A new cytotoxic and anti-fungal C-glycosylated benz[α]anthraquinone from the broth of endophytic Streptomyces blastomycetica strain F4-20

Endophytes reside in the tissues of living plants without exerting any pathogenic effects.¹ Endophytes produce a great number of secondary metabolites with diverse chemical structures and various biological activities, which have been implicated in the protection of their hosts against pathogens and herbivores.² Endophytic microorganisms are an excellent source of structurally diverse molecules with potential therapeutic value.

In this study, a new C-glycosylated benz[α]anthraquinone, dehydroxyaquayamycin B (1), along with two known alkaloids, teleocidin B2 (2) and N-methyl-l-valyl-l-tryptophanol (3), was isolated from the endophytic Streptomyces blastomycetica strain F4-20 (Figure 1). The new compound was tested for cytotoxic and anti-fungal activities. The strain F4-20 was isolated from the root of Tripterygium wilfordii Hook. f., a medicinal plant in China, by spreading on actinomyces isolation agar from Difco (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), consisting of 0.05% dipotassium phosphate, 0.0001% ferrous sulfate, 0.01% magnesium sulfate, 0.05% sodium chloride, 0.1% potassium nitrate, 0.2% soluble starch and 1.5% agar, pH 7.2–7.4, and incubated at 28 °C for 7–14 days. The strain was identified as a member of the genus Streptomyces because its 16S rRNA sequence exhibited 99% similarity to Streptomyces spectabilis NRRL B-5480, and has been deposited in the Research and Development Center of Biorational Pesticide, Northwest A&F University, with the accession no. F4-20.

Figure 1

Chemical structures of dehydroxyaquayamycin B (1), teleocidin B2 and N-methyl-l-valyl-l-tryptophanol.

The strain was cultivated on the optimized solid medium containing glucose (Bei Jing Ao Bo Xing, Beijing, China) 20 g, potato 200 g, beef extract 10 g, KH₂PO₄ 1 g, ammonium sulfate 1 g, NaNO₃ 1 g, NH₄Cl 1 g and agar 17 g in 1.0 L tap water, pH 8.0–8.5. The spore suspension was then filtered through six layers of sterile filter cheesecloth and adjusted to 10⁷–10⁸CFU ml⁻¹. A 2.0 ml of the spore suspension was inoculated into a 250-ml flask containing 100 ml of seed medium consisting of glucose (Bei Jing Ao Bo Xing) 4 g, malt extract powder (Cormwin, Beijing, China) 10 g and yeast autolysate (Bei Jing Ao Bo Xing) 4 g in 1.0 L tap water, pH 7.3, and incubated at 28 °C for 24 h, with shaking at 140 r.p.m. Then, each 8.0 ml of the cultured seed liquid were transferred into 340 250-ml Erlenmeyer flasks containing 150 ml of the sterile fermentation medium consisting of glucose (Bei Jing Ao Bo Xing) 0.4%, malt extract powder (Cormwin, Beijing, China) 1% and yeast autolysate (Bei Jing Ao Bo Xing) 0.4%, pH 7.3. Fermentation was carried out at 28 °C for 7 days on a rotary shaker at 140 r.p.m.

The final 50 L of broth was filtered and evaporated under reduced pressure to 1 L at 55 °C and the resulting concentrate was extracted three times using an equal volume of EtOAc. The EtOAc-soluble fraction (9.5 g) was applied to silica gel column (200–300 mesh; Qingdao Marine Chemical, Qingdao, China) eluting with a CHCl₃-MeOH gradient (10:0, 20:1, 9:1, 8:2, 7:3, 1:1 and 0:1) to give five fractions A–E. The separation of fraction C (2.4 g) over silica gel column (200–300 mesh; Qingdao Marine Chemical) was eluted with petroleum ether–acetone (30:1–4:1) to yield fractions C-1–C-7. Fraction C-3 (0.21 g) was subjected to a reversed-phase column (RP-18) eluting with MeOH–water (20–90%) to afford four subfractions (C-3-1–C-3-4). C-3-2 (0.08 g) was subjected to semipreparative reversed-phase HPLC (Shimadzu LC20A apparatus equipped with a UV detector and a Hypersil BDS C₁₈ (Thermo, Shanghai, China; 250 × 10 mm²)) to give 1 (29.7 mg) and 2 (2.4 mg). C-3-3 (0.017 g) was subjected to semipreparative reversed-phase HPLC to give 3 (1.4 mg).

Compound 1 was isolated as an optically active amorphous red solid. Its molecular formula C₃₇H₄₂O₁₁ was determined by the ESI-HRMS (API QSTAR Pulsar mass spectrometer; VG, Manchester, UK), owing to the presence of a pseudomolecular ion peak at m/z 685.2623 [M+Na]⁺ (calcd. for C₃₇H₄₂O₁₁Na, 685.2625). The IR spectrum (Tensor 27 FT-IR spectrometer with KBr pellets) showed absorption bands at 3387, 1686, 1590, 1281 and 1268 cm⁻¹, indicative of the existence of hydroxyl and carbonyl groups. Analysis of the ¹³C and DEPT NMR spectra (DRX-500, ¹H: 500 MHz; ¹³C: 125 MHz) revealed the presence of two ketone carbonyl groups (δ_C 188.25, C-7 and δ_C 189.93, C-12), 10 sp² quaternary, 6 sp² methine, 10 sp³ methine, 5 sp³ methylene and 4 sp³ methyl carbons. The ¹H NMR spectrum of 1 also showed 19 well-resolved resonances that comprised of 6 sp²-hybridized methines, 10 sp³-hybridized methines, 5 sp³ methylene and 4 sp³-hybridized methyls, consistent with an angucycline core and three glycosidic residues. Two broad singlets at δ_H 12.70 and 11.45, representing peri-hydroxy groups, and two 1,2,3,4-tetrasubstituted aromatic moieties were revealed by two AB systems (δ_H 7.92 and δ_H 7.97, δ_H 8.18 and δ_H 8.36). Two additional broad aromatic signals, each ¹H, at δ 7.19 and 7.30, showed another highly substituted aromatic ring with two m-coupled aromatic protons. Furthermore, a singlet of an aromatic-bound methyl group was observed at δ_H 2.54. The ¹H and ¹³C NMR also revealed the presence of three saccharide moieties (three anomeric ¹H singlets, δ_H 5.10–4.89) (Table 1). The HMBC spectrum displayed correlations from the sp³ methine proton at δ_H 4.96 (H-2′) to two carbons at δ_C 137.9 (C-9) and 133.3 (C-10), suggesting a C-glycosidic linkage. Further examination of these NMR data exhibited comparability of dehydroxyaquayamycin,³ which was originally obtained upon successive hydrogenation and acidification of aquayamycin. The only difference between compound 1 and dehydroxyaquayamycin was the longer saccharide moiety at C-9 position.

Table 1 ¹H and ¹³C NMR spectral data (in acetone-d₆) of dehydroxyaquayamycin B

The saccharide moieties were deduced from detailed analyses of the one- and two-dimensional NMR data (¹H-¹H COSY, HMQC, HMBC and NOESY spectra) of 1. The anomeric proton at δ 4.96 (d, J=11.2 Hz, H-2′) showed large coupling constant and thus represented β-d-glycoside moieties. The remaining two anomeric protons at δ_H 5.10 (brs, H-2″) and δ_H 4.89 (brs, H-2‴) were α-glycosidically linked l-sugars. The COSY spectrum revealed the spin systems extending from H-2′ to H-7′ and HMBC spectrum displayed correlations from the sp³ methine proton at δ_H 4.87 (H-2″) to two carbons at δ_C 137.9 (C-9) and 133.3 (C-10). The latter suggested that this β-d-glycoside moiety was linked at the C-9 position. The dd peak at δ_H 3.25 (H-5′) gave coupling constants of 8.87 and 8.87 Hz, indicating the axial orientation. Hence, the methyl at C-7′ and two hydroxyl protons at C-4′ and C-5′ are in equatorial positions. NOESY spectrum, showing the cross-peak correlations from H_ax-2′ to H_eq-4′ and H_ax-6′, also confirmed the above assumption. Thus, the first hexose moiety that linked to C-9 by a C-glycosidic linkage was identified as a β-d-olivose unit. The COSY spectrum also revealed the spin systems extending from H-2″ to H-7″. Wide single peaks at 3.60 (H-5″) indicated the equatorial orientation of this proton. In consideration of NOESY cross-peak correlations from H_ax-3″ to H_eq-2″ and H_ax-6″, this hexose moiety could be deduced to l-rhodinose. The last sugar showed the same signal patterns and connectivity as l-rhodinose. HMBC correlations were used to establish the structure of the side chains and their points of attachment. The anomeric proton (δ_H 4.96) of the olivose moiety showed an HMBC correlation to C-9 (δ_C 137.9) and 133.3 (C-10) of the aglycone, whereas an HMBC correlation between the anomeric proton of the second l-rhodinose moiety (δ_H 5.10) and the carbon at δ_C 76.21(C-5′), indicating that the second as rhodinose unit was attached to C-5′ of the olivose. The anomeric protons of the third l-rhodinose moiety (δ_H 4.89) showed HMBC correlations to C-5″ (δ_C 74.56) of the second l-rhodinose unit, indicating that the third as a rhodinose unit was attached to C-5″ of the second l-rhodinose (Figure 2). Thus, the gross structure of 1 was assigned as shown in Figure 1, and this new compound was named as dehydroxyaquayamycin B.

Figure 2

HMBC connectivities (→), ¹H,¹H COSY correlations (bold lines) and diagnostic NOESY couplings (←→) of dehydroxyaquayamycin B (1).

Cytotoxicity of compound 1 against cancer cell lines BGC-823 and HeLa were tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Kit (Promega, Madison, WI). In these tests, dehydroxyaquayamycin B showed strong inhibitory activity on the proliferation of BGC-823 and HeLa cells with the half maximal inhibitory concentration (IC₅₀) value of 0.71 and 1.34 μg ml⁻¹, respectively.

Fungicidal activities of the compound 1 against Valsa mali, Colletotrichum orbiculare, Fusarium graminearum, Rhizoctonia cerealis, Botrytis cinerea, Sclerotinia sclerotiorum and Penicillium italicum were evaluated in vitro using the mycelium growth rate method⁴ with 50 μg ml⁻¹ (Table 2). The results are given in Table 2. Results indicated dehydroxyaquayamycin B showed appreciable anti-fungal activity against V. mali, C. orbiculare and F. graminearum with the inhibition rate of 41.5%, 58.3% and 51.0%, respectively.

Table 2 Fungicidal activities of 1 with 50 μg ml⁻¹

Dehydroxyaquayamycin B is a benz[α]anthracene glycoside with a C-glycosidic substituent on C-9 position. Benz[α]anthraquinones have been isolated from various actinomycetes, especially in the genus Streptomyces. The first benz[α]anthraquinones, tetrangomycin and tetrangulol, were isolated from Streptomyces rimosus in 1966. These type of compounds have gathered attention because of their structural diversity and significant biological activity. Biological activities of benz[α]anthraquinones such as anti-Helicobacter pylori,⁵ anti-fungal,⁶ anti-Gram-positive bacterial,^{7, 8} anticancer,⁹ anti-bacterial¹⁰ activities have been reported. For example, urdamycins, isolated from Streptomyces fradiae strain Tu 2717, displayed biological activities including inhibition of platelet aggregation, anti-microbial activity for Gram-positive bacteria and anticancer against stem cells of murine L1210 leukemia.⁹

Naturally occurring C-glucosylated benz[α]anthraqinones at C-9 such as YM-181741 from Streptomyces sp. showed anti-Helicobacter pylori activity with an minimum inhibitory concentration value of 0.2 mg ml⁻¹, but was inactive against Gram-positive and -negative bacteria.⁵ Urdamycinones E, G and dehydroxyaquayamycin exhibited antimalarial and antitubercular activities.³

Dehydroxyaquayamycin B exhibited considerably cytotoxic activities on the proliferation of BGC-823 and HeLa cells with the IC₅₀ value of 0.71 and 1.34 μg ml⁻¹, respectively. It should be noted that naturally occurring C-glucosylated benz[α]anthraqinones such as urdamycinone E, urdamycinone G, dehydroxyaquayamycin and urdamycin E also possess strong cytotoxic activities with IC₅₀ value of 0.092 and 0.242 μg ml⁻¹ against NCI-H187 cells, revealing a promising potential of C-glucosylated benz[α]anthraqinones as new lead compounds for antitumor. Dehydroxyaquayamycin B also showed an appreciable anti-fungal activity against V. mali, C. orbiculare and F. graminearum, and this was the first report of fungicidal activities of C-glycosylated benz[α]anthraquinones.

In conclusion, the natural product dehydroxyaquayamycin B is a new benz[α]anthracene glycoside that was isolated from the the broth of endophytic S. blastomycetica strain F4-20. Dehydroxyaquayamycin B showed considerable cytotoxic activities. The results presented in this paper highlighted endophytic actinomycetes as a rich source of bioactive compounds, and this was the first report of the secondary metabolites of Streptomyces blastomycetica genus.