1. Introduction to Endophytic actinobacteria

Endophytic actinobacteria affiliated with medicinal plants have been shown to have the potential to inhibit or kill pathogenic bacteria, fungi, and viruses. Thus, they are considered a significant source for the development of new antimicrobial products ^[1]. In our earlier studies, we demonstrated that endophytic bacteria could be a potent source for secondary metabolites with bioactive potential ^[2][3][4][5]. Additionally, bioactive compounds with cytotoxic and antioxidant properties produced by endophytic actinobacteria associated with plants have also been reported. Among these are endophytic actinobacteria, which exhibit cytotoxicity against several cancer cell lines ^[6][7]. Tanvir et al. ^[8], reported antioxidant activity in most of the endophytic actinobacteria (66.6%) that were recovered from different medicinal plants. Presently, researchers are continuing to search for novel actinobacteria with antioxidant properties for therapeutic use. Mirabilis jalapa L., a member of the Nyctaginaceae, is a traditional medicinal plant whose plant parts can be used to make a drink that is orally consumed 2–3 times a day (10–15 mL) for the treatment of kidney and urinary infections ^[9]. Rozina ^[10] reported that M. jalapa had several pharmacological functions, including antimicrobial, antimalarial, antioxidant, cytotoxicity, and antifungal properties.

In the previous study, six endophytic isolates (Streptomyces sp. strain DBT33; Streptomyces sp. strain DBT34; Brevibacterium sp. strain DBT35; Streptomyces thermocarboxydus strain DBT36; Actinomycete strain DBT37 and Streptomyces sp. strain DBT39) obtained from M. jalapa were tested for antimicrobial activities against three bacterial pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans). Isolate DBT35 showed significant antimicrobial activities against S. aureus (13.7 mm) and P. aeruginosa (10.1 mm), whereas BPSAC39 (10.6 mm) and BPSAC37 (9.6 mm) exhibited acute activities against P. aeruginosa and C. albicans, respectively. Although isolate DBT34 showed lower antimicrobial activities against all bacterial pathogens, isolate DBT34 showed strong antifungal activities against plant fungal pathogens Rhizoctonia solani, Fusarium graminearum and Fusarium oxysporum. In addition, isolate DBT34 showed maximum PGP activity in comparison to other isolates. The potent isolate DBT34 was used for an in-vivo plant growth promotion study under greenhouse conditions in an effort to enhance the growth of Capsicum annuum L. ^[3][4]. Notably, however, no systematic study has been conducted to understand the cytotoxicity, antioxidant, and antidiabetic potential of the endophytic actinobacteria associated with M. jalapa. Hence, we have selected the strain DBT34 to be evaluated for its antioxidant potential to alleviate the oxidative stress in their host plants by scavenging the ROS. Additionally, DBT34 was also selected to experimentally assess its anticancer ability against liver hepatocellular cancer cells (HepG2) lines and also antidiabetic capabilities. Also, a methanolic extract of isolate DBT34 was used to identify the volatile compounds (VOCs) that are potentially related to functional mitigation of stress in plants. The majority of researchers have reported antioxidant and antidiabetic potential from diverse bacteria, but there have been few reports from actinobacteria. Hence, we have added to evaluate an endophytic Streptomyces sp. isolate which has the potential for antioxidant, cytotoxicity, and antidiabetic capabilities. 

2. Conclusion

A phylogenetic tree was constructed using the neighbor-joining method to evaluate the similarity of Streptomyces sp. strain DBT34 with other species of Streptomyces. Results indicated that the sequence of strain DBT34 was highly analogous to Streptomyces glauciniger type strain CGMCC 41858 (99.51%) followed by Streptomyces erringtonii type strain I36 (98.92%) and Streptomyces avellaneus type strain NBRC13451 (97.35%). In a similar analysis, Tan et al. ^[11][12] reported that Streptomyces sp. strain MUM256 exhibited a maximum 16S-rRNA gene sequence identity to Streptomyces albidoflavus DSM40455T (99.7%) and Streptomyces hydrogenans NBRC13475T (99.7%).

Streptomyces sp. strain DBT34 extract was evaluated for its ability to scavenge superoxide anion (O2•−)radicals, which can impact the synthesis of other ROS intermediates. SOD-like activity mitigates the accumulation of ROS intermediates by converting superoxide anion radicals to the lesser toxic entity, hydrogen peroxide. Hydrogen peroxide can then be neutralized by catalase, which turns into a neutral form [24]. Therefore, the present study evaluated catalase and SOD-like activity present in the methanolic extracts of DBT34 at a concentration of 50 µg extract/mL. These results are comparable to the level of inhibition reported for a microbial extract by Tan et al. ^[13]. In that study, the level of SOD-like activity was evaluated in methanolic extracts of Streptomyces sp. (MUM212). Their results indicated that SOD-like activity in methanolic extracts at a concentration range of 0.25 to 4 mg/mL exhibited a percentage of inhibition of superoxide radical formation ranging from 17 to 37%. Similarly, catalase-like activity was observed in methanolic extracts of Gnaphalium polycaulon Pers. plants ^[14].

Phenolic compounds are a significant group of antioxidants that also scavenge ROS. Cruz De Carvalho, ^[15] suggested that microorganisms produce ROS oxygen species as byproducts during the metabolism process. High levels of free radicals can induce oxidative stress and many other side effects, including the promotion of cancer ^[16]. It has been well established that free oxygen radicals can contribute to the development of a variety of diseases, including cancer, as well as neurodegenerative and cardiovascular diseases, which can be treated with the use of novel antioxidant compounds. In the present study, the total phenolic content in the methanolic extract of Streptomyces sp. strain DBT34 was estimated at 94.21 µg of GAE/mg of DW. These data represent a higher level than what was previously reported by Kaur et al. ^[17], who stated that an ethyl acetate extract of Streptomyces sp. strain OEAE (isolated from the soil) possessed a level of TPC of 84.3 mg of GAE/g DW. Lertcanawanichakul et al. ^[18] stated that the maximum amount of total phenolics (0.24 GAE ± 0.02 mg/g DW) was found in an ethyl acetate extract of Streptomyces sp. strain KB1-ET. Additionally, the maximum TFC in KB1-ET was 112.6 µg quercetin/mg DW. Our results represent the first report of the total phenolic content (TPC) and total flavonoid content (TFC) in a methanolic extract of Streptomyces sp. with the highest TPC and TFC exhibit the highest antioxidant activity ^[19].