Isolation of actinobacteria
A total of five soil samples have been obtained from rhizosphere of medicinal plants i.e., Asparagus racemosus, Withania somnifera, Salvia officinalis, Rouwolfia serprntina, and Ocimum sanctum from different locations of Lucknow, Uttar Pradesh, and kept in sterilized plastic bags at 4ºC (Lee et al. 2014). Eleven actinobacterial isolates representing different colony morphologies were isolated and cultivated on actinomycetes isolation agar for five to seven days at ±28℃ (Sharma and Thakur 2020).
Preliminary Antimicrobial screening of Actinobacteria
Primary screening was done by single streak of positive isolate of actinobacteria against pathogenic and Multi drug resistant (MDR) microbes streak at 90° on Mueller Hinton agar (Elbendary et al. 2018). The actinobacterial isolates were streaked as a parallel line on Mueller Hinton agar for bacteria and potato dextrose agar plates for fungi and incubated at 28˚C for 4-6 days (Ganesan et al. 2017). When actinobacterial strains properly grown on media, selected pathogenic bacterial strains i.e., Staphylococcus aureus (ATCC-6538), Pseudomonas aeruginosa (NCIM-5029), Salmonella abony (ATCC-6017), Klebsiella pneumoniae (NCIM-2957), Bacillus subtilis (MTCC-441) and Escherichia coli (MDR) (ATCC-25923) and pathogenic fungi i.e., Aspergillus niger (ITCC 545), Aspergillus flavus (MTCC277), Aspergillus parasiticus (MTCC-2796) were streaked at right angles to the previous streak of actinobacteria and incubated at 30˚C. The A measurement of the zone of inhibition was taken after 72 hours for fungi and 24 hours for bacteria. Seven actinobacterial isolates were found positive after primary screening and subjected to molecular characterization.
Molecular characterization of positive isolates
The selected isolates from the secondary screening were subjected to molecular characterization using 16S rRNA sequence amplification performed at Bio-kart India Pvt. Ltd, Bangalore. The 16S ribosomal sequence amplification was conducted using the primers F243 (5’GGATGAGCCCGCGGCCTA3’) and 1378R (5’CGGTGTGTACAAGGCCCGG 3’). Subsequently, the construction of phylogenetic tree by MEGA6 software, by applying the neighbor-joining DNA distance algorithm. The results of the microbial characterization revealed two isolates, i.e., LA2(R) Microbacterium Proteolyticum (MN560041) and LA2(O) Streptomycetes rochei (Zothanpuia 2015; Ganesan et al. 2017).
Production of secondary metabolites
The primarily screened actinobacteria with antimicrobial activities were used for the extraction of secondary metabolite. Production of secondary metabolites from actinobacteria was done by submerged state fermentation (SmF) (Salim et al. 2017). The isolates LA2(R) Microbacterium Proteolyticum and LA2(O) Streptomycetes rochei were inoculated in 500 ml flask containing 100 ml of ISP-2 medium (g/L): yeast extract, 4.0; malt extract, 10.0; dextrose, 4.0; Agar, 20.0; distilled water, 1000 mL and pH 7.3. The flasks with inoculated strains were incubated for 5-7 days at 200 rpm in rotary shaker. The cultures was centrifuged at 10,000 rpm after the growth, and its supernatant was used for future experimentations (Abd-Elnaby et al. 2016; Singh and Dubey 2020).
Green synthesis of silver nanoparticle
Silver nanoparticles in-vitro production, was carried out by using the 1 mM aqueous solution of 50 μl AgNO3, that was pre-mixed with 50 ml supernatants of actinobacteria at 8.5 pH (Abd-Elnaby et al. 2016). In rotary shaker at 200 rpm, and suspension was incubated in the dark at 37°C. for 5 days. To evaluate whether bacteria are involved in nanoparticle creation, the control tests involved running the process using un-inoculated medium and AgNO3 solution. Silver ions reduction was tested by taking samples at specified intervals, using a UV–Vis spectrophotometer, and monitoring the UV–Vis spectrum. The color of silver nitrate solution changed to yellowish brown in each reaction vessel, which was then incubated with actinobacteria supernatant (Azman et al. 2017; Al-Sheddi et al. 2018).
Characterization of synthesized silver nanoparticle
To demonstrate the formation of extracellular silver nanoparticles, UV–visible spectroscopy and transmission electron microscopy were used to examine synthesized AgNPs. The preliminary synthesis of AgNPs was authenticated using a UV–visible spectrophotometer (Shimadzu dual beam-model UV-1601 PC) with a wavelength of 1 nm. A drop of AgNps (suspension) was dried on a carbon-coated TEM copper grid at a step-up voltage of 80 kV with a TEM TecnaiTM G2 Spirit Bio-TWIN (FEI, Hillsboro, OR, USA) to assess the proportions of the inorganic core.
FT-IR spectroscopy of biogenic AgNPs
Existence of diverse functional groups was inveterate by Perkin-Elmer Spectrum 2 FT-IR (PerkinElmer Inc., Waltham, MA, USA), at the surface of AgNps. In this method, samples are held by a worldwide attenuated complete reflectance sampling instrument, which is diminished with respect to the entire surface, and is scanned through a transmission technique with a resolution of 4 cm-1 above the wave number range of 4,000–4500 cm-1. The FTIR aim is to measure how much light, at each wavelength, absorbed by the samples (Barapatre et al. 2016).
Adenocarcinoma cells from the National Centre for Cell Science (NCCS) in Pune, India, were obtained and Incubated at 37°C with 5 percent Carbon dioxide in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM/F12, HiMedia-AT155) medium supplemented with 10 percent foetal bovine serum (FBS, HiMedia-RM10432) and 1percent of antibiotic or antimycotic solution (HiMedia-A002) (Ravikumar et al. 2012).
To check the feasibility of cells, an MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was accomplished. It is a colorimetric assay used to determine the activity of cellular enzymes responsible for reduction of the tetrazolium dye MTT to formazan crystals, which results in a purple color. 1x104 cells/well were seeded in 96-well plate and were cultivated to about 70% confluency. Different concentrations of chemicals were used to treat the cells, secondary metabolite of LA2(R) and LA2(O) and their silver synthesized nanoparticles LA2(RN) and LA2(ON) and 5-FU for 24 hours. Further MTT was performed as described earlier by Hasan et. al. (Hasan et al. 2020).
ROS determination cell line of lung cancer
A solution of 2,7-dichlorofluorescein diacetate was used to measure the level of intracellular ROS production (carboxy-DCFDA, Molecular Probes-D369, Invitrogen). In a 24-well plate, cells were plated and treated with compounds (LA2(R), LA2(O), LA2(RN), LA2(ON) and 5-FU) for 24 hours. Image J software was used to evaluate the fluorescence strength of stained cells (Hasan et al. 2020).
DAPI staining to verify the impact of compounds on DNA integrity
4, 6-diamidine-2-phenylindole (DAPI), Molecular Probes-D1306, Invitrogen, staining was done as stated by the manufacturer. Briefly, the cells were cultured and treated with LA2(R), LA2(O), LA2(RN), LA2(ON) and 5-FU, in a 24-well plate, for 24 h. Image J software was used to evaluate fluorescence strength of stained cells (Hasan et al. 2020).
MMP staining (Mito Tracker Red) was used for the determination of the influence of compounds on membrane potential of mitochondria. After 24-hour exposure to compounds (LA2(R), LA2(O), LA2(RN), LA2(ON) and 5-FU). Cells (treated and untreated both) became stained after being washed twice in PBS for 30 minutes with 300 nM Mito Tracker Red CMXRos fluorescent dye (Molecular Probes-M7512, Invitrogen) at room temperature in the dark (Hasan et al. 2020). Fluorescent images after washing in PBS were obtained using a Thermo-Scientific EvosFLc Microscope at an excitation wavelength of 579 nm and an emission wavelength of 599 nm. By using the Image J software, we were able to determine the fluorescence intensity of the stained cells (Abd-Elnaby et al. 2016).
One-way ANOVA was performed using Graph Pad prism 5 for statistical analysis on the data. The results were provided as the mean standard deviation of at least three independent measurements (nonsignificant (ns), *p 0.05, ** p 0.01, *** p 0.001 against the untreated control).