1. INTRODUCTION:

Bimetallic nanomaterials and nanoparticles also known as alloy nanomaterials or nanoparticles are the next step to advance the nanoparticle research in science and technology. Nanoparticles and nanomaterials have already proven to be a valuable source in medicinal and industrial applications and are continuing to contribute greatly in the field of disease treatment, drug-delivery, environmental protection, etc.

Bimetallic nanoparticles could potentially take the benefits of nanoparticles research to the next level. Bimetallic nanoparticles combines two different metal atoms to bring about a combinational alloy, that incorporates the physiological and biological properties of the individual components, and yet provides much higher biological activity, biocompatibility and structural integrity and novelty.

Bimetallic nanoparticles are considered as valuable for their suppleness in catalytic, optical and electronic properties. Beyene et. al (2010) and Singh et.al (2009) reported that bimetallic nanoparticles do not only differ in size and effect, but also in their pure components composition¹. Several studies have been reported in synthesizing various bimetallic nanoparticles Pd-Pt, Pt-Co,Ni-Mo, Ag-Cu, Au-Ag, etc. Oxidation of styrene was done using AgCu catalyst as this provides superior catalytic activity in different oxidation reactions². Bimetallic NPs provides hundreds of combinations of existing metal elements and could bring about a revolution in the medicinal research.

Silver nanoparticle comprises several biomedical applications such as antimicrobial, drug delivery, artificial implants and are also being added to wound dressings, antiseptic sprays and topical creams^3,4. Balajiet.al (2014) reported that the silver nanoparticle showed number of pharmacological activities like antifungal, antiviral, antibacterial and anti-plasmodium activity⁵. Refractive index sensors, spectroscopy, photo-thermal, high sensitivity bio-molecular detection and diagnostics are some applications of Silver and copper nanoparticles⁶. In bioanalytical and medical areas Cu nanoparticles are used as nanoprobes and when this nanoparticles are functionalized with thermoplastics it results in many applications such as antiseptics, textiles, cleaning, intra-hospital coatings and paintings^7,8.

Organic dyes are extensively degraded with the help of Ag nanoparticle by photocatalytic reaction and redox potential technique.Chinnashanmugamet.al (2017) reported that when Ag nanoparticle are stabilized with EPS, it provides an efficient degradation of textile dyes, congo red and methyl orange compared to bacterial degradation using microbes like Lactobacillus fermentum, Halomonas spp. and Lactobacillus acidophilus⁹. Due to their desired properties, nanochemicals are used in methods like photo catalysis, chemical oxidation and disinfection in order to remove pathogens and pollutants from waste water. On the other hand, organic and inorganic compounds can be parallely removed from wastewater by doping Ag nanoparticle with other metallic nanoparticle¹⁰.

Usually nanoparticles are synthesized with the help of microbes but it possess several disadvantages like maintenance of microbial culture ,difficult in purification and it also requires to identify whether it synthesis intracellular or extracellular¹¹. Also Sutanuka Pattanayak et al reported that nanoparticle synthesized by radiation, photochemical, chemical, electrochemical methods, which produce hazardous by-products, with high energy demand and difficulty in purification steps¹². Thus the use of green synthesis method for producing nanoparticle is preferred as an eco-friendly approach, since it is nontoxic, ecofriendly, ease of development, cost effective and considered as safe for human therapeutic use ¹³. Additionally the extracts from plants also act as reducing and capping agents for synthesing nanoparticle¹⁴. In comparison with green synthesis, the physical and chemical methods results in low yields of nanoparticle¹⁵.

Objective of this study was to develop an eco-friendly, nontoxic and cost-effective method for synthesizing Bimetallic Nanomaterial (AgCuO) using aqueous extract from Cordia sebestena leaf extract. The synthesized Nanomaterial were studied for their dye degradation i.e., Methylene Blue (MB) degradation and also their potential antibacterial activity.

2. MATERIALS AND METHOD:

2.1. Preparation of leaf extract:

Leaves of Cordia sebestena were collected from VIT University, Vellore, Tamil Nadu, India. Fresh leaves were collected and washed with sterile distilled water to remove the dust particles. Then the leaves were dried and powdered .2grams of powdered leaves was dissolved in 20ml distilled water and boiled .the content was filtered using Whatman filter paper No. 1 and aqueous extract was obtained ¹⁶.

2.2. Phytochemical Analysis:

The plant extract were qualitatively analyzed for the presence of phytochemical constituents like Alkaloid, Anthraquinones, Steroids, Flavonoids, Phenolic compound, Tannin and Saponin using standard methods¹⁷¹⁸¹⁹.

2.2.1. Alcohol extract:

A few amount of leaves powder was take and it is dissolved withmethanol. Allow it to stand for 30 minutes, after 30min the content was filtered using what manfilter paper No.1. Then the content was kept in water bath for evaporation. after it is cooled add 6ml of chloroform and use it for following test.

2.2.2. Test for Alkaloid

Few drops of Dragendorff’s reagent was added to 2-4ml of alcohol extract. appearances of orange brown precipitate indicate the presence of alkaloid.

2.2.3. Test for Anthraquinones

In the test tube, take 2ml of filtrate and add 2ml of ammonia solution. Gently shake for 30sec. Appearance of pink colour indicates the presence of anthraquinone.

2.2.4. Test for Steroids:

In a test tube, take 2ml of filtrate and add concentrated H2SO4 to the wall of tube. Appearance of the red color indicates the presence of steroids.

2.2.5. Acid extract:

Add 2ml of HCl to the leaf powder and mix it well. After 20minutes the content was filtered using Whatman filter paper No.1 and extract were used to flavanoid.

2.2.6. Test for Flavonoids:

Few drops of sodium hydroxide solution were added to acidextract. Appearance of intense yellow color and changes to colorless by addition of dilute acid indicates the presence of flavanoids.

2.2.7. Water extract:

Leaves powder was added to 10ml of distilled water and mixed well. Then the content was boiled for few minutes and filtered.

2.2.8. Test for Phenolic compound:

Appearance of dark green colour indicates the presence of phenol when few drops of ferric chloride were added to the sample solution.

2.2.9. Test for Tannin:

To the crude extract, 2ml of 2% Ferric chloride was added. The presence of tannin can be confirmed if the extract changes from blue-green to black colour.

2.2.10. Test for Saponin:

Crude extract was taken in the test tube and diluted with 3ml of distilled water. The content was shaken vigorously, if it forms stable foam it indicates the presence of saponin

2.3. Synthesis of AgCu dual metallic Nanomaterial:

CuSo4 (1M) solution was prepared by dissolving 1.59grams of CuSo4 crystal in 100ml of distilled water and 1Molarity of silver nitrate solution was prepared by dissolving 1.69grams of silver nitrate in 100ml of distilled water. From the above solutions, AgCu dual mixture was prepared by taking 50ml from each solution in a conical flask. To this combination 1ml of aqueous extract of Cordia sebestena and 100µl of PEG was added that acts as a capping agent. The whole procedure was carried out under dark condition. Then this mixture was heated for 15 minutes using microwave oven. The whole solution was kept undisturbed until it reaches room temperature. Once it is cooled, centrifugation was carried out at 12000rpm for 10minutes at 4c .After centrifugation the supernatant was discarded carefully and to the pellet add 10ml methanol and distilled water respectively. It is centrifuged at 7000rpm for 10min in order to washout unwanted particles. The pellet was washed several times with methanol and kept for drying to obtain pure nanomaterial. Next day the dried sample was collected and weighed to be 30mgs of AgCuO bimetallic nanomaterial^20–22.

2.4. Characterization studies:

Presence of silver in the obtained particles was confirmed by presence of characteristic Ag absorbance in UV spectrum (between 300nm – 500nm) using ELICO Double beam SL-210 UV Vis Spectrophotometer, VIT University, India.

The obtained particles were suspended in water and analyzed for size, using HORIBA Scientific Nano PARTICA SZ-100 Dynamic Light Scattering (DLS), VIT University, India. Zeta potential of the particles was also studied in the same device.

Size and shape of the particles were observed using scanning electron microscopy, Zeiss EVO 18 Research (SEM), VIT University. The samples are scanned at the operating voltage of 10 KV with a working distance of 12.5 mm from low to higher magnification ranges.

2.5. Dye degradation:

Methylene blue stock solution was prepared by dissolving powdered methylene blue in distilled water (10µg/ml). This solution was directly added with the synthesized particles and observed for color change. The post-reaction solution (decolorized) was then subjected for UV spectrum and GC-MS analysis ⁹.

2.5.1. GC-MS analysis:

Sample volume of 500µl (Control Methylene Blue and Post-Reaction Mixture) was submitted for GC-MS analysis. Instrumentation used was GC-MS JOEL from VIT sophisticated analytic lab. GCMATE-II GC-MS, Agilent Technologies 6890N Network GC system for GC was used for this purpose. The column temperature was 235˚C, injector temperature was 240˚C and helium was used as carrier gas. Experiment was carried out for a period of 30min. The components were resolved by the gas chromatogram and the individual compounds were analyzed by the mass spectrum coupled with the GC. The molecular weight of the resolved components in the sample were identified from the mass spectrum data ²³.

2.6. Antibacterial activity of dual metallic nanomaterial:

The Antibacterial activity of AgCuO nanomaterial was evaluated against pathogenic bacteria such as Escherichia coli and Staphylococcus aureus using well diffusion method.The bacterial culture was prepared using nutrient broth.With the overnight bacterial culture, bacterial lawn was prepared on Mueller–Hinton agar. Agar plate was punched with a sterile cork borer and each well was filled with 100µl of AgCuO dual nanomaterial prepared at different concentrations 100mg, 50mg and 25mg. The antibiotic amoxicillin disc was used as the positive control, then the plates were incubated at 37c for 24hrs ^20,24.

3. RESULT:

3.1. Phytochemical analysis of Cordia sebestena leaves

Qualitative analysis of the phytochemical groups present in the leaves showed that, the leaves are rich in flavanoids, tannins, saponins, and alkaloids. However, sterols, triterpenes and anthroquinone glycosides are absent in the leaf extracts. Results of the phytochemical analysis is tabulated in Table.1.

Table.1: Qualitative analysis of phytochemical constituents

Phytochemical	Inference
Flavanoids	+
Tannins	+
Saponin	+
Phenol	+
Alkaloids	+
Sterols, Triterpenes	_
Anthroquinone glycoside	_

3.2. Nanomaterial Synthesis and Characterization:

Nanomaterial synthesis was confirmed by color change of the reaction mixture. After which, the mixture was centrifuged and the pellet was washed with acetone twice and dried. Three different particles were synthesized i.e., Ag, Cu and AgCu.

3.3. Scanning Electron Microscope (SEM) and EDX Analysis:

SEM image analysis showed that, the synthesized Ag and Cu particles were in the micrometer scale and that the AgCu particles were in nanometer scale. Morphology of the particles were unique for all 3 particles. Figure.1 shows the morphology of the three synthesized particles from SEM analysis. Ag particles appeared to be slices of stacks, with rectangular shape and rough surface. Cu particles were hexagonal rod shaped with smooth surface. AgCu particles were clusters of spiky structures with smooth surface, ranging less than 1µm size. Among the 3 particles, AgCu bimetallic particles were considered as Nanomaterial, based on its size range.

Energy disperse X-ray spectroscopy (EDX) analysis confirmed the inorganic elements present in the synthesized particles. Figure.2 shows the EDX plot of the synthesized particles. Ag particles consisted of Ag, C, Cl, and O elements. Cu particles consisted of Cu, Cl, C, and O elements. AgCuO particles consisted both Ag and Cu elements, in addition to S, Cl and O elements. This confirmed that the synthesized AgCuO particles consisted of both silver and copper metal atoms in its structure.

3.4. Dynamic Light Scattering (DLS) Analysis of AgCuO NPs:

The particle size of the synthesized AgCuO Bimetallic Nanomaterial was identified to be 671.6nm ± 37.3nm through dynamic light scattering (DLS) analysis. It was also observed that the particle size distribution was homogenous in nature, with only a single size range was observed at 670nm. This confirms that the synthesized particles are all almost uniform in size with an average size of 671.6nm. Figure.3 shows the particle size plot from the DLS analysis for AgCuO Bimetallic Nanomaterial.

3.5. Methylene Blue Degradation by AgCuO Bimetallic Nanomaterial:

The synthesized particles were all tested for their ability to decolorize/degrade methylene blue. Methylene blue was used as control to compare the UV absorption. Among the three particles, Ag particles and AgCuO Nanomaterial showed positive discoloration. However UV spectrum scan showed that, AgCuO Nanomaterial degraded methylene blue most effectively. Cu particles did not affect the methylene blue, as the UV spectrum did not have any difference from the control sample. As shown in Figure.4. The discoloration process was spontaneous in less than 1min of mixing of dye and particles. No external energy was used to initiate or catalyze the reaction. Post-reaction, the particles were recovered by centrifugation and were further tested for dye degradation. The recovered AgCuO Nanomaterial were able to decolorize the methylene blue for upto 5 reactions. This showed that 1mg of AgCuO Nanomaterial were able to decolorize a total of 50ml of methylene blue (10µg/ml) and is reusable after centrifugation and acetone wash.

Figure.1: SEM morphology of synthesized particles.

A) Ag particles; B) Cu particles; C) AgCu particles.

Figure.2: EDX spectrum of synthesized particles.

A) Ag Particles; B) CuO particles; C) AgCuO particles.

Figure.3: Particle size analysis of AgCuO Bimetallic Nanomaterial.

3.6. Mechanism of Methylene Blue Degradation by AgCuO Nanomaterial:

To identify the mechanism of degradation, the methylene blue (MB) control sample and the AgCuO Nanomaterial’s post-reaction product (decolorized methylene blue) were subjected for GC-MS analysis. Chromatogram of the GC analysis is shown in Figure.5. It was preliminarily observed that, MB produced a single broad peak in the gas chromatogram at a retention time of 10.42min. While, the post-reaction produced two peaks merged/very close to each other at retention times of 11.76min and 12.36min respectively. The GC-MS software analysis revealed that the single peak observed is due to superimposition of the two peaks. This clearly suggested that, the single sharp peak of MB is being degraded into two different peaks, suggesting production of two by-products/fragments of MB.

To further identify the by-products/fragments of MB, mass spectrum of the two peaks were analyzed. Mass-spectrum of the two peaks are give in Figure.6. It was observed that the two peaks at 11.76min and 12.36min RT, consisted of 119.06g/mol and 133.08g/mol molecular weight respectively. This molecular weight was cross verified with the structure of MB and its derivative fragments. Based on the fragmentation analysis, the mechanism of MB degradation was predicted. The mechanism of degradation of MB by AgCuO Nanomaterialis represented in Figure.7.

AgCuO Nanomaterial attacks the Sulphur group ‘S’ present in MB and results in production of AgCuO-S complex, this breaks down the MB skeleton into two fragments, i.e., N,N-dimethylcyclohexa-2,5-dienamine and N,N-dimethylcyclohexa-2,5-diene-1,4-diamine with molecular weights of 119.06g/mol and 133.08g/mol respectively. Thus based on the GC-MS analysis, the mechanism of MB degradation by AgCuO Nanomaterial was identified.

Figure.4: Discoloration of methylene blue by synthesized particles. A) Ag particles decolorized the methylene blue; B) CuO particles did not catalyze any discoloration; C) AgCuO Nanomaterial decolorized the methylene blue; D) UV spectrum absorption of control methylene blue and post-reaction solution with all 3 particles.

Figure.5: Gas chromatogram of Control Methylene Blue and AgCuO Nanomaterial Post-Reaction Mixture.

Figure.6: Mass-Spectrum analysis of peaks present in Gas-Chromatogram of AgCuO Nanomaterial post-reaction products.

Figure.7: Mechanism of Methylene Blue degradation by AgCuO Nanomaterial

3.7. Antibacterial Activity of Synthesized Particles:

The antibacterial activity of the synthesized particles were analyzed by using agar-well diffusion method against two bacterial pathogens Eschericia coli and Staphylococcus aureus. Ag particles and AgCuO Nanomaterial showed significant antibacterial activity, however, CuO particles did not show any antagonism against the studied pathogens. Results of antibacterial activity are shown in Table.2 and Figure.8. AgCuO Bimetallic Nanomaterial showed a maximum of 16mm zone of inhibition against S. aureus at 1mg/ml concentration. Ag particles showed a maximum of 13mm zone of inhibition against S. aureus at a concentration of 1mg/ml.

Table.2: Antibacterial activity of synthesized particles against E. coli and S. aureus.

	Concentration	*E. coli*	*S. aureus*
Ag particles	250µg	9mm	9mm
	500µg	10mm	11mm
	1000µg	12mm	13mm
AgCuO Np	250µg	11mm	11mm
	500µg	13mm	13mm
	1000µg	15mm	16mm
Ampicillin	10µg	18mm	21mm

Figure.8: Antibacterial activity against S.aureus.

A) Ag particles against S.aureus; B) AgCuO Nanomaterial against S. aureus.

4. DISCUSSION AND CONCLUSION:

Green synthesis of AgCuO Bimetallic Nanomaterial was achieved by using C. sebestena leaf extract as reducing agent and PEG as capping agent carried out with microwave assistance. Presence of phenols, flavonoids, tannins, alkaloids and saponins in the leaves suggest that it could act as a strong reducing agent for synthesis of Nanomaterial. Color change in the reaction mixture indicated that the metal salts were reduced to produce their respective particles of smaller size.

The synthesized AgCuO Bimetallic Nanomaterial was characterized using SEM, EDX and DLS analysis. The particle was identified to contain both Ag and Cu elements, with average particle size of 671nm and was found to have a morphology of clusters of “sharp spikes with smooth surface”. This morphology increases the volume to surface area ratio, that is advantageous in nanomaterial/nanotechnology research.

Methylene Blue degradation by AgCuO Bimetallic Nanomaterial study, suggested that the synthesized AgCuO Nanomaterial is a good catalyst for fragmentation of the MB dye. The GC-MS analysis identified that the AgCuO Nanomaterial attacks the Sulphur atom present in MB and breaks the molecule into two fragments (N,N-dimethylcyclohexa-2,5-dienamine) and(N,N-dimethylcyclohexa-2,5-diene-1,4-diamine). This breakdown decolorizes the methylene blue and also makes it biodegradable for natural recycling. Hence, is a suitable process to treat industrial effluents containing methyleneblue.

The AgCuO Biometallic Nanomaterial also demonstrates significant antibacterial activity, providing additional benefits in killing of bacteria present in the industrial effluents, thereby reducing the environmental impact.

5. ACKNOWLEDGEMENT:

The authors thank St. Joseph’s College (Autonomous) for their support in carrying out this research work.

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Received on 07.10.2019 Modified on 10.12.2019

Research J. Pharm. and Tech. 2020; 13(7): 3122-3128.

DOI: 10.5958/0974-360X.2020.00552.1