INTRODUCTION:

Nanotechnology is the newest and one of the most promising areas of research in modern medical science. It literally means any technology on nano scale that has applications in the real world. It encompasses with synthesis, strategy and manipulation of particle’s structure ranging from approximately 1 to 100nm in size ^[1]. Nanotechnology has a profound impact on our economy and society in the early 21^st century.

Various types of nanomaterials like copper, zinc, magnesium, titanium, alginate, gold and silver have come up. Among several noble metal nanoparticles, silver nanoparticles have attained a special focus. Silver nanoparticles (AgNs) are receiving broad interest for a wide number of applications such as in optics, selective coatings for solar energy absorption, biolabeling, catalysts, and antibacterial agents owing to their unique properties^[2]. In antibacterial application, for instance, apart from being effective, AgNs still remain a popular choice due to their non toxicity towards human in comparison to other metals or materials ^[3].

But, scarcity leads them to be expensive and limits their applications. To compensate this problem, various synthesis methods were developed^[4-7]. Many conventional methods for synthesizing silver nanoparticles require different chemicals which are expensive and hazardous to mankind. So, green synthesis of AgNs is desirable approach to provide an economic, eco-friendly, and cleaner synthesis route^[8].

Considering the vast potentiality of plants as sources of anti-bacterial agents, current investigation aims to apply a biological green technique for the synthesis of silver nanoparticles as an alternative to conventional methods. In this regard, present study was designed to prepare silver nano particles using eco-friendly method i.e., by using aqueous extract of seeds of Sorghum bicolor and screen it for anti-bacterial activity.

MATERIALS AND METHODS:

Collection and authentication of plant material: Sorghum bicolor seeds were purchased from local market and authenticated by botanist Dr. K. Madhava Chetty, Asst. Professor, Department of botany, S.V University, Tirupati and with voucher number -2301 specimen was deposited in S.V. University, Botany Department, Tirupati.

Preparation of aqueous extract:

The seeds were ground into coarse powder by using a mixer blender. To 10g of powder 90ml of distilled water is added. Boiled for 1 hour and filtered. Thus aqueous extract was prepared.

Preliminary Phytochemical studies:

Preliminary phytochemical studies were carried out as per standard methods.

Preparation of silver nanoparticles:

To 10ml of aqueous extract, 90ml of 0.001M silver nitrate was added and heated until colour change (brown colour) was observed. The colour change indicates the formation of silver nanoparticles. Then the solution was centrifuged at 10,000rpm for ten minutes and the process was repeated for three times and the pellet obtained was dried in Freeze drier and used for further experimentation.

Characterisation of Silver NanoParticles:

UV-Vis Spectra analysis:

The reduction of pure Ag+ ions in supernatant solution was monitored by measuring the UV-Vis spectrum of the reaction medium after diluting a small aliquot of the sample with distilled water.

FT-IR:

For FTIR measurements, the silver nanoparticles solution was centrifuged at 15,000 rpm for 10 min. The pellet was washed three times with 20ml of de-ionized water to get rid of the free proteins/enzymes that are not capping the silver nanoparticles. The samples were dried and grinded with KBr pellets and analyzed on a Brucker FT-IR.

SEM:

Scanning Electron Microscopic (SEM) analysis was done using Hitachi S-4500 SEM machine. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 min.

TEM:

Transmission electron microscopy (TEM) analysis of the synthesized silver nanoparticles was performed on a LEO model 912AB instrument at an accelerating voltage of 100kV. A drop of the nanoparticle suspension was placed on carbon coated copper grids and allowing the solvent to evaporate prior to analysis.

Screening of anti-bacterial activity:

Silver nano particles prepared with aqueous extract were screened at concentrations 50, 100, 150, 200µg/ml for in vitro anti-bacterial activity against two gram positive bacterial strains i.e., Staphylococcus aureus, Bacillus subtilis and one gram negative bacterial strain Escherichia coli using cup-plate method in nutrient agar media by measuring the zone of inhibition^[9]. Streptomycin at a concentration of 10mg/ml was used as positive control and tested for anti-bacterial activity. Anti-bacterial activity of aqueous extract of seeds of Sorghum bicolor was tested using different concentrations i. e., 25, 50, 100mg/ml. 0.001M Silver nitrate solution was prepared and tested for anti-bacterial activity.

Bacterial organisms were obtained from Department of Microbiology, SPMVV, Tirupati. Cultures of test organisms were maintained on nutrient agar slants and were sub cultured in prior to testing. Nutrient agar medium containing Petri dishes were prepared. Test organism was inoculated by spread plate method using L-shaped rod. After spreading the plates are allowed to dry for 10 minutes. Then wells or cups were made with the help of cork borer of diameter 6 mm. The test solutions at different concentrations were added to the respective well aseptically and labeled accordingly. After incubation of plates at 37±1⁰C for 24 hours the diameter of zone of inhibition surrounding each of the wells was measured with the help of an antibiotic zone reader. All the experiments were carried out in triplicate ^[10].

RESULTS:

Preliminary phyto-chemical studies of aqueous extract of Sorghum bicolor:

The preliminary phytochemical studies of aqueous extract of seeds of Sorghum bicolor revealed the presence of various phytoconstituents as shown in Table 1.

Characterisation of silver nano particles:

Silver nanostructure exhibits interesting optical properties directly related to surface Plasmon resonance (SPR), which is highly dependent on the morphology of the samples. The SPR band in nanoparticles solution remain close to 410nm (Figure 1), suggesting that the nanoparticles were dispersed in the aqueous solution with no evidence for aggregation in UV-Vis absorption spectrum.

The FTIR spectrum (Figure 2) of silver nanoparticles showed strong IR bands characteristic of hydroxyl (3411.01 cm-1), alkanes (2,924.04 and 2,085.61 cm-1), C=O (1,632.01 cm-1) and phenols (1,092.30 cm-1) functional groups (Figure 2). The FTIR analysis strongly supported the capping behaviour of bioreduced silver nanoparticles synthesized by seed extract of Sorghum bicolor which in turn imparted the high stability of the synthesized silver nanoparticles.

The scanning electron microscope image of the silver nanoparticles was shown in the Figure 3. It indicated the well dispersed particles in the size range from 20nm -200nm. Further the TEM result revealed that silver nanoparticles were in spherical shape as shown in Figure 4.

Screening of anti-bacterial activity of silver nanoparticles:

When compared to aqueous extract of Sorghum bicolor and silver nitrate solution, the prepared phyto silver nanoparticles had shown more zone of inhibition against bacterial strains in microgram levels where as aqueous extract had shown antibacterial activity at relatively high concentrations (Table 2,3 and 4)

Table 1: Results of preliminary phytochemical studies

Phytochemicals	Name of the test	Result
Alkaloids	Dragendroff’s test	-ve
Proteins and Amino acids	Ninhydrin test	+ve
Saponins	Froath test	+ve
Flavonoids	Shinoda test	+ve
Glycosides	Borntragger’s test	+ve
Steroids	LibermannBurchard test	-ve
Tannins	Lead Acetate test	+ve
Triterpenoids	Salkowski Test	-ve
Carbohydrates	Molisch’s Test	+ve
Fats	Saponification Test	+ve
Phenols	Fecl₃test	+ve
Gums and mucilages	Swelling test	-ve

Table 2: Anti bacterial activity of aqueous extract of seeds of Sorghum bicolor

Name of the tested Bacteria	zone of Inhibition (in cm)
Name of the tested Bacteria	25 (mg/ml)	50 (mg/ml)	100 (mg/ml)
Staphylococcus aureus	1.1	1.5	2
Bacillus subtitis	_	0.5	1.3
Escherichia coli	__	_	0.5

Table 3: Anti bacterial activity of 0.001 M silver nitrate solution

Name of the tested bacteria	Zone of Inhibition (in cm)
Staphylococcus aureus	2.0
Bacillus subtitis	1.8
Escherichia coli	1.4

Table 4: Antibacterial activity of silver nano particles

Name of the tested bacteria	Zone of Inhibition (in cm)
Name of the tested bacteria	50 (µg/ml)	100 (µg/ml)	150 (µg/ml)	200 (µg/ml)
Staphylococcus aureus	2.0	2.4	2.6	3.0
Bacillus subtitis	1.9	2.2	2.4	2.8
Escherichia coli	1.2	1.5	1.6	1.9

Figure 1: UV-Visible spectrum of prepared silver nanoparticles

Figure 2: FTIR spectra of prepared silver nanoparticles

Figure 3: SEM images of prepared Silver nanoparticles

Figure 4: TEM images of prepared silver nanoparticles

DISCUSSION:

In the current investigation, simple green pot synthesis of silver nanoparticles by reducing the silver ions present in the solution of silver nitrate with aqueous seed extract of Sorghum bicolor was carried out.

A facile, environmentally benevolent green synthetic route is used for synthesis of silver Nanoparticle. The Phytofabrication of silver nanoparticle by using seed extract of Sorghum bicolor without involvement of any toxic chemicals. The metal ions reduction occurred very rapidly, and the reduction of Ag ions was completed within 4 hours which was observed by the colour change from yellow to dark brown. This was further confirmed by UV spectroscopy. In accordance with earlier reports on green synthesis of nanoparticles UV-vis absorption showed band close to 410 nm which suggests the formation and no aggregation of the silver nanoparticles ^[11].

FTIR analysis confirmed that the bio-reduction of Ag+ ions to silver nanoparticles are due to the reduction by capping material of plant extract. In TEM analysis it was demonstrated that silver nanoparticles are spherical in shape and further by scanning electron microscope (SEM) the particle size of prepared nanoparticles was found to be in the range of 20 to 200nm. Synthesized silver nano particles were screened for anti-bacterial activity at 50, 100, 150, 200 µg/ml concentrations against two Gram positive bacteria (Staphylococcus aureus, Bacillus subtilis) and one Gram negative bacteria (Escherichia coli).

When compared to aqueous extract and silver nitrate solution, the synthesized Silver nano particles exhibited more zone of inhibition against tested bacterial strains. Further silver nanoparticles showed good anti-bacterial activity even in microgram concentration where as aqueous extract has shown the activity in milligrams. The activity exhibited may be due to the attachment of the nanoparticles to the cell membrane and their penetration inside the bacteria^[12]. The bacterial membrane contains sulfur-containing proteins and the silver nanoparticles interact with these proteins in the cell as well as with the phosphorus-containing compounds like DNA ^[13]. Highest bactericidal activity of prepared nanoparticles may possibly caused by synergistic antibacterial effects of AgNPs and naturally-occurring phytoconstituents in the seeds of Sorghum bicolor.

CONCLUSION:

This study can be applied for rapid, cost effective and eco-friendly green synthesis of silver nanoparticles using seeds of Sorghum bicolor. Further the prepared silver nanoparticles exhibited good anti-bacterial activity and can be suggested to be employed as anti bacterial agent.

REFERENCES:

1. Bhushan B. Introduction to Nanotechnology, Springer Handbook of Nanotechnology 2010; pp.1-12.

2. Zainal A, Rosiyah Y, Shamala D et al. Green Synthesis of Silver Nanoparticles Using Apple Extract and Its Antibacterial Properties. Advances in Materials Science and Engineering. 2016; 1-6.

3. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials. Biotechnology Advances. 2009; 27(1): 76-83.

4. Caswell KK, Bender CM, Murphy CJ. Seedless, surfactantless wet chemical synthesis of silver nanowires. Nano Letters. 2003; 3(5): 667-669.

5. Yin Z,. Li Z. Zhong B. GatesYet al. Synthesis and characterization of stable aqueous dispersions of silver nanoparticles through the Tollens process. Journal of Materials Chemistry. 2002; 12(3): 522527.

6. Khanna PK, Singh N, Charan S, Subbarao VVS et al. Synthesis and characterization of Ag/PVA nanocomposite by chemical reduction method. Materials Chemistry and Physics. 2005; 93(1): 117-121.

7. Wiley B, Herricks T, Sun Y, Xia Y. Polyol synthesis of silver nanoparticles: use of chloride and oxygen to promote the formation of single-crystal, truncated cubes and tetrahedrons. Nano Letters. 2004; 4(9): 1733-1739.

8. Singh A . Green synthesis of silver nanoparticles using Argemone mexicana leaf extract and evaluation of their antimicrobial activities. Digest Journal of Nanomaterials and Biostructures.2010; 5(2): 483-489.

9. Mahmoud WM, Abdelmoneim TS, Elazzazy AM. The Impact of Silver Nanoparticles Produced by Bacillus pumilus As Antimicrobial and Nematicide. Front Microbiol. 2016;7:1746.

10. Shubha HS, Hiremath RS. Evaluation of antimicrobial activity of Rasaka Bhasma. Ayu. 2010;31(2):260–262.

11. Abiola. Green synthesis of silver nanoparticles using terrestrial fern (Glechinia pectinata (wild) persl.) characterization and antimicrobial studies, Heliyon, 2019, 5(4):e01543.

12. Siddiqi KS, Husen A, Rao RAK. A review on biosynthesis of silver nanoparticles and their biocidal properties. J Nanobiotechnology. 2018;16(1):14.

13. Agnihotri S, Mukherji S, Mukherji S. Size-controlled silver nanoparticles synthesized over the range 5–100 Nm using the same protocol and their antibacterial efficacy. RSC Adv. 2014; 4:3974–3983.

Received on 08.12.2019 Modified on 20.02.2020

Research J. Pharm. and Tech. 2020; 13(12):5935-5938.

DOI: 10.5958/0974-360X.2020.01036.7