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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 2  |  Page : 222-228

Green synthesis of silver nanoparticles using Eranthemum Pulchellum (Blue Sage) aqueous leaves extract: Characterization, evaluation of antifungal and antioxidant properties


1 Department of Chemistry, S P Jain College, Veer Kunwar Singh University, Sasaram, Bihar, India
2 Department of Chemistry, Veer Kunwar Singh University, Ara, Bihar, India
3 Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Department of Chemistry, K P College, BNMU, Madhepura, Bihar, India
5 School of Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission25-Apr-2021
Date of Acceptance03-May-2021
Date of Web Publication16-Jun-2021

Correspondence Address:
Shyam Raj Yadav
Department of Chemistry, S P Jain College, Veer Kunwar Singh University , Sasaram - 821 115, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_63_21

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  Abstract 


Background: Nanoparticles modulate several physiochemical and biological properties. In this regard, silver nanoparticles (AgNPs) have shown remarkable applications. The present research work comprises green synthesis of AgNPs using aqueous leaves extract of Eranthemum pulchellum and evaluation of its efficacy as antifungal and antioxidant agents. Methods: Synthesized AgNPs have been characterized by Fourier Transforms Infrared (FTIR) Spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and ultraviolet-visible spectrophotometry. Qualitative phytochemical analysis of aqueous leaves extract of E. pulchellum and FTIR spectrum of the synthesized AgNPs suggest about the presence of different phytochemicals and functional groups, respectively, which are responsible for reducing silver ions as well as capping and stabilizing synthesized nanoparticles. Results: The wavelength of maximum absorbance of AgNPs solution near to 439 nm indicates the spherical morphology. XRD infers about average crystalline size of the synthesized AgNPs to be ~12 nm. Selected area electron diffraction pattern of the synthesized AgNPs shows four visible diffraction rings corresponding to (111), (200), (220), and (311) set of planes which are attributed to face centered cubic metallic silver. The size and spherical shape of the synthesized AgNPs have been further determined by TEM. The synthesized AgNPs have shown a significant antifungal activity against Aspergillus flavus (AF-LHP-NS7) strain with minimum inhibitory concentration value of 200 μg/mL. The synthesized AgNPs have also shown strong antioxidant efficacy through Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid assay with IC50 value of 462.56 μg/mL. Conclusions: The present study shows a green and facile synthesis of AgNPs. Leaves extract of E. pulchellum has been first time utilized as efficient reductant for the AgNPs synthesis. These AgNPs have shown potent antifungal and antioxidant activity.

Keywords: Antifungal, antioxidant, green synthesis, phytochemicals, reducing agent, silver nanoparticles


How to cite this article:
Prakash J, Shekhar H, Yadav SR, Dwivedy AK, Patel VK, Tiwari S, Vishwakarma NK. Green synthesis of silver nanoparticles using Eranthemum Pulchellum (Blue Sage) aqueous leaves extract: Characterization, evaluation of antifungal and antioxidant properties. Biomed Biotechnol Res J 2021;5:222-8

How to cite this URL:
Prakash J, Shekhar H, Yadav SR, Dwivedy AK, Patel VK, Tiwari S, Vishwakarma NK. Green synthesis of silver nanoparticles using Eranthemum Pulchellum (Blue Sage) aqueous leaves extract: Characterization, evaluation of antifungal and antioxidant properties. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Jul 23];5:222-8. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/222/318439




  Introduction Top


Nanoparticles impart several vital physiochemical and biological properties because of their nano range size which leads to high surface area having low volume. Among the various metal nanoparticles, silver nanoparticles (AgNPs) have been extensively exploited for the purposes of various technological and medicinal areas. AgNPs have been found highly efficacious for the development of potent drug candidate for the treatment of diabetes, microbial infections, aging, cancer, inflammation as well as for the targeted drug delivery.[1],[2],[3],[4],[5],[6],[7],[8],[9] In the literature, there are several physical and chemical methods reported for the synthesis of AgNPs, but the green chemistry-mediated approach for the synthesis of AgNPs does not incorporate toxic and expensive chemicals.[10],[11],[12] Green approaches utilizing phytochemicals have several advantages because these phytochemicals not only act as reducing and capping agent but also stabilizes synthesized nanoparticles as well as augment biological efficacy.[13],[14] Eranthemum pulchellum (blue sage) is widely distributed in various parts of India, China, and Nepal. Extract of leaves, stem, and root of this plant is utilized as antimicrobial and antiseptic agents.[15] Iridoid glucoside, an essential constituent for plant defense mechanism against pathogens and insects, has been also isolated from E. pulchellum stem.[16]

In continuation of phytochemical-mediated green synthesis of AgNPs, herein we report a simple, efficient, and economically viable method that furnishes stabilized AgNPs in aqueous media utilizing aqueous leaves extract of E. pulchellum. Further, AgNPs have been characterized and evaluated for its antifungal and antioxidant properties.


  Methods Top


All required chemicals were purchased from Merck to Sigma-Aldrich. All chemicals were used without further purification.

Identification of plant

Leaves of the E. pulchellum [Figure 1] were collected from Kaimur hills in Rohtas district of Bihar (India). This plant belongs to the Acanthaceae family. Identity of the test plant was confirmed by Prof. N. K. Dubey, Department of Botany, Banaras Hindu University, Varanasi (India).
Figure 1: Eranthemum pulchellum (blue sage) plant

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Preparation of plant leaves extract

Leaves of the E. pulchellum were washed three times with double distilled water. These leaves were dried in open air under shaded environment for 15 days at room temperature. Dried leaves were further washed with double distilled water and chopped in small pieces. 10 g of these chopped leaves were added in 50 mL of water and heated at 50°C for 45 min. Extract was cooled to room temperature and filtered using Whatman (Grade-1) filter paper. This filtered aqueous leaves extract was used for the synthesis of AgNPs.

Biosynthesis of silver nanoparticles

In a 250 mL Erlenmeyer flask, 50 mL of aqueous silver nitrate (10 mM) was added. To this aqueous silver nitrate solution 50 mL of filtered aqueous leaves extract was added drop wise under continuous stirring at room temperature (28°C ± 2°C). After 90 min, a color change of reaction mixture from pale yellow to dark brown [Figure 2] with suspended particles indicated the formation of AgNPs. Further, the reaction mixture was centrifuged at 10,000 rpm for 15 min to obtain dark gray precipitate of AgNPs. These obtained AgNPs were further washed thrice with double distilled water to remove associated impurities and dried at room temperature.
Figure 2: Synthesis of silver nanoparticles

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Characterization techniques of silver nanoparticles

Ultraviolet-visible (UV-VIS) spectra and Fourier Transforms infrared (FTIR) spectra were recorded with UV-VIS Spectrophotometer-2373 and PerkinElmer (spectrum 2, UK) instruments, respectively. Powder X-ray diffraction (XRD) was performed with the D8 Bruker, Germany, and samples were scanned over a range of 2θ values, 5°–80°. Transmission electron microscopy (TEM) images, selected area electron diffraction (SAED), and energy dispersive X-ray (EDX) spectrum were recorded on FEI TECHNAI G[2] win instrument (Model No. 9432 050 22121), operating at an acceleration voltage of 200 kV.

Antifungal assay of synthesized silver nanoparticles

Antifungal assay of synthesized AgNPs was performed against AF-LHP-NS7 strain by previously reported method.[17] Before the experiment, a stock solution of 10 mg/mL of AgNPs was prepared in dimethylsulfoxide. Requisite amount of AgNPs solution (200 μg/mL–1000 μg/mL) was added to the potato dextrose agar media containing 10 μL spore suspension of AF-LHP-NS7 strain. All samples were kept in incubator at 25°C ± 2°C for 7 days. Minimum concentration of AgNPs that inhibited the growth of fungus is considered as its minimum inhibitory concentration (MIC). Further, minimum fungicidal concentration (MFC) of AgNPs was also analyzed.[18]

Antioxidant efficacy through Azino-bis-3- ethylbenzothiazoline-6-sulfonic acid assay

Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) scavenging potential of AgNPs was determined by literature method of Sridhar and Charles with slight modification. ABTS•+ stock solution was prepared mixing 7 mM ABTS•+ aqueous solution and 2.45 mM potassium persulfate aqueous solution in equal amount and kept them to react for overnight.[19] Different concentration of AgNPs was added to the reaction mixture and absorbance was taken at 734 nm. Antioxidant efficacy was determined in terms of IC50 value using the formula as follows:

% Radical scavenging potential = (ABlank-ASample)/ABlank × 100

where, A = Absorbance at 734 nm.


  Results Top


Phytochemical analysis

Qualitative phytochemical analysis of aqueous leaves extract of E. pulchellum was performed according to the standard methodologies reported in literature.[20],[21] Obtained results are shown in [Table 1].
Table 1: Phytochemical analysis of aqueous leaves extract of Eranthemum pulchellum

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Biosynthesis of silver nanoparticles

A color change of reaction mixture from pale yellow to dark brown [Figure 2] and [Figure 3] with suspended particles after the addition of plant leaves extract indicated the formation of AgNPs.
Figure 3: Progress of silver nanoparticles formation at different time interval

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Ultraviolet-visible spectroscopy analysis of synthesized silver nanoparticles

During monitoring of progress of the reaction, it was observed that initial color of the silver nitrate solution changed from colorless to light yellow with the wavelength of maximum absorbance near to 439 nm [Figure 4]. The color of reaction mixture further changed to reddish brown with time designated the formation of AgNPs.[22]
Figure 4: Absorbance of reaction mixture at different concentration of leaves extract after 10 min

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Fourier transforms infrared spectroscopy analysis

FTIR spectrum of the synthesized AgNPs is shown in [Figure 5]. This spectrum highlights the major peaks obtained from interaction of silver ions and functional groups associated with phytochemicals in aqueous leaves extract. These interactions play a major role in stabilizing, capping, and reduction of silver ions.
Figure 5: Fourier transforms infrared spectrum of synthesized silver nanoparticles

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Powder X-ray diffraction analysis

[Figure 6] shows the XRD analysis of the synthesized AgNPs. The crystalline diffraction intensities were recorded from 20 to 80 at 2θ angles. Four strong characteristic Bragg's reflection peaks were observed at 2θ angles of 37.78°, 45.78°, 64.12°, and 76.91° corresponding to the (111), (200), (220), and (311) set of lattice planes.[23] Beside above four peaks, other obtained peaks depict the role of phytochemicals in aqueous leaves extract in reducing, capping, and stabilizing the synthesized AgNPs.[24]
Figure 6: X-ray diffraction pattern of synthesized silver nanoparticles

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Transmission electron microscopy analysis of synthesized silver nanoparticles

TEM image [Figure 7]a and [Figure 7]b infers that synthesized AgNPs are of spherical/distorted spherical shape of the size 5 nm–17 nm with mostly without agglomeration.
Figure 7: (a and b) Transmission electron microscopy image of silver nanoparticles (c) selected area electron diffraction pattern of silver nanoparticles (d) energy dispersive X-ray analysis pattern of silver nanoparticles (e) Silver nanoparticles average size distribution curve

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Antifungal assay of synthesized silver nanoparticles

Synthesized AgNPs was found to have efficacious antifungal activity [Table 2]. MIC of AgNPs against AF-LHP-NS7 strain was found to be 200 μg/mL.
Table 2: Representing antifungal activity of silver nanoparticles

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Antioxidant activity of silver nanoparticles

AgNPs was found to have strong free radical scavenging activity as its IC50 value was 462.56 μg/mL obtained by ABTS•+ assay [Figure 7].


  Discussion Top


Phytochemical-mediated synthesis of AgNPs has been performed by a facile, efficient, and biocompatible method. Phytochemicals present in aqueous leaves extract play a vital role in reducing silver ions and stabilizing, capping of synthesized AgNPs.

[Figure 3] depicts the UV-vis Spectra for the formation of AgNPs at different time intervals by taking 2 mL AgNO3 (10 mM) solution and 100 μL aqueous leaves extract. It has been found that, with the increase in reaction time, the absorbance of the solution also increases. It has been reported that absorbance of AgNPs solution near to 450 nm corresponds to size of AgNPs in the range of 10–100 nm with spherical shape.[25] [Figure 4] depicts about absorbance of reaction mixture at different concentration of leaves extract. This figure shows that the extent of reaction is increasing with the increase in concentration of aqueous leaves extract. With the increase of amount of aqueous leaves extract, maximum absorbance underwent a red shift indicating an increase of the size of the AgNPs.[22]

The FTIR spectrum [Figure 5] reveals the broad absorption peak at 3372 cm−1 is attributed to the O-H stretching of the hydroxyl group. The absorption bands at 2919 and 2843 cm−1 correspond to C-H stretching vibration. The absorption bands obtained at 1586, 1375, and 1238 cm−1 correspond to medium C = N, C–O, and C–N stretching vibrations, respectively. The small absorption peak at 3750 cm−1 can be attributed to N-H stretching vibration.

XRD spectrum [Figure 6] with four characteristic peaks depicts the polycrystalline nature of metallic silver. Average crystalline size of the AgNPs was calculated using Scherrer formula, D = 0.94 λ/β cosθ, where D = average crystalline size, λ = X-ray wavelength of 1.54 Å, β = full wavelength half maximum, and θ = Bragg diffraction angle. The average crystalline size of the synthesized AgNPs was found to be ~12 nm corresponding to (111) plane.[26]

The distribution size histogram [Figure 7]e shows that most of the synthesized AgNPs are of ~10 nm size. Similar to XRD analysis, SAED pattern [Figure 7]c of the synthesized AgNPs shows four visible diffraction rings corresponding to (111), (200), (220), and (311) set of planes which are attributed to face centered cubic metallic silver. [Figure 7]d shows the EDX spectrum of synthesized AgNPs that infers about the presence of metallic silver and other associated elements (C, N, and O) from phytochemicals.

The potent antifungal activity [Table 2] of AgNPs with MIC value of 200 μg/mL against AF-LHP-NS7 strain was also found to be MFC at which AF-LHP-NS7 strain of fungus was completely killed. The potent antifungal activity of AgNPs may be due to its most probable mode of actions such as disruption of fungal plasma membrane, mitochondrial membrane potential, and leakage of vital cellular ions.

High antioxidant efficacy [Figure 8] of AgNPs would also be acting as one of the most important factors contributing toward its antifungal properties.[27] Further, potent antioxidant efficacy of AgNPs also recommends it as a novel green alternative of several harmful synthetic antioxidant compounds such as butylated hydroxyl anisole and butylated hydroxyl toluene.[28]
Figure 8: Antioxidant activity of silver nanoparticles

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Qualitative phytochemical analysis of aqueous leaves extract confirms the presence of tannin, flavonoids, and phenols [Table 1]. Several articles have reported the role of these classes of bio-molecules as a reducing agent for the reduction of Ag + to Ag[0].[29],[30] A plausible mechanism based on phenolic moiety-mediated formation of AgNPs is proposed in [Figure 9].
Figure 9: Plausible mechanism for the formation of silver nanoparticles

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  Conclusions Top


Based on UV-vis, XRD, FTIR, TEM-EDX, SAED analysis, the AgNPs synthesized using E. pulchellum leaves extract showed the spherical shape and 5–17 nm size along with phytochemical capping over nanoparticle. The potent antifungal and antioxidant activity establish the synthesized AgNPs as efficacious bioactive agent. Moreover, the utilization of E. pulchellum leaves extract as reductant is a novel and highly useful method for AgNPs synthesis.

Acknowledgment

The authors (JP and SRY) are thankful to the Principal, S P Jain College, Sasaram for providing all required amenities and infrastructure to carry out this research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2]



 

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