Polyketides From the Endophytic Fungus Cladosporium sp. Isolated From the Mangrove Plant Excoecaria agallocha

Five new polyketides (2–6) and ten known compounds (1 and 7–15) were obtained from the fermentation products of the endophytic fungus Cladosporium sp. OUCMDZ-302 with the mangrove plant, Excoecaria agallocha (Euphorbiaceae). The new structures of 2–6 were established on the basis of ECD, specific rotation and spectroscopic data as well as the chemical calculation. Compound 7 showed cytotoxicity against H1975 cell line with an IC50 value of 10.0 μM. Compounds 4 and 8–10 showed radical scavenging activity against DPPH with the IC50 values of 2.65, 0.24, 5.66, and 6.67 μM, respectively. In addition, the absolute configuration of compound 1 was solidly determined by X-ray and sugar analysis of the acidic hydrolysates for the first time as well as those of compounds 8–10 in this paper.


Fungal Material
The strain Cladosporium sp. OUCMDZ-302 was isolated from the surface sterilized stems of the mangrove plant E. agallocha grown in Wenchang, Hainan, China. Briefly, the stems were washed with tap water and sterile distilled water in sequence. The stems with clean surface were further sterilized in a sequence of 75% ethanol for 2 min, 0.1% of HgCl 2 for 3 min, and sterile distilled water. The outer bark was removed, and the inner bark was cut into small pieces that were then placed on a potato dextrose agar (PDA) plate and cultured at 28 • C for 3 days. A single colony was transferred to PDA media and was identified according to its morphological characteristics ( Figure S35) by Prof. Kui Hong, Wuhan University. A voucher specimen is deposited in our laboratory at −80 • C. The working strain was prepared on PDA slants and stored at 4 • C.

Fermentation and Extraction
The producing fungal strain Cladosporium sp. OUCMDZ-302 was inoculated into a 500 mL cylindrical flask containing 100 mL of seawater consisting of 2% maltose, 2% mannitol, 1% glucose, 1% monosodium glutamate, 0.3% yeast extract, 0.1% corn flour, 0.05% KH 2 PO 4 , 0.03% MgSO 4 · 7H 2 O (pH 6.5) and cultured at 28 • C for 48 h on a rotary shaker at 120 rpm. The seed culture was transferred into three hundred and fifty 500 mL conical flasks (200 mL/flask) containing the same medium, and performed at 28 • C for 7 days on rotary shakers at 160 rpm. The whole fermentation broth (70 L) was filtered through cheese cloth to separate the mycelia from filtrate. The filtrate was concentrated to about one-quarter of the original volume under reduced pressure and then extracted three times with equal volumes of ethyl acetate (EtOAc) and concentrated to dryness. The mycelia were extracted three times with acetone and concentrated to an aqueous solution. The aqueous solution was subsequently extracted three times with equal volumes of EtOAc and concentrated. Both EtOAc extractions were combined to give 45 g of the extract.

Anti-oxidant Activities
The anti-oxidant activities of compounds 1-14 were evaluated by DPPH assay in vitro (Wang et al., 2007). Vitamin C was used as the positive control with an IC 50 value of 3.29 µM.

Antimicrobial Assays
The antimicrobial activities of compounds 1-14 against E. coli, E. aerogenes, P. aeruginosa, B. subtilis, and C. albicans were evaluated by an agar dilution method (Zaika, 1988). Ciprofloxacin lactate and ketoconazole was used as the positive controls for bacteria and fungi with MIC values of 4.0, 0.5, 32.0, 16.0, 4.1 µg/mL, respectively.

Identification of Compounds
Compounds 1 and 2 were first isolated as an isomeric mixture whose molecular formula was determined to be C 15 (Figure 2 and Figures S4-S6) spectra established a pentose moiety and a benzopyrane moiety similar to those of 5,7-dihydroxy-2methylchroman-4-one (11) (Rao et al., 1994). The upfield shift of C-7 (−1.5 ppm) and the key HMBC correlations between the anomeric proton (δ H−1 ′ 5.66/5.68) and C-7 (δ 165.1/165.0) indicated that 1 and 2 were 7-O-pentosides of 11. Acidic hydrolysis of the mixture of 1 and 2 with 2 M HCl yielded (±)-11 and D-ribose that was identified by GC-MS analysis of the reaction products with L-cysteine methyl ester and Me 3 SiCl (Figures S33, S34) (Deyrup et al., 2007). These data indicated that 1 and 2 are a pair of epimers at C-2. Separation of 2-epimeric mixture of 1 and 2 was achieved on a chiral column using MeOH-MeCN-EtOH as eluent. And then, NMR data of optically-pure 1 (Figures S7, S8) and 2 (Figures S9, S10) were obtained. Xray single crystal diffraction of 1 revealed the α-glycosidic bond and 2R-configuration (Figure 3). The ECD Cotton effects of compounds 1 and 2 were opposite in sign (Figure 4), confirming the opposite configuration of C-2. Thus, the structures of 1 and 2 were unambiguously elucidated as (2R)-and (2S)-7-O-α-Dribofuranosyl-5-hydroxy-2-methylchroman-4-one, respectively. This is the first time that to solidify the absolute configuration of compound 1, although it was reported last year (Hu et al., 2017).

Biogenetic Origin
These compounds were postulated to be biosynthesized by the polyketide pathway from acetyl coenzyme A (Figure 9). The acetyl-CoA units underwent condensation, cyclization, dehydration and hydrogenation to produce compounds 11 and 12. Compound 11 formed compounds 1 and 2 by glycosidation. (S)-12 underwent oxidation and reduction to yield compound 3. The reduction of (S)-12 produced compound 10 that was transformed to compound 9 followed by methylation. (S)-12 was subjected to Baeyer-Villiger oxidation followed by methanolysis and hydrolysis to yield compounds 4 and 8, respectively. Compounds 5 and 6 were formed from different lengths of acetyl-CoA units by condensation, reduction, dehydration, and decarboxylation. The condensation of acetyl-CoA units followed by cyclization and reduction formed compound 15 that was transformed to compound 14 after enolization and dehydration.

CONCLUSIONS
Five new polyketides were isolated and identified from the fermentation of the mangrove fungus Cladosporium sp. OUCMDZ-302 with Excoecaria agallocha. The new compound 4 showed DPPH radical scavenging activity with an IC 50 value of 2.65 µM.