Phytofabrication of Silver Nanoparticles using aqueous leaf extract of Aegle marmelos and its Characterization

 

A. Najitha Banu¹*, Anand Singh¹, A.M. Raut², Johnson Wahengbam²

¹Department of Zoology, School of Bioengineering and Biosciences,

Lovely Professional University, Phagwara, Punjab, India.

²Department of Entomology, Szent Istvan University.Budapest, Hungary.

 

ABSTRACT:

Nanotechnology has proved a boon as it brings a revolutionary change in the world with the tremendous advancements in the field of science such as engineering, biotechnology, analytical chemistry, and agriculture. The synthesis of the metal nanoparticle is a developing area of exploration in present-day material science and innovation. Among the techniques involved in the synthesis of metallic nanoparticles, the biological methods or the green synthesis are reliable due to its cost-effective, environment friendly, and use of non-harmful materials in the processing. Therefore, the current work is focused on the biosynthesis of silver nanoparticles with the leaf extract of holy plant Aegle marmelos by utilizing 1mM silver nitrate solution. From the bio-reduction reaction mixture, the synthesis of silver nanoparticles was studied by UV-Visible spectrophotometer at the range of 200 nm to 800nm and discovered maximum absorbance at 460nm. Further, the SNPs were analyzed for the protein or other organic groups by Fourier Transformed Infrared spectrophotometer (FTIR). The dried silver nanoparticles were further characterized by utilizing a scanning electron microscope (SEM) to observe the actual size, shape, and distribution of particles. This result showed the actual size of the bioengineered silver nanoparticles ranges from 25-30nm. The EDX result showed a peak of Ag that confirmed its presence in the suspension. The main objective of this study is to develop a fast, environmentally friendly, and advantageous technique for the synthesis of silver nanoparticles. By standardizing the biosynthesis techniques, silver nanoparticles can be synthesized in bulk with a safe environment and it may be utilized for different clinical applications including control of pathogenic microorganisms and so on.

 

 

 

INTRODUCTION:

Nanotechnology is a branch of science and technology which deals with nanoparticles that have size of 1 to 100nm in one or more dimensions, is taking over various fields. Nanoparticles are being extensively used for different purposes, such as in making fluorescent labels, drug delivery, tissue engineering, tumor destruction, detection of pathogens and proteins and phagokinetic studies¹.

 

 

 

Though there are natural nanoparticles present in the environment engineered nanoparticles are being widely used. These engineered nanoparticles are different from the natural nanoparticles and may impart varied effects when they come in contact with living cells. The engineered nanoparticles can be classified into carbon-based, silicon ceramic, polymer-based, metal or metal oxide materials².

 

Pharmaceutical nanotechnology is also an emerging branch of pharmaceutical sciences and has provided various tools, including targeted drug delivery system. It is much better than the conventional methods because it increases the rate of dissolution of drug, enhance solubility and often a very less amount of it is required as compared to those of conventional methods³. As the use of nanoparticles in increasing, intentional as well as unintentional exposure to these particles is becoming inevitable and it has been studied over years that these nanoparticles are highly toxic to humans. Nanoparticles of metallic substances impart toxicity. Nanoparticles of aluminum oxide, copper oxide, silica, silver and zinc have evidently decreased cell viability, increased oxidative stress and have even caused cell death in certain cases⁴. The use of nanotechnology for building nano scale items in innovative work divisions is developing⁵.

 

The synthesis of plant nanoparticles is referred to as Green Chemistry approach that joins nanotechnology and biotechnology. Plant-based union of nanoparticles is conversely quicker, more secure and lighter; works at low temperatures; and requires just unobtrusive and naturally safe segments⁶. Plant-based nanoparticles have pulled in more consideration because of developing enthusiasm for naturally cognizant items. What's more, the union of nanoparticles utilizing plants offers different preferences, for example, the use of more secure solvents, diminished utilization of risky reagents, milder reaction conditions, plausibility, and their versatility being used for therapeutic, careful, and pharmaceutical applications⁷.

 

Over the last few decades, the applications of nanotechnology in various fields such as medicine have been extensively explored as a broad area in the field of pharmacology. Medicinal plants have been used as the chief source of treatment and disease management almost in all countries of the world because the herbal medicines are inexpensive, easily available and do not possess side effects^8,9. Medicinal plants play an important role to improve the immunological response against much pathology due to the presence of a wide variety of secondary metabolites, which are associated with therapeutic efficacy against various diseases and disorders¹⁰. However the delivery and efficacy of many herbal drugs is often limited to reach the site of therapeutic action and they require few modifications such as changing the molecular structure of the drug or their proper distribution by incorporation in carrier system¹¹. Therefore, the present study was carried out the process of synthesis of green silver nanoparticles using aqueous extract of A. marmelos leaf extract.

 

A. marmelos is commonly known as Indian bael and found all over the India¹². The therapeutic value of this plant has been discribed by almost all the ancient Ayurvedic treatises. It has chemicals like caumarins, steroids, alkaloids, tannins etc¹³. Nanoparticles are the spearheads of nanotechnology because of its high surface area to volume ratio. This peculiar property makes the more efficiency of the pesticides. The metallic silver nanoparticles have enormous antimicrobial and antiseptic properties Further the silver nanoparticles penetrate into the body of lower organisms easily as compared to mammals. Besides this silver nanoparticles is non hazardous. The major aim of the present work is to design a cost effective metallic silver nanoparticles using an aqueous extract of the A. marmelos leaves and its characterization by UV-visible spectrophotometer, (FTIR) Fourier transform infrared spectroscopy, XRD, FESEM and EDAX for the morphological structure and the purity of the bioengineered silver nanoparticles.

 

MATERIALS AND METHODS:

Collection and Plant extraction:

The leaves of A. marmelos were collected from the herbal garden, Lovely Professional University, Phagwara, Punjab. Fresh and new leaves of A. marmelos were preferred because of their succulence. Collected leaves were washed thoroughly with simple tap water twice followed by the distilled water. The leaves were kept on the filter paper for drying at room temperature. Ten grams of dried leaves were chopped into fine pieces with the help of surgical blade or knife. The Erlenmeyer flask containing 10mg of finely chopped leaves and 100 ml of distilled water was allowed to boil on the heating mantle for 5-10minutes. After the hand bearable temperature the flask containing the pale yellow extract was allowed to cool at the room temperature. The extract was filtered in another conical flask with the help of funnel and whatman filter paper. The filtrate was stored at 4°C for the further use.

 

Phytofabrication of silver nanoparticles (AgNPs) using A. marmelos aqueous extract:

Silver nitrate is white color, odorless crystalline solid. One mili molar clear solution of silver nitrate was mending by adding 0.017g of silver nitrate in 100ml of de-ionized water.  One mili molar clear solution of silver nitrate (AgNO3) was prepared using de-ionised water and kept in a conical flask which is properly covered with black paper to avoid photo-oxidation reaction. The flask containing 10ml of plant extract was kept on magnetic stirrer and 90ml silver nitrate solution was slowly added to it. The time of color change was also monitored using a stop watch. The reduction of the silver ions to silver atoms was roughly monitored by visualization of the solution. The conversion of the reaction mixture pale yellow to dark brown color was clearly indicating the synthesis of AgNPs.

 

Characterization of silver nanoparticles:

UV-Visible Spectroscopy:

The optical properties of dark brown aliquots of synthesized AgNPs were monitored by UV-Vis absorption spectroscopy using an Elico-double beam-SL-210 Mumbai, India in the agriculture lab, Lovely Professional University, Phagwara, Punjab. It is a very effective technique for the primary characterization of the biologically synthesized silver nanoparticles. The double distilled water was taken as the reference. An aliquot of synthesized nano sample was placed in a quartz cell of 2cm³ for scanning the absorption spectrum from 200-800nm.

 

Purification of AgNPs:

Prepared nano samples were allowed to centrifuge at 10,000RPM for 10 minutes. The pellet was then washed with distilled water and again centrifuged for 10 minutes at 10,000RPM. After getting the purified nano pellet the supernatant was discarded and pellet was washed with the double distilled water followed by drying in the incubator. The dried powder was scrapped from the petri plate. The dried sample was kept in hot air oven so that the sample remains impoverished for further characterization such as FTIR, XRD, SEM and EDX analysis.

 

Fouier Transform Infrared Spectroscopy (FTIR):

The dried pellets were kept in the hot air oven for evaporation of any moisture. FTIR was performed to identify the major functional groups of the synthesized silver nanoparticles. For FTIR (FTIR-84005, SHIMADZU, Japan) KBr disc procedure was used. The pellet was prepared using pelletizer. The mixture of nano sample and KBr (1:5) was grinded in charcoal mortar and pestle. The mixture was then placed under the dye and one quintal pressure was applied. After some time the pressure was released slowly. The pellet was removed and collected for FTIR spectroscopy.

 

FESEM and EDX analysis:

For both the SEM and EDAX analysis fine and shiny powder of AgNPs were used. For this purpose, the prepared sample was allowed to dry overnight in hot air oven at 50°C. About 1 gram of powdered sample of phytofabricated AgNPs was submitted to the Central Instrumentation Laboratory of LPU Phagwara, Punjab for the SEM and EDAX analysis.

 

RESULTS:

Phytofabrication of AgNPs:

The aqueous extract was prepared by using fresh leaves of A. marmelos (Figure 1). Light yellow aqueous extract act as a starting material for nanoparticles synthesis (Figure 2).

 

 

Figure 1: Fresh leaves of Aegles marmelos

 

Figure2: Aqueous extract of Aegle’s marmelos

 

Synthesis of silver nanoparticles:

The crystal clear solution of 1 mM AgNO₃ when mixed with the light yellow colour plant extract gives brown colour suspension after 2 minutes. This colour change is the preliminary indication and formation of AgNPs. The colour of the AgNPs gradually darkens after 24hrs of incubation. According to the present literature metallic Silver nanoparticles have dark yellow or brown colour. When an aqueous solution of plant extract was mixed with the aqueous Silver nitrate solution, the colour change was observed from light yellow to yellowish-brown due to reduction of Silver ions into Silver nanoparticles. The Surface Plasmon Resonance phenomenon is responsible for the colour change (Figure 3).

 

 

Figure 3: The colour changes from yellow to brown indicates the synthesis of silver nanoparticles.

 

Characterization of Phtofabricated AGNPs:

UV-Visible Spectroscopy:

The Silver nanoparticles appears brown in colour in the aqueous medium as a result of surface plasmon vibrations. The phytofabricated AgNPs was confirmed by using UV-Visible spectrophotometer in a range of wavelength from 200 to 800 nm. This is a very appropriate method for the study of optical properties of the nanoparticles. The spectroscopic band of synthesized AgNPs solution was found to be close to 460 nm which confirms the synthesis of AgNPs. The peak become more prominent after 3 hours (Figure 4).

 

Figure 4: UV-visible spectra of AgNPS

 

FTIR analysis of AgNPs:

FTIR gives the information about the functional groups present in the biologically synthesized Silver nanoparticles. This study is helpful for understanding the transformation of simple silver nitrate salt (AgNO₃) into metallic AgNPs by the action of different phytochemical components. Through FTIR analysis of phytofabricated AgNPs, confirms the phytochemical components which are present in the aqueous extract playing the dual role reducing and capping agent. FTIR spectrum clearly indicates that phytofabrication of the AgNPs is mediated by the plant extract.

 

FTIR investigation of synthesised Silver nanoparticles depicts absorption peak at 3311.89, 1633.76, 1574.93, 1524.78, 1434.12, 1347.32, 1254.74, 1098.5, 1036.77, 506.33 outline stretch and strong peak at 506.33, 1098.5, 1434.12 and 1633.76 repported to alkyl halide ( C-CI .C-F) and amide, alkenes. It shows stretch free strong and sharp vibration in alcoholic group. Stretch with two bands and multiple bonds were seen in 1524.78. It depicts the presence of alkenes, C-N and nitro compounds. Stretch and variable vibration at 2237.88 depicts alkynes. Absorption at 1347.32 shows alkane groups with pending peak and variable intensity. This graph depicts the presence of nitro, amide and alkynes groups. From FTIR data, it can be concluded that some of the bioorganic compounds from Aegle leaf extract formed a strong coating/capping on the nanoparticles particularly for silver nanoparticles (Table 1 and Figure 5).

 

Table 1: FTIR function groups with vibration

Absorption	Functional Group	Vibration	Intensity
3311.89	O-H	Stretch free	Strong, Sharp
1633.76	Alkynes	Stretch	Variable
1574.93	Amide, C=C	Stretch	Strong
1024.78	C=C, C=N, Nitro	Stretch, two bands	Multiple bonds, Medium, Weak
1434.12	C-H	Bending	Variable
1098.5	C-F	Stretch	Strong
1036.77	C-H	Bending	Strong
506.33	C-Cl	Stretch	Strong

 

Scanning electron microscope (SEM) analysis:

The shape and morphology of the biologically synthesized nanoparticles were identified by scanning electron microscope analysis. The nanoparticles were examined under various magnifications such as 15,000X, 30,000X, 45,000X and 55,000X SEM images of the biologically synthesized silver nanoparticles are shown. It shows relatively spherical-shaped nanoparticles. Accumulation of two or more reducing moieties bound on the surface of the pre formed nuclei of particles could have contributed to the formation of elongated large spherical nanoparticles (Figure 6).

 

 

 

 

Figure 5: FTIR spectra Silver nanoparticles

 

Figure 6: SEM images of Silver nanoparticles

 

EDX analysis:

From the EDAX study the presence elemental silver is confirmed. The EDX result showed a sharp peak of silver at 3 keV which confirmed  the reduction of Ag ions to Ag atom. Other peaks may be due to the attached groups from the leaf extract on the surface of nanoparticle. The highest peak of 80.1 is seen for the silver. Some other small peaks of C, O, and Cl is also seen (Figure 7).

 

 

Figure 7: EXD spectra of A. marmelos AgNPs

 

DISCUSSION:

In the present study the silver nanoparticles (AgNO₃) was phytofabricated through biological method using A. marmelos leaf extract. The synthesized silver nanoparticles using bael extract and reported the presence of alkaloids, tannins, caumarins, and steroids¹⁴. The colour change from yellow to brown changed after 3hours. The surface plasmon resonance band occurs at 450nm. The study was correlated with current experiment which shows the presence of steroids. flavonoids, cumarins, and tanins. The colour change was after 10 min. The absorption peak was recorded at 460 nm which is somewhat near to 450nm. Whereas the presence of many functional groups was investigated in Indian bael extract and its nanoparticles having alcohol, alkyl halides and nitro groups by FTIR spectroscopy.

 

The leaves and fruits of the plant also have medicinal values and were used to treat dyspepsia, diabetes, asthma, cough, and sinusitis. Similarly Nithya et al.,¹⁵ reported that apsorption peak was recorded at 423nm. Fruits of the plant contain several bioactive compounds which include luvangetin, marmelosin, auraptene, marmelide, psoralen and tannin.

 

The bio-engineered silver nanoparticles sample was further analyzed by SEM. Scanning electron microscopy  analysis showed that A. marmelos mediated AgNPs are shepical in shape. Devaraj et al.,¹⁶ observed SEM image reveal high-density AgNPs synthesized by A. marmelos leaf extract. A. marmelos leaf extract respond well to the treatment with silver AgNO₃. Moreover the EDX analysis have revealed absorption of strong silver signal along with supplementary elements such as Carbon, Oxygen, and Chlorine. EDX spectra clear that AgNO₃ reduced into Ag with the help of A.marmelos extract. Green synthesis of silver nanoparticles by the assist of  plants is a cost effective and safe practice. A. marmelos manifest great capability to synthesize silver nanoparticles (AgNPs) at particular temperature conditions on incubation.

 

Patil et al.,¹⁷observed the antimicrobial and antibacterial properties of bio-engineered silver nanoparticles. This result showed the average particle size of 15 -30nm and spherical structure of stable silver nanoparticles. The present study also showed the average particle size of 20 to 30 nm and spherical structure of stable silver nanoparticles.

 

CONCLUSION:

Nanotechnology is a green technology having tremendous properties in every field. It also has a potential for reducing microbial infection. This is a new era of pharmaceutical science in which A. marmelos leaves were used for the synthesis of AgNPs due to the presence of phytochemical components cumarins, alkaloids, steroids, and tenins which help in reducing silver ions to atoms. Further, in future synthesised AgNPs were examining against multidrug resistant microbes. In coming years there is great scope of research to exploit nanotechnology for medical and Pharmaceuticals. Green synthesis of nanoparticles has an efficient advantage over conventional methods and eco-friendly approach.

 

ACKNOWLEDGEMENT:

The authors gratefully acknowledge the Management, Lovely Professional University, Punjab, India, for providing the facilities to perform the research works in the Laboratory.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 21.01.2022           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(6):2709-2714.