 0.02, MeOH); CD (MeOH, 0.3 mg/ml): 206 (+3.8), 214 (−33.5), 231 (−5.3), 247 (+12.9), 262 (−2.3), 288 (+0.9), 308 (+0.6) nm; UV (MeOH) λ_max (log ε) 213 (5.33), 310 (4.26) nm; IR ν_max 3,236, 2,926, 1715, 1,614, 1,470, 1,427, 1,268, 1,233, 1,198, 1,162, 1,017, 977, 903, 885, 795, 752, 739 cm⁻¹. For ¹H and ¹³C NMR, see Table 1; HRESIMS: m/z 373.1281 [M + H]⁺ (calcd for C₂₀H₂₁O₇; 373.1282).

Cytorhizophin F (3): yellow powder [α]²⁵_D = +127 (c 0.02, MeOH); CD (MeOH, 0.15 mg/ml): 207 (+108.0), 238 (−2.4), 286 (−5.2) nm; UV (MeOH) λ_max (log ε) 284 (4.15) nm; IR ν_max 3,415, 2,953, 1,616, 1,597, 1,472, 1,285, 1,225, 1,140, 1,225, 1,015, 926, 887, 864, 826, 795, 753, 738 cm⁻¹. For ¹H and ¹³C NMR, see Table 2; HRESIMS: m/z 359.1497 [M + H]⁺ (calcd for C₂₀H₂₃O₆, 359.1489).

TABLE 2

TABLE 2. ¹H (600 MHz) and¹³C (150 MHz) NMR data of 3 and 4 in CD₃COCD₃.

Cytorhizophin G (4): yellow powder [α]²⁵_D = ‒76 (c 0.08, MeOH); CD (MeOH, 0.10 mg/ml): 207 (‒75.3), 238 (+2.7), 287 (+4.7) nm; UV (MeOH) λ_max (log ε) 282 (4.07) nm; IR ν_max 3,290, 1,616, 1,474, 1,472, 1,225, 1,015, 887, 795 cm⁻¹. For ¹H and ¹³C NMR, see Table 2; HRESIMS: m/z 359.1493 [M + H]⁺ (calcd for C₂₀H₂₃O₆, 359.1489).

Cytorhizophin H (5): pale yellow powder [α]²⁵_D = ‒117.5 (c 0.06, MeOH); CD (MeOH, 0.2 mg/ml): 202 (+42.2), 214 (−33.5), 235 (−4.2), 243 (−8.7) nm; UV (MeOH) λ_max (log ε) 311 (4.01), 282 (3.90) nm; IR ν_max 3,303, 2,828, 1715, 1,462, 1,423, 1,281, 1,236, 1,192, 1,144, 1,045, 1,011, 902, 786 cm⁻¹. For ¹H and ¹³C NMR, see Table 3; HRESIMS: m/z 373.1285 [M + H]⁺ (calcd for C₂₀H₂₁O₇, 373.1282).

TABLE 3

TABLE 3. ¹H (600 MHz) and¹³C (150 MHz) NMR data of 5 and 6 in CD₃COCD₃.

Cytorhizophin I (6): pale yellow powder [α]²⁵_D = ‒39.3 (c 0.02, MeOH); CD (MeOH, 0.2 mg/ml): 200 (−38.5), 214 (−30.2), 237 (−3.3), 243 (+7.4) nm; UV (MeOH) λ_max (log ε) 310 (3.87), 284 (3.84) nm; IR ν_max 3,302, 1,472, 1,238, 1,016, 903, 677, 600, 592, 556 cm⁻¹. For ¹H and ¹³C NMR, see Table 3; HRESIMS: m/z 373.1284 [M + H]⁺ (calcd for C₂₀H₂₁O₇, 373.1282).

DPPH Photometric Assay

The DPPH photometric assay were carried out according to our previously established method (Zhong et al., 2020).

Results and Discussion

Cytorhizophin D (1) was purified as a yellow powder, and the molecular formula of 1 had been established as C₂₀H₂₀O₇ by HRESIMS with an obvious ion peak discovered at m/z 373.1285 ([M + H]⁺, calcd for C₂₀H₂₁O₇, 373.1282). The IR spectrum of 1 displayed prominent resonance bands at 3,230 and 1,716 cm⁻¹, clarifying the existence of hydroxy and carbonyl functionality. The ¹H NMR spectrum of 1 exhibited four downshifted protons at δ_H 6.74 (1H, s, H-12), δ_H 6.98 (1H, t, J = 8.2 Hz, H-2), 6.38 (1H, d, J = 8.2 Hz, H-1), and 6.38 (1H, d, J = 8.2 Hz, H-3), which were responsive for two independent benzenoid rings. Moreover, the signals for three methyl groups [δ_H 1.14 (3H, s), 1.20 (3H, s), 2.31 (3H, s)] signals were also observed in its ¹H NMR spectrum. Additionally, the ¹³C NMR data (Table 1) coupling with HSQC data of 1 further identified 20 carbon signals, which could be readily differentiated to three methyls, two methylenes, six methines, as well as nine quaternary carbons with a carbonyl moiety (δ_C 171.7).

Two spin systems of C-1/C-2/C-3 and C-2′/C-3′ were successfully assigned by carefully analysis of the ¹H-¹H COSY spectrum of 1 (Figure 2). As referring to the fragment C-1/C-2/C-3, the critical HMBC correlative signals from H-1 to C-3 and C-5, H-2 to C-4 and C-6 in conjunction with consideration of the overlapping NMR data of C-1/C-3 as well as C-2/C-4 confirmed the presence of a symmetric aromatic ring. Moreover, the conclusive HMBC correlative signals from H₃-15 to C-10, C-11, and C-12 as well as H-12 to C-8, C-10 and C-14 revealed the existence of the ring B. The linkage of rings A and B via C-7 methine was successfully verified by the unambiguous HMBC correlations from H-7 to C-4, C-6, C-9, and C-13. The five-membered lactone ring C was then established with the aid of the conclusive HMBC correlations from the critical proton H-7 to C-13 and C-14. Moreover, the key HMBC correlations from H-7 to C-9, and H-12 to C-14 concluded that the lactone ring C was fused with the benzene ring B to construct the key phthalide core.

FIGURE 2

FIGURE 2. ¹H-¹H COSYs and key HMBCs of 1-4.

Furthermore, with the aid of the spin fragment C-2′/C-3′, a highly oxygenated isopentyl unit was strongly suggested to connect with the phthalide core through an ether bond in 1, answering for the informative HMBC correlations from the methyl protons H₃-4′ to C-1′, C-2′ together with H₃-5′ to C-1′, C-2′. Moreover, there was an epoxy ring in the isopentyl unit, which could be further concluded through the high-field shift of C-2′ (δ_C 56.8) and C-3′ (δ_C 43.9) together with the molecular formula. Because the lack of direct HMBC correlations from the isopentyl unit and the ring B, the location of isopentyl moiety could be readily assigned at C-10 position by comparing the carbon resonance shifts of the C-9 (δ_C 152.0) and C-11 (δ_C 142.7) along with the definitive NOESY correlations from methyls H₃-4′ and H₃-5′ to H₃-15. Consequentially, the planar structure of 1 was finally elucidated as outlined in Figure 1.

Cytorhizophin E (2) was obtained as a yellow powder and found to possess a molecular formula of C₂₀H₂₀O₇ based on the HRESIMS ion peak at m/z 373.1281 [M + H]⁺, indicating eleven indices of hydrogen deficiency. The IR spectrum of 2 was quite similar to that of 1. The inspection of the NMR data (Table 1) of 2 with those of 1 demonstrated that 2 displayed close similarity with 1. The obvious differences were the chemical shifts of the H-7 (δ_H 7.16 ppm for 1 versus 7.12 ppm for 2), H-15 (δ_H 2.31 ppm for 1 versus 2.28 ppm for 2), H-2′ (δ_H 3.13 ppm for 1 versus 3.17 ppm for 2), H-3′ (δ_H 2.69, 2.55 ppm for 1 versus 2.61, 2.51 ppm for 2), H₃-4′ (δ_H 1.20 ppm for 1 versus 1.22 ppm for 2), and H₃-5′ (δ_H 1.14 ppm for 1 versus 1.17 ppm for 2), which strongly concluded that the compounds 1 and 2 should be a pair of diastereoisomers.

To determine the absolute configuration of 1 and 2, the experimental and TDDFT calculated circular dichroism (CD) spectra at cam-b3lyp/def2svp level were performed. The calculated ECD spectrum with 7R configuration showed very excellent similarities to those of the experimental CD spectrum of 1 as shown in Figure 3. Thus, the absolute configuration at C-7 of 1 was rationally assigned as R. Moreover, the calculated ECD Cotton effects of the 7S enantiomer were well agreement with those in the experimental ECD spectrum of 2. Cytorhizophins D (1) and E (2) possessed a couple of distant stereogenic centers at C-7 and C-2′ positions, which made the establishment for the absolute configuration of C-2′ to be challenged. The absolute configuration of C-2′ was deducted as R as that of the co-isolated compound rhizophol B, which was confirmed by X-ray diffraction (Liu Z. et al., 2019). Therefore, the absolute stereochemistries for 1 and 2 were clarified as 7R,2′R and 7S,2′R.

FIGURE 3

FIGURE 3. Experimental and calculated ECD spectra of 1 and 2.

Cytorhizophin F (3) was also afforded as a yellow powder. The molecular formula of 3 was confirmed as C₂₀H₂₂O₆ by its (+)-HRESIMS m/z 359.1497 [M + H]⁺. The 1D NMR spectroscopic data of the natural product 3 showed a collection of typical resonance signals responsive for a 1,2,3-trisubstituted benzene ring and an isopentyl unit, which showed very close similarity to the structure of 1. After a detail inspection and interpretation of 1D NMR spectra of 3, it could readily disclose that its planar structure should be closely similar to that of 1, and the major difference between them was the absence of carbonyl group in compound 3. This conclusion further strengthened on the basis of the signals for the O-substituted methylene [δ_C 72.3, δ_H 5.10 (dd, J = 2.4, 12.2 Hz), 5.22 (dd, J = 2.4, 12.2 Hz)] in 3 instead of a carbonyl functionality in 1. Moreover, the informative HMBC correlative signals from H-7 to C-4, C-6, C-9, C-13, and C-14 could further strengthen this deduction. Thus, the planar structure of 3 was completely elucidated as depicted in Figure 1.

Cytorhizophin G (4) was obtained to be a yellow powder and had same molecular formula with that of 3 as determined by its HREIMS ion peak at m/z 359.1493 [M + H]⁺, revealing ten degrees of unsaturation. Obviously, the ¹³C NMR spectroscopic data and HSQC spectrum of 4 collectively suggested 20 carbon signals, and all of them showed very similar chemical shifts to those of 3. Their little differences between chemical shifts implied that they shared the same planar structure. For compound 4, the Cotton effects in the ECD spectrum were almost direct contrary to those of 3, suggesting that 4 should share the opposite configuration at C-7 position comparing with 3, attributed to the slight contribution of C-2′ chiral center. The configuration of chiral center for C-7 was further confirmed by ECD calculations in Figure 4. The results showed that the theoretical ECD curve for 7R agreed with the experimental plot of 3, 7S was matched with the experimental plot of 4. Therefore, the absolute configurations of 3 and 4 were successfully established as 7R,2′R, 7S,2′R, correspondingly.

FIGURE 4

FIGURE 4. Experimental and calculated ECD spectra of 3 and 4.

FIGURE 5

FIGURE 5. Key ¹H-¹H COSY, and HMBC correlations of 5 and 6.

Cytorhizophin H (5) was isolated as a pale yellow powder and assigned an HRESIMS ion peak at m/z 373.1285 [M + H]⁺ (C₂₀H₂₁O₇, calcd 373.1282), which perfectly agreed the molecular formula of C₂₀H₂₀O₇ and showed 11 degrees of hydrogen deficiency. The ¹H NMR spectrum of 5 exhibited four aromatic protons [δ_H 7.28 (1H, s, H-12), 7.07 (1H, t, J = 8.1 Hz, H-2), 6.76 (1H, d, J = 8.1 Hz, H-1), 6.38 (1H, d, J = 8.1 Hz, H-3)] as well as the characteristic proton resonance signals of three methyls [δ_H 2.24 (3H, s, H₃-15), 1.44 (3H, s, H₃-4′), 1.38 (3H, s, H₃-5′)].

Analysis of 1D as well as 2D NMR spectra including COSY, HSQC, and HMBC could readily finish the preliminary construction of the planar structure of 5 as shown in Figure 5. Firstly, the obvious HMBC correlations from H-1 to C-3 and C-5, H-2 to C-4 and C-6 along with the pivotal spin system C-1/C-2/C-3 successfully evidenced the presence of a 1,2,3-trisubstituted aromatic ring A. Secondly, as referring to the other spin system of C-1′/C-2′, the existence of a highly oxygenated C-5 isopentyl unit was then verified with the aid of the HMBC correlative signals from H₃-4′ to C-2′ and C-3′, H₃-5′ to C-2′ and C-3′. Moreover, the location of isopentyl functionality had been assigned to attach at C-6 position in the ring A through the C-1′-O-C-6 ether bond, attributable to the decisive HMBC cross-peak from H-1′ to C-6. Additionally, the resulting penta-substituted ring B was finally established and clarified by the HMBC correlations from H-12 to C-8, C-10 and C-14, H₃-15 to C-10, C-11, and C-12. Taking the degrees of unsaturation into account, the assignment of a benzopyran ring between C-4 and C-9 via a fused oxygen bridge was eventually verified. Moreover, the asymmetrical ¹H and ¹³C NMR signals for the typical trisubstituted aromatic ring C further strengthened the conclusion. Therefore, the planar structure of 5 was determined and established to possess a fascinating 6/6/6/5 tetracyclic ring system fusing as unusual furo [4,3,2-kl]xanthen-2 (10bH)-one skeleton and showed in Figure 1.

Cytorhizophin I (6) was separated as a pale yellow powder and assigned an HRESIMS ion peak at m/z 373.1285 [M + H]⁺ (calcd for C₂₀H₂₁O₇, 373.1282), which perfectly agreed with the molecular formula of C₂₀H₂₀O₇ and showed 11 degrees of hydrogen deficiency. The ¹H NMR spectrum of 6 exhibited four aromatic protons [δ_H 6.75 (1H, s, H-10), 7.10 (1H, t, J = 8.1 Hz, H-2), 6.62 (1H, d, J = 8.1 Hz, H-1), and 6.47 (1H, d, J = 8.1 Hz, H-3)], suggesting the existence of two phenyl rings. Interestingly, close comparison of the NMR data of compounds 5 and 6 as shown in Table 3 indicated that these two compounds ought to share a significantly similar core structure. The further HMBC correlations analysis collectively pointed to that the compounds 5 and 6 were a pair of diastereoisomers.

The natural products 5 and 6 possessed two distant stereocenters C-7 and C-2′. In order to establish their absolute configurations of diastereoisomers 5 and 6, the effort towards theoretical ECD calculation at b3lyp/6-311 + g (d,p) level were performed. The results revealed that the theoretical ECD plots of 7S and 7R matched with the experimental spectra of 5 and 6, respectively, which allowed to establish the absolute configurations of 7S for 5 and 7R for 6 (Figure 6). Unsatisfactorily, the absolute configuration for C-2′ was failed to be assigned by the little amount and the lack of CD contribution.

FIGURE 6

FIGURE 6. Experimental and calculated ECD spectra of 5 and 6.

Due to the structural novelty cytorhizophins H-I (5-6) with fascinating 6/6/6/5 tetracyclic furo [4,3,2-kl]xanthen-2 (10bH)-one skeletons, their biogenetic pathways were proposed as shown in Scheme 1. Cytorhizophins H-I (5-6) were bio-originated from the monodictyphenone (7), the following selective oxidation, reduction, and hemiacetalization transformations would result the critical intermediate i, which underwent selective oxidative lactonization and dehydrated to generate the key precursors ii and iii, respectively. Then, the selective prenylation of iii further gave rise to cytorhizophins H-I (5-6).

SCHEME 1

SCHEME 1. Plausible Biosynthetic Pathways of 5 and 6.

The characteristic of polyhydroxy groups in these new compounds logically suggested that they might possess antioxidant activity. The further experimental testing confirmed that compounds 1-6 indeed showed significant antioxidant activity as evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay and described in the Experimental part (Coteele et al., 1996; Mensor et al., 2001). Compounds 1-4 showed remarkable DPPH radical scavenging activities with EC₅₀ values ranging from 5.86 to 26.80 μM, which are better than that of the positive control ascorbic acid (EC₅₀ of 25.53 μM). Compounds 5 and 6 were found to be weak DPPH scavengers at a concentration of 100 μM (Table 4). From a comparison of the structures of compounds 1-4 with compounds 5 and 6, it could be readily found that the opening of the middle ring might play the predominant roles in enhancing their DPPH scavenging capacity.

TABLE 4

TABLE 4. Antioxidant activities of compounds 1-6.

Conclusion

The chemical research on the endophytic fungus Cytospora rhizophorae has disclosed a new range of antioxidative ingredients, involving six novel phthalan derivatives named as cytorhizophins D-I (1-6). Among them, cytorhizophins D-E (1-2) and F-G (3-4) were two pairs of diastereoisomers, and all of them featuring a 1-phenyl-1,3-dihydroisobenzofuran scaffold with a highly oxygenated O-linked isopentenyl unit; whereas cytorhizophins H-I (5-6) represent the first examples of phthalide family with a fascinating 6/6/6/5 tetracyclic ring system fusing as unprecedented furo [4,3,2-kl]xanthen-2 (10bH)-one skeleton. Compounds 1-4 showed significant DPPH radical scavenging activities with EC₅₀ values ranging from 5.86 to 26.80 μM, which are much better than that of the positive control ascorbic acid (EC₅₀ of 25.53 μM). Therefore, the preliminary results revealed that cytorhizophins D-G might be served as promising lead compounds for the development of bio-available potent anti-oxidant drugs. The detailed potential mechanisms to explain the antioxidant action of these compounds is now underway and will be reported in due course.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, KU529867.

Author Contributions

HL, YZ, and HW conducted and collected the experiment data. HL performed the experiments of compound isolation. ZL carried out the ECD calculations. HL and HT finished the structure identification of the isolated compounds. YC evaluated activities of all the isolates. HL and HT interpreted the data, and wrote the paper. HL, HT, and WZ revised the manuscript. HT and WZ conceived and designed the experiments. All authors read and approved the final manuscript.

Funding

Financial support for this research was provided by the Guangdong Special Support Program (2019TQ05Y375), Guangdong Provincial Project for Science and Technology (No. 2021A1515011549), GDAS’ Project of Science and Technology Development (2020GDASYL-20200104012), and Youth Innovation Promotion Association of CAS (2020342). We sincerely thank. Can Li of central laboratory of Southern Medical University for NMR measurements.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.826615/full#supplementary-material

References

Aitken, R., De Iuliis, G., Gibb, Z., and Baker, M. (2012). The Simmet Lecture: New Horizons on an Old Landscape - Oxidative Stress, DNA Damage and Apoptosis in the Male Germ Line. Reprod. Domest. Anim. 47, 7–14. doi:10.1111/j.1439-0531.2012.02049.x

CrossRef Full Text | Google Scholar

Bodoira, R., and Maestri, D. (2020). Phenolic Compounds from Nuts: Extraction, Chemical Profiles, and Bioactivity. J. Agric. Food Chem. 68, 927–942. doi:10.1021/acs.jafc.9b07160

PubMed Abstract | CrossRef Full Text | Google Scholar

Breimer, L. H. (1990). Molecular Mechanisms of Oxygen Radical Carcinogenesis and Mutagenesis: the Role of DNA Base Damage. Mol. Carcinog. 3, 188–197. doi:10.1002/mc.2940030405

PubMed Abstract | CrossRef Full Text | Google Scholar

Cerutti, P. A. (1985). Prooxidant States and Tumor Promotion. Science 227, 375–381. doi:10.1126/science.2981433

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, S., Li, H., Chen, Y., Li, S., Xu, J., Guo, H., et al. (2019). Three New Diterpenes and Two New Sesquiterpenoids from the Endophytic Fungus Trichoderma Koningiopsis A729. Bioorg. Chem. 86, 368–374. doi:10.1016/j.bioorg.2019.02.005

PubMed Abstract | CrossRef Full Text | Google Scholar

Cotelle, N., Bernier, J. L., Catteau, J. P., Pommery, J., Wallet, J. C., and Gaydou, E. M. (1996). Antioxidant Properties of Hydroxy-Flavones. Free Radic. Biol. Med. 20, 35–43. doi:10.1016/0891-5849(95)02014-4

PubMed Abstract | CrossRef Full Text | Google Scholar

Dao, D. Q., Phan, T. T. T., Nguyen, T. L. A., Trinh, P. T. H., Tran, T. T. V., Lee, J. S., et al. (2020). Insight into Antioxidant and Photoprotective Properties of Natural Compounds from marine Fungus. J. Chem. Inf. Model. 60, 1329–1351. doi:10.1021/acs.jcim.9b00964

PubMed Abstract | CrossRef Full Text | Google Scholar

Ding, M., Li, J.-J., Leonard, S. S., Ye, J.-P., Shi, X., Colburn, N. H., et al. (1999). Vanadate-induced Activation of Activator Protein-1: Role of Reactive Oxygen Species. Carcinogenesis 20, 663–668. doi:10.1093/carcin/20.4.663

PubMed Abstract | CrossRef Full Text | Google Scholar

El-Hawary, S. S. E.-D., El Zalabani, S. M., Selim, N. M., Ibrahim, M. A., Wahba, F. A., El Badawy, S. A., et al. (2019). Phenolic Constituents of Chrysophyllum Oliviforme L. Leaf Down-Regulate TGF-β Expression and Ameliorate CCl4-Induced Liver Fibrosis: Evidence from In Vivo and In Silico Studies. Antioxidants 8, 646. doi:10.3390/antiox8120646

PubMed Abstract | CrossRef Full Text | Google Scholar

Fotso, S., Mahmud, T., Zabriskie, T. M., Santosa, D. A., SulastriProteau, P. J., and Protea, P. J. (2008). Rearranged and Unrearranged Angucyclinones from Indonesian Streptomyces Spp. J. Antibiot. 61, 449–456. doi:10.1038/ja.2008.61

CrossRef Full Text | Google Scholar

Grisham, M. B., Jourd'Heuil, D., and Wink, D. A. (2000). Review Article: Chronic Inflammation and Reactive Oxygen and Nitrogen Metabolism - Implications in DNA Damage and Mutagenesis. Aliment. Pharmacol. Ther. Suppl. 14, 3–9. doi:10.1046/j.1365-2036.2000.014s1003.x

CrossRef Full Text | Google Scholar

Halliwell, B. (1987). Oxidants and Human Disease: Some New Concepts 1. FASEB j. 1, 358–364. doi:10.1096/fasebj.1.5.2824268

PubMed Abstract | CrossRef Full Text | Google Scholar

Harper, J. K., Arif, A. M., Ford, E. J., Strobel, G. A., Porco, J. A., Tomer, D. P., et al. (2003). Pestacin: a 1,3-dihydro Isobenzofuran from Pestalotiopsis Microspora Possessing Antioxidant and Antimycotic Activities. Tetrahedron 59, 2471–2476. doi:10.1016/s0040-4020(03)00255-2

CrossRef Full Text | Google Scholar

Kang, H.-S., Jun, E.-M., Park, S.-H., Heo, S.-J., Lee, T.-S., Yoo, I.-D., et al. (2007). Cyathusals A, B, and C, Antioxidants from the Fermented Mushroom Cyathus Stercoreus. Cyathus Stercoreusj. Nat. Prod. 70, 1043–1045. doi:10.1021/np060637h

PubMed Abstract | CrossRef Full Text | Google Scholar

Kapoor, M., Dhawan, S. N., Mor, S., Bhatia, S. C., Gupta, S. C., and Hundal, M. S. (2003). Stereoselective Synthesis of Z-3-Alkoxy-2-[(4′-Methoxyphenyl)methylidene]-1(3h)-Isobenzofuranones. Tetrahedron 59, 5027–5031. doi:10.1016/s0040-4020(03)00757-9

CrossRef Full Text | Google Scholar

Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ischiropoulos, H., and Beckman, J. S. (1992). Peroxynitrite, a Cloaked Oxidant Formed by Nitric Oxide and Superoxide. Chem. Res. Toxicol. 5, 834–842. doi:10.1021/tx00030a017

PubMed Abstract | CrossRef Full Text | Google Scholar

Kusio, J., Sitkowska, K., Konopko, A., and Litwinienko, G. (2020). Hydroxycinnamyl Derived BODIPY as a Lipophilic Fluorescence Probe for Peroxyl Radicals. Antioxidants 9, 88. doi:10.3390/antiox9010088

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, H.-X., Tan, H.-B., Chen, K., Zhao, L.-Y., Chen, Y.-C., Li, S.-N., et al. (2019b). Cytosporins A-D, Novel Benzophenone Derivatives from the Endophytic Fungus Cytospora Rhizophorae A761. Org. Biomol. Chem. 17, 2346–2350. doi:10.1039/c8ob03223h

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, H.-X., Tan, H.-B., Liu, Y., Chen, Y.-C., Li, S.-N., Sun, Z.-H., et al. (2017). Three New Highly-Oxygenated Metabolites from the Endophytic Fungus Cytospora Rhizophorae A761. Fitoterapia 117, 1–5. doi:10.1016/j.fitote.2016.12.005

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, H., Tan, H., Chen, Y., Guo, X., Wang, W., Guo, H., et al. (2019a). Cytorhizins A-D, Four Highly Structure-Combined Benzophenones from the Endophytic Fungus Cytospora Rhizophorae. Org. Lett. 21, 1063–1067. doi:10.1021/acs.orglett.8b04107

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, H., Tan, H., Wang, W., Zhang, W., Chen, Y., Li, S., et al. (2019c). Cytorhizophins A and B, Benzophenone-Hemiterpene Adducts from the Endophytic Fungus Cytospora Rhizophorae. Org. Chem. Front. 6, 591–596. doi:10.1039/c8qo01306c

CrossRef Full Text | Google Scholar

Liu, Z., Tan, H., Chen, K., Chen, Y., Zhang, W., Chen, S., et al. (2019d). Rhizophols A and B, Antioxidant and Axially Chiral Benzophenones from the Endophytic Fungus Cytospora Rhizophorae. Org. Biomol. Chem. 17, 10009–10012. doi:10.1039/c9ob02282a

PubMed Abstract | CrossRef Full Text | Google Scholar

López-Alarcón, C., and Denicola, A. (2013). Evaluating the Antioxidant Capacity of Natural Products: A Review on Chemical and Cellular-Based Assays. Anal. Chim. Acta 763, 1–10. doi:10.1016/j.aca.2012.11.051

PubMed Abstract | CrossRef Full Text | Google Scholar

Mensor, L. L., Menezes, F. S., Leitão, G. G., Reis, A. S., Santos, T. C. d., Coube, C. S., et al. (2001). Screening of Brazilian Plant Extracts for Antioxidant Activity by the Use of DPPH Free Radical Method. Phytother. Res. 15, 127–130. doi:10.1002/ptr.687

PubMed Abstract | CrossRef Full Text | Google Scholar

Mussard, E., Cesaro, A., Lespessailles, E., Legrain, B., Berteina-Raboin, S., and Toumi, H. (2019). Andrographolide, a Natural Antioxidant: An Update. Antioxidants 8, 571. doi:10.3390/antiox8120571

PubMed Abstract | CrossRef Full Text | Google Scholar

Naito, S., and Kaneko, Y. (1969). Two New Phenolic Reductones from. Tetrahedron Lett. 10, 4675–4678. doi:10.1016/s0040-4039(01)88780-3

CrossRef Full Text | Google Scholar

Quiñones, M., Miguel, M., and Aleixandre, A. (2012). The Ployphenols, Naturally Occurring Compounds with Beneficial Effects on Cardiovascular Disease. Nutr. Hosp. 27, 76–89.

PubMed Abstract | Google Scholar

Russell, E. G., and Cotter, T. G. (2015). “New Insight into the Role of Reactive Oxygen Species (ROS) in Cellular Signal-Transduction Processes,” in International Review of Cell and Molecular Biology (Cambridge, MA, USA: Academic Press), Vol. 319, 221–254. doi:10.1016/bs.ircmb.2015.07.004

PubMed Abstract | CrossRef Full Text | Google Scholar

Strobel, G., Ford, E., Worapong, J., Harper, J. K., Arif, A. M., Grant, D. M., et al. (2002). Isopestacin, an Isobenzofuranone from Pestalotiopsis Microspora, Possessing Antifungal and Antioxidant Activities. Phytochemistry 60, 179–183. doi:10.1016/s0031-9422(02)00062-6

PubMed Abstract | CrossRef Full Text | Google Scholar

Taniguchi, N., Pickett, C. B., and Griffith, O. W. (1993). Oxy Radicals and Antioxidative Responses in Cancer: 12th Sapporo Cancer Seminar. Cancer Res. 53, 3207–3210.

PubMed Abstract | Google Scholar

Wen, L., Zhao, Y., Jiang, Y., Yu, L., Zeng, X., Yang, J., et al. (2017). Identification of a Flavonoid C -glycoside as Potent Antioxidant. Free Radic. Biol. Med. 110, 92–101. doi:10.1016/j.freeradbiomed.2017.05.027

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhong, W., Chen, Y., Mai, Z., WeiWang, X. J., Wang, J., Zeng, Q., et al. (2020). Euroticins A and B, Two Pairs of Highly Constructed Salicylaldehyde Derivative Enantiomers from a marine-derived Fungus Eurotium Sp. SCSIO F452. J. Org. Chem. 85, 12754–12759. doi:10.1021/acs.joc.0c01407

PubMed Abstract | CrossRef Full Text | Google Scholar