World Journal of Pharmaceutical Sciences Ficus benghalensis ...

World Journal of Pharmaceutical Sciences ISSN (Print): 2321-3310; ISSN (Online): 2321-3086 Published by Atom and Cell Publishers © All Rights Reserved Available online at: http://www.wjpsonline.org/ Original Article

Ficus benghalensis mediates synthesis of silver nanoparticles: the green approach yields NPs that are its anti-bacterial and anti-oxidant N. Vasanth1, G. Melchias2*, P. Kumaravel3 Department of Botany, St. Joseph’s College (Autonomous), Tiruchirappalli, Tamil Nadu, India. Department of Biotechnology, St. Joseph’s College (Autonomous), Tiruchirappalli, Tamil Nadu, India.

1,2 3

Received: 25-05-2016 / Revised: 10-06-2016 / Accepted: 25-06-2016 / Published: 26-06-2016

ABSTRACT The current study emphasizes the process of synthesis of nanoparticles from the natural residue that is rapid and eco-friendly. So we present a simple and eco-friendly biosynthesis of silver nanoparticles using Ficus benghalensis fruit extract as reducing agent, rapid reduction of silver ions is observed leading to the formation of silver nanoparticles in solution. The synthesized nanoparticles are characterized using UV-visible spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction. The silver nanoparticles exhibit significantly maximum zones of inhibition against human pathogens and it also enhance the seed germination growth of Pisum sativum, Cicer arietinum, and Vigna radiate. Key words: Silver nanoparticles, Antimicrobial activity, Seed germination.

INTRODUCTION Nanotechnology is a broad interdisciplinary research arena that keeps growing rapidly worldwide. The science is being considered interesting for research in which the creation and utilization of materials with structural features between those of atoms and bulk materials with at least one dimension in the nano range focused as fundamental element. Silver nanoparticles (AgNPs) are the most widely used among metal nanoparticles these nanoparticles can be synthesized using different approaches, including physical and chemical methods; but these are very aggressive in nature and not suitable for environment [1]. In recent years, the use of elements of microbial and plant origins have provided eco-friendly routes to the nanoparticle (NP) synthesis [2-6]. The metal nanoparticles can be produced using various methods like chemical reduction, electrochemical and radiation [7-9]. Current research is focusing to have variety of natural materials prepare noble metal nanoparticles, such as synthesis of nanoparticles using microorganism [10-12], enzyme [13-14], plant extract [15] and these are said to be eco-friendly and alternatives to the chemical and physical methods that harm nature. The environment mediated green synthesis

of metallic nanoparticles used in higher plants [16]. Similar formation of gold and silver nanoparticles are produced by living plants [17]. Silver nitrate (AgNO3) exhibits numerous properties depending upon their size, morphology, and distribution [18]. Antimicrobial properties are one of the most important and applicable properties of silver nanoparticles that is used in medical, industry and agriculture fields. Most studies showed effective activity of silver nanoparticles in human photogenic bacteria [19]. Therefore, we have formulated a simple, rapid and green route for the synthesis of AgNPs employing a renewable, biodegradable, non-toxic residue from the fruit extract of Ficus benghalensis. The whole process was a biologically benign aqueous medium at ambient temperature and pressure and without using any form of external chemical reducing and stabilizing agents. MATERIALS AND METHODS Chemicals: Silver nitrate used as a substrate for the synthesis of silver nanoparticles. AgNO3 was purchased from Himedia laboratories Pvt. Ltd., Mumbai, India. The Water has been obtained through glass double distillation [20].

*Corresponding Author Address: Dr. G. Melchias, Dean, School of Biological Sciences, St. Joseph’s College (Autonomous), Tiruchirappalli - 620 002, Tamil Nadu, India. E-mail: [email protected]

Melchias et al., World J Pharm Sci 2016; 4(7): 1-12

Collection of plants: The fruit extract of plant sample was collected from the (Thoothukudi) southern part of Tamil Nadu. India.

fruit extract. The reaction mixture was incubated in the dark for 30 min. The absorbance at 517 nm was measured. The Ascorbic acid (2g in 5ml water) was used as standard. The lower absorbance of the reaction mixture indicated a higher percentage of scavenging activity. DPPH scavenging activity is calculated using the following equation:

Preparation of Plant extracts: To prepare Aqueous extract of Ficus benghalensis was prepared by taking 20g of leaves washed thoroughly with distilled water and dried for 5 days at room temperature. Then the leaves were powered and 1g of leaf powder was taken in a 250 ml Erlenmeyer flask with 100 ml sterile distilled water. The sample was boiled for 5min at 70°C before finally filtering it. The filtrate thus obtained was stored at 4 °C and used.

 Ac - As  DPPH scavenging activity (%)     100  Ac  The Ac and As are the intensity of peak at 517 nm for control (DPPH) and supernatant DPPH solvent, respectively.

Synthesis of AgNPs: A 5 ml aliquot of the extract was mixed with 95 ml of aqueous 1mM silver nitrate (AgNO3) for reduction of the silver nitrate into Ag+ ions and kept at room temperature for up to 24 hours. About 10 minutes after mixing the color of the solution began changing into reddish brown indicating the formation of silver nanoparticles. The bio-reduced AgNPs was screened using UV-VIS absorbance.

Anti-microbial assay: The antibacterial activities of synthesized silver nanoparticles were carried out by disc diffusion method [21-23]. The antibacterial activity was done on ten human pathogenic (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Serratia marcescens, Vibrio cholerae, Shigella sannei, Klebsiella pneumonia, Proteus vulgaris, Salmonella typhi and Proteus mirabilis) by the standard disc diffusion method. Nutrient agar medium plates were prepared, sterilized and solidified. After solidification bacterial cultures were swabbed on these plates. The discs were soaked in silver nitrate solution and solution containing silver nanoparticles of each type separately. The sterile discs were dipped in silver nanoparticles solution (10 mg/ml) and placed in the nutrient agar plate and kept for incubation at 370C for 24 hours. The inhibition was measured and compared with standard antibiotic disc and silver nitrate solution. Zones of inhibition for NPs and silver nitrate and streptomycin as antibiotic were measured. The experiments were repeated thrice values of zone diameter were presented.

UV-Visible spectra analysis: The AgNPs were characterized in a Perkin-Elmer UV-VIS spectrophotometer Lambda-19 to know the kinetic behaviour of the AgNPs. The scanning range for the sample was 200-800 nm with a scan speed of 480 nm/min. Fourier Transform Infrared Spectroscopy (FTIR): Infra-red spectroscopy was used to determine and predict possible physicochemical interactions between the components in a formulation using FTIR. The measurements were taken for the AgNPs synthesized after 24 hrs of reaction and were done using a FTIR SHIMADZU 8400S with a wavelength range of 400~4000 nm where the samples were incorporated with KBr pellets to acquire the spectra. The results were compared for shift in functional peaks of critical value.

Effect of biosynthesized silver nanoparticles on seed germination: The seeds were purchased from the local market, Tiruchirappalli, TN. Seeds were surface sterilized with 1% mercuric chloride solution for 1 min. and rinsed several time in sterile distilled water. The following analyses are experimented with three different varieties of seeds like Pisum sativum, Cicer arietinum and Vigna radiate.

XRD analysis: In order to determine the XRD patterns of AgNPs the process were recorded on Xray diffractometer (x’pert pananalytical) instrument operating at a voltage of 40 kV and current of 30 mA with Cu K (α) radiation to determine the crystalline phase and material identification. The samples were taken in lids and put under instrument for analysis.

Percentage of Seed germination Seed germination (%) = (Number of germinated seeds/ Number of total seeds) ×100

Antioxidant Activity: Antioxidant activity of the AgNPs was assayed by DPPH method. One milliliter of 0.1 mM DPPH (in ethanol) was added to different concentrations (100, 200, 300,400 and 500 µg ml-1) of silver nanoparticles synthesized by

Length of root and shoot: Root length was taken from the point below the hypocotyls to the end of the tip of the root. Shoot length was measured from the base of the root-hypocotyls transmission zone 2


up to the base of the cotyledons. The root and shoot length was measured with the help of a scale.

brown color within 10 min of reaction. The Ag nanoparticles produced by the Ficus benghalensis fruit was observed to be very stable in the solution, even 3 months after their synthesis, which validates the application of Ficus benghalensis as biomaterials for the synthesis of Nano-sized Ag particles. The silver particles were observed to be extremely stable even after 6 weeks (Figure 1).

Fresh and Dry weight: The fresh weight of root and shoot of seedlings was determined by weigh the root and shoot separately on electric balance. After the fresh weight taken then the seedlings was kept in a hot air oven at 80° C for 48 hrs then the weight of dry matter was recorded.

UV-Visible spectra analysis: The formation of the silver nanoparticles is confirmed by measuring the surface plasmon absorption spectrum of the silver nanoparticles, using UV-visible spectrometry [24]. It is well known that silver nanoparticles exhibit a yellowish-brown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles [25]. Reduction of silver ions to silver nanoparticles could be followed by a color change and UV-Vis spectroscopy. The technique outlined above has proven to be very useful for the analysis of nanoparticles [26, 27]. Therefore the silver nanoparticles were characterized by UV-Vis Spectroscopy, one of the most widely used techniques for structural characterization of silver nanoparticles [28].

Determination of relative water content (RWC): RWC was calculated by using the method of Barrs and Weatherly, (1962). The seedlings were weighed and then dipped in distilled water for 4 hours. The seedlings were then blotted dry and weighed prior to oven drying at 80 0C for 24 hours. RWC = Fresh weight Dry weight / Turgid weight Dry weight*100 RESULT AND DISCUSSION Biosynthesis of nano-scale silver particles: On challenging, fruit extracts of Ficus benghalensis and aqueous AgNO3 (1mM) solution changed from yellowish green to brown, the final color appeared immediately. The entire reaction mixture turned to

Figure 1: Biologically Synthesized AgNPs from Ficus benghalensis UV-Vis spectral analysis 3

443

0.338

Absorbance(A.U.)

2

1

0

400

600

800

Wavelength(nm)

Figure 2: UV-Vis measurements of Ag NPs from Ficus benghalensis

3


Hence nanoparticles have characteristic optical absorption spectrums in the UV-vis region. The conduction band and valence band lie very close to each other in which electrons move freely. These free electrons give rise to a SPR absorption band, occurring due to the collective oscillation of electrons of silver nanoparticles in resonance with the light wave [29]. The higher the concentration of the extract being added, the higher was the possibility of the green reduction mechanism of the silver nitrate occurring, which would lead to the formation of well defined and stable silver nanoparticles. Hence UV–vis absorption spectrum of the fruit extract cum silver nanoparticles showed the surface Plasmon resonance derived from the silver nanoparticles at around 443nm (Figure 2).

spectra are analyzed. The fruit extract displays a number of absorption peaks (Table 1, Figure 3). The absorption peaks are 3432.72, 2363.07, 2338.25, 2076.83, 1637.43, 1461.27, 1156.25, 1078.08, 714.08 cm-1. The initial peak at 3432.72 is represents O-H stretch of the Alcohol/phenol compound. From 2363.07 to 2076.83 cm-1 corresponds to the Si-OR as Silicon. FTIR study indicates that the O-H stretch, Alcohol/phenol and amine -N-H bond of fruit extract are mainly involved in reduction of Ag+ to Ago nanoparticles. A FTIR spectrum of synthesized silver nanoparticles by this green method is analyzed. A number of absorption peaks at 3432.72, 2363.07, 2338.25, 2076.83, 1637.43, 1461.27, 1156.25, 1078.08, and 714.08 cm-1 indicating the biomaterial bind to the silver nanoparticles through Alcohol/phenol, Silicon, Amines, Alkenes, Halogen. Thus indicating the fruit extract act as a reducing and capping agent for silver particles.

Fourier transform infrared spectroscopy (FTIR) In order to investigate the functional groups of fruit extract, a FTIR study was carried out and the

Table 1: FTIR Peak value and its functional groups of Ficus benghalensis Composition AgNO3: Extract 95:5 (ml)

24hours- Time duration

Frequency range

Bond

Compound

3432.72 2363.07 2338.25 2076.83 1637.43 1461.27

3200-3550 1000-1100 2100-2360 1000-11000 1580-1650 1350-1470

Alcohol/phenol Silicon Silicon Silicon Amines Alkenes

1156.25 1078.08 714.08

1000-1400 1000-1400 600-800

O-H stretch Si-OR Si-OR Si-OR N-H bond CH2 & CH3 deformation C-F stretch C-F stretch C-d stretch

Halogen Halogen Halogen

ACIC St.Joseph's College ( Autonomous) T richy-2 Spectrum Name: IR-Bot-C.sp 100.0 95 90 1156.25

85

2363.07 2338.25

80

1078.08

75

1461.27 2076.83

70 65 60 55

714.08

50 %T 45 40 35 30 25 20 1637.43

15 10 5

3432.72

0.0 4000.0

3000

2000

1500 cm-1

Figure 3: FTIR pattern of Ficus benghalensis

4

1000

500 400.0


β Cos θβ Π) by determining Bragg’s reflection, where “k” is Scherrer constant, λ is the wavelength of the X-rays, β and θβ are full width half maximum of the Bragg angle [33]. The dry powders of the silver nanoparticles were used for XRD analysis. The diffracted intensities were recorded 2 theta angles. To study the crystalline nature of the silver nanoparticles of Ficus benghalensis, the XRD analysis was done (Figure 4). The broadening of X-ray peaks observed is primarily due to the small particle size. The mean size of silver nanoparticles was calculated using the Debye-Scherrer’s equation. The observed peak broadening and noise were probably related to the effect of nanosize particles and the presence of various crystalline biological macromolecules in the plant extracts. The obtained results illustrate that silver ions had indeed been reduced to Agº [34].

Analysis of FTIR studies were confirmed that the carbonyl group from the amino acid residues and proteins has the stronger ability to bind metal indicating that the proteins could possibly from the metal nanoparticles (i.e., capping of silver nanoparticles) to prevent agglomeration and thereby stabilize the medium. This suggests that the biological molecules could possibly perform dual functions of formation and stabilization of silver nanoparticles in the aqueous medium XRD analysis of AgNPs: The biosynthesis of silver nano particles from Ficus benghalensis was observed in the XRD .The XRD spectrum showed three distinct diffraction peaks and indexed with (111), (220) , (222) and (420) of the cubic facecentered silver. The average grain size of the silver nanoparticles formed in the process was estimated from the Debye-Scherrer equation [(d = (kλ×180)/

900

(200)

Intensity (AU)

600

(220)

(420)

(222)

300

(111)

0

25

30

35

40

45

50

55

60

65

70

75

80

Position 2Theta( in degree) XRD pattern of Biosynthesised silver nanoparticles

Figure 4: XRD images of SNPs generated using Ficus benghalensis DPPH free Radical scavenging Activity: The anti-oxidant property of synthesized nanoparticles was determined by DPPH free radical scavenging assay method. Antiradical activity assay is based on the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Due to the presence of an odd electron it gives a strong absorption maximum at 517 nm. The antioxidant activity also increases with an increase in the concentration and time [35]. DPPH assay has

been widely used to determine the free radical scavenging activity of various plants and hence the Ficus benghalensis results show the maximum scavenging activity compared with ascorbic acid (Figure 5). It is evident from the results that the antioxidant potential of the silver nanoparticles might be acquired from the plant extract which was used for reducing silver nitrate to elemental silver [36].

5


Figure 5: DPPH radical scavenging activity of Ficus benghalensis the silver nanoparticles interact with these proteins in the cell as well as with the phosphorus containing compounds like DNA. When silver nanoparticles enter the bacterial cell it forms a low molecular weight region in the center of the bacteria to which the bacteria conglomerates thus, protecting the DNA from the silver ions. The nanoparticles preferably attack the respiratory chain, cell division finally leading to cell death. The nanoparticles release silver ions in the bacterial cells, which enhance their bactericidal activity [30, 33].

Antimicrobial assay The silver nanoparticles synthesized by Ficus benghalensis were found to have highest antimicrobial activity against Bacillus subtilis, Vibrio cholerae, Salmonella typhi and lesser antimicrobial activity against Staphylococcus aureus, Escherichia coli, Shigella sannei, Klebsiella pneumonia, and Proteus mirabilis (Figure 6, 7 & 8). However these silver nanoparticles get attached to the cell membrane and also penetrated inside the bacteria. The bacterial membrane contains sulfur containing proteins and

6


Figure 6: Antimicrobial activity of silver nanoparticles medited by Ficus benghalensis.

7


Figure 7: Antimicrobial activity of silver nanoparticles medited by Ficus benghalensis.

8


Figure 8: Antibacterial activity of AgNPs extracts of Ficus benghalensis against human pathogenic bacteria using Disc diffusion method. Seed germination: The use of Biosynthesized silver nanoparticles to increase the root/shoot length as well as chlorophyll and protein content in Pisum sativum, Cicer arietinum and Vigna radiate. Maximum increase in total protein content was observed at 1000 mg/L in leaves and at 100 mg/L in roots, respectively, as compared to without AgNPs. These growth aspects show that it is due to AgNPs and it does not induce any toxic effect plant

[31]. The AgNPs resulted in reduction in root growth when seeds were soaked in distilled water (control) and incubated in nanoparticles while root growth enhanced on soaking in nanoparticles (Table 2, Figure 9). Similar results were observed when Phaselous radiatus, Sorghum bicolor and Lolium multiflorum were subjected to silver nanoparticles resulted in enhanced root growth, root length and biomass were observed.

Table 2: Effect of control, AgNPs on seed germination, Seedling vigor index, growth, fresh, dry weight and relative water content of Ficus benghalensis. Pisum sativum Treatments Seed Seedling growth (cms) Fresh Weight Dry weight Relative germination (gms) (gms) Water (%) Content Root Shoot (%) AgNPs

100

0.22±1.6

0.79±3

52.1±548

50.05±241.6

Control

60

0.25±0.26

0.5±0.5

112±559

32.4±207

Cicer arietinum Treatments Seed germination (%)

Seedling growth (cms) Root

Shoot

Fresh Weight (gms)

Dry weight (gms)

Relative Water Content (%) 67.39

AgNPs

100

1.78±2.6

0.74±2.6

83.67±542

34.9±245

Control

80

0.57±0.8

1.29±1.4

148.6±513.4

19.20±201.4

9

11.111


Vigna radiata Treatments

Seed germination (%)

Seedling growth (cms) Root

Shoot

Fresh Weight (gms)

Dry weight (gms)

Relative Water Content (%) 18.51

AgNPs

100

1.30±2.3

1.22±3.5

60.9±343

17.7±213

Control

80

0.82±1.1

0.54±0.9

73.9±231

73.9±231

Pisum sativum L.

A

B

Cicer arietinum L.

B

A Vigna radiate L.

B

A A: (AgNPs soaked seeds)

B: (Water soaked seeds)

Figure 9: Effect of biologically synthesized silver nanoparticles on seed germination

10


biologically synthesized silver nanoparticles could be of immense use in medical field for their efficient antimicrobial function. Germination is important for determining the final plant density if planted seeds germinate completely and vigorously. AgNPs have been implicated nowadays in agriculture to improve crops. The current study showed that exposure to AgNPs soaked seeds enhance seed germination and seedling growth when compared with water soaked seedlings, so this types of nanoparticles which plays a vital role in the agricultural field.

CONCLUSION The silver nanoparticles have been produced by Ficus benghalensis, which is an economical, efficient and eco-friendly process. UV-vis spectrophotometer, FTIR and XRD, techniques have confirmed the reduction of silver nitrate to silver nanoparticles. The zones of inhibition were formed in the antimicrobial screening test indicated, that the silver nanoparticles synthesized in this process has the efficient antimicrobial activity against pathogenic bacteria. The

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