Introduction

We have isolated many rare actinomycete strains from the roots of plants over the past few years and discovered a new genus and several new species of actinomycetes.1, 2, 3, 4, 5 To enable the discovery of new compounds from the cultured broth of the endophytic actinomycete isolates, we have exploited biological screening programs, looking for antibacterial, antifungal, cytotoxic and antiprotozoal activities, as well as a chemical screening approach using LC/UV and a LC/MS/MS system. During the course of chemical screening, new compounds possessing antitrypanosomal activity, namely spoxazomicins A–C, were discovered using Dragendorff’s reagent from the cultured broth of Streptosporangium oxazolinicum K07-0460T.6 Further research using LC/UV and LC/MS has identified compounds with unique UV and MS spectra, isolated from the cultured broth of an endophytic actinomycete strain, Polymorphospora rubra K07-0510. The new compounds, designated trehangelins A (1), B (2) and C (3), were isolated and the structures were elucidated (Figure 1). The trehangelins showed potent inhibitory activity against hemolysis of red blood cells (RBCs) induced by light-activated pheophorbide a.

Figure 1
figure 1

Structures of trehangelins A, B and C isolated from a cultured broth of strain K07-0510.

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This report details the taxonomy of the producing strain, fermentation, isolation, physico-chemical properties, structural elucidation and some biological activities of the compounds 1, 2 and 3.

Results

Taxonomy of the producing strain, Polymorphospora rubra K07-0510

Strain K07-0510 was isolated from the roots of a plant, a variety of orchid, collected on Iriomote Island, Okinawa, Japan. The strain grew well on yeast extract–malt extract agar, inorganic salts–starch agar and tyrosine agar but did not grow on oatmeal agar, glucose–nitrate agar or glucose–peptone agar. The colony color was reddish orange. Aerial mycelia were produced on GPM agar (glucose 1.0%, peptone 0.5%, meat extract 0.5%, NaCl 0.3%, agar 1.2%, pH 7.0) of the test media, and the aerial mass color was white. Short spore chains were formed, and the spores with the smooth surface were cylindrical in shape, 0.7∼1.1 μm long × 0.5∼1.0 μm wide in size (Figure 2). No soluble pigment was produced. Whole-cell hydrolysates contained meso-DAP (diaminopimelic acid). The predominant menaquinones of the strain were MK-10 (H6) and MK-9 (H6), whereas MK-10 (H8), MK-10 (H4), MK-9 (H8) and MK-9 (H4) were detected as minor menaquinones. The 16S rRNA gene sequence (1200 nucleotides) was determined and analyzed using the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/). The result indicated that the strain K07-0510 is closest to Polymorphospora rubra TT97-42T (AB223089, similarity; 99.9%), which is the only valid species in the genus Polymorphospora.7 On the basis of the morphological and cultural properties and 16S rRNA gene sequence similarity, strain K07-0510 is identified as P. rubra.

Figure 2
figure 2

Scanning electron micrograph of strain K07-0510 grown on GPM agar at 27 °C for 2 weeks.

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Isolation of the bioactive compounds

The procedure for isolation of compounds 1, 2 and 3 is summarized in Scheme 1. The 9-day old cultured broth (1.8 l) was added to an equal amount of ethanol and then filtered. The filtrate was concentrated under reduced pressure to remove ethanol and the aqueous solution was extracted three times with ethyl acetate (total volume of 1.8 l). The organic layer was concentrated under reduced pressure to yield a crude material (662 mg). The ethyl acetate extract was applied to silica gel column chromatography that was eluted with each 500 ml of a mixture of CHCl3–MeOH (100:0, 100:1, 50:1, 10:1, 1:1, 0:100) in this order. The CHCl3–MeOH (10:1 and 1:1) fractions (470.0 mg) were applied to an octadecylsilyl silica gel (ODS) column chromatography, which was eluted with 100 ml each of 20, 30 and 100% MeOH. Trehangelins B (2, r.t.=16 min, 4.4 mg), C (3, r.t.=17 min, 1.3 mg) and A (1, r.t.=18 min, 59.0 mg) were isolated, each as a yellowish white powder, by HPLC (Inertsil ODS-4, 10 i.d. × 250 mm) with 5% CH3CN aq. (flow rate, 5.0 ml min−1; detection, UV 254 nm) from the crude mixture of 30% MeOH eluate in ODS column chromatography (186.4 mg).

Physico-chemical properties of trehangelins A (1), B (2) and C (3)

The physico-chemical properties of compounds 1, 2 and 3 are summarized in Table 1. They are readily soluble in H2O and MeOH. Compounds 1, 2 and 3 showed absorption maxima at 216–218 nm in UV spectra. The IR absorption at 1700 cm−1 suggested the presence of carbonyl groups. The similarity in physico-chemical properties strongly suggested that these three compounds are structurally related.

Table 1 Physico-chemical properties of trehangelins A (1), B (2) and C (3)
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Structure elucidation of compound 1

The molecular formula of 1 was elucidated by ESI-MS to be C22H34O13, requiring six degrees of unsaturation. The 1H and 13C NMR spectra data measured in CD3OD of 1 are listed in Table 2. The 13C NMR and HSQC spectra indicated only 11 carbons, half the expected number, implying a symmetric structure, which were classified into one ester carbonyl carbon (δC 169.6), one sp2 quaternary carbon, one sp2 methine carbon, five oxygenated sp3 methine carbons, including one sp3 anomeric carbon (δC 95.1), one oxygenated sp3 methylene carbon and two olefinic methyl carbons. In 1H–1H COSY experiments, the spin systems were from H-1 to H-5 and from 8-CH3 to 9-CH3 (Figure 3). The HMBC correlations from H-1 (δH 5.21) to C-3 (δC 76.5) and C-5 (δC 73.9), from H-2 (δH 3.71) to C-4 (δC 70.0) and from H-3 (δH 5.48) to C-5 indicated the linkage between C-1 and C-5 through an oxygen atom to form a tetrahydropyran ring. The HMBC correlations from H2-6 (δH 3.73 and 3.80) to C-4 and from H-4 (δH 3.56) to C-6 (δC 62.2) indicated that the oxygenated sp3 methylene carbon in 6-position was attached to C-5 to form a carbohydrate moiety. A small vicinal coupling constant was observed between H-1 and H-2 (J=3.6 Hz), and large coupling constants were observed between H-2 and H-3 (J=10.0 Hz), between H-3 and H-4 (J=9.6 Hz) and between H-4 and H-5 (J=10.0 Hz). Moreover, the ROESY correlations were observed for H-2/H-4 and H-3/H-5 (Figure 4). These results indicated that the carbohydrate moiety of 1 is a α-glucopyranose. The HMBC correlations from H-1 (δH 5.21) to C-1′ (δC 95.1) indicated the linkage between C-1 and C-1′ of two glucopyranose units through an oxygen atom to form an α, α-1,1-glycoside bond, the same as trehalose. This was supported by characteristic IR absorption at 850 cm−1 and chemical shift of anomeric carbon (δC 95.1).8

Table 2 1H-NMR and 13C-NMR data of trehangelins A (1), B (2) and C (3) in CD3OD (400 MHz)
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Figure 3
figure 3

COSY and HMBC correlations of trehangelins A (1), B (2) and C (3).

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Figure 4
figure 4

Relative configuration of trehangelins A (1), B (2) and C (3).

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The HMBC correlations from H-9 (δH 6.11) to 8-CH3 (δC 20.8), from 8-CH3 (δH 1.95) to C-7 (δC 169.9), C-8 (δC 129.6) and C-9 (δC 138.2) and from 9-CH3 (δH 2.01) to C-8 and C-9 (Figure 3), together with the ROESY correlations for H-9/8-CH3 (Figure 4) indicated that 1 had an angelic acid (Z-2-methyl-2-butenoic acid) moiety. Finally, the HMBC correlation from H-3 to C-7 indicated linkage between C-3 and C-7 through an oxygen atom to form an ester bond. Therefore, the structure of 1 was elucidated as a 3,3′-diangeloyl trehalose, and 1 was designated as trehangelin A (Figure 1).

The absolute stereochemistry of the trehalose moiety of 1 was determined as described in the following. The trehalose purified from hydrolysate by the treatment of hydrazine hydrate was identified as D-(+)-trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside) by 1H NMR spectra; δH 5.15 (d, 2H, J 4.0 Hz), δH 3.85-3.72 (m, 8H), δH 3.61 (dd, 2H, J 4.0, 10.0 Hz), and δH 3.41 (t, 2H, J 9.2 Hz), ESI-MS; calcd for C12H22O11Na [M+Na]+ 365.1060, found [M+Na]+ 365.1034, and optical rotation; [α]D27= +170.8 (c=1, H2O).9

Structure elucidation of compound 2

The molecular formula of 2 was elucidated by ESI-MS to be C22H34O13 and identical to that of 1. The 1H and 13C NMR spectra data measured in CD3OD of 2 are listed in Table 2 and they were similar to those of 1. The 13C NMR and HSQC spectra showed 22 carbons, which indicated that the structure of 2 was asymmetric. Although the chemical shifts at H-2 (δH 3.68) and H-3 (δH 5.30) in 2 were almost the same as the chemical shifts at H-2 (δH 3.71) and H-3 (δH 5.48) in 1, the chemical shifts of H-2′ (δH 4.76) and H-3′ (δH 4.13) in 2 were actually different from those of 1. The HMBC correlations from H-3 (δH 5.30) to C-7 (δC 169.6) and H-2′ (δH 4.76) to C-7′ (δC 168.8) indicated linkage of C-3 and C-7 through an oxygen atom and C-2′ and C-7′ through an oxygen atom to form ester bonds (Figure 3). Therefore, the structure of 2 was elucidated as a 3,2′-diangeloyl trehalose, and 2 was designated as trehangelin B (Figure 1).

Structure elucidation of compound 3

The molecular formula of 3 was elucidated by ESI-MS to be C22H34O13 and similar to that of 1. The 1H and 13C NMR spectra data measured in CD3OD of 3 are listed in Table 2 and they were similar to those of 1. The 13C NMR and HSQC spectra identified only 11 carbons, which indicated that the structure of 3 was symmetric. However, the H-3 (δH 5.48) and H-4 (δH 3.56) in 1 were shifted at H-3 (δH 4.02) and H-4 (δH 4.91) in 3, respectively. The HMBC correlation from H-4 (δH 4.91) to C-7 (δC 168.8) indicated linkage of C-4 and C-7 through an oxygen atom to form an ester bond (Figure 3). Therefore, the structure of 3 was elucidated as a 4,4′-diangeloyl trehalose, and 3 was designated as trehangelin C (Figure 1).

Biological activities

The inhibitory effects of compounds 1, 2 and 3 on the hemolysis of RBCs induced by light-activated pheophorbide a were evaluated. All three compounds had no effect on RBCs under dark at a final concentration of 1 mg ml−1. However, as shown in Figure 5, compounds 1, 2 and 3 showed inhibitory activity against the hemolysis in a concentration-dependent manner, and the IC50 values of compound 1, 2 and 3 were estimated to be 0.1 mg ml−1 (0.2 mM), >1 mg ml−1 (>2 mM) and 0.4 mg ml−1 (0.8 mM), respectively. The compounds 1 and 3, those with a symmetric structure, showed more potent inhibitory activities than that of ascorbic acid (IC50 value, 0.4 mg ml−1 (2.3 mM)), but compound 2, which has an asymmetric structure, showed only slight inhibitory activity. On the other hand, no significant cytotoxic activities of compounds 1, 2 and 3 against the several cell lines were observed even at 100 μg ml−1 (Table 3).

Figure 5
figure 5

Inhibitory effects of trehangelins A, B and C on hemolysis of red blood cells by light-activated pheophorbide a. ●, trehangelin A; ▪, trehangelin B; ▴, trehangelin C; ○, ascorbic acid.

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Table 3 Cytotoxic activities of trehangelins A, B and C against several human cell lines
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Discussion

A endophytic rare actinomycete stain, P. rubra K07-0510, was isolated from roots of an orchid. This strain produced three new compounds 1, 2 and 3. All the three compounds consisted of a trehalose moiety and two angelic acid moieties.

Trehalose was first found from an ergot of rye by Wiggers in 1832.10 Trehalose is known to be one of the sources of energy in many living organisms and can be found in many organisms, including bacteria, fungi, insects, plants and invertebrates. Trehalose has protective activity against various natural stresses, such as dryness, freezing and osmopressure. Recently, it was reported that trehalose was capable of suppressing degradation of fatty acid by heat oxidation.11 The protective effect of trehalose on heat oxidation of linoleic acid was demonstrated to be an interaction between the positions 3 and 6 of trehalose molecules and the positions 9, 10, 12 and 13 on olefin residue of the linoleic acid molecule, using NMR analysis. The results suggest that the interaction with trehalose stabilizes the olefin residue of linoleic acid against heat oxidation.

In our study, the inhibitory effects of compounds 1, 2 and 3 on the hemolysis of RBCs induced by light-activated pheophorbide a were investigated. It is known that pheophorbide a activated with light leads to the production of a singlet oxygen and that cells suffer damage through oxidation of the cell membrane.12, 13 Compounds 1, 2 and 3 demonstrated inhibitory activity against the hemolysis of RBCs induced by light-activated pheophorbide a. If this inhibitory effect of these compounds is the same as the inhibitory action of trehalose against the heat oxidation of linoleic acid, it is considered that the compound 1, which contains two angelic acid moieties at 3 and 3′ positions of trehalose, should have decreased activity compared with compounds 2 or 3, which have two angelic acid moieties at position 2, 3 or 4. However, compound 1 had more potent inhibitory activity than compounds 2 and 3. Therefore, the inhibitory action of trehangelins on the hemolysis of RBCs induced by light-activated pheophorbide a may occur via a different mechanism than the mode of action of trehalose on the heat oxidation of linoleic acid.

Furthermore, the symmetric compounds 1 and 3 showed more potent activities than the asymmetric compound 2. Therefore, the positions of angelic acids bound to a trehalose molecule and the structural symmetry may be important for the hemolysis inhibition of RBCs induced by light-activated pheophorbide a.

Angelic acid is isolated from plants such as Alkanna tinctoria,14 carrots15 or Euphorbia species.16 In addition, angelic acid esters included in Roman chamomile, a popular herb, are used as an aromatherapy essential oil. It is known that angelic acid esters are especially oriented towards mood and the psyche, with cephalic and psychotropic characteristics.17 Thus, compounds 1, 2 and 3, consisting of trehalose and angelic acid, may have many other uses. In addition, we are particularly interested in knowing whether there is any physiological effects of these compounds produced by an endophytic actinomycete strain, P. rubra K07-0510, on the plants they are associated with. To our knowledge, this is the first report of the metabolites produced by the genus Polymorphospora.

Methods

General experiments

All solvents (EtOAc, CHCl3, EtOH, MeOH and CH3CN) were purchased from Kanto Chemical (Tokyo, Japan). Silica gel and Pegasil ODS were purchased from Merck (Darmstadt, Germany) and Senshu Scientific Co. (Tokyo, Japan), respectively. The Inertsil ODS-4 column was obtained from GL Science Inc. (Tokyo, Japan).

NMR spectra were measured in CD3OD using a Varian XL-400 spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts were expressed in p.p.m. and were referenced to CD3OD (3.31 p.p.m.) in the 1H NMR spectra and to CD3OD (49.0 p.p.m.) in the 13C NMR spectra. ESI-MS spectra were measured on a QSTAR Elite Hybrid MS/MS spectrometer (AB Sciex Co., MA, USA). IR spectra (KBr) were taken on a Horiba FT-210 Fourier transform IR spectrometer (Horiba Ltd, Kyoto, Japan). UV spectra were measured with a Hitachi U-2810 spectrophotometer (Hitachi, Tokyo, Japan). Optical rotation was measured on a JASCO model DIP-181 polarimeter (JASCO Co., Tokyo, Japan).

Taxonomic studies

The International Streptomyces Project media recommended by Shirling and Gottlieb18 and media recommended by Waksman19 were used to investigate cultural characteristics. Cultures were observed after incubation for 3 weeks at 27 °C. The morphological characteristics were observed by scanning electron microscope (JSM-5600, JEOL, Tokyo, Japan) after incubation on GPM agar for 2 weeks at 27 °C and fixation with 4% osmium tetroxide vapor. Isomers of DAP in whole-cell hydrolysates were determined by TLC, following the standard methods of Becker et al.20 and Hasegawa et al.21 Menaquinones were extracted and purified by the method of Collins et al.,22 then analyzed by LC/MS (JSM-T 100LP, JEOL) with a CAPCELL PAK C18 column (Shiseido, Tokyo, Japan) eluted with methanol/2-propanol (7:3). 16S rRNA gene sequence was amplified by PCR and sequenced on a DNA sequencer (Applied Biosystems 3130 Genetic Analyzer, Applied Biosystems, Foster, CA, USA) using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems), according to the manufacturer’s instructions.

Fermentation

Strain K07-0510 was grown and maintained on agar slants consisting of 1.0% starch, 0.3% NZ amine, 0.1% yeast extract, 0.1% meat extract, 1.2% agar and 0.3% CaCO3. A loop of spores of strain K07-0510 was inoculated into 100 ml of seed medium, consisting of 2.4% starch, 0.1% glucose, 0.3% peptone, 0.3% meat extract, 0.5% yeast extract and 0.4% CaCO3 (adjusted to pH 7.0 before sterilization) in a 500-ml Erlenmeyer flask. The flask was incubated on a rotary shaker (210 r.p.m.) at 27 °C for 3 days. A 1-ml portion of the seed culture was transferred to 500-ml Erlenmeyer flasks (total 18 l), each containing 100 ml of production medium, consisting of 2.0% soluble starch, 0.5% glycerol, 1.0% defatted wheat germ, 0.3% Ehlrich meat extract from Katsuwonus pelamis (Kyokuto Pharmaceutical, Inc., Tokyo, Japan), 0.3% dry yeast, 0.3% CaCO3 (adjusted to pH 7.0 before sterilization) and fermentation was carried out on a rotary shaker (210 r.p.m.) at 27 °C for 9 days.

Hydrazinolysis of trehangelin A (1)

Hydrazinolysis of the peripheral ester groups of compound 1 was performed for the determination of an absolute configuration of trehalose.23 Compound 1 (30 mg) in MeOH (3 ml) was treated with hydrazine hydrate (3 ml) for 72 h. The reaction mixture was concentrated by evaporation under vacuum, and trehalose was separated by partition between H2O and CHCl3 (each 3 ml). Trehalose (16.5 mg) was obtained from the H2O layer. Parts of trehalose were dissolved in D2O for measurement of 1H NMR spectroscopy, in H2O for optical rotation and in MeOH for MS spectrometry.

Biological assays

The inhibitory activities of compounds 1, 2 and 3 against photo-oxidative hemolysis of RBCs induced by light-activated pheophorbide a were measured in vitro. In addition, cytotoxic activities of compounds 1, 2 and 3 were evaluated.

Hemolytic assay: The hemolysis of RBCs induced by light-activated pheophorbide a was measured by a modified method of light irradiation system as previously described.12 Rabbit RBCs were obtained from Nippon Bio-Supply Center (Tokyo, Japan) and used within 7 days. The RBCs were washed three times with sterile phosphate-buffered saline (PBS) and made to a final concentration of 5% (v/v) in PBS. For the hemolysis inhibition assay, 20 μl aliquots of sample in PBS (at a final concentration of 0, 0.125, 0.25, 0.5, and 1 mg ml−1) and the 100 μl of 5% (v/v) suspension of RBCs in PBS were added (in triplicate) to the wells of round-bottom 96-well microplates (Corning Inc., Corning, NY, USA). After incubation for 5 min at 37 °C, the reaction was initiated by the addition of 80 μl pheophorbide a (at a final concentration of 5 μM). The plate was illuminated for 30 min under a light from an Eye Lamp (PRS500W, Iwasaki Electric Co., Ltd, Tokyo, Japan) or placed in the dark. In addition, a clear plastic water container was put between the lamp and the plate with the RBCs to prevent any temperature increase. After illumination, the RBCs were centrifuged at 900 g for 10 min. A 100 μl of each supernatant was transferred to a flat-bottom microplate (Corning Inc.). Hemolysis was determined by measuring the absorbance at 570 nm, which is due to hemoglobin released from RBCs, using a microplate reader (model ELx 808, BioTek Instruments, Inc., Winooski, VT, USA).

Cell viability assay: Cell viabilities of compounds 1, 2 and 3 were measured by Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) in four different cell lines, namely HEK-293FT (human embryonic kidney cell line), Panc-1 (human pancreatic cancer cell line), NCI-H1299 (human non-small cell lung carcinoma cell line) and HT-29 (human colon carcinoma cell line). Briefly, 2 × 104 cells were seeded in 96-well plates and cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries Ltd, Osaka, Japan) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. After culturing overnight, compounds 1, 2 and 3 in dimethyl sulfoxide or dimethyl sulfoxide alone were added into each well (at final concentrations of 100 μg ml−1). After 48 h of incubation at 37 °C, WST-8 solution was added to each well and incubated at 37 °C for 4 h.

After mixing the microplate, the cell viability was determined by measuring the absorbance at 450 nm. The absorbance of control cells (treated with 0.3% dimethyl sulfoxide) was considered as 100%. The rate of cell growth inhibition was calculated using the following formula: mean value of ((control group−treated group)/control group) × 100%. The effect of each concentration was determined in duplicate.

scheme 1

Purification of trehangelins A (1), B (2) and C (3).