| Abstract |
Infectious (pathogen-associated) and non-infectious (non-pathogen-associated) diseases are causing major problems in worldwide. To combat these diseases various synthetic compounds have been used. But the scientists are continuously making efforts in searching new eco-friendly potential drugs that act efficiently against both pathogenic and non-pathogenic diseases. In the current research, Ajugabracteosa has been selected to synthesize silver nanoparticles because green synthesis has a low range of toxicity, eco-friendly, and increasing the interest in biomedical research such as in wound healing, drug delivery, molecular imaging and preparation of implantable biomaterial.Therefore, the aim of present study was to synthesize, characterize, and evaluate the biological activities (both in vitro and in vivo) of green synthesized silver nanoparticles (ABAgNPs) and A. bracteosa extract (ABaqu). The silver nanoparticles synthesis (ABAgNPs) was carried out via maceration and boiling methods. The characterization was done using UV-Viz spectroscopy, scanning electron microscopy, and Fourier-transform infrared spectroscopy. After synthesis, both qualitative and quantitative phytochemical screening of ABAgNPs and ABaquwere carried out. Thin-layer chromatography and spraying techniques were done for the phytochemicals detection. Total phenolic and flavonoid contents were determined. Antioxidant activity of ABAgNPs and ABaquwas measured through both ABTS•+ decolorization and DPPH scavenging assays. Minimum inhibitory concentrations of ABaqu extracts and silver nanoparticles ABAgNPs (0.02 g/ml, 0.04 g/ml, 0.06 g/ml, 0.08 g/ml, and 0.10 g/ml) were tested against bacterial pathogens 14 bacterial pathogens (Escherichia coli,Staphylococcus aureus,Serratiamarcesscens, xxvii Klebsiellapneumoniae, Pseudomonas aeruginosaStaphylococcus epidermidis(clinical samples associated), Lactobacillus delbruekiand Lactobacillus curvatus (yogurt associated),Staphylococcuswarneri(red meat associated), Enterobacteramnigenus,Salmonella typhimurium, and Shigellaflexneri(spoiled fish samples) via broth dilution method. Antibacterial efficacy of ABAgNPs and ABaqu were evaluated via agar well diffusion assay. Time kill assay, biofilm inhibition effect (crystal violet assay), cell proliferation inhibition effect (MTT assay), antibiogram analysis and synergistic effect, TLC bio-autography, and TLC spot screening was done against seven bacterial pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Lactobacillus curvatus, Klebsiella pneumonia, Escherichiacoli and Pseudomonas aeruginosa which were effectively inhibited by ABAgNPs and ABaqu at 0.10 mg/ml concentration. Protein kinase inhibitorassay, Brine shrimp toxicityassay, Alpha-glucosidase inhibition activity, in vivo biological activities (acute oral toxicity, acute dermal toxicity, wound healing effect, anti-inflammatory effect, anti-cancerous effect, and antidiabetic effect) of ABAgNPs and ABaqu were carried out in Swiss albino mice (Balb/c). The color conversion from yellowish-brown to dark brown indicated the preliminary synthesis of ABAgNPs. Further, UV-Viz spectroscopy confirmed the ABAgNPs synthesis at 400 wavelengths, SEM image indicated the generated tubelike nanoparticles particles, separable as well as several aggregates of nanoparticles with a diameter ranging from 5 µm up to 50 µm. ABAgNPsshowed the presence of phytochemicals such as proteins, phenols, alcohols, amino acids, and polysaccharides viaFTIR spectroscopy.ABAgNPs and ABaqu showed the presence of alkaloids, saponins, tannins, quinones, free amino acids, phenols, glycosides, steroids, xxviii flavonoids, and terpenoids which are involved in reduction and capping of ABAgNPs. In the case of free radical scavenger activity, the highest antioxidant potential effect in ABaqu was recorded as 13.64% (ABTS) and 14.62% (DPPH), though ABAgNPs showed 4.86% (ABTS) and 4.85% (DPPH) antioxidant potential. The maximum antibacterial activity of synthesized silver nanoparticles ABAgNPs and ABaqu were observed at 0.10 mg/ml.ABAgNPs showed maximum inhibition of Klebsiellapneumoniae(15.0 ± 0.0 mm), Escherichia coli (15.0 ± 0.0 mm), Streptococcus pyogenes(17.0 ± 0.0 mm), Staphylococcus aureus(15.0 ± 0.0 mm), and Pseudomonas aeruginosa(2) (17.0 ± 0.0 mm) at 0.1 mg/ml concentration. Similarly, ABaqu showed maximum inhibition of Pseudomonas aeruginosa(2) (15.0 ± 0.0 mm) at 0.1 mg/ml.The time-kill activity showed that Gram-positive bacteria and Gram-negative bacteria were killed after4 h and 1 h of incubation. A strong synergistic effect of chloramphenicol and kanamycin in combination with ABaqu and ABAgNPs was recorded. It was found that ABAgNPs+Kan, ABaqu+KanABAgNPs+Chl, and ABaqu+Chl, showed maximum inhibition against tested bacteria. Biofilm inhibition assay, cell proliferation inhibition assay, TLC bioautography, and TLC spot screening persist the results of agar well diffusion and broth dilution assays. Oral toxicity results indicated that there was no apparent change or damage to the kidney, liver, and heart of ABaqu (1000 mg/kg) and ABAgNPs (1000 mg/kg) treated groups compared to the vehicle control group (DH2O) and also no significant change noted in body weight, organ weight and physical appearance. Our study confirmed that both the ABaqu and ABAgNPs were nontoxic to the kidney, heart, and liver at 1000 mg/kg and the toxic dose may be higher and there was no mortality xxix observed during the treatment at tested dose level. Dermal toxicity showed that there were no adverse effects, changes, and alteration in the skin of treatment groups as well as the control vehicle group (petroleum jelly) recorded. Therefore, the ABaqu and ABAgNPs at the tested concentration are safe for use in topical medical applications. In our study, the enhanced wound contraction in ABaqu, ABAgNp, and the Nitrofuranose treated groups from 7thto 11thdays was observed. Both of the ointments (ABaqu and ABAgNp) showed significant wound closure than the open wound in all days as in the case of the Nitrofuranose treated group. The anti-inflammatory activity in formalin-induced paw edema model illustrated the potential use of silver nanoparticles ABAgNPs and ABaqu as a reducing or inflammation inhibiting agents due to the release of acute inflammatory mediators. It was observed that Ajugabractoesaexhibited good α- glucosidase inhibition activity under in vitro conditions. In vivo anti-diabetic activity showed that both ABaqu and ABAgNPs showed greater anti-hyperglycemic potential in alloxan-induced diabetic mice at 400 mg/kg dose. The anti-cancerous activity demonstrated that ABaqu and ABAgNPsshowed a significant anti-cancerous effect in CCl4-induced mice compared to other treatments. The clear mechanism of cytotoxic potential of ABAgNPs is still not well known, but we can say that ABAgNPs disrupt the cell cycle, causes the cytotoxicity effect due to the interaction of proteins functional groups with silver, and generate reactive oxygen species, which could cause DNA damage andlead to cancerous cells death. Therefore, overall results of our study showed that ABaqu and ABAgNPs inhibited the growth of both Gram-positive and Gram-negative bacteria within few xxx hours and dose-dependent mode. Both ABaqu and ABAgNPs are not toxic at 1000 mg/kg, 400 mg/kg, and 200 mg/kg dose and possessed strong anti-diabetic, antiinflammatory, wound-healing, and anti-cancerous effect due to presence of phytochemical constituents. This research demonstrated that ABAgNPs are found to be safe, economic, and environmentally friendly, hence ABAgNPs and ABaqu extract could be considered as a therapeutic drug against both infectious and non-infectious associated diseases. |
