Microbes have played a very important role in the production of natural antimicrobial drugs [1]. So far, more than 23,000 biologically active compounds produced by microbes have been reported [2]. Furthermore, 45% of the reported microbial metabolites is produced by actinomycetes, with ~75% of metabolites obtained from the genus Streptomyces [1, 3]. However, as the increasing problem of drug resistance and the human’s growing desire of health, the development of new antibiotics and anticancer agents has become a major problem to be solved [4]. In the continuation of our chemical and biological screenings of extracts libraries from endophytes (mainly actinomycetes) [5,6,7], the extract of Streptomyces sp. KIB-H1083, which is an endophyte isolated from traditional Chinese medicinal plant Diaphasiastrum veitchii, indicated antibacterial activity against Bacillus subtilis and Staphylococcus aureus. In this context, we describe the isolation, structure elucidation, cytotoxicity, and antimicrobial evaluation of two new glycosylated piericidins, glucopiericidinol A3 (1) and 7-demethyl-glucopiericidin A (2), as well as four known analogs from the fermentation broth of strain KIB-H1083.

The fermentation broth of strain KIB-H1083 was centrifuged to obtain supernatant and mycelia cake, which were extracted with EtOAc and acetone, respectively. Both extracts were combined and then purified using various chromatographic techniques. Two new glycosylated piericidin derivatives, glucopiericidinol A3 (1) and 7-demethyl-glucopiericidin A (2), as well as four known congeners BE-14324-113 (3) [8], glucopiericidin A (4) [9], glucopiericidinol A (5) [10], and piericidin A (6) [11] were obtained.

Compound 1 was obtained as faint yellow oil, with the molecular formula, C37H57NO15 (10 double-bond equivalents), as derived from high-resolution ESI mass spectrometry ([M + Na]+ at m/z 778.3633, calcd 778.3620). The IR spectrum exhibited absorption bands for hydroxyl (3420 cm−1) and aromatic ring (1606, 1575, and 1471 cm−1) functional groups. The 1H and 13C NMR (Table 1) spectra of 1 revealed the presence of 37 carbon atoms. The substructures of C-1 to C-2, C-4 to C-6, C-8 to C-10, C-12 to C-13, C-1′′ to C-6′′, and C-1′′′ to C-6′′′ was identified by the 1H-1H COSY spectrum (Fig. 1). In the HMBC experiment, correlations were observed from H3-17 to C-2, C-3, C-4, from H3-16 to C-6, C-7, C-8, and from H3-14 to C-10, C-11, C-12, thereby revealed the connection of these partial structures. Further HMBC correlations from H3-6′ to C-1′, C-2′, and C-3′, from H3-7′ to C-4′, and from H3-8′ to C-5′ defined a fully substituted pyridine ring. HMBC correlations from H2-1 to C-1′ and C-2′ revealed that C-1 to C-13 chain was connected to the pyridine ring at C-1′ (Fig. 1). The 13C-chemical shifts of C-3 and C-10 (74.3 and 94.0 ppm, respectively) suggested that both carbons were oxygenated. This was supported by the similarities of 1H and 13C NMR signals between 1 and the known compounds, glucopiericidinol A (5). [10] However, a set of signals at δC 100.4 (C-1′′′), 70.7 (C-2′′′), 71.8 (C-3′′′), 71.1 (C-4′′′), 72.3 (C-5′′′), and 62.8 (C-6′′′), which did not existed in the 13C NMR spectrum of 5, was exhibited in that of 1. On the other hand, the molecular weight variation (Δ 162) between 1 and 5 suggested that 1 possessed one more glycosyl group than 5. The 1H and 13C NMR data of 1 exhibited specific signals for the α-galactopyranose of 1 [δH 4.84 (d, J = 3.0 Hz, H-1′′′), 3.74 (m, H-2′′′), 3.75 (m, H-3′′′), 3.88 (m, H-4′′′), 3.85 (m, H-5′′′), and 3.69 (d, J = 6.0 Hz, H-6′′′). [12, 13] The galactopyranose in 1 is bonded to C-6′′ according to the correlations in the HMBC spectrum from H-1′′′ to C-6′′ (δC 67.3). The geometry of the disubstituted olefins ∆1 and ∆5 was determined to be E type, respectively, from the large vicinal proton coupling constant (J1, 2 = 15.3 Hz and J5, 6 = 15.5 Hz). The geometry of the trisubstituted olefins ∆7 and ∆11 was determined by ROESY experiments (Fig. 1). ROE correlations H-6/H-8 and H-10/H-12 indicated the geometry of ∆7 and ∆11 were all E type. Thus, the structure of 1 (6′′-O-α-galactopyranosyl glucopiericidinol A) was identified as shown in Fig. 2.

Table 1 1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data for compounds 1 and 2 in CD3OD
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Fig. 1
figure 1

Key 2D NMR correlations of compound 1

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Fig. 2
figure 2

The chemical structures of compounds 1–6

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7-demethyl-glucopiericidin A (2) was also obtained as faint yellow oil. Compound 2 was found to possess the molecular formula C36H55NO14 from the HRESIMS data (m/z 726.3698 [M + H] +, calcd for 726.3695), indicating an unsaturation index of ten. The 1H and 13C NMR spectra of 2 (Table 1) were quite similar to those of 1. Through the comparison of 13C NMR data at pyridine ring and sugar moiety, the structure of pyridine ring and sugar moiety was concluded to be the same with that of 1. However, a signal at δH 1.75 (s, H-16) observed in the 1H NMR spectrum of glucopiericidinol A3 (1) disappeared in that of 7-demethyl-glucopiericidin A (2). On the other hand, a signal at δH 6.05 (m, H-7), which was not existed in the 1H NMR spectrum of glucopiericidinol A3 (1), was exhibited in that of 7-demethyl-glucopiericidin A (2). Further HMBC correlation from H2-1 to C-2, C-3, from H-2 to C-1, C-4, C-17, and from H3-17 to C-2, C-3, C-4 revealed the side chain as shown in Fig. 2. This was supported by the NMR data similarities between 2 and 7-demethylpiericidin A1 indicated that both compound 2 and 7-demethylpiericidin A1 contained the same polyketide chain [14], and the HMBC from δH 4.24 to δC 93.8 showed the position (C-10) of the sugar moiety. Thus, the structure of 2 was identified as shown in Fig. 2.

Cytotoxic activities of the six piericidins (1–6) were evaluated against HL-60, SMMC-7721, A-549, MCF-7, and SW480 tumor cell lines by MTS method [15]. Compounds 1, 3, and 6 were inactive against all six tumor cell lines (IC50 values >40 μm). Compound 2 exhibited moderate cytotoxicity against SMMC-7721, A-549, and MCF-7 cell lines with the IC50 values of 21.66, 2.78, and 10.88 μm, respectively. Compounds 4 and 5 showed significant cytotoxicity to HL-60, SMMC-7721, A-549, and MCF-7 cell lines with the IC50 values ranging from 0.34 to 0.65 μm and from 2.08 to 9.92 μm, respectively (Table 2). It’s the first report for the cytotoxicity of compounds 4 and 5 to HL-60, SMMC-7721, A-549, and MCF-7 cell lines.

Table 2 Cytotoxic activities (IC50 [μm]) of compounds 1–6
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Compounds 1–6 were also assessed for their in vitro antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Xanthomonas oryzae pv. oryzicola, and Penicillium decumbens by the filter paper method [16]. The result demonstrated that compounds 1, 4, and 6 exhibited potent inhibitory activities against Staphylococcus aureus and Bacillus subtilis (Table 3). At the same time, compound 6 showed powerful inhibitory activities against Xanthomonas oryzae pv. oryzicola and Penicillium decumbens.

Table 3 Antimicrobial activities (zone [mm]) of compounds 1–6
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In this study, we have isolated and characterized two new glycosylated piericidins, glucopiericidinol A3 (1) and 7-demethyl-glucopiericidin A (2), together with four known piericidins from a plant-derived endophytic Streptomyces sp. KIB-H1083. All the isolates were evaluated for their cytotoxic and antimicrobial activities. Piericidins are some of the most common compounds exiting in the metabolite of actinomycetes. More than 40 piericidins, including nine piericidin glycosides, have been isolated to date [17]. Previous studies showed that piericidins are potent inhibitors of both mitochondrial and bacterial NADH-ubiquinone oxidoreductase (complex I), therefore leading to their strong antimicrobial activity. In addition, structure activity relationships studies indicate that the sugar component of the piericidin glycosides is important in modulating their physiological activities [18]. In this study, piericidin A (6) with no sugar moiety showed the most powerful antimicrobial activity without cytotoxicity, however, glucopiericidin A (4) has powerful antitumor activity. Above observation may indicate that the antimicrobial activity and cytotoxicity of different piericidins have distinct modes of action.

Glucopiericidinol A 3 (1). Faint yellow oil. [α]24.2 D + 35.6 (c 0.19, MeOH). UV (MeOH): 295 (3.89), and 220 (4.46) nm. IR (KBr): 3420, 2927, 1709, 1626, 1606, 1575, 1471, 1411, 1385, 1307, 1259, 1229, 1194, 1150, 1077, 1038, and 974 cm−1.1H- and 13C-NMR data, see Table 1. HRESIMS: m/z 778.3633 [M + Na] + (calcd for C37H57NO15Na+, 778.3620).

7-demethyl-glucopiericidin A (2). Faint yellow oil. [α]18.7 D + 61.9 (c 0.05, MeOH). UV (MeOH): 268 (1.49), 228 (2.05), and 204 (2.19) nm. IR (KBr): 3417, 3407, 2927, 1630, 1610, 1587, 1473, 1412, 1355, 1251, 1191, 1151, 1126, 1076, 1044, and 973 cm−1. 1H- and 13C-NMR data, see Table 1. HRESIMS: m/z 726.3698 [M + Na]+ (calcd for C36H55NO14Na+, 726.3695).