Bioactive Secondary Metabolites from the Culture of the Mangrove-Derived Fungus Daldinia eschscholtzii HJ004

Two new polyketides, 8-O-methylnodulisporin F (1) and nodulisporin H (2), two new naphthoquinones, 5-hydroxy-2-methoxy-6,7-dimethyl-1,4-naphthoquinone (3) and 5-hydroxy-2-methoxynaphtho[9–c]furan-1,4-dione (4), and a new naphthofuran 1,3,8-trimethoxynaphtho[9–c]furan (5), along with five known compounds 4-O-methyl eleutherol (6), 2-acetyl-7-methoxybenzofuran (7), (-)-orthosporin (8), diaporthin (9), and 6-hydroxymellein (10), were obtained from the EtOAc extract of the mangrove-derived fungus Daldinia eschscholtzii HJ004. The structures of the isolated compounds were elucidated by extensive NMR and MS analyses, while the absolute configurations of the stereogenic carbons were established based on experimental and calculated electronic circular dichroism spectra. Compounds 4 and 7 displayed a potent inhibitory activity against α-glucosidase with the IC50 values of 5.7 and 1.1 μg/mL, respectively. Compounds 1 and 2 showed a moderate antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) and Bacillus cereus, with minimum inhibitory concentration (MIC) values ranging from 6.25 to 12.5 μg/mL. Compound 3 exhibited antibacterial activity against B. cereus with the MIC value of 12.5 μg/mL.


Introduction
Diabetes poses a serious threat to cardiovascular diseases, and is the eleventh common cause of disability worldwide. According to the 8th edition of the International Diabetes Federation Diabetes Atlas 2017, more than 425 million people worldwide are suffering from diabetes, and China alone accounts for one-third of that [1,2]. Around 90% of all cases of diabetes were Type 2 diabetes mellitus [3][4][5]. α-Glucosidase, a crucial enzyme that breaks down complex carbohydrates for absorption, plays a key role in the treatment of diabetes. α-Glucosidase inhibitors such as natural products acarbose and voglibose can reduce the impact of carbohydrates on blood glucose level and prevent the Mar. Drugs 2019, 17, 710; doi:10.3390/md17120710 www.mdpi.com/journal/marinedrugs digestion of carbohydrates, indicating that natural compounds play an important role in a discovery of anti-diabetic drugs [6,7]. Mangrove-derived fungi can produce secondary metabolites with novel structures and biological activities [8].

Results and Discussion
Compound 1 was isolated as a white amorphous powder. Its molecular formula C21H24O5 (ten degrees of unsaturation) was determined by the one carbonyl (δC 208.1) carbons. The above NMR data suggested that the structure of 1 was very similar to that of nodulisporin F [20] except for the presence of the methoxyl group (δH 3.63/δC 55.7). That the methoxyl group was located at C-8′ was confirmed by the HMBC correlation from 8′-OMe

Results and Discussion
Compound 1 was isolated as a white amorphous powder. Its molecular formula C 21  one carbonyl (δ C 208.1) carbons. The above NMR data suggested that the structure of 1 was very similar to that of nodulisporin F [20] except for the presence of the methoxyl group (δ H 3.63/δ C 55.7).
That the methoxyl group was located at C-8 was confirmed by the HMBC correlation from 8 -OMe (δ H 3.63) to C-8 (δ C 158.1) and the NOESY correlation from H-7 (δ H 6.42) to 8 -OMe ( Figures S6 and S7). The 2D NMR data allowed to elucidate the complete planar structure of 1 ( Figure 2 and Figures S4-S6).
(δH 3.63) to C-8′ (δC 158.1) and the NOESY correlation from H-7′ (δH 6.42) to 8′-OMe ( Figures S6 and  S7). The 2D NMR data allowed to elucidate the complete planar structure of 1 ( Figure 2 and Figures  The relative configurations of the C-1′ and C-3′ estereogenic centers were deduced by the coupling constants (J1′,2′a = 5.2 Hz and J3′,2′a = 11.6 Hz) in the 1 H NMR spectrum. The small coupling constant of 5.2 Hz between H-1′ and H-2′a established that H-1′ was in equatorial orientation. By contrast, the coupling constant for J3′,2′a = 11.6 Hz indicated the axial orientation of H-3′, and the equatorial position of the Me-3′. These results implied that H-1′ and H-3′ were on the opposite sides. Besides, the NOESY spectrum of 1 showed correlations between H-1′ and H-2′a, as well as between H-3′ and H-2′b ( Figure S7). Furthermore, the chemical shifts and coupling constants of H-1′/ H-2′/ H-3′/ H-4′ were close to those of nodulisporin F [20], which suggested a similar configuration. Thus, the relative configurations of 1 were confirmed as 1′R*,3′S*, and 1 was named 8-O-methylnodulisporin F.
Compound 2 was also isolated as a white amorphous powder with the same molecular formula  (Table 1 and Figure S9). The large coupling constants of J1′,2′a= 10.4 Hz and J3′,2′a = 11.2 Hz confirmed that H-1′ and H-3′ were both in the axial orientation. Additionally, the NOESY spectrum of 2 showed correlations between H-1′ and H-2′b, as well as between H-3′ and H-2′b ( Figure S15). These results indicated the relative configurations of 2 as 1′R*,3′R*, and 2 was named as nodulisporin H. Therefore, 1 and 2 are C-3′ epimers.
The absolute configurations of C-1′ and C-3′ in 1 and 2 were determined by comparison of experimental and calculated ECD spectra. The calculated ECD curve of 1′R,3′S matched well the experimental ECD curve of 1, while the calculated ECD spectrum of the 1′R,3′R matched well the experimental ECD spectrum of 2 ( Figure 3).  The relative configurations of the C-1 and C-3 estereogenic centers were deduced by the coupling constants (J 1 ,2 a = 5.2 Hz and J 3 ,2 a = 11.6 Hz) in the 1 H NMR spectrum. The small coupling constant of 5.2 Hz between H-1 and H-2 a established that H-1 was in equatorial orientation. By contrast, the coupling constant for J 3 ,2 a = 11.6 Hz indicated the axial orientation of H-3 , and the equatorial position of the Me-3 . These results implied that H-1 and H-3 were on the opposite sides. Besides, the NOESY spectrum of 1 showed correlations between H-1 and H-2 a, as well as between H-3 and H-2 b ( Figure S7). Furthermore, the chemical shifts and coupling constants of H-1 / H-2 / H-3 / H-4 were close to those of nodulisporin F [20], which suggested a similar configuration. Thus, the relative configurations of 1 were confirmed as 1 R*,3 S*, and 1 was named 8-O-methylnodulisporin F.  (Table 1 and Figure S9). The large coupling constants of J 1 ,2 a = 10.4 Hz and J 3 ,2 a = 11.2 Hz confirmed that H-1 and H-3 were both in the axial orientation. Additionally, the NOESY spectrum of 2 showed correlations between H-1 and H-2 b, as well as between H-3 and H-2 b ( Figure S15). These results indicated the relative configurations of 2 as 1 R*,3 R*, and 2 was named as nodulisporin H. Therefore, 1 and 2 are C-3 epimers.
Compounds 1 and 2 showed moderate antibacterial activity against Staphylococcus aureus, Methicillin-resistant S. aureus MRSA and Bacillus cereus, with minimum inhibitory concentration (MIC) values ranging from 6.25 to 12.5 µg/mL (Table 3). Compound 3 exhibited antibacterial activity against B. cereus with the MIC value of 12.5 µg/mL ( Table 3). The other compounds showed no antibacterial activity against six pathogenic bacteria.

General Experimental Procedures
Both 1D and 2D NMR spectra were measured on a Bruker AV-400 (Bruker Corporation, Fällanden, Switzerland) instrument by using CDCl 3 as a solvent with TMS as the internal standard. The other experimental procedures were performed as previously described in the literature [9].

Fungal Material
The strain HJ004 was isolated from the stem of mangrove Brguiera sexangula var. rhynchopetala, collected in the South China Sea, and was identified as Daldinia eschscholtzii with the GeneBank (NCBI) accession number MH059553 [9]. This strain was deposited at the Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University, Haikou, China.

Extraction and Isolation
The fermentation was carried out statically in 45 L of potato glucose liquid medium at 25 • C for one month. The fermented broths were filtered through cheesecloth, and the filtrate was extracted with EtOAc (3 × 45 L, 48 h each). The EtOAc extracts were combined and concentrated under reduced pressure to yield a residue of 64.0 g, which was fractionated by vacuum liquid chromatography (VLC) with a petroleum ether-EtOAc-MeOH gradient, to yield five fractions (Frs. 1−5). Fr. 2 was subjected to a Sephadex LH-20 (300 g) column chromatography (CC) eluting with petroleum ether-CHCl 3 -MeOH (1:1:1, v/v/v), and then further purified by using semi-preparative HPLC (85% MeOH-H 2 O) to give 1 (2.0 mg) and 2 (

Biological Assay
The α-glucosidase inhibitory activity of 1-10 was determined using the procedure reported by Sawada et al. [22], with modifications for carrying out in 96-well plates, and acarbose was used as a positive control.