Microindolinone A, a Novel 4,5,6,7-Tetrahydroindole, from the Deep-Sea-Derived Actinomycete Microbacterium sp. MCCC 1A11207

A novel indole, microindolinone A (1), was isolated from a deep-sea-derived actinomycete Microbacterium sp. MCCC 1A11207, together with 18 known compounds (2–19). By detailed analysis of the 1H, 13C, HSQC, COSY, HMBC, high resolution electron spray ionization mass spectrum (HRESIMS), and circular dichroism (CD) data, the absolute configuration of 1 was elucidated as 5R-hydroxy-4,5,6,7-tetrahydroindole-4-one. It is noteworthy that 1 is the second example of a saturated indole isolated from nature.


Introduction
Actinomycetes are Gram-positive bacteria known for their ability to produce structurally novel secondary metabolites with various biological activities [1]. The best-known compound is salinosporamide A [2,3]. Very recently, the representative examples included pyrazolofluostatins and aminorifamycins isolated from marine Micromonospora species [4,5].
The genus Microbacterium of the Microbacteriaceae family was first proposed by Orla-Jensen in 1919. Up to now, there are 97 species reported from diverse habitats including land, ocean, air, blood, etc. However, only four compounds were reported from this genus [6,7]. In our current research for novel compounds from deep-sea-derived microorganisms [8][9][10], the actinomycete Microbacterium sp. MCCC 1A11207, isolated from southwestern Indian Ocean sediment, was subjected to a systematic chemical examination. Consequently, one new and 18 known compounds were obtained. Herein, we report the isolation, structural elucidation, and bioactivities of these compounds.

Results and Discussion
Microbacterium sp. MCCC 1A11207 was cultured in a 50 L fermentor containing 30 L A3 medium for 10 days. Then, the fermentation broth was centrifuged. The supernatant was extracted with EtOAc and the mycelium was extracted with MeOH. They were concentrated and combined to give the crude extract (17 g). By repeated column chromatography (CC) over silica gel, octadecylsilyl (ODS), and Sephadex LH-20, 19 compounds were obtained (Figure 1).

Anti-Allergic Activity of 1
Microindolinone A (1) was further subjected to anti-allergic bioactivity on IgE-mediated rat mast RBL-2H3 cells. However, it did not show any positive effects under the concentration of 20 µg/mL, while the positive control loratadine exhibited a significant inhibition rate of 37% (Table 3).

General Experimental Procedures
HRESIMS spectra were obtained from a Xevo G2 Q-TOF mass spectrometer (Waters). Optical rotations were conducted by a Rudolph IV Autopol automatic polarimeter. NMR spectra were recorded on a Bruker 400 MHz spectrometer. Materials for column chromatography were silica gel (Qingdao Marine Chemistry Co. Ltd., Qingdao, China), ODS (50 µm, Daiso, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Pre-coated silica gel plates were used for thin-layer chromatography (TLC) analysis.

Bacterial Material
The strain MCCC 1A11207 was isolated from a deep-sea sediment of the southwestern Indian Ocean (−1603 m) in 2014. By comparison of its 16S rRNA gene sequence with those of validly published names from the GenBank database via the BLAST program, the strain showed the highest similarity (98.03%) to Microbacterium amylolyticum N5 T . Therefore, it was identified to be Microbacterium sp. MCCC 1A11207. A voucher strain of the actinomycete was deposited in the Marine Culture Collection of China with the accession number of MCCC 1A11207.

Cultivation and Extraction
The strain was cultured on the 2216E medium at 28 • C for 3 days and the colony was inoculated to 250 mL Erlenmeyer flasks containing 50 mL A3 medium compositing with 15 g bacterial peptone, 5 g soybean peptone, 15 g soluble starch, 30 g marine salt, and 1 L tap water, and then was cultured in a rotary shaker with 180 rpm at 28 • C for 3 days as the spores' medium. The large-scale fermentation was performed by a 50 L fermentor containing 30 L A3 medium with the 5% seed culture, and the fermentation continued at 28 • C with 180 rpm for 10 days. Then, the fermentation broth was centrifuged (16,000 rpm) to get supernatant and mycelium. The supernatant was extracted with EtOAc three times, and then concentrated under reduced pressure to provide the crude extract A. The mycelium was extracted with MeOH twice. After removing MeOH, the residue was re-extracted with EtOAc three times to get extract B under reduced vacuum. Extracts A and B were combined to give the total crude extract.

Anti-Proliferative Assay
According to previously reported protocols [34], the cytotoxicity test was carried out using the MTT assay on RBL-2H3 cells. In brief, RBL-2H3 cells were seeded into 96-well cell culture plates. Then, six different concentrations of 1, ranging from 0.625 to 20 µg/mL, were added. After 24 h, the cells were treated with 20 µL MTT solution. The cytotoxicity was quantified by measuring the absorbance at 570 nm. The cell viability was calculated using the following equation: Cell viability (%) = [(As − Av)/(Ac − Av) × 100%, where As is the absorbance of the sample, Av is the absorbance of the vehicle, and Ac is the absorbance of the control.

Anti-Allergic Test
The anti-allergic activity, indexed by the β-hexosaminidase release, was measured for the efficiency of the RBL-2H3 cell degranulation inhibition rate using IgE-mediated mast cell allergic reaction [8,35]. In short, RBL-2H3 cells were seeded into 96-well cell culture plates (1 × 10 5 cells/well) to incubate with dinitrophenol (DNP) specific IgE overnight. IgE-sensitized RBL-2H3 cells were pre-treated with compound 1 (20 µg/mL) for 1 h and stimulated with dinitrophenyl-bovine serum albumin (DNP-BSA) (500 ng/mL). The negative control group was added to 200 µL phosphate-buffered saline (PBS) buffer. The β-hexosaminidase activity was quantified by measuring the fluorescence intensity of the hydrolyzed substrate in a fluorometer. The degranulation efficiency was calculated using the following formula: Degranulation efficiency (%) = Fsup/(Fsup + Flys) × 100%, where Fsup is the fluorescence value of the supernatant and Flys is the fluorescence value of cell lysates. The inhibition rate was calculated based on the following formula: Inhibition rate (%) = (Positive − Sample)/(Positive − Negative) × 100%, where Positive is the degranulation efficiency of the DNP-BSA stimulated group, Sample is the degranulation efficiency of the sample group, and Negative is the degranulation efficiency of the vehicle group.

Statistical Analysis
Anti-proliferative and anti-allergic experiments were conducted three times. Results are presented as means ± SD. One-way analysis of variance (one-way ANOVA) comparison tests of SPSS was used to evaluate the statistical significances of the differences between experimental groups. Differences were considered statistically significant for p < 0.05 using Duncan's multiple range tests between groups.

Conclusions
From the deep-sea-derived rare actinomycete Microbacterium sp. MCCC 1A11207, 19 secondary metabolites were isolated and identified. The new compound, microindolinone A (1), was determined as 5R-hydroxy-4,5,6,7-tetrahydroindole-4-one. It was the second example of the tetrahydroindole found in nature. Its absolute configuration was determined for the first time.