Biosynthesis of Silver Nanoparticles using Medicinal Plant Anacyclus pyrethrum and its Antibacterial Efficacy

 

Sapna Tyagi1*, Tanveer Alam2, MohdAzhar Khan3, Hina Tarannum4, Neha Chauhan5

1,2DepartmentofChemistry,K.L.D.A.V.P.G.College,Roorkee-247667,India

3,5DepartmentofBiotechnology,ShooliniUniversity,Solan-173230,India

4College of Sciences, Department of Chemistry, Yanbu, Taibah University, Kingdom of Saudi Arabia

*Corresponding Author E-mail: sapnatyagi88@gmail.com

 

ABSTRACT:

Biologically synthesized silver nanoparticles are extensively used in the field of medicines, chemistry, zoology, botany, physics and other area of research. AgNPs are well known to have inhibitory and bactericidal activity. In the present research, biogenic green synthesis of silver nanoparticles (AgNPs) using medicinally important plant Anacyclus pyrethrum extract has been carried out. No additional toxic chemical was used in the process of reduction and capping of nanoparticles. Synthesized AgNPswas characterized by UV-Vis spectroscopy, X-rays diffraction, FTIR and Transmission electron microscopy. TEM images represents that the synthesized nanoparticles showed spherical geometry with an average size of 14 to 34 nm. FTIR studies shows the responsible functional groups of biomolecules present in the plant extract for reduction and capping of nanoparticles. The XRD spectra shows that the AgNPs are of face centered cubic (FCC) structure. Silver nanoparticles synthesized by Anacyclusphythrumalso have a great potential against bacteria P. aeruginosa, followed by S. Typhi, and K. Pneumonia, respectively. So that plant mediated synthesis of nanoparticles has been suggested as a cost effective and environment friendly process and alternative to chemical and physical methods.

 

KEYWORDS:Anschlusspyrethrum, Green synthesis, Nanoparticles, XRD, FTIR.

 

 


INTRODUCTION:

Nanotechnologyismostoftenrefertoasthemanufactureandmanipulationofpurpose-madestructureswhichareatleastsmallerthan100nm[1].Nanotechnologyismanilydefinedasageneral-purposetechnologybecausenanoparticleshaveasignificantimpactonalmostallindustriesandallareasofsociety.Thegreenapproachforsynthesisofnanoparticleswillofferlasting,cleaner,safer,andenvironmentallyproductsforthehome,forcommunications,medicine,transportation,agriculture,andallindustriesingeneral[2-3].

 

Nano range microscopic particles are the excellent research area of interest because they are bridging the gap between the bulk material and molecular or atomic structure level.

 

By literature review, many physical and chemical methods such as sol-gel process, chemical precipitation, reverse micelle method, etc. were found for the synthesis of metallic nanoparticles [4].

 

Nowadays, biological and green methods are used for the production of nanoparticles by using medicinal plants, fungi, and bacteria. Physiochemical methods required lots of hazardous and toxic chemicals and are highly expensive and time-consuming process [5]. The new green approach is developed to overcome this kind of problems by the researchers. Nature has blessed us with an enormous wealth of herbal plants which are widely distributed all over the world as a source of therapeutic agent [6] and for the prevention and cure of various diseases [7]. Therefore, in this study an attempt has been made to explore such plants and form the Nanoparticlesto make a significant contribution to the society. Plants seem to be the Nano factories for the production of nanoparticles [8]. Plants extracts contain various types of secondary metabolites having reducing as well as capping agent’s properties for the production of nanoparticles. It is a very reliable and environment-friendly process without using any toxic chemicals. Silver nanoparticles have wide uses in different regions such as in nanobiotechnologicalresearch[9-10], sensors[11], catalysis[12], cell electrodes[13], low-cost paper batteries [14] etc. They are used as antimicrobial agents in wound dressings [15-17] as topical creams to prevent wound infections [18], and as anticancer agents [19].

 

Anacyclus pyrethrum (common name Akarkara) is a medicinally crucialperennial herb that belongs to family Asteraceae found in Indian subcontinent region, in the Himalayas, in northern India, North in Arabian countries. It is mainly known for its aphrodisiac effect on the body, and it also helps in the detoxification of excess waste and fluids from the body. Chemical obtained from this plant are pyrethrin, Inulin, alkyl amides, Anayclin, sesamin,andhydrocarolin. The alkyl amides are responsible for its aphrodisiac and neuroprotective properties.

 

Anacyclus pyrethrum root is used for atoothache, rheumatic and neuralgic affections and rhinitis. Ayurveda Pharmacopoeia of India indicates the use of the root in sciatica, paralysis, hemiplegic and amenorrhea [20]. Further, in our study these biologically synthesized nanoparticles were studied against different bacterial species to evaluate their antimicrobial efficacy. So that the main aim of this study is the focused on formulation of nanomaterials with the help of plant extracts which includes the enhancement of solubility, protection from toxicity, enhancement of pharmacology activity and protection from physical and chemical degradation.

 

2. MATERIALS AND METHODS:

2.1 Collection of Plant material:

The healthy plant materials of Anacyclus pyrethrum were collected from Patanjali herbal garden, HaridwarUttarakhand. All the analytical reagents were purchased from Fisher chemicals. Double distilled water was used throughout the experiments.

 

2.2 Preparation of plant extract:

For the green synthesis of silver nanoparticles, 10gm of roots were thoroughly washed with running tap water, followed by distilled water and dried at room temperature. Dried and powdered roots material was boiled with 100ml of Millipore water at 60o for 30 min as reported earlier. The extract was cooled to room temperature and filtered by Whatman filter paper No.1 and finally stored at 4oC for further experiment.

 

 

Figure1.Shows theplantandrootsofAnacycluspyrethrum

 

2.3 Preparation of silver nanoparticles:

For the synthesis of silver nanoparticles, 10 ml of aqueous roots extract was added to 90 ml of AgNO3 (2 mM) solution and was kept for 48 hours for the formation of AgNPs. The color changes of root extract indicated the formation of AgNPs. The reaction mixture was centrifuged at 10,000 rpm for 15 minutes. A pellet was collected followed by redispersion of pellets of AgNPs in deionized water to get rid of any uncoordinated biological impurities.

 

2.4 UV-VIS spectra analysis:

UV-VIS spectrum of synthesized AgNPs was carried out at different interval of time. The bioreduction of Ag+ to Ag0 and stability using roots extracts was monitored by periodic sampling of aliquots (1 ml) of aqueous component. The AgNPs show the Plasmon resonance at 430 nm[22]. From the study, it has been found that silver nanoparticles show the characteristic SPR at a wavelength in the range of 400-450 nm. Broadening of peak indicated that the particles are polydispersed[23].

 

2.5. X-ray diffraction analysis of AgNPs:

The synthesis and crystalline nature of nanoparticles were confirmed by X-ray diffraction (XRD) method. The particles size of AgNPs was confirmed by using Debye Sherrer's equation.

 

D=0.94λ/βcosθ:

 

Where D is the average crystalline domain size perpendicular to the reflecting planes, λ is the wavelength of X-rays, β is the full width at half maximum (FWHM), and θ is the diffraction angle.

 

2.6.TEManalysisofsynthesizedAgNPs:

Structural Morphology and size of the synthesized silver nanoparticles were identified by TEM images using PUSA TEM 91/N-III, JEOL TEM 1011 TEM 100 KVA instrument. A thin film of the sample was prepared on a carbon coated copper grid by just dropping a tiny amount of the sample on the grid and drying under thelamp. Hence the size, shape and phase composition of particles were studied by TEM.

 

2.7.FTIR:

FT-IR analysis was carried out by using PerkinElmer FT-IR C91158 Spectrum to identify the bioactive compounds of Anacyclus pyrethrum roots extract to reduce Ag+ ions and capping of the silver nanoparticles associated with synthesized AgNPs and spectrum was recorded from 4000 cm-1 to 400 cm-1.

 

2.8.AntibacterialpropertyofsynthesizedAgNPs:

The bacterial strains (Salmonella typhi (MTCC-734), Klebsiella pneumoniae (MTCC-39), Staphylococcus aureus (MTCC-737) and Pseudomonas aeruginosa (MTCC-741) were collected from IMTECH, Chandigarh. The microorganisms were sub-cultured in nutrient broth and incubated at 37°C for 24 hrsbefore the experiment.

 

Evaluationofantimicrobialactivityof Nanoparticles:

Antimicrobial activity of various nanoparticles was assessed by well diffusion method [24]. The turbidity of the subcultured microorganisms was adjusted with sterile distilled water using 0.5 McFarland as standard (~1.5 X 108 cells/ml). Mueller Hinton Agar (HiMedia) was prepared by dissolving readymade agar powder in distilled water. Agar plates were prepared and inoculated with the test microorganisms by spread plate method. The plates were left undisturbed for 30 min at room temperature. The powdered nanoparticles were weighed and dissolved in Dimethyl Sulfoxide (DMSO) and used in triplicates. Then, the solution was added in the wells in a constant concentration of 50µl/well. The standard of antibiotic Chloramphenicol (HiMedia) was also prepared as a positive control. DMSO was also added to a well as negative control to make sure that the solvent used for dissolving the extracts do not have antimicrobial activity. Then, the plates were incubated at 37°C for 24h in an upright position. After incubation, the zone of inhibition was measured and compared with the standard antibiotic zone.

 

Minimum Inhibitory Concentration:

The MIC assay was performed for those nanoparticles which were active by well diffusion susceptibility assay (inhibition zone >10 mm) [25, 26]. The MIC values were determined by microdilution method. The plates were prepared by dispensing 100μl of nutrient broth into each well. 100μl was taken from the stock solution of tested nanoparticles (concentration of 50mg/ml) and added into the first well of the plate. Then, two-fold serial dilutions were performed by using a micropipette. The obtained concentration range was from 50 mg/ml, and then added 50μl of inoculum to each well except negative control. The positive control of antibiotic (Chloramphenicol), negative control (Nutrient broth), Broth alone and the inoculums alone were also put in the experiment. The test plates were incubated at 37°C for 24h. The lowest sample concentration showing clear well and inhibited complete growth were taken as MIC value [27].

 

3. RESULT AND DISCUSSION:

3.1UV-Visiblespectroscopy:

UV-Visible spectroscopy is one of the key tools to study the synthesis of metal nanoparticles in aqueous solution. The reduction of silver metal ions inanaqueous solution by using roots extract of Anacyclus pyrethrum was monitored by the color change, i.e., colorless to reddish brown with the help of UV–Vis spectroscopy.

 

The characteristic SPR of colloidal Ag nanoparticles ranges between 300 to 500 nm. (Fig. 1). UV-Vis absorption spectra (fig. 1) represented that the broad SPR contained one peak at 420 nm. It is well known that AgNPs exhibit different colors depending on the size of the AgNPs and these are due to the excitation of SPR in the AgNPs. The SPR absorbance is sensitive to the plant extract concentration nature, size and shape of particles present in the solution.

 

 

Figure 1.Uv-Visible peak of AgNPs Solution at 420 nm

 

3.2 X-ray diffraction analysis of synthesized AgNPs:

Figure 2 shows the XRD diffraction pattern corresponding to reduced silver. Intense peaks were observed at 2θ value is equal to 38.37, 44.60, 64.91, 77.99 and could be recognized to the 111, 200, 220 and 311 crystallography planes of the face centered cubic (fcc) silver crystal, respectively. The average crystalline size is calculated using Debye Scherrer formula and the calculated average size of silver nanoparticles is 14 nm.

 


 

Position [°2Theta] (Copper (Cu))

 

Figure 2. XRD peak pattern of synthesized silver nanoparticles

 


3.3. FTIR:

FTIR spectrum of the roots extract shows peaks at about 3412, 2916, 2328, 1640, 1351, 1255 and 1039 cm-1. Peak at 3412 cm-1 arises due to N-H stretching of anamino group or is indicative of O-H group due to the presence of alcohols, phenols. Peak at 2916 cm-1indicates the presence of C-H bond stretching of the alkyl group. Peaks at 1640 cm-1are associated with N-H bond in amino acid. Peak at 1351 cm-1 represents C-N stretch vibration as well as to amide I bands of proteins in the roots extract. The band at 1255 cm-1 confirms the presence of C–O groups from polyols. C-O stretch assigned to alcohols represented by peak at 1039 cm-1

 

Figure 3.represents the FTIR Analysis of silver nanoparticles

 

3.4.TEManalysisofsynthesizedAgNPs:

TEM micrograph revealed that the particles are spherical, oval and well dispersed without agglomeration (fig.3). The particles size of synthesizes silver nanoparticles from Anacyclus pyrethrum roots extract is in the range of 19-34 nm. Various reports have provided evidence of extracellular synthesis of silver nanoparticles by TEM images.

 

3.5 Antibacterial property of synthesized AgNPs:

AgNPs have lately received agreat deal of attention and concern due to their antibacterial activity. In the present study, the biologically synthesized AgNPs from Anacyclus pyrethrum showed excellent antimicrobial activity against test microorganisms. (table1. The antibacterial activity of silver nanoparticles against the human pathogens showed varied levels of inhibition. The zone of inhibition of AgNPs for P. aeruginosa, K. pneumonea, and S. Typhi. were 11.2 ± 0.5; 10.0 ±1.2, 10.6 ±1.5 0.2 mm, respectively. As shown in Table-1, the present study revealed that AgNPs possess potential antibacterial activity against, but it was inactive against S. aureus.

 

 

3a

 

 

3b

Figure 4 TEM images of silver nanoparticles


Table-1:Antibacterialeffectofnanoparticlesagainstseveralbacterialstrains

S. No.

Bacterial strains

S. typhi

S. aureus

P. aeruginosa

K. pneumoniae

-C

+C

Nanoparticles

 

 

Zones of inhibition in mm

 

 

1

NP-3

10.6±1.5

-

11.2±0.5

10.0±1.2

-

25±0

NP=nanoparticles;mm=millimeter;+C=Positivecontrol;-C=Negativecontrol;-=Nozoneofinhibition

 

Table-2: Minimum inhibitory concentrations (MIC in mg/ml) of nanoparticles against bacterial strains.

S. No.

Bacterial strains

S. typhi

S. aureus

P. aeruginosa

K. pneumoniae

Nanoparticles

 

 

MIC in mg/ml

 

1.

NP-3

12.5

-

1.6

1.6

 

Fig.5:-HistogramofantibacterialactivityofnanoparticlesofAnacycluspyrethrum.Clearly,itcanbeseenthattheantibacterialpotentialofnanoparticlesagainstS.typhiismuchhigherthanotherstwobacteria.

 


The results obtained by measurement of the zone of inhibition were presented in table-1. As shown in table 1 and fig. 4, the nanoparticles showed a maximum zone of inhibition against P. aeruginosa, followed by S. Typhi, and K. Pneumoniae respectively.

 

Determination of MIC:

The maximum inhibitory concentration of the AgNPswas estimated by microdilution method was used. The lowestconcentration of AgNPs at which there is no visible growth of the organism is mentioned in Table 2.

 

CONCLUSION:

Silver nanoparticles were successfully synthesized by using green, cost-effective and environmentally friendly manner. The reduction of silver ions by using Anacyclus pyrethrum roots compound and stabilization of the AgNPs were thought to occur due to the surface plasmon resonance with the ingredients present in the plants roots extract. Formation of spherical shaped and well dispersed silver nanoparticles with an average particle size of 19 to 34 nm was identified with the help of TEM and XRD techniques. The FTIR data showed the biomolecules present in synthesized nanoparticles were acting as capping as well as reducing agents. The results explain that synthesized AgNPs showed antimicrobialactivity against Salmonella typhi, Klebsiellapneumoniae, and Pseudomonas aeruginosaand exhibit potential for medicines and other therapeutic applications.

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Received on 08.02.2018         Modified on 22.03.2018

Accepted on 20.04.2018         © AJRC All right reserved

Asian J. Research Chem. 2018; 11(3):515-520.

DOI:10.5958/0974-4150.2018.00092.5