INTRODUCTION:

Nano technologies are a Potential alternative for treatment of various diseases because they have unique biological effect based on their structure and shape^[1]. In the last few years FDA has approved many pharmaceutical companies for the development of nanotechnology mediated drugs. In the recent years metal nanoparticles have gained attention in the field of drug delivery due to their unique properties^[2-4].

Metal nanoparticles are of great importance because of their high surface area and a high fraction of surface atoms. Metal nanoparticles like gold and silver are of particular interest due to distinctive properties, such as good electrical conductivity, chemical stability, catalytic and antibacterial activity^[5-7]. The inorganic metal nanoparticles can fight against human pathogens and fight against the dangerous diseases like cancer. Because of the extraordinary features of gold nanoparticles like monodispersivity and adjustable core size, they play crucial role for gene and drug delivery systems as promising scaffolds. These metal nanoparticles have wide scope in detection and destruction of cancer cells. The cancer cells and the organic moieties like proteins and flavonoids can easily be adsorbed to the Metal nanoparticles surface of particular shapes carry better photon detecting ability in contrast with photo-thermal-dyes. The size and shape of the nanoparticles play important role in deciding the level of sensitivity detection. Chemical, physical, and biological methods have been developed to synthesis nanoparticles but chemical and physical methods are involved in the production of toxic byproducts which are hazardous moreover the methods are very expensive [8]. To synthesis stable metal nanoparticles with controlled size and shape, there has been search for inexpensive, safe, and reliable and “green” approach. Plants provide a better platform for nanoparticles synthesis as they are free from toxic chemicals as well as contain natural capping agents [9]. Among various plants, we have chosen Desmodium Gangeticum leaves extract for the present study since it has several pharmacological effects such as anti-Cancer, anti-diabetic, anti-hyperlipidemia, antioxidant, and hypotensive activities^[10-13]. Phytochemical screening indicated the presence of chemicals such as alkaloids, tannins, phenols, flavonoids, terpenoids. Desmodium gangeticum have marked anti-oxidant activities in its Leaf parts^[14,15]. The present investigation include rapid, cost effective, user friendly (Green Synthesis), stable and one step process of plant mediated gold nanoparticles for their biological screenings.

MATERIALS AND METHODS:

Fresh leaves of Desmodium gangeticum were collected from the Botanical garden of Dr Y.S Parmar University. Analytical grade gold chloride was procured from Hi-media Labs

Biosynthesis of Gold nanoparticles using Desmodium gangeticum leaf extract:

Analytical grade gold chloride is prepared in various concentration is used for the experiment. Fresh plant leaf (10g) cut in to small size were taken in a wide neck Borosil flask and washed thoroughly using double distilled water. Then 200mL of double distilled water is poured in to the flask and subjected to microwave heating for 3 min to subdue the enzymes and proteins which interferes the reduction process. The solution is then filtered in hot condition using 10µm mesh to remove the solid fibrous residues. The clear filtrate is the extracellular extract of the leaf is used for the nanoparticles synthesis. 10^-3M concentrations of gold chloride solution were prepared and interacted with the plant extract (1:1) mixing ratio at 27°C in a rotary shaker at 120rpm. Immediately after the addition plant extract to gold chloride aqueous solution, a light golden color of the reaction mixture changes to dark purple color within 5 min. This change of color indicates that the formation of HAuCl₄ has taken place^[16].

Characterization:

UV-visible spectroscopy:

UV-visible spectroscopy is a simple and quite a sensitive technique that can be used to detect the formation of Gold nanoparticles^11-12. The reduction of Gold ions to the nanoparticle form was monitored by measuring the UV-visible spectra of the solutions after diluting the sample with millipore water 20 times. The spectra were recorded on UV-visible double beam spectrophotometer from 400 to 700nm.

X-ray diffraction:

The purified powders obtained after 4 h of interaction under laboratory conditions was subjected to X-ray diffraction analysis. The generator was operated at 40 kv and with a 30mA current. The scanning range was selected between 10 and 100^θ angles.

Particle size analysis:

The particle size range of the nanoparticles along with its polydispersity was determined using SALD-7500 particle size analyzer. Particle size was arrived based on measuring the time dependent fluctuation of scattering of laser light by the nanoparticles undergoing Brownian motion.

HR-TEM analysis:

The characterization of nano particles was carried out by high resolution transmission electron microscopy (HRTEM) using lyophilized samples. TEM samples of the metal nanoparticles synthesized were prepared by placing drops of the product solution onto carbon-coated copper grids. Grid was completely dried and examined by TEM at 200 kv.

FESEM-EDS:

Silver nanoparticles were examined by FESEM equipped with an energy dispersive spectrometer (EDS). Analyzed samples were dried at room conditions for 5 days and small fragments were placed on pin stubs and then coated with carbon under vacuum.

Antioxidant activity:

DPPH scavenging assay [17,18]:

To examine the concentration effect of the Mono-and bimetallic Au/Ag NPs, 20µl, 50µl, 100µl, 150µl and 200 µl of the nanoparticles were mixed with 2mL of 100µl M DPPH solutions. The samples were vortexes and allowed to scavenge DPPH in dark for 30 min. The absorbance of the supernatants after centrifugation at 9200×g for 2 min was measured at 517nm in UV-vis spectrophotometer. In all the cases, measurements were done in triplicates. The scavenging percentage was calculated using the formula:

(AC-As)

DPPH scavenging = ----------------------- × 100

Where, AC and AS are absorption of blank DPPH and DPPH subjected to interact with the sample at 517 nm, respectively.

RESULTS AND DISCUSSIONS:

Adding the above mentioned extract of Desmodium Gangeticum to HAuCl4 (10-3 M) aqueous solution, light golden color of the reaction mixture changes to dark purple color within 5 min. This change of color indicates that the formation of AuNP has taken place. The absorbance intensity of the reaction mixture increases exponentially with time. Aqueous HAuCl4 (10-3 M) solution was tested for verification with an exposure to microwave for a period of 5min and it was found that it did not show any peak in the visible region of the spectra. SPR developed as a clear visible peak at 525 nm confirms the influence of aqueous Desmodium Gangeticum leaf extract in reducing Au3+ ions to AuNP from aqueous HAuCl4 solution (shown in Fig 1. Absorbance intensity increases steadily as a function of reaction time. Change in reaction mixture color is attributed to excitation of internal metal surface plasmon vibrations of the nanoparticles of the gold [19].

Figure 1: UV-VIS Spectra indicating microwave-assisted Gold nanoparticles synthesis recorded as a function of time

XRD investigation was carried out to confirm the crystalline nature of gold nanoparticles and result has been shown in figure 2. Prominent peaks at (111), (200), (220) and (311) are the Gold nanoparticles peaks and match the standard JCPDS file 04-0783 pattern.

Figure 2: XRD Patterns of crystalline Gold nanoparticle

Figure 3: EDX of Gold nanoparticles

The results of EDX analysis are shown in Fig. 3. Elemental gold can be seen in the graph presented by the EDX analysis in support of XRD results, which indicated the reduction of gold ions to elemental gold.

Figure 4: FTIR Analysis of Biosynthesize Gold nanoparticles

The stretch of -OH group was observed at wavenumber 3371 cm−1, C-H group at 2946 cm−1, C=C aromatic group at 1609 cm−1, -C-O aromatic ring at 1402 cm−1, and C-OC group at 1093 cm−1. (Shown in Figure 4)

We can presume that the flavonoids [20] and Terpenoids [21] which are abundant in Desmodium Gangeticum leaves show characteristic absorption peaks appear to be responsible for accelerated reduction and capping process. Terpenoids are poorly water soluble and hence may not be among prime moieties involved in the reduction process. It can be understood that the flavonoids could be adsorbed on the metal surface by interaction with carbonyl groups

The bio-moiety capping which also confers the stability of silver nanoparticles colloidal solution produced by this modus is noteworthy.

FESEM images are shown in figure 5. If observed carefully a layer of bio moiety is seen covering the metal surface of all nanoparticles. This organic moiety might have taken part the reduction of AU+ ions to Gold nanoparticles.

Figure 5: FESEM Images of Gold nanoparticles

Figure 6 shows TEM image which gives a clear understanding of the morphology of functionalized Gold nanoparticles. TEM images showed that particles are mostly triangular and spherical in shape, whereas some particles showed hexagonal shapes as well. The sizes found for NPs are roughly in the range of 12–20nm.

Figure 6: (A) HR-TEM Image of Gold nanoparticles and (B) Size distribution histogram Gold nanoparticles

Figure 7: (Table1) Antioxidant activity of Biosynthesized Gold Nanoparticles

S. No	Concentration µg/ml	% Inhibition
S. No	Concentration µg/ml	Std	DGG
1	200	76.45736	65.4904
2	150	58.29441	62.63622
3	100	39.21467	49.92216
4	50	28.52448	44.43868
5	20	21.65715	36.49888

The antioxidant methods confirmed that the Gold nanoparticles have significant antioxidant activity as compared to Standard drug.

CONCLUSION:

To conclude, the GNPs were produced by the use of the extract of Desmodium Gangeticum as reducing and capping agent. In this study, it was observed that the reaction is rapid and is completed within few minutes at room temperature. This method is green and environmentally friendly. Thus, the synthesized GNPs could have a high potential for use in biological applications. This method is inexpensive and highly recommended to be used in large-scale production of GNPs

ACKNOWLEDGEMENT:

Authors are thankful to the authority of Bahra University for fulfill the research work by providing the essential infrastructure.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 20.09.2019 Modified on 17.10.2019

Research J. Pharm. and Tech 2020; 13(6): 2685-2689.

DOI: 10.5958/0974-360X.2020.00477.1