Plant Assisted Synthesis of Silver Nanoparticles Using Boerhaavia diffusa Leaves Extract and Evolution of Antibacterial Activity

 

U.Kanagavalli¹, A. Mohamed Sadiq¹, Sathishkumar¹, S.Rajeshkumar²

¹PG & Research Department of Biochemistry, Adhiparasakthi College of Arts and Science, G.B. Nagar, Kalavai – 632506.

² Research Scientist, School of Bio-Sciences and Technology, VIT University, Vellore - 632014, TN, India

 

ABSTRACT:

In recent years, green synthesis of silver nanoparticles has gained much interest from chemist and researchers. This has to achieving through numerous sources of cost-effective non-hazardous reducing and stabilizing compounds utilized in preparing silver nanoparticles (AgNPs). The synthesis of nanoparticles from plant sources has proved to be an effective and alternative method for the novel production of nanoparticles. This study investigates an efficient and sustainable route of AgNPs preparation from 1 mM aqueous AgNO₃ using leaf extracts of Boerhaavia diffusa well adorned for their wide availability and medicinal property. The synthesized silver nanoparticles were characterized by UV–visible (UV–Vis) spectroscopy, Fourier infrared spectroscopy (FT-IR), and Scanning Electron Microscopy (SEM) coupled with Energy Dispersive Spectroscopy (EDS). UV–vis spectra of AgNPs and visual perception of brownish yellow color from colorless reaction mixture confirmed the AgNPs formation. Involvement of functional groups of Boerhaavia diffusa leaf extract in the reduction and capping process of nanoparticles was well displayed in Fourier transform infrared spectroscopy (FTIR). An investigation of the shape and size of nanoparticle was done by X-ray diffraction and scanning electron microscopic studies. The antibacterial effect of the silver nanoparticles was tested against pathogenic bacterial strains like Pseudomonas sp, Klebsiella pneumonia, Salmonella sp, Escherichia coli and Staphylococcus sp.

 

 

 

INTRODUCTION:

The green nanotechnology is term tends with uses of green process in chemistry and green chemical technology in synthesis of nanoparticles with eco-friendly manner. This technique receiving have much attention and much consideration as a result of global level enigmas associated with environmental      concerns [1].

Techniques to synthesis of method of nanoparticles like physical and chemical methods use toxic chemicals or high energy requirements which are rather difficult and including wasteful purification [2,3]. Eco-friendly method of nanoparticles synthesis provides much more advantages over the physical and chemical methods as they are simple, one step, cost effective and relatively reproducible and often results in more stable materials.

 

Eco-friendly methods has achieved by using microbes, plants, sugars and polymers. Plant extracts and microorganisms act as reductants and capping agents could be turn fascinating technology [4-6]. The herbal extracts have come up nanofactory for gold and silver metal nanoparticle synthesis. Its use in nanoparticle synthesis is powerfully advantageous over microorganism due to the ease of scale up, less biohazard, eco-friendly and embellish the process of maintaining cell cultures. It is believed to be the best as well as providing natural capping agents for stabilization of silver nanoparticles [7].

 

Microorganisms can also be utilized to produce nanoparticles [8-11] but the rate of syntheses is slow compared to routes involving plants mediated synthesis [12]. Although, the potential of higher plants as source for this purpose is still largely unexplored. Very recently plant extract of Vitis vinifera [13], Coleus aromaticus [14], Cissus quadrangularis [15],  Morinda tinctoria [16], Piper nigrum [17], Elettaria Cardamomom [18], marigold flower [19], Ziziphora tenuior [20], Abutilon indicum [21], Sesuvium portulacastrum [22], latex extract of Thevetia peruviana [23], Punica granatum [24], Acorous calamus rhizome extract [25], and tea leaf [26] reported synthesis of silver nanoparticles using plant extracts as an alternative to the conventional methods.

 

Silver is a transition metal and is the one of the most commercialized nano-material used in various fields like medicine, electronics, catalysis optics, mechanics, and sensing [27]. Silver nanoparticles have been used in field of high sensitivity biomolecular detection, and biosensors and it has been accepted to have powerful inhibitory and bactericidal effects along with the antifungal, anti-inflammatory and anti-angiogenesis activities. In this study, it has established that an aqueous extract of Boerhaavia diffusa leaves were used in reduction of Ag(I) and in the formation of stable silver nanoparticle and identified the effect of antimicrobial activity.  Boerhaavia diffusa belonging to the family of the Nyctaginaceae, is mainly a diffused perennial herbaceous creeping weed of India. It is also known as punarnava. Boerhaavia diffusa is up to 1 m long or more, having spreading branches. The leaves are simple, thick, fleshy, and hairy, arranged in unequal pairs, green and glabrous above and usually white underneath. This herb is has a diuretic, anticancer, hepatoprotective, immunomodulatory, antiamoebic and antiestrogenic agent. The ancient use of the plant includes relieving problems like epilepsy, hysteria, gastritis, jaundice, fever, convulsion, asthma, dysentery and diarrhea [28].

 

MATERIALS AND METHODS:

Silver nitrate, Luria Bertani agar, and nutrient broth were purchased from Himedia, Mumbai. Leaves of Boerhaavia diffusa were collected from Adhiparasakthi Agricultural College, Kalavai, Tamilnadu, India.

Preparation of Leaf Extract

About 10g of fresh leaves of Boerhaavia diffusa was thoroughly washed 2-3 times with distilled water for surface cleaning, and surface sterilized with 0.1% HgCl₂ for 1min to reduce microbial contamination. The sterile leaves were cut into fine pieces and boiled with 100 mL of double distilled water for 15min at 60°C and filtered through Whatman number 1 filter paper and stored at 4°C in refrigerator for 2 weeks.

 

Synthesis of silver nanoparticles

In the typical synthesis of silver nanoparticles, 10 mL of leaf extract was treated with 90 mL of 1 mM silver nitrate solution and kept in room temperature. Subsequently the synthesis of silver nanoparticles was initially identified by brown colour formation and further monitored by measuring UV-Vis spectra of the reaction mixture.

 

Characterization of synthesized silver nanoparticles

Synthesis of silver nanoparticles was initially characterized by position of SPR band by measuring double beam UV-Vis spectroscopy at different wavelengths from 360 to 700 nm. Shape and size were analysed by using SEM (Philip model CM 200). Elemental composition was performed by EDX (Philips XL-30). FTIR spectrum of silver nanoparticles was obtained on a Shimadzu instrument with the sample as KBR pellet in the wave number region of 500–4,000cm⁻¹.

 

Antibacterial Activity of Synthesized Silver Nanoparticles

The antibacterial activity of synthesized silver nanoparticles was performed by agar well diffusion method against pathogenic bacteria, Pseudomonas sp, Klebsiella pneumonia, Salmonella sp, Escherichia coli and Staphylococcus sp.  Fresh overnight culture of each strain was swabbed uniformly onto the individuals’ plates containing sterile Luria Bertani agar and 3 wells were made with the diameter of 6 mm. Then 20, 40 and 50 µL of purified silver nanoparticles solutions were poured into each well and commercial antibiotic disc Novamycin placed as control and incubate for 24 h at 37°C. After incubation the different levels of zonation formed around the well and it was measured. This experiment was repeated for three times.

 

RESULT AND DISCUSSION:

Visual Observation

Silver nanoparticles formation was primarily identified by colour change visually Boerhaavia diffusa leaf extract was treated with silver nitrate aqueous solution showed a colour change from yellow to brown within 2 min (Figure 1a, b and c). The colour change was clear indication for the formation of silver nanoparticles. This brown colour of silver nanoparticles arises due to the surface plasmon vibrations in the aqueous solution [29, 30].

 

Figure 1 (a) 1 mM silver nitrate aqueous solution (b) leaf extract added into silver nitrate solution shows brown colour at 0 min (c) dark brown colour formation within 12 hrs of incubation

 

UV-visible spectrum

The reduction of silver ions from silver nitrate to silver nanoparticles was monitored by measuring the absorbance using UV-vis spectrophotometer (figure 2). The absorbance was recorded from 370-510 nm at resolution of 1 nm for detection of phyto-synthesized silver nanoparticles. Figure 2 shows UV spectrum of synthesized silver nanoparticles. The UV-vis spectroscopy method can be used to track the size evolution of silver nanoparticles based on localized surface plasmon resonance band exhibited at different wavelengths. The optical properties of silver nanoparticles are related to excitation of plasmon resonance or inter band transmission particularly on the size effect. The spectra show narrow peak at 420 nm which indicates the formation of disaggregated nanoparticles. This single and strong band indicates that the particles are isotropic in shape and uniform size [31, 32]. The completion of nanoparticle synthesis is visually identified by appearance of precipitation in the bottom of the flask.

 

 

Figure 2: UV-Vis spectrum of effect of reaction time on silver nanoparticles synthesis by leaves extract of Boerhaavia diffusa.

Scanning Electron Microscopy:

The SEM image (Figure 3) showing the high density Ag-NPs synthesized by using the leaf extract of Boerhaavia diffusa further confirmed the development of silver nanostructures. Obtained nanoparticle showed that Ag-NPs are spherical shaped and monodispersed and well distributed with aggregation in the size range about 50–70 nm (scale bar 500 nm). Similarly monodispersed silver nanoparticle was reported by using the extract of Piper nigrum [17].

 

 

Figure 3: Morphology of silver nanoparticles showed by SEM.

 

Energy Dispersive X-Ray (EDX)

 Analysis through energy dispersive X-ray (EDX) spectrometers confirmed the crystalline and elemental composition of the synthesized silver nanoparticles. The vertical axis displays the number of X-ray counts whilst the horizontal axis displays energy in keV. The EDX spectrum (Figure 4) observed a strong signal from the silver atoms in the nanoparticles at 3 keV and weak signal from “O.” This weak signal received from the organic constituents of plant extract [33].

 

 

Figure 4: Elemental analysis of synthesized silver nanoparticles using leaf extract Boerhaavia diffusa

 

 Fourier Transform Infrared Spectroscopy

The dual role of the plant extract as a reducing and capping agent and presence of some functional groups was confirmed by FT-IR analysis of silver nanoparticle. Figure 5 shows that the FT-IR image of Boerhaavia diffusa leaf mediated synthesized silver nanoparticles indicates presence of biomolecules involved in the reduction process. The minor peak found at 3336.67cm^-1 represents–OH stretch of alcohols and phenols; the peak at 1635.06 cm^-1 is due to the –OH stretch of carboxylic acids. FT-IR reveals that carboxyl and amine groups may be involved in the reduction and stabilizing mechanism. The phytochemicals present in the plant extract responsible for nanoparticle synthesis [9].

 

 

Figure 5: FT-IR spectrum of silver nanoparticles synthesized by Boerhaavia diffusa leaf extract

 

Antibacterial Activity of Silver Nanoparticles:

Silver nanoparticles are very good antibacterial activity against many bacteria isolated from many sources [34, 35]. Antibacterial activity of synthesized silver nanoparticles was performed against Staphylococcus sp, Salmonella sp, Pseudomonas sp, Klebsiella pneumonia, and E. coli by well diffusion method (Figure 6). The antibacterial activity of synthesized silver nanoparticles was compared with Novamycin is a commercial antibiotic disc. The zone of inhibition was measured and denoted in millimeter (mm) in diameter. The zone of inhibition was tabulated by performing triplicate experiments (Figure 7). Among the three concentrations, increased 60 µL silver nanoparticles concentrations highly inhibit the growth of pathogenic bacteria. The increasing concentration of nanoparticle will be the zone of inhibition against pathogenic bacteria. Highest inhibition was noted against E. coli and Pseudomonas sp. Low inhibition activity of synthesized silver nanoparticles was noted against Staphylococcus sp. 

 

Figure 6: Plates of antibacterial activity of silver nanoparticles against different pathogenic microorganisms.

 

Figure 7: Zone of inhibition against pathogenic bacteria using green synthesized silver nanoparticles.

 

CONCLUSION:

Phytoconstituents present in plant extracts can be used to reduce metal ions to nanoparticles in a single-step and eco-friendly green chemical synthesis process. The involved reducing agents include the various water soluble plant metabolites in the green synthesis process. Extracts of a diverse range of Boerhaavia diffusa Linn have been successfully used in making nanoparticles. UV-vis Spectra and visual shows perception peak at 420 nm and of brownish yellow colour from colourless reaction mixture confirmed the silver nanoparticle formation, respectively. SEM and EDX analysis shows that polydispersed and crystalline structured nanoparticles synthesized using extract of Boerhaavia diffusa Linn. FTIR spectroscopy revealed that silver nanoparticles were functionalized with biomolecules that have alcohols, phenols and carboxyl groups. Thus, this eco-friendly synthesized silver nanoparticles acts as an excellent antibacterial activity against highly pathogenic microorganisms.  These reports reveals that the silver nanoparticles are very good antibacterial agents may play a vital role in lot of commercial products related with antibacterial and antifungal property in pharmaceutical and chemical industry.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest

 

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Accepted on 03.08.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(8):1064-1068.