Background
Reactive Oxygen Species (ROS) are constantly generated as a byproduct during the electron transport chain in mitochondria [
1], the bane to all aerobic species. It is plausible that at low concentration, ROS play an important role in cell signaling including apoptosis and gene expression [
2]. A balance between production and removal of ROS is paramount to the survival of all aerobic life forms. However, the disequilibrium of oxidation status due to accumulation of free radicals creates an oxidative stress in intracellular milieu [
3]. Oxidative stress has been implicated in physiological aging [
4], diabetes [
5], development of neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease [
6], cardiovascular diseases [
7] and cancer [
8].
In order to antagonize the oxidative stress, antioxidants play an important role in the survival of aerobic species. Antioxidants act as electron donors to the highly reactive species and make them stable [
9]. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) have been used but their use is being restricted due to their low solubility, moderate antioxidant activity and negative health effects [
10]. In view of the importance of antioxidants, demand for natural antioxidants with potential beneficial effects on human health is increasing [
11,
12]. Thus far, plants have been the main source of natural antioxidants owing to their high antioxidant content [
13,
14]. However, microbial species are known as an immense reservoir of pharmaceutically active compounds and has gained increasing attention in drug discovery [
15,
16].
In the field of microbial drug discovery, actinobacteria have been greatly studied for their ability to make a wide range of novel and highly potent bioactive compounds; accounting for 45% of all the discovered bioactive metabolites [
17,
18]. The dominant and best-studied genus of this phylum is
Streptomyces which has a remarkable contribution to mankind since the golden era of drug discovery was initiated with the discovery of streptomycin from
Streptomyces griseus [
12,
19,
20]. At present,
Streptomyces accounts for 70–80% of relevant bioactive metabolites produced by more than 500 species with diverse biological activities such as antibacterial, antifungal, antioxidant, anticancer, anti-inflammatory and anti-parasitic [
21]. However, there are very limited studies on
Streptomyces with respect to phenolic compounds as antioxidants. So, there is need to screen more streptomycetes with potent free radical scavenging activity. Rhizospheric
Streptomyces have great importance in search of novel species and new bioactive compounds with diverse biological activities. Due to the presence of various complex interactions in the rhizosphere, microorganisms have coevolved with plants and show similar type of structure and function [
22]. Various studies reported the potential of
Streptomyces spp. from one such valuable region [
23‐
25].
In the light of this, during our screening programme for isolation of rhizospheric
Streptomyces spp. exhibiting different bioactivities, a potent streptomycete isolate indexed as TES17 was isolated from rhizospheric soil of tea (
Camellia sinensis L.; family Theaceae) plant collected from Palampur (Himachal Pradesh, India). Tea is the most popular and widely consumed beverage in world second to water [
26]. The numerous health benefits associated with tea consumption have been attributed to the free radical-scavenging capabilities of the most abundant compounds such as tea catechins (up to 30% of dry weight), quercetin and myricetin [
27‐
29].
Keeping this in mind, the tea rhizospheric soil strain TES17 was evaluated for antioxidant activities using various in vitro free radical scavenging assays and identified using polyphasic approach. To further support, DNA damage protective activity of Streptomyces TES17 was assessed using an in vitro DNA nicking assay, and cytotoxicity of the extract was evaluated on lung cancer cell line. The chemical constituents of the extract responsible for antioxidant activity were determined through ultra high-pressure liquid chromatography (UPLC) analysis.
Discussion
The ‘rhizosphere’, narrow and specific zone forms unique microhabitat from a bulk of the soil in terms of high nutrient availability, optimum pH and input of organic materials derived from root exudates [
48]. These conditions favor the abundance of diverse microbial community in the rhizosphere. In well-studied rhizosphere, root exudates play an important role in enhanced biomass and activity of microorganisms [
22,
49]. The complex interactions between plants and microorganisms in the rhizosphere for carbon sequestration, ecosystem functioning and nutrient cycling, lead to the production of novel bioactive metabolites [
50]. Among microorganisms,
Streptomyces, a largest known genus of actinobacteria which commonly inhabit rhizosphere soil, has been greatly explored as producer of a wide variety of unique metabolites with interesting biological activities [
20,
51]. Therefore, it is an efficient approach to explore
Streptomyces spp. from such environment for the discovery of novel bioactive compounds.
In the present study,
Streptomyces strain TES17 isolated from rhizosphere of tea (
Camellia sinensis) was characterized through the polyphasic approach which included morphological, physiological, biochemical molecular characterization, and phylogenetic analysis. It formed branched short spore chains having a rough surface as observed via SEM studies. The strain was capable to survive under unfeasible growth conditions because it could tolerate NaCl concentration of 2.5%, temperature up to 50 °C and pH 12.0. Furthermore, strain TES17 also could have the potential to gain attention in industrial sector due to the production of industrially important enzymes such as cellulase, amylase, and β-galactosidase. The type and quantity of secondary metabolites are greatly influenced by the availability of substrates given during the growth [
52]. The results revealed that strain was capable to utilize different carbon sources such as glucose, arabinose, malonate, xylose, rhamnose, and cellobiose. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TES17 is closely related to
Streptomyces cellulosae (AB184265) as they showed 100% sequence similarity and formed a distinct clade at the bootstrap value of 64% which was significantly higher than the threshold value of 50%. However, phenotypically the strain TES17 is different from reference strain
Streptomyces cellulosae (AB184265) in terms of having rough spore surface [
53]. Overall, the strain TES17 could belong to same species as
S. cellulosae (AB184265) based on 16S rRNA sequencing. To the best of our knowledge,
S. cellulosae strain TES17 has not been reported earlier for the antioxidant, DNA damage protective and anticancer activities.
Oxidative stress and other neurodegenerative diseases are associated with accumulation of free radicals or ROS [
54,
55]. The process of oxidation may occur via different types of radicals which have different types of reaction mechanisms based on their interaction with surrounding molecules such as electron donation, reducing radicals, and electron acceptance [
55,
56]. Hence, to assess the overall antioxidant potential of the extract, a total of six assays which elicit different mechanism of actions were performed. These assays demonstrated that the TES17 extract exhibited DPPH radical scavenging activity, ABTS radical scavenging activity, superoxide anion scavenging activity, reducing power of ferric ions and molybdate ions and ability to inhibit lipid peroxidation.
Simplest and robust methods to screen antioxidant activity are DPPH and ABTS assays. These assays involve stable free DPPH and ABTS• + radicals. Hydrogen donating potential of antioxidants is responsible for their effects on DPPH and ABTS radicals. The transfer of hydrogen atom or electron by the antioxidant molecule to the DPPH and ABTS radical leads to the decolorization of violet and bluish-green color, respectively [
57]. Besides the hydrogen donating ability, TES17 extract also has the tendency to scavenge the superoxide anion radical (O
2•-). Excessive generation of O
2•- (directly or indirectly) involves in the formation of other highly reactive hydrogen peroxides (H
2O
2), notorious hydroxyl radicals (OH
•), peroxynitrite (ONOO
−) or singlet oxygen species during the process of aging and pathological events which ultimately contribute to oxidative stress and carcinogenesis [
58]. Thus, the increased production rate of O
2•- overwhelms the capacity of superoxide dismutase enzyme of an internal defense system. So, the need to control O
2•- production to prevent oxidative stress is of great importance [
59].
Some of the assays including total reducing power, total antioxidant activity, total phenol content and total flavonoid content are the direct measure of presence of the phenolic compounds. Total reducing power of extract was determined by measuring the reduction of potassium ferricyanide (Fe
3+) to potassium ferrocyanide (Fe
2+) which further undergoes reaction with ferric chloride to produce ferric ferrous complex (which has intense blue color). The higher reducing potential of the extract was indicated by the increase in absorbance. Similarly, in molybdate ion reduction assay antioxidant compound reduces Mo (VI) to Mo (V) that results in the formation of green colored phosphate/ Mo (V) complex at acidic pH that determines the process of donation of electrons [
42,
60].
The targets of ROS are mainly proteins, DNA, RNA molecules, sugars and lipids [
61]. Lipids having many numbers of C=C bonds undergo easier oxidative deterioration resulting in the formation of monounsaturated (MUFA) and saturated fatty acids (SFA), more resistant to radicals than polyunsaturated fatty acids (PUFA). The process of lipid peroxidation is initiated by an attack towards a fatty acid’s side chain by a hydroxyl radical (OH
•) produced by the interaction between hydrogen peroxide and iron metal ions present in Fenton reaction. This process abstracted a hydrogen atom from a methylene carbon which further undergoes molecular rearrangements and forms a peroxyl radical. The latter facilitates the production of the carcinogenic and mutagenic product MDA [
55]. The reduction in MDA level by inhibiting the ferryl-perferryl complex and quenching the OH
• confirmed the significant role of TES17 extract in inhibiting lipid peroxidation [
62].
To further support the antioxidant potential, this study demonstrated the DNA protective effect of TES17 extract using oxidative stress induced DNA damage model. In DNA nicking assay, O
2•- radical is produced by the autoxidation of Fe (II) which further generates OH
• by a rapid reaction of H
2O
2 in the presence of ascorbic acid as catalyst at pH 7.4. Ascorbate plays the role of reducing Fe (III) to Fe (II) making Fenton reaction to take place [
63,
64]. A decrease in the single stranded or double stranded nicked (Form II) and linear forms of DNA (Form III), and simultaneous increase in the native supercoiled form (Form I) in the presence of extract confirmed protective effect of TES17 against ROS induced DNA damage.
Oxidative stress ultimately initiates cancer progression by various modifications in the biological molecules which eventually lead to increased mutation rate [
65]. Various antioxidant assays revealed that the extract TES17 produced such bioactive compounds which could be further used as chemopreventive drugs to reduce cancer. An ideal chemotherapeutic drug should have high specificity i.e. able to differentiate cancer and normal cells. However, many of the drugs in use are still lacking in the drug specificity as they kill both cancer as well as normal cells [
66,
67]. This study investigated the specificity of TES17 extract which revealed that the extract was highly toxic to A549 lung cancer cell line (25.3 ± 1.52 to 22.72 ± 0.34% viable cells) as compared to normal cell line (87.71 ± 6.66 to 85.41 ± 3.14% viable cells) at the different tested concentrations. These key findings could provide useful information for future development of
S. cellulosae strain TES17 as the producer strain of anticancer drugs.
The correlation studies between the antioxidant assays, the total phenolics and favonoids in the TES17 extract suggested that phenolic compounds (both phenolic acids and flavonoids) made a significant contribution to the antioxidant potential of
Streptomyces TES17. Further confirmation of phenolic compounds in the TES17 extract was done using HPLC analysis which is a powerful analytical tool and widely used for the detection of phenolic compounds based on the particular retention time [
68]. Phenolic acids are derivatives of hydroxybenzoic acids and hydroxycinnamic acids such as gallic acid, caffeic acid and coumaric acid [
69]. Second class of phenolic compounds are flavonoids which are mainly characterized as containing two phenolic rings (Ring A and Ring B) linked by another oxygenated heterocycle which is three carbon bridge (Ring C), forming a common diphenylpropane (C6-C3-C6) skeleton structure. Based upon the saturation level of the C ring, flavonoids are classified into different subclasses
viz. flavonols, flavanols, anthocyanins, isoflavonoids, flavanones and flavones. Individual compounds within a subclass differ in the substitution pattern of hydroxyl groups present in the A and B rings that influence the free radical-scavenging properties of the phenolic compounds [
70]. Some of the most important flavonoids are catechin, rutin, quercetin and kaempferol. Both phenolic acids and other flavonoids act as antioxidants by chelating the ions and scavenging he free radicals particularly, superoxide (O
2●-), peroxyl and hydroxyl radicals (OH
●) and hence inhibit both DNA damage and lipid peroxidation, which can cause membrane damage.
UPLC analysis of extract of
S. cellulosae strain TES17, isolated from tea rhizosphere, revealed that among nine phenolic compounds [
viz. catechin, epicatechin, quercetin and kaempferol (flavonoids), gallic acid, caffeic acid and coumaric acid (phenolic acids) and umbelliferone (coumarine)], catechins were the principal phenolic compounds. Catechins are reported to be the main phenolic compounds responsible for antioxidant activity of the tea. The principal catechins present in tea leaves are epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), gallocatechin (GC), epicatechin (EC), and catechin [
28,
71]. These results suggest that there could be gene transfer between tea plant and rhizospheric microflora which further elaborate the potential of
Streptomyces TES17 in search of phenolic compounds with interesting bioactivities.