Two New Metabolites from the Endophytic Fungus Alternaria sp. A744 Derived from Morinda officinalis

Two new compounds isobenzofuranone A (1) and indandione B (2), together with eleven known compounds (3–13) were isolated from liquid cultures of an endophytic fungus Alternaria sp., which was obtained from the medicinal plant Morinda officinalis. Among them, the indandione (2) showed a rarely occurring indanone skeleton in natural products. Their structures were elucidated mainly on the basis of extensive spectroscopic data analysis. All of the compounds were evaluated with cytotoxic and α-glucosidase inhibitory activity assays. Compounds 11 and 12 showed significant inhibitory activities against four tumor cell lines; MCF-7, HepG-2, NCI-H460 and SF-268, with IC50 values in the range of 1.91–9.67 μM, and compounds 4, 5, 9, 10, 12 and 13 showed excellent inhibitory activities against α-glucosidase with IC50 values in the range of 12.05–166.13 μM.


Introduction
Endophytic fungi have been considered a rich source of structurally novel and bioactive diverse metabolites that have become interesting and significant resources for drug discovery [1][2][3]. Morinda officinalis, known as one of the 'top four south authentic traditional Chinese medicines', has obvious regional characteristics. Its roots contain plant sterols, anthraquinones, flavonoids, vitamin C, sugar, resin and other ingredients, and they are widely used to treat impotence, spermatorrhea, rheumatism and female infertility [4]. However, there are few systematic reports on endophytic fungus resources from this plant and their active components. During our ongoing search aimed at structurally unique and bioactive substances from endophytic fungi [5][6][7][8], we conducted a chemical analysis on a fraction of the broth extract of the fungus Alternaria sp. A744 derived from M. officinalis, which led to the isolation of two new secondary metabolites along with eleven known compounds ( Figure 1). All the compounds were evaluated for their cytotoxic and α-glucosidase inhibitory activities via assays. Herein, the details of the isolation, structural elucidation and bioassay are described.

Structural Elucidation of New Compounds
Isobenzofuranone A (1), colorless oil, had a molecular formula of C11H10O5 on the basis of negative high-resolution electrospray ionization mass spectra (HR-ESI-MS) ([M − H] − m/z 221.0460, calcd. for 221.0450), corresponding to seven degrees of unsaturation (see Figures S1-S9). The infrared spectroscopy (IR) spectrum exhibited absorption bands at 3435 (hydroxyl group) and 1732 (carbonyl group) cm −1 . The 1 H-NMR spectroscopic data of 1 (Table 1)  . The COSY correlations between H-4 (δH 7.00) and H-5 (δH 7.55), H-5 and H-6 (δH 6.9) confirmed the presence of 1,2,3-trisubstituted benzene moiety. The benzene ring along with two carbonyl groups accounted for six unsaturation degrees, while the remaining degrees of unsaturation indicated that an additional ring must be present in the molecule. Meanwhile, a signal of 13 C-NMR resonance at δC 171.5 and an absorption in the IR spectrum at 1732 cm −1 suggested the presence of a lactone carbonyl group. These data showed a great resemblance to the known compound isoochracinic acid [9], the only difference between them is that a hydroxyl group at position C-10 in the known compound was replaced by a methoxy group in 1. Furthermore, the heteronuclear multiple bond correlation (HMBC) correlations ( Figure 2) from methine proton H-3 (δH 5.82) to C-1 and C-7a, H2-8 to C-1 and C-3a, as well as H-6 to C-7a and C-1 secured the connection of C-3 and C-1 to the aromatic ring. Simultaneously, the relative downfield chemical shift of C-3 (δC 78.9) revealed a connection with oxygen, thereby forming a lactone ring. The HMBC cross-peaks from H-3 and the proton of methoxy H3-10 (δH 3.72) to C-9 determined the presence of a methyl acetate unit in 1. The critical COSY signals ( Figure 2) from H-3 to H2-8 suggested that the methylene group was directly connected to the lactone ring at C-3. Thus, the planar structure of 1 was determined, as shown in Figure 1.

Structural Elucidation of New Compounds
Isobenzofuranone A (1), colorless oil, had a molecular formula of C 11 H 10 O 5 on the basis of negative high-resolution electrospray ionization mass spectra (HR-ESI-MS) ([M − H] − m/z 221.0460, calcd. for 221.0450), corresponding to seven degrees of unsaturation (see Figures S1-S9). The infrared spectroscopy (IR) spectrum exhibited absorption bands at 3435 (hydroxyl group) and 1732 (carbonyl group) cm −1 . The 1 H-NMR spectroscopic data of 1 (Table 1)  The benzene ring along with two carbonyl groups accounted for six unsaturation degrees, while the remaining degrees of unsaturation indicated that an additional ring must be present in the molecule. Meanwhile, a signal of 13 C-NMR resonance at δ C 171.5 and an absorption in the IR spectrum at 1732 cm −1 suggested the presence of a lactone carbonyl group. These data showed a great resemblance to the known compound isoochracinic acid [9], the only difference between them is that a hydroxyl group at position C-10 in the known compound was replaced by a methoxy group in 1. Furthermore, the heteronuclear multiple bond correlation (HMBC) correlations ( Figure 2) from methine proton H-3 (δ H 5.82) to C-1 and C-7a, H 2 -8 to C-1 and C-3a, as well as H-6 to C-7a and C-1 secured the connection of C-3 and C-1 to the aromatic ring. Simultaneously, the relative downfield chemical shift of C-3 (δ C 78.9) revealed a connection with oxygen, thereby forming a lactone ring. The HMBC cross-peaks from H-3 and the proton of methoxy H 3 -10 (δ H 3.72) to C-9 determined the presence of a methyl acetate unit in 1. The critical COSY signals ( Figure 2) from H-3 to H 2 -8 suggested that the methylene group was directly connected to the lactone ring at C-3. Thus, the planar structure of 1 was determined, as shown in Figure 1.   . The 13 C-NMR spectrum and the HMQC revealed 12 carbon resonances attributed to one methyl, one methylene, three methines, and seven quaternary carbons. The abovementioned information was quite similar to that of the known compound indanostatin, which was isolated from Streptomyces sp. [10]. They all have a typical indandione five-membered ring structure, except for the absence of a hydroxyl group at position C-4 and a methyl group at position C-5 on the benzene ring in 2. The HMBC correlations from H-4 to C-3, C-6 and C-7a, H-5 to C-3a and C-7, H-6 to C-3a, C-7a, and C-1, along with the HMBC cross-peaks between H-8 to C-1, C-2, and C-3, secured the presence of an indandione five-membered ring. Moreover, the HMBC correlations ( Figure 2) from H2-8 and H3-10 to C-9 implied the presence of a 2-oxopropyl unit in 2. Finally, the HMBC cross-peaks of methylene protons with C-1, C-2 and C-3 suggested that the 2-oxopropyl group was connected with the indandione five-membered ring at C-2. Therefore, the planar structure of 2 was assigned, as shown in Figure 1.

In Vitro Cytotoxicity Assay
The in vitro cytotoxic activity of compounds 1-13 was investigated against four tumor cell lines, including MCF-7, HepG-2, NCI-H460 and SF-268, by the SRB (Sulforhodamine B) method with cisplatin as the positive control. As outlined in Table 2, compounds 11 and 12 showed significant inhibitory activities against the four tumor cell lines with IC 50 values in the range of 1.91-9.67 µM.

Fungal Material
The endophytic fungal strain A744 was isolated from the twigs of Morinda officinalis, which was collected from Gaoyao city, Guangdong province of China, in January 2015. The strain A744 was identified by sequence analysis of rDNA ITS (internal transcribed spacer) region. The sequence of the ITS region of the strain has been submitted to GenBank (Accession No. KF706672). By using BLAST (nucleotide sequence comparison program) to search the GenBank database, A744 has 100% similarity to Alternaria sp. MY-2011 (Accession No. JN038490). The strain was preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology.

α-Glucosidase Inhibitory Activity Assay
An assay of α-glucosidase inhibitory activity was evaluated according the method previously published in Reference [22].

Conclusions
In this study, thirteen compounds, including two new ones, were isolated from Alternaria sp., an endophytic fungus from Morinda officinalis. All the structures were established by extensive spectroscopic analysis. The isolates were evaluated their cytotoxicities against four human tumor cell lines and α-glucosidase inhibitory activity assays. Compounds 11 and 12 exhibited significant inhibitory activities with IC 50 values in the range of 1.91-9.67 µM. Compounds 4, 5, 9, 10, 12 and 13 showed excellent inhibitory activity against α-glucosidase, which might be useful for further developing α-glucosidase inhibitor.
Supplementary Materials: Supplementary material relating to this article can be accessed online.