New Helvolic Acid Derivatives with Antibacterial Activities from Sarocladium oryzae DX-THL3, an Endophytic Fungus from Dongxiang Wild Rice (Oryza rufipogon Griff.)

Three new helvolic acid derivatives (named sarocladilactone A (1), sarocladilactone B (2) and sarocladic acid A (3a)), together with five known compounds (6,16-diacetoxy-25-hy- droxy-3,7-dioxy-29-nordammara-1,17(20)-dien-21-oic acid (3b), helvolic acid (4), helvolinic acid (5), 6-desacetoxy-helvolic acid (6) and 1,2-dihydrohelvolic acid (7)), were isolated from the endophytic fungus DX-THL3, obtained from the leaf of Dongxiang wild rice (Oryza rufipogon Griff.). The structures of the new compounds were elucidated via HR-MS, extensive 1D and 2D NMR analysis and comparison with reported data. Compounds 1, 2, 4, 5, 6 and 7 exhibited potent antibacterial activities. In particular, sarocladilactone B (2), helvolinic acid (5) and 6-desacetoxy-helvolic acid (6) exhibited strongly Staphylococcus aureus inhibitory activity with minimum inhibitory concentration (MIC) values of 4, 1 and 4 μg/mL, respectively. The structure–activity relationship (SAR) of these compounds was primarily summarized.


Introduction
Endophytic fungi have been recognized as a rich source of potent bioactive natural products with diverse structural groups, and a large number of metabolites, such as alkaloids, steroids, terpenoids, peptides, polyketones, flavonoids and xanthones, are continuously being isolated from them [1,2]. The various natural products possess unique structures and bioactivities, thus representing a huge reservoir that offers an enormous amount of potential for exploitation in the agricultural and industrial areas [3].
Strain DX-THL3 was isolated from the leaf of Dongxiang wild rice (Oryza rufipogon Griff.) in the Jiangxi Province of China, and identified as Sarocladium oryzae [4]. S. oryzae is an Ascomycetes fungus that causes sheath rot disease in rice [5]. Several studies have reported that S. oryzae produces helvolic acid and cerulenin secondary metabolites, which have been reported as phytotoxins [6,7]. Helvolic acid was originally isolated from the culture filtrates of Aspergillus fumigatus mut. helvola Yuill [8], and the confirmation of its structure was completed by Shigeo Iwasaki [9]. Nowadays, helvolic acid is a representative fusidane-type antibiotic, which exhibits potent activity against Gram-positive bacteria [10]. Fusidane-type antibiotics have recently attracted renewed attention for their lack of cross resistance with other commonly used antibiotics [11]. Up until now, helvolic acid was generally found in marine-derived or terrestrial fungi of the Aspergillus spp., and in other endophytic fungi, such as Fusarium sp. [12], Penicillium sp. [13] and Sarocladium sp. [6].

Identification of Fungus Identification
The strain of DX-THL3 was identifed as Sarocladium oryzae (located in GenBank under the accession number KF558875) based on DNA amplification and the internal transcribed spacer (ITS) sequence ( Figure S1).
Compounds 3a and 3b manifested as a white mixture solid, and the amount of compound 3a was a little higher than that of 3b. The negative HRESIMS showed a pair of molecular ions (588.3439, 586.3267) and their quasimolecular ions (587.3377, 585.3232), indicating they share the same skeleton with a difference of 2 Da, and the formulae of C 33 H 48 O 9 /C 33 H 46 O 9 were given. From the 1 H, 13 C NMR and DEPT spectra, the two compounds are also helvolic acid derivatives (Table 1). Combination with extensive 2D NMR analysis, the signals were finely sorted and assigned. The result implied that the difference between 3a and 3b lies only in the unsaturation of C 1 -C 2 . Additionally, they have a distinctly oxygenated quaternary carbon at δ C 69.8 (C-25), and two acetoxyl group, two keto carbonyl, one carboxyl, and one or two olefinic bond, and compound 3b is identical to the reported (4S,5S,6S,8S,9S,10R,13R,14S,16S,17Z) 6,16-diacetoxy-25-hydroxy-3,7-dioxy-29nordammara-1,17 (20)-dien-21-oic acid, which was confirmed via the 1 H-1 H COSY, HMBC and ROESY spectra [13]. Thus, compound 3a was elucidated as a 1,2-dihydro derivative of 3b, and given the trivial name sarocladic acid A (Figures 2 and 3).
Compounds 5 and 6 were firstly reported as hydrolyzed products from helvolic acid (4), named helvolinic acid and 6-deacetoxyhelvolic acid, respectively. They exhibit a similar antibiotic spectrum. Meanwhile, they were separated from a cuture broth of Cephalosporium caerulens, indicated they are precursors of 4 [19], with no detailed NMR spectroscopic data. Herein, the 1 H and 13 C NMR data of compounds 5 and 6 were fully elucidated and assigned.
Compound 7, named 1,2-dihydrohelvolic acid, was previously reported by Zaman [16] and Lee [20], respectively, and only the NMR data recorded in pyridine-d 5 are displayed, while the data recorded in methanol-d 4 are not available. Herein, the significant 1 H and 13 C NMR data are listed in Table 2.

Biological Activities
The antibacterial activities of the isolated compounds 1-7 (compounds 3a and 3b were not tested due to the presence of mixtures) against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Xanthomonas oryzae pv.oryzicola were evaluated using the 2-fold dilution assay. Compounds 1, 2, 4, 5, 6 and 7 showed antibacterial activity against S. aureus with MIC values of 64, 4, 8, 1, 4 and 16 µg/mL (Table 3), respectively (with tobramycin as the positive control, MIC 1 µg/mL), while compound 5 also showed antibacterial activity against B. subtilis with an MIC value of 64 µg/mL (with tobramycin as the positive control, MIC 64 µg/mL). Compounds 2, 5 and 7 showed some potent antibacterial activity against E. coli with MIC 64 µg/mL ( Table 3). All of the compounds were inactive against X. oryzae pv.oryzicola at 128 µg/mL. It was reported that helvolic acid and its derivatives exhibited better antibacterial activities, mainly against Gram-positive bacteria [21,22]. On the basis of the structural differences between compounds 4-7 and compounds 1 and 2, comparing the MIC values of compounds 4 and 7, it can be concluded that the α,βunsaturated ketone unit in ring-A contributes the activity about 2-fold. The corresponding acetoxy substituent at C-6 in helvolic acid was replaced by a hydroxyl group (compound 5), for which activity is significantly increased 8-fold versus compound 4. The acetoxyl deficiency at C-6 of helvolic acid (compound 6) is significantly increased 2-fold versus compound 4.
As for compound 2, there is a C-21/C-16 lactone ring present, which will theoretically lessen the antibacterial activity [15], while for an α-orientation hydroxyl substitution at C-7 instead of an ortho-hydroxy carbonyl in Ring-B, the MIC value is less than that of compound 4.
To summarize, regarding the antibacterial structure-activity relationship (SAR) of helvolic acid and its derivatives, it can be concluded that the presence of a C-21/C-16 lactone ring and a C-21/C-24 lactone ring significantly reduced the antibacterial activity. Remarkably, compound 5, with a hydroxyl group replacing the corresponding acetoxy substituent at C-6 in helvolic acid, exhibited stronger antibacterial activity against S. aureus than the antibacterial drug helvolic acid.

Fungus Material
The endophytic fungus DX-THL3, isolated from the healthy leaves of Dongxiang wild rice, was collected from a nature reserve in Dongxiang County, Jiangxi Province, China, in November 2013. This strain was deposited with the culture collection of the Key Laboratory of Protection and Utilization of Subtropical Plant Resources of Jiangxi Province, Jiangxi Normal University.

Identification of Strain DX-THL3
The endophytic fungus DX-THL3 was identified using both morphological characters and phylogenetic data. DX-THL3 was grown on the surface of potato dextrose agar (PDA) medium at 28 • C for 2 weeks, followed by identification based on the morphology of the fungal colony and the characteristics of the spores. Mycelia, conidia and pycnidia of the DX-THL3 strain were observed with a light microscope (BA300, Motic, Xiamen, China).
On the other hand, the DX-THL3 fungal strain was incubated in PDA for 7 days at 28 • C. Mycelia were scraped from the plate and ground to a powder under liquid nitrogen. Genomic DNA was then extracted using the Cetyltrimethylammonium Bromide (CTAB) method. The DNA was subjected to PCR amplification using the primers ITS1 (5 -TTCGTAGGTGAACCT GCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATAT GC-3 ). The PCR reaction was performed in 50 µL of reaction mixture containing 25 µL of 2× Taq PCR Mastermix (Tiangen Biotech Beijing Co Ltd., Beijing, China), 2 µL of forward primer (10 µM), 2 mL of reverse primer (10 µM), 2 µL of template DNA and 19 µL of sterile doubledistilled water. The PCR cycling protocol consisted of an initial denaturation at 94 • C for 3 min, 30 cycles of denaturation, annealing and elongation at 94 • C for 30 s, 60 • C for 30 s and 72 • C for 1 min. This protocol was followed by a final elongation step of 72 • C for 5 min. As a negative control, the template DNA was replaced by sterile double-distilled water. The PCR amplified products were analyzed using gel electrophoresis at approximately 500 bp-800 bp. The purification and sequencing of the PCR products was performed by Shanghai Invitrogen Company Ltd. (Shanghai, China). The corresponding sequence was analyzed through the basic local alignment search tool (BLAST). A neighbor-joining (NJ) phylogenetic tree was constructed in MEGA 6.0, using 1000 bootstrap replicates.
The ITS-rDNA of DX-THL3 was submitted to GenBank, and the accession number is KF558875.

Fermentation, Extraction and Metabolite Isolation
The endophytic fungus DX-THL3 was grown on PDA (200 g of potato, 20 g of glucose and 20 g of agar per liter of seawater) at 28 • C for 4 days, after which the agar was cut into pieces. The pieces (with a diameter of 0.5 cm) of mycelial agar were inoculated into 500 mL Erlenmeyer flasks (which each containing 200 mL of PDB medium) for 5 days on a rotary shaker (150 rpm). Then, the seed liquid was transferred into 500 Erlenmeyer flasks containing 150 mL of PDB medium each. The flasks were then incubated at 28 • C on a rotary shaker (180 rpm) for 15 days.
After 15 days of cultivation, the cultures (30 L) were filtered through cheesecloth to separate the mycelial mass from the aqueous layer. The filtrate was then extracted three times with an equal volume of ethyl acetate, followed by the removal of the ethyl acetate under vacuum to yield a crude extract (4.

Antibacterial Assay
The antibacterial activity in vitro against Gram-positive bacteria (Staphylococcus aureus (ATCC 29213) and Bacillus subtilis (ATCC 7508)) and Gram-negative bacteria (Escherichia coli (ATCC 25922) and X. oryzae pv.oryzicola) was evaluated via the 2-fold dilution assay in 96-well microtiter plates [23]. The bacterial strains were inoculated on Luria broth and incubated for 24 h at 37 • C; then, the cells were collected with normal saline and were diluted with broth to achieve 1.0 × 10 7 mL −1 . The cell broth was further diluted 10-fold before being adding into 96-well microtiter plates. 200 µL of mixtures was transferred into the first test well of each line in the 96-well while 100 µL was added in the other wells in the same line. Compounds were dissolved into DMSO and adjusted to 10 mg mL −1 . Compounds were added into the first well at a concentration of 256 µg mL −1 , then 100 µL was transferred to the second well until the final concentration of the twelfth well was 0.5 µg mL −1 . Streptomycin sulphate and tobramycin were used as positive controls and DMSO was used as the negative control. The 96-well plates were incubated at 37 • C for 24 h, and the optical density (OD) was tested at 600 nm while using a microplate reader. The minimum inhibitory concentration (MIC) was defined as the minimal concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after 24 h incubation.

Conclusions
In summary, we discovered eight helvolic acid derivatives from the endophytic fungus Sarocladium oryzae of the Dongxiang wild rice (Oryza rufipogon Griff.) leaf, among which compounds 1, 2 and 3a are new. An antibacterial assay showed that compounds 4-7 exhibited somewhat antibacterial activities against S. aureus, which is consistent with previous results [19,21,22]. The new compound 2 is more potent against S. aureus than compounds 4 and 7. Compound 5 showed the strongest amount of activity against S. aureus, which may serve for antibacterical agent use. Thus, for the antibacterial SAR of helvolic acid and its derivatives, it can be concluded that an α,β-unsaturated ketone unit in ring-A, an ortho-hydroxy carbonyl or an α-orientation hydroxyl substitution at C-7 in ring-B contribute to antibacterial activity. Meanwhile, the presence of a C-21/C-16 lactone or a C-21/C-24 lactone will significantly reduce antibacterial activity.