*3.4. Biological Activity*

The cytotoxic activities of compounds 1–8 against K562, HCT-116, and HepG2 cancer cell lines are showed in Table 3. Eight compounds restrained proliferation of the tested cells and compound **1** showed the highest cytotoxic activity, and the average IC50 values were lower than 10.0 μg/mL.


**Table 3.** The cytotoxicity of compounds 1–8.

The result of minimum inhibitory concentrations (MICs) showed that compound **1** showed good activities against Gram-positive bacteria *Staphylococcus aureus*, *Sarcina lutea*, and *Bacillus subtilis*, and the Gram-negative bacteria *Klebsiella pneumoniae* in vitro (Table 4). Compound **8** showed weak antibacterial activity against two Gram-positive bacterium and the minimum inhibitory concentrations (MICs) of compounds **2**–**7** were determined to be >10 mg/mL, so they had no activity against these tested pathogens.



#### **4. Discussion**

In this research, the results of morphological, physiological, and biochemical tests showed that strain NEAU-wh3-1 has typical characteristics of the genus *Embleya* [41]. Such as containing LL-diaminopimelic acid as the cell wall peptidoglycan, MK-9(H4), and MK-9(H6) as the major menaquinones, phosphatidylethanolamine (PE) as the predominant phospholipid and arabinose in the whole sugars. Moreover, strain NEAU-wh3-1 formed spiral spore chains and the spore surface was rough, which are consistent with *E. scabrispora* DSM 41855<sup>T</sup> and *E. hyalina* MB891-A1T [39,41]. In addition, the phylogenetic trees constructed from the 16S rRNA gene sequences and the concatenated sequences alignment (1979 bp) of five housekeeping genes all suggested that the isolate should be assigned to the genus *Embleya*.

However, some obvious differences could also be found between strain NEAU-wh3-1 and its closely related strains regarding several phenotypic and chemotaxonomic characteristics (Table 1). The isolate was able to grow at 4 ◦C, in contrast to its closely related strains, which were not. The composition of phospholipids and menaquinones of strain NEAU-wh3-1 was also different from its related species, *E. scabrispora* DSM 41855T, *E. hyalina* MB891-A1T and *S. lasii* 5H-CA11T. Most notably, the whole-cell sugars of strain NEAU-wh3-1 was found to contain arabinose, glucose, and ribose,

while *E. scabrispora* DSM 41855T only contains arabinose, *E. hyalina* MB891-A1<sup>T</sup> contains arabinose and glucose and *S. lasii* 5H-CA11T contains glucose and ribose, which also could distinguish the strain from its closely related strains. Other phenotypic differences include the temperature and pH range of growth, patterns of carbon and nitrogen utilization, hydrolysis of cellulose, starch, and Tweens (20, 40, and 80), liquefaction of gelatin, peptonization of milk, production of H2S, and urease and reduction of nitrate. Therefore, it is evident from the phenotypic, genotypic, and chemotaxonomic data that strain NEAU-wh3-1 may represent a novel species of the genus *Embleya*.

The genus *Embleya*, recently transferred from genus *Streptomyces*, is a new member of the family *Streptomycetaceae* [39,40]. At present, it contains only two species: *Embleya scabrispora*, could produce hitachimycin with antitumor, antibacterial, and antiprotozoal activities [44–46]; and *Embleya hyaline*, could produce nybomycin which is an effective agent against antibiotic-resistant *Staphylococcus aureus* and is called a reverse antibiotic [48]. During the study of the chemical properties of the active ingredients of strain NEAU-wh3-1, eight active compounds were obtained, including one macrolide dilactone, five piericidins, one β-hydroxy acetamides, an analogue of monocarboxylic acid ionophore, which were observed to fit into at least three types based on their molecular skeletons. This shows to some extent that this strain has the ability to produce metabolites with a wide variety of different skeletal structures. Out of these compounds, Piericidins (**3**-**7**) were a class of polyene alpha-pyridone heterocyclic antibiotics, among them, Piericidin A1(**5**) was first reported [81], which was isolated from *Streptomyces mobaraensis*. Piericidins exhibit interesting biological activities, in particular antitrypanosomal [82]. In our research, compounds **3**-**7** exhibited different degrees of cytotoxicity on three types of tumor cells, but they did not show any antibacterial activity, which was consistent with previous reports [78,81]. As a Piericidins-producing strain, NEAU-wh3-1 has certain application potential in pest control. Conglobatin (**2**) is an unusual 16-membered macrocyclic diolide originally isolated from a polyether-producing strain of *Streptomyces conglobatus* ATCC 31005<sup>T</sup> and was reported to be essentially devoid of antifungal, antibacterial, antitumor, and antiprotozoal activity at that time [75]. However, in recent research, FW-04-806 is identical in structure to conglobatin, and it has been reported to inhibit the growth of a human chronic myelocytic leukemia K562 cell line with an IC50 of 6.66 μg/mL, further study also investigated the effects of FW-04-806 on SKBR3 and MCF-7, respectively [83]. Its mechanism of action appears to be novel, via direct binding to the N-terminal domain of Hsp90 and disruption of its interaction with co-chaperone Cdc37 [84]. In our antitumor activity test, Conglobatin (**2**) showed good bioactivity against two tumor cell lines, supporting it at least partially accounted for the cytotoxic activity of the strain NEAU-wh3-1 extract. BE-52211 D (**8**) was a cytotoxic metabolite from a strain of *Streptomyces* and had been reported to have moderate cytotoxicity against human hepatocellular liver carcinoma cells HepG2, human leukemia cells K562, and human colon carcinoma cells HCT-116 with the IC50 values of >10 μg/mL [80], which is consistent with the result in the present study. Compound **1** structurally related to zincophorin, which was also referred to as M144255 or griseochellin and is a polyoxygenated ionophoric antibiotic isolating from *Streptomyces griseus* in 1984 [17]. It has been reported to possess strong in vivo activity against Gram-positive bacteria and have strong cytotoxicity against human lung carcinoma cells A549 and Madin-Darby canine kidney cells MDCK [18]. No biological activity has been reported against Gram-negative bacteria, yeasts, and fungi [85]. The second member in the zincophorin family named CP-78545, was found in the culture broth of *Streptomyces* sp. N731-45. The structural difference between them is that CP-78545 has an extra terminal double bond; but they have similar spectrum and potency on biological properties except for the antitumor activity (no reports) [86]. In our antitumor and antimicrobial assays, compound **1** showed the highest antitumor activity against three human cell lines and good antibacterial activity against Gram-negative bacteria. To our knowledge, this is the first report of this kind of compound with antibacterial activity against Gram-negative bacteria. This study has enriched the activity spectrum of Zincophorins.
