Abstract
In the present study, for the first time, biomimetization of hydroxyapatite (HA) with Azadirachta indica (AI) was proposed and established its antioxidant, antibacterial, and anti-inflammatory potential on lipopolysaccharide (LPS). The ethanolic extract of AI was found rich with phenolics and flavonoids, and determined their concentration as 8.98 ± 1.41 mg gallic acid equivalents/g and 5.46 ± 0.84 mg catechin equivalents/g, respectively. The HA was prepared by sol-gel method from calcium nitrate tetrahydrate and orthophosphoric acid, and successfully biomimetization was performed with ethanolic extract of AI. The FTIR analysis settled that as-synthesized HA-AI composite was comprised of both HA and AI. The XRD pattern and Zeta potential revealed that the HA-AI composite was crystalline and negative in charge (−24.0 mV). The average-size distribution, shape, and size of the HA-AI composite was determined as 238.90 d.nm, spherical, and 117.90 nm from size distribution, SEM, and HR-TEM analysis, respectively. The SEM-EDX concluded that the HA-AI composite was comprised of elements of HA as well as AI. The HA-AI composite presented potential antioxidant activity and its EC50 values (dose required to inhibit about half of the radicals) for ABTS and DPPH assays were determined as 115.72 ± 2.33 and 128.51 ± 1.04 μg/ml, respectively. The HA-AI composite showed potent antibacterial activity, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) towards S. aureus (ATCC 700699) and E. coli (ATCC 10536) were correspondingly determined as 266.7 ± 28.87 and 600.0 ± 50.0 μg/ml, and 400.0 ± 86.6 and 816.7 ± 76.38 μg/ml. Most importantly, HA-AI composite presented the potential anti-inflammatory response toward lipopolysaccharide (LPS) in RAW 264.7 cells. The dose of 250 μg/ml of HA-AI composite has shown optimum protection against LPS-induced stress (1 μg/ml) by scavenging oxidants and regulating mitochondrial membrane potential (MMP), inflammatory and apoptotic factors. Thus, this study concluded that the impartation of potential biofunctional features to HA from plant sources through biomimetic approach is much beneficial and could find potential application in dentistry and orthopedic.
Highlights
The earth is an affluent source of biominerals, such as calcium carbonates, calcium phosphates, iron hydroxides, and iron oxyhydroxides of unicellular and multicellular organisms that occur in the form of shell, ivory, teeth, magnetic crystals, etc. (Driessens and Verbeeck, 1990; Dhami et al, 2013)
The obtained ethanolic stem extract of Azadirachta indica (AI) was found affluent with phenolics and flavonoids, and their concentrations were noted as 8.98 ± 1.41 mg GAE/g and 5.46 ± 0.84 mg CCE/g, respectively (Figure 1)
Our study suggests that HA successfully gained antimicrobial activity from AI through biomimetic and, thereby, could find a potential role as an antimicrobial agent in orthopedic and dental implants
Summary
The earth is an affluent source of biominerals, such as calcium carbonates, calcium phosphates, iron hydroxides, and iron oxyhydroxides of unicellular and multicellular organisms that occur in the form of shell, ivory, teeth, magnetic crystals, etc. (Driessens and Verbeeck, 1990; Dhami et al, 2013). The cell viability assays, i.e., MTT and live/dead cell dual staining assays revealed that the HA-AI composite was effective in protecting the cells from LPS-induced inflammatory death, and this characteristic was determined to be dose-dependent (Figures 7E,F).
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