INTRODUCTION:

Nanotechnology field is growing rapidly due to broad range of applications, including food packaging materials, cosmetic products and delivering the therapeutic agents to advance the various treatments¹. Indeed, inorganic nanoparticles have distinctive features due to their small size and a large surface to mass ratio. So far, different types of metallic nanoparticles have been prepared such as gold, silver, platinum and pelladium nanomaterials that have gained remarkable attention due to their high performance in many scientific fields². Gold nanoparticles are of 1-100 nm in size, which is in the sub wavelength of visible light.

They are widely used in biology and medicine owing to their unique physiochemical properties, including high chemical stability, biocompatibility, small size, surface modification and optical properties^3,4. Further, they are stable against oxidation under physiological condition without any major risk of leaching toxic substances⁵. Mostly, nanoparticles can be synthesized using different approaches, including decomposition, wet chemical procedures, electrochemical methods, and microwave assisted techniques^6,7. But, the chemicals used in those approaches are usually highly toxic and flammable, which limit their applications as they may pose risk to human beings⁸. Thus, there is an increasing need to promote safe, inexpensive and eco-friendly methods to synthesize metallic nanoparticles without using hazardous chemicals⁹. Recent past, the green synthesis methods have gained much attention as an alternative approach to synthesize metal nanoparticles because usage of nontoxic chemicals, renewable materials, or environmentally benign solvents and also which can eliminate or reduce the generation of toxic substances¹⁰. The biosynthesis of metal nanoparticles using plant extracts is eco-friendly and cost-effective for large-scale production. Earlier studies have shown that the biomolecules, such as polyphenols, phenolic acids, terpenoids, alkaloids, sugars, and proteins, in plant extracts may work as both reducing and capping agents in green synthesis of nanoparticles¹¹.

Caesalpinia bonducella (L) Roxb. (Family: Caesalpiniaceae) commonly known as Fever nut; bonduc nut; Nata Karanja (Hindi), is a prickly shrub found throughout the hotter regions of India, srilanka and Myanmar. Various parts of the this plant have reported to poses multiple therapeutic properties like antidiuretic, antipyretic, antibacterial¹², antiviral¹³, antiamoebic¹⁴, antihelmenthic, anticonvulsant¹⁵, anti-anaphylytic, anti-diarrheal¹⁶, antiasthmatic¹⁷, hepatoprotective and antioxidant activities¹⁸. Keeping these properties in mind, the present work was designed to synthesize gold nanoparticles using green chemistry method. The properties of the nanoparticles were measured using a variety of techniques including, UV-Vis spectroscopy, FTIR spectroscopy, X-Ray diffraction, and TEM. Further, the antimicrobial properties of the nanoparticles were tested against both gram-positive and negative bacterias such as Escherichia coli and Staphylococcus aureus.

MATERIALS AND METHODS:

CHEMICALS:

Chloroauric acid (HAuCl₄3H₂O) was purchased from Merck, India and all other chemicals used were of analytical grade.

Preparation of C. Bonducella leaf extract:

Fresh leaves of Caesalpinia bonducella were collected and identified taxonomically. They were washed thoroughly in running tap water followed by distilled water. 5gms of 2 day air dried leaves were taken in a round bottom flask fitted with a condenser and heated for 30 mins with 100ml of distilled water at 40⁰C. It was cooled and filtered through Whatman No.1 filter paper and kept at 4⁰C in refrigeration for further use.

Synthesis of gold nanoparticles:

5ml of leaf extract and 2ml of 1mM Chloroauric acid were taken in a boiling tube. The reaction mixture was irradiated in a domestic microwave oven with a 2.45GHz frequency operating at a power of 300W for 4 mins. The confirmation of Gold nanoparticles formation was done by using the UV-Vis spectra.

Characterization techniques:

The synthesized Gold nanoparticles were characterized by using UV-Vis spectroscopy (UV-3600, Shimadzu), FTIR spectra (IR Affinity-1), X-ray diffraction ((Rigaku, Miniflex) and Transmission electron microscopy (JEOL 2000 FX-II TEM) techniques.

Antimicrobial potential evaluation:

The synthesized silver nanoparticles antimicrobial potential was tested against both gram-positive and negative bacterias using disc diffusion method¹⁹. Microorganisms were procured from Dept. Of Microbiology, Osmania University, Telangana, India. Sterile and solidified Muller Hinton agar (20ml) plates were swabbed with gram-positive (S. aureus) and gram-negative (E. coli) bacterias. The antibacterial activity was tested using (A) leaf extract (10µg/ml), (B) Streptomycin (5µg/ml), (C) AuNPs (5µg/ml) and (D) AuNPs (10µg/ml) in agar plates. Streptomycin was used as positive control. After 24 hrs of incubation period, the zone of inhibition was measured in nm to assess the antibacterial activity.

RESULTS AND DISCUSSION:

UV-Vis spectrometer:

UV-Vis spectroscopy is a simple and sensitive technique. It is mainly used to determine the size and stability of nanoparticles. Generally, the formation of AuNPs is detected by using UV-Visible spectroscopy and the change in colour is attributed to the SPR occurrence. In the present study, the role of leaf extract in the synthesis of nanoparticles was studied using different concentrations (0.1-0.5%) of leaf extract solution containing 2mM of HAuCl₄ (Fig. 1). the obtained UV-Visible absorption spectra showed a maximum peak in the wavelength range of around 518-538nm (Fig. 1), which is ascribed to the SPR band for AuNPs. The production of GNPs with different concentrations of HAuCl₄ at the fixed concentration of gum was shown in Figure 2. It reveals that the amount of nanoparticles formation increases with an increase in the concentration of HAuCl₄^20,21.

Figure 1: UV-visible spectra of AuNPs synthesized with different concentrations of C. bonducella leaf extract (i-1.0%, ii-0.75%, iii: 0.5%, iv-0.25% and v-0.1%).

Figure 2: UV-visible spectra of AuNPs synthesized with different concentration of HAuCl₄(i-1mM, ii-0.75 mM, iii: 0.5 mM, iv-0.25 mM, v-0.1 mM)

FTIR:

Figure 3a and b show the FTIR spectra of leaf extract and leaf extract capped AuNPs respectively. Figure 3 reveals that the FTIR spectra of leaf extracts major peaks located at 3403, 2946, 1797, 1683, 1489, and 1109 cm^-1. The absorption bands of leaf extract capped GNPs were noticed at 3212, 2945, 1784, 1625, 1445, and 1106 cm^-1. These peaks were shifted from 3403 to 3212, 1797 to 1784, 1683 to 1625 and 1489 to 1445 cm^-1 when compared to the leaf extract and other peaks were found to remain unchanged. These results specify that the hydroxyl and carboxyl groups were involved in the synthesis and stabilization of GNPs¹⁴.

Fig. 3: FTIR spectra of the leaf extract (a) and leaf extract capped AuNPs (b)

XRD

The XRD analysis was used to confirm the crystalline nature of synthesized gold nanoparticles. In the present study, various Bragg's reflections are clearly visible in XRD pattern are corresponding to the (111), (200), (220) and (311) set of lattice planes (Fig.4). On the basis of these Bragg's reflections it is clear that the synthesized gold nanoparticles are face centered cubic and essentially crystalline in nature. The existence of diffraction peaks was matched to the standard data files (JCPDS card No. 04-0784) for all reflections.

Figure 4: XRD pattern of synthesized AuNPs.

TEM:

The size and shape of the GNPs were confirmed by TEM analysis. Figure 5 indicates that GNPs were spherical in shapes and well dispersed. To get size distributions of AuNPs, approximately 58 particles were counted and then constructed histograms. Figure .6 indicates a histogram of the particle size distribution of AuNPs. Most of the particles were in the size around 11 nm.

Figure 5: TEM images of synthesized GNPs

Figure 6: Particle size distribution histogram of AuNPs

Anti-microbial activity:

The antibacterial activity of AuNPs and leaf extract alone was studied against Gram positive and negative strains of bacterias such as E.coli and S. aureus respectively using disc diffusion method (Fig. 7). The diameter of zone of inhibition values was recorded and analysed. The zone of inhibition around the disc was considered as a measure of the effectiveness of AuNPs. The zone of inhibition was observed around 09 mm diameter for 5µg Ml-1 AuNPs and 13mm diameter for 10µg Ml-1 AuNPs for both Gram positive and negative bacterias. The leaf extract alone was shown no inhibition zone. The results indicate that the zone of inhibition was increased with increased in concentration of AuNPs. Further, the zone of inhibition was more against gram positive bacteria than gram negative bacteria. This may be due to thick cell wall in gram negative bacteria. The results are corroborated with the earlier reports^22,23. The inhibitory zone was attributed to the phytochemicals, flavonoids, alkaloids and polyphenols present in the aqueous plant extract that capped with gold nanoparticles. Thus, the anti-microbial properties can be tapped in various medical applications and consumer product industries.

Figure 7: Anti-bacterial activity of AuNPs against E.coli and S.aureus after 24 hours of incubation. I-IV represents 10µg/ml of leaf extract, 5µg/ml of Streptomycin, 5µg/ml of AuNPs and 10µg/ml of AuNPs solutions.

CONCLUSION:

Biosynthesis of Gold nanoparticles using C. bonducella was demonstrated to be a simple, low-cost and non-toxic method. C. bonducella acts as both reducing and stabilizing agent. The nanoparticles exhibited a surface Plasmon resonance at around 518-538nm. The XRD studies showed that the AuNPs have a face-centred cubic lattice and crystalline in nature. FTIR analysis showed that both carbonyl and hydroxyl groups involved in the synthesis and stabilization of gold nanoparticles. The spherical shape and the nano-regime size of the gold nanoparticles were proven by TEM images. These AuNPs showed an antimicrobial activity against Gram-positive and Gram-negative bacterias. Thus, this study indicates that the green synthesized gold nanoparticles seem to be promising and effective antibacterial agents. In vivo studies are needed for the clinical adaptation of the results.

ACKNOWLEDGMENTS:

The authors thank the department of chemistry and CFRD, Osmania University for providing instrumental facilities. The authors also thank the Centre for Nanotechnology, University of Hyderabad for allowing to use their TEM facility.

CONFLICT OF INTEREST:

The authors declare no conflict of interest

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Received on 08.03.2020 Modified on 27.04.2020

Research J. Pharm. and Tech. 2021; 14(2):1037-1040.

DOI: 10.5958/0974-360X.2021.00185.2