Secondary Metabolites of The Endophytic Fungus Alternaria alternata JS0515 Isolated from Vitex rotundifolia and Their Effects on Pyruvate Dehydrogenase Activity

The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2–8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 μM, respectively.


Introduction
Endophytes are microorganisms that live within the internal tissues of plants, and they form symbiotic relationships with their host plants [1]. The functional diversity of endophytic fungi is notable among plant-associated microbes [2,3]. Endophytic fungi have been identified as sources of various bioactive metabolites with interesting structures, which are potential candidates for drug development [4][5][6]. Endophytic fungi have been reported to protect their host plants by producing diverse biologically active secondary metabolites with antiviral, antifungal, and antibacterial properties [7].
Halophytes are plants that have adapted to growing in highly saline water, and they comprise only 2% of all plant species [8]. The relationships between halophytes and their endophytes could help the plants adapt to highly saline conditions [9]. Vitex rotundifolia Linne fil. (Verbenaceae) is a halophyte that is widely distributed along the coast of East Asia [10]. Various chemicals have been isolated from V. rotundifolia, including lignans, diterpenes, lactones, glycerols, flavonoids, and iridoids [11]. Its fruit has been used as a folk remedy to treat asthma, chronic bronchitis, colds, ocular pain, female hormonal imbalance, headaches, migraines, and gastrointestinal infections [12,13]. Previous studies have identified a range of bioactivities of V. rotundifolia, including antioxidative, anticancer, and antiproliferative activity [14,15].
The endophytic fungi Cochliobolus geniculatus, Curvularia sp., Nemania primolutea, Paecilomyces sp., Phoma sp., and Nemania primolutea have been isolated from the leaves of V. rotundifolia grown in the coastal regions of the Malaysian Peninsula. In particular, C. geniculatus, Curvularia sp., Paecilomyces sp., and Phoma sp. exhibit antibacterial activity [16]. An endophytic fungus isolated from rhizomes of V. rotundifolia grown in the coastal region of Korea has a growth-promoting effect in Waito-C rice [17].
The pyruvate dehydrogenase complex (PDC) is a multienzyme complex and a crucial metabolic gatekeeper: it is the convergence point between glycolysis and the tricarboxylic acid (TCA) cycle for ATP generation. Its pyruvate dehydrogenase (PDH) E1α subunit catalyzes the oxidative decarboxylation of pyruvate into acetyl-CoA in the mitochondria [23][24][25][26][27]. PDH E1α activity is inhibited by the phosphorylation of its serine residues. Suppression of PDH activity is associated with various metabolic disorders, including obesity, non-alcoholic fatty liver disease, diabetes, and cancer [28][29][30][31][32][33]. In this study, twelve secondary metabolites were isolated from the ethyl acetate extracts of A. alternata (JS0515). The twelve secondary metabolites were then evaluated as PDH activators in a cellular PDH activity assay using AD-293 cells. We are thankful to the reviewers for reminding us of this crucial information. The human AD-293 cell line is a derivative of the commonly used HEK293 cell line. HEK293 cell line is often used in the inhibition of pyruvate dehydrogenase.

Bioassays
The pyruvate dehydrogenase E1α subunit is a characteristic marker of PDH kinase activity. The phosphorylation of PDH E1α serine (Ser300) residues in AD-293 cells cultured with either 3 or 11 was quantified via immunofluorescence measurement. Cell viability was evaluated using a colorimetric MTT assay. The results showed that these tested compounds have no cytotoxicity at the concentrations tested. IC50 concentrations were determined by normalization against 5-mM dichloroacetic acid (DCA), which is a PDH kinase inhibitor. The amount of phosphorylated p-PDH E1α (Ser300) was reduced after treatment with 3 ( Figure 3). Compound 3 precipitated during cell treatment, but no cytotoxicity was indicated. Phosphorylation of PDH E1α was reduced by 11 as well, and no morphological side effects were observed (Figure 3).

Bioassays
The pyruvate dehydrogenase E1α subunit is a characteristic marker of PDH kinase activity. The phosphorylation of PDH E1α serine (Ser300) residues in AD-293 cells cultured with either 3 or 11 was quantified via immunofluorescence measurement. Cell viability was evaluated using a colorimetric MTT assay. The results showed that these tested compounds have no cytotoxicity at the concentrations tested. IC 50 concentrations were determined by normalization against 5-mM dichloroacetic acid (DCA), which is a PDH kinase inhibitor. The amount of phosphorylated p-PDH E1α (Ser300) was reduced after treatment with 3 ( Figure 3). Compound 3 precipitated during cell treatment, but no cytotoxicity was indicated. Phosphorylation of PDH E1α was reduced by 11 as well, and no morphological side effects were observed (Figure 3). The phosphorylation of PDHE 1α was measured by immunofluorescence and IC50 was calculated by normalizing against a pharmacological inhibitor control (DCA 5 mM). Compounds 3 and 11 of 100 μg/μL inhibited the PDH phosphorylation with IC50 values of 32.58, and 27.82 μM, respectively ( Figure S8). Under the same conditions, DCA inhibited PDH phosphorylation at an IC50 concentration of 1 mM ( Figure S8). Based on these results, compounds 3 and 11 increased PDH activity. Alternariol (3) is a protein kinase and xanthine oxidase inhibitor. Compound 3 exhibits cytotoxicity in L5178Y mouse lymphoma cells, [43,44] and 2 and 3 can kill human colon carcinoma cells [45]. Compound 3 and alternariol 5-O-methyl ether (4) induce cytochrome P450 1A1 activity in murine hepatoma cells and cause apoptosis [46]. The compounds 3, 4, altenuene (6), and 2epialtenuene (7) also exhibit cytotoxic activity [43]. Compounds 3, 4, 6, 7, and altertoxin I (11) are known to be toxic to brine shrimp [47][48][49].

Conclusion
In this study, twelve secondary metabolites including a new compound (alternatiol; 1), were isolated from the fungal strain A. alternata JS0515. To our knowledge, this is the first chemical investigation of A. alternata JS0515. In addition, compounds 3 and 11 increased PDH activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. Overall, the results suggest that altenusin and isochromanone derivatives from JS0515 can possibly be used to treat some various metabolic disorders. In a future study, we will go into compound 3 and 11 induces the apoptosis of cancer cells by through inhibition of PDH phosphorylation.

Conclusions
In this study, twelve secondary metabolites including a new compound (alternatiol; 1), were isolated from the fungal strain A. alternata JS0515. To our knowledge, this is the first chemical investigation of A. alternata JS0515. In addition, compounds 3 and 11 increased PDH activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. Overall, the results suggest that altenusin and isochromanone derivatives from JS0515 can possibly be used to treat some various metabolic disorders. In a future study, we will go into compound 3 and 11 induces the apoptosis of cancer cells by through inhibition of PDH phosphorylation.

Isolation of The Fungal Strain
The fungal strain (JS515) was isolated from the beach vitex (V. rotundifolia), which was collected from a swamp in Suncheon, South Korea (34 • 83'79" N, 127 • 44'95" E) in September, 2011. Rhizome tissues were cut into small pieces (0.5 × 0.5 cm) and sterilized with 2% sodium hypochlorite for 1 min and 70% ethanol for 1 min, and then washed with sterilized distilled water. Fungal strains were cultured from plant tissues after about seven days of incubation on malt extract agar (MEA, Difco) supplemented with 50 ppm kanamycin, 50 ppm chloramphenicol, and 50 ppm Rose Bengal at 22 • C. The growing colony cut edges off and then pieces were transferred to fresh potato dextrose agar (PDA, Difco) for pure culture before being stored as 20% glycerol stocks in a liquid nitrogen tank at the Wildlife Genetic Resources Bank at the National Institute of Biological Resources (Incheon, Korea) before use.

Cultivation and Extraction of The Fungal Strain
The JS0515 strain was cultivated according to two methods. The first method was cultivated on solid rice medium (80 g rice per 120 mL distilled water in a 500 mL Erlenmeyer flask was autoclaved) at room temperature. After three weeks, the fungal cultures were extracted with ethyl acetate (200 mL per Erlenmeyer flasks) in an ultrasonic sonomatic cleaning bath for 1 hour three times. The EtOAc extracts were then evaporated in vacuo. The EtOAc extracts were partitioned with n-hexane and acetonitrile to eliminate oily constituents. Then, a portion of aceonitrile was evaporated in vacuo to give an extract (335.5 mg). The second method was cultivated on PDB medium (12 g potato dextrose per 500 mL distilled water in a 1 L Erlenmeyer flask) at room temperature. After 3 weeks, the culture was extracted with EtOAc three times and then evaporated under reduced pressure to obtain the extract (860.0 mg).

Pyruvate Dehydrogenase Complex (PDH) Cellular Activity
We added 0.2 % Gelatin to the black 96-well plate with clear bottom and incubated for 1hr. After then, the plate was washed with growth media. Human AD-293 cells, derivative of the HEK293 cells, were seeded into black 96-well plates with clear bottom and grown for 24 hours. Compounds were then added and incubated for 24 hours. The cells were then fixed with 2% paraformaldehyde, permeabilized. Anti-PDHE1 pSer300 (Merk Millipore, AP1064, Darmstadt, Germany) was added and incubated overnight. Next, the cells were washed and Alexa fluor 488, goat anti-rabbit ab (Invitrogen, A11008, Waltham, MA, USA) was added with Hoechst 33258 (Invitrogen, H3569, Waltham, MA, USA) and incubated for two hours. Finally, cells were washed and the plates were measured in Operetta (PerkinElmer, Waltham, MA, USA). The raw data were normalized for the pharmacological inhibitory control (5 mM dichloroacetate (DCA)) and percent effect values using the software package Harmony High-Content Imaging and Analysis Software 3.1. The Deose response curves were generated by plotting the percent effect values and calculated IC 50 via GraphPad Prism 6.

Statistical Analysis
All values are expressed as means ± standard error of the mean. The statistical significance threshold (p < 0.05 for all analyses) was assessed by one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons using Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA).