Ergosterols from the Culture Broth of Marine Streptomyces anandii H41-59

An actinomycete strain, H41-59, isolated from sea sediment in a mangrove district, was identified as Streptomyces anandii on the basis of 16S rDNA gene sequence analysis as well as the investigation of its morphological, physiological and biochemical characteristics. Three new ergosterols, ananstreps A–C (1–3), along with ten known ones (4–13), were isolated from the culture broth of this strain. The gross structures of these new compounds were elucidated on the basis of extensive analysis of spectroscopic data, including HR-ESI-MS, and NMR. The cytotoxicities of these isolates against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268, and human lung cancer cell line NCI-H460 and their antibacterial activities in inhibiting the growth of Candida albicans and some other pathogenic microorganisms were tested. Compounds 3–8, 10 and 11 displayed cytotoxicity with IC50 values in a range from 13.0 to 27.8 μg/mL. However, all the tested compounds showed no activity on C. albicans and other bacteria at the test concentration of 1 mg/mL with the paper disc diffusion method.


Introduction
In recent years, many researchers have paid attention to marine-derived microorganisms because of the increasing rate of rediscovery of known compounds on terrestrial land [1]. Marine microbial secondary metabolites are a prolific source of novel natural products, including sterols and other related metabolites with potent cytotoxic or antimicrobial activities [2,3]. Ergosterols are a class of components possessing many hydroxyl groups, a tetracyclic skeleton and a short alkyl chain. They are a sort of critical component in membranes, involved in many biological functions and play an important role in regulating fluidity of membrane, controlling cellular cycle, and organizing membranes for signal transduction and protein trafficking [4,5]. To the best of our knowledge, ergosterols were usually found in fungi, sponges, and corals, and it is rarely to discover new ergosterols from Streptomyces species [6,7]. Streptomyces are a genus of Gram-positive, filamentous bacteria usually dwelled in soil. They are one of the most diverse in species and show the ability to produce clinical useful compounds with different structures, such as streptomycins, actinomycins, adriamycin, vancomycin, and tacrolimus [8][9][10].
On our present study, a strain of actinomycete H41-59, isolated from sea sediment at a mangrove district of South China Sea, was identified as Streptomyces anandii. Application of multiple chromatographic procedures and modern spectral methods led to the isolation and identification of thirteen sterols including three new sterols (1-3). Their cytotoxicities against three cancer cell lines and their antibiotic activities inhibiting the growth of C. albicans and some pathogenic bacteria were evaluated. Herein, we describe the structure determination of three new compounds, the isolation and bioactivity assay of these isolated compounds from the ethanol extract of fermented broth of strain H41-59, and try to hypothesize the biosynthetic pathway of these sterols.

Characterization of the Compounds
An ethyl acetate (EtOAc) partition from the 95% ethanol (EtOH) extract of mycelium of Streptomyces anandii strain H41-59 was subjected to repeated silica gel column chromatography and then purified by semi-preparative reverse phase HPLC separation to yield thirteen compounds (1-13, Figure 1). On the basis of the NMR analysis and the comparison with reported data, three of them were identified as new sterols (1-3).
Mar. Drugs 2016, 14,84 and their antibiotic activities inhibiting the growth of C. albicans and some pathogenic bacteria were evaluated. Herein, we describe the structure determination of three new compounds, the isolation and bioactivity assay of these isolated compounds from the ethanol extract of fermented broth of strain H41-59, and try to hypothesize the biosynthetic pathway of these sterols.

Characterization of the Compounds
An ethyl acetate (EtOAc) partition from the 95% ethanol (EtOH) extract of mycelium of Streptomyces anandii strain H41-59 was subjected to repeated silica gel column chromatography and then purified by semi-preparative reverse phase HPLC separation to yield thirteen compounds (1-13, Figure 1). On the basis of the NMR analysis and the comparison with reported data, three of them were identified as new sterols (1-3  -27), two oxymethines at δ 66.0 (C-3) and 72.1 (C-6), and an oxygenated quaternary carbon at δ 74.5 (C-5) existed in the 13 C NMR spectrum, which were further confirmed by DEPT and HSQC spectra. All the spectroscopic data indicated that 1 was a tetrahydroxylated sterol, similar to those of the previously isolated compounds 4 and 5 except for the signals of the side chain.
Compared with those of known compound 5 [11,12], there were visible changes at δ 36.9 (C-24), 40. The configuration of C-20, C-24 and C-25 can not be defined only by NOESY experiment. However, there is only one single epimer which was isolated up to now. The stereochemistry of 1 at the chiral centers C-3, C-5, C-6, C-18, C-19 and C-17 were confirmed as the same as 5 on the basis of comparison of the NMR spectral data of 1 with those of compounds containing analogous side chain [11][12][13], and were further confirmed by NOESY spectral data. Accordingly, the structure of 1 was determined to be ergosta-7,22-diene-3β,5α,6β,27-tetraol and named as ananstrep A.
Why there are so many ergosterols isolated from the ethanol extract of mycelium of strain H41-59? As we all know, ergosterol is a chemical constituent mainly found in fungi, and there is no solid evidence to show that the actinomycete biosynthesizes ergosterol carbon skeleton. However, cholesterol oxidase (ChO), one of the key enzymes of microbial sterol metabolism, was usually observed among Gram-positive G + C-rich actinobacteria, e.g., Streptomyces [24,25], Nocardia [26], Mycobacterium [27], Gordonia [28], Rhodococcus [29], and some genes encoding different ChOs have been cloned from some actinomycete. Therefore, actinobacteria are capable of effecting diverse types of sterol transformation, such as hydroxylation, dehydrogenation, double bond isomerization and partial degradation of the side chain or sterol nucleus [30]. Accordingly, it is not far-fetched to imagine that one or some known ergosterols originated in yeast powder, a nitrogen source of culture medium, might be transformed into other ergosterols including three new ones, and the plausible biotransformation was hypothesized as shown as Scheme 1 [31][32][33]. Mar. Drugs 2016, 14,84 Accordingly, it is not far-fetched to imagine that one or some known ergosterols originated in yeast powder, a nitrogen source of culture medium, might be transformed into other ergosterols including three new ones, and the plausible biotransformation was hypothesized as shown as Scheme 1 [31][32][33].

Biological Activity
The isolated metabolites 1-13 showed no bioactivity against Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus sp. and Dickeya zeae at 1 mg/mL, while the crude extract displayed a moderate activity against five test strains. The antibacterial activity of the extract may come from the unisolated minor components in the extract. The cytotoxicity against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268, and human lung cancer cell line NCI-H460 has Scheme 1. Plausible biosynthetic pathway of 1-13.

Biological Activity
The isolated metabolites 1-13 showed no bioactivity against Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus sp. and Dickeya zeae at 1 mg/mL, while the crude extract displayed a moderate activity against five test strains. The antibacterial activity of the extract may come from the unisolated minor components in the extract. The cytotoxicity against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268, and human lung cancer cell line NCI-H460 has also been investigated using an MTT assay. As is described in Table 3, all the isolated compounds showed to some extent cytotoxicity against three cancer cell lines. According to the cytotoxicity result, it is worthy to note that those 8(9) -sterols and 8(14) -sterols such as compounds 3, 8, 10 and 11 were likely to display better effect against three tumor cell lines. Among them, compound 3, possessing a 8(9) moiety, indicated much better effect against SF-268, MCF-7 and NCI-H460 with IC 50 value of 13.0, 18.1 and 23.5 µg/mL, respectively.

General Experimental Procedures
Melting points were recorded on an X-5 micro-MP apparatus (Huayan Corporation, Shanghai, China), uncorrected. UV spectra were determined on a JASCO V-550 UV/VIS spectrometer (JASCO Corporation, Tokyo, Japan). IR spectra were carried out with a Nicolet Impact 410-FTIR instrument (Thermo, San Jose, CA, USA) in KBr pellets. The optical rotations were measured with a JASCO digital polarimeter (JASCO Corporation, Tokyo, Japan). HR-ESI-MS were acquired on an Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectrawere performed on Bruker AV-300 and AV-600 spectrometer (Bruker Instrument, Inc., Zurich, Switzerland), tetramethylsilane (TMS) was used as the internal standard for 1 H NMR, and referencing to the NMR solvent used for 13 C NMR. Chemical shifts were evaluated in δ (ppm). HPLC was performed on an Agilent 1200 HPLC system equipped with an diode array detector, using a column A (Ultimate XB-C18, 5 µM, 4.6ˆ250 mm, Welch, Potamac, MA, USA) for analysis and a column B (Ultimate XB-C18, 5 µm, 10ˆ250 mm, Welch, Potamac, MA, USA) for semi-preparative purification. Open column chromatography was performed on silica gel (300-400 mesh, Qingdao, Haiyang Chemical Group Corporation, Qingdao, China). Sephadex LH-20 (25-100 mm) was purchased from Pharmacia (Uppsala, Sweden). HSGF254 silica gel TLC plates (0.2 mm thickness, 200ˆ200 mm, Qingdao Marine Chemical, City, China) were used as routine analysis of fractions. Strains Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus sp., and Dickeya zeae were obtained from the Institute of New Drug Research in our college.

Strain Isolation and Identification
The actinomycete strain H41-59 was isolated in 2001 from a marine sediment sample collected at the Zhapo mangrove site, on the Hailing island of Yangjiang, Guangdong province, China, and kept in a sandy soil tube and stored at about 4˝C refrigerator before use. Strain H41-59 was activated on Gauserime synthetic agar medium at 30˝C. Studies on its morphological, physiological and biochemical characteristic indicated that this strain belonged to the genus Streptomyces. Analysis of its 16S rDNA gene sequence let us identify this strain as Streptomyces anandiidue by its 99.9% similarity with the sequence of S. anandii deposited in GenBank under accession number GU350497.1. A voucher specimen of strain H41-59 was preserved in the Guangdong Key Laboratory of New Technique for Plant Protection, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou, China.

Fermentation and Extraction
Strain H41-59 was grown under shaking condition at 28˝C and 170 rpm for two days in four 1 L Erlenmeyer flasks containing 250 ml of the seed medium (composed of yeast powder 30 g, corn starch 30 g, crude salt 2.5 g, CaCO 3 1.5 g, KNO 3 1 g, MgSO 4 0.6 g, K 2 HPO 4 0.9 g, FeSO 4 0.02 g, H 2 O 1L, adjusting pH to 7.4 with NaOH solution), then inoculated into production medium (20 L) in a 30-L fermentor and incubated for five days at 28˝C. After subjecting to 20 L scale fermentation four times up to total volume of 80 L, the culture broth was centrifuged at 4416 g and then the mycelium was percolated using 95% ethanol (EtOH). After removal of EtOH with a rotatory evaporator under vacuum at 50˝C, the aqueous solution was diluted with distilled water, then added on macroporous resin HP2MGL column (Mitsubishi Chemical, Japan) to absorb secondary metabolites. The column was successively washed with water, 50% EtOH, and 95% EtOH. The 95% EtOH elution was concentrated using rotatory evaporator in vacuum and under 50˝C. Then, the residual material was suspended in water and extracted with ethyl acetate (EtOAc). Finally, the EtOAc solution was evaporated to give a residue (50 g).