Biosynthesis of silver nanoparticles using Garcinia kola and its antimicrobial potential

1 Department of Chemistry, Federal University of Lafia, Lafia, Nasarawa State, Nigeria. 2 Department of Physics, Federal University Lafia, Lafia, Nasarawa State, Nigeria. 3 Department of Chemistry, College of Agriculture, Engineering and Science, University of KwazuluNatal, South Africa. 4 Deparment of Chemistry Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. 5 Chemistry Department, Covenant University, Ota, Ogun State, Nigeria.


INTRODUCTION
Nanotechnology is a growing field that has drawn the attention of researchers in different disciplines.Metal nanoparticles with controlled size and shape form the basis for advanced functional materials for electronic, sensor, optical devices (Gurunathan et al., 2009).Metal nanoparticles synthesis is achieved majorly using physical, chemical and biological approaches.Laser ablation (Tsuji et al., 2003), Arc discharged method (Tien *Corresponding author.E-mail: labuloayomide@gmail.com, labulo@yahoo.com.Tel: + 2348062295936.et al., 2008), irradiation (Yong et al., 1999), polyol process (Sun and Xia, 2002), polymers (Hebeish et al., 2011;Chengcai et al., 2005;Narendra and Khanna, 2007), co-precipitation (Yang et al., 2003), sol-gel (Lu et al., 2002), bacteria (Korbekandi et al., 2012), fungi (Ahmad et al., 2003;Balaji et al., 2009) have been used in the synthesis of nanoparticles.The use of plant extracts, enzymes, microorganisms, polymers, sugars and vitamins in nanoparticles formation has made nanotechnology a promising area of research (Oxana et al., 2013).Among the noble metals (e.g., Ag, Pt, Au and Pd), silver (Ag) is the metal of choice for potential applications in the field of biological systems, living organisms and medicine (Jain et al., 2009).The use of plant extract in synthesizing silver nanoparticles is ecofriendly, energy saving and can be easily scaled up.Biologically synthesized silver nanoparticles using medicinal plants or plant extracts include locust bean gum (Chandrakant et al., 2013), Andrographis paniculata (Venkata et al., 2014), lingonberry and cranberry juices (Judita et al., 2014), Morinda citrifolia (Gnanasekar et al., 2012), Alternanthera dentata (Deenadayalan et al., 2014), guava leaves extract (Shinde et al., 2014), Gardenia jasminoides (Lü et al., 2014), Jatropha seedcake (Anjali et al., 2013), Mimusops elengi (Hoskote et al., 2014), Abutilon indicum (Ashokkumar et al., 2015), Ziziphora tenuior (Babak and Gholamhoseinpoor, 2015), Ficus carica (Bulent et al., 2015), Acacia auriculiformis (Pradnya et al., 2014), Cocos nucifera (Mariselvama et al., 2014).Pineapple leave extract has also been used for the green synthesis of silver nanoparticles and its high antibacterial activities have been reported (Elemike et al., 2014).Recently our research group studied the growth kinetic of silver nanoparticles under the influence of plant biodiversity (Dare et al., 2015) for pharmaceutical and biological applications have been reported.G. kola Heckel (Clusiaceae), commonly known as bitter kola (English) and orogbo (Yoruba) is a widespread tree of evergreen forest valued in Nigeria for its medicinal nuts which has led to its exploitation in the natural forests in recent times (Ibikunle and Emmanuel, 2011).The phytochemicals obtained from G. kola as documented in literature include biflavonoids such as kolaflavonone, and 2 hydroxyflavonoids, xanthones, kolanone, ameakoflavone, 2,4-methylenecyclartenol, coumarine and prenylatebenzophenones, oleoresin, the chromanols, garcioic and garcinal (Adesuyi et al., 2012).Others are tannin, saponins, alkaloids, and cardiac glycoside.The proximate analyses showed that the sample has high level of carbohydrate, little amount of crude fibre and protein respectively and negligible amount of ash content and crude fat.This composition shows that the sample could be a good source of carbohydrate, dietary fibre and protein (He et al., 2007).Biomolecules with carbonyl, hydroxyl, and amine functional groups have the potential for metal ion reduction and capping the newly formed particles during their growth processes (Adesuyi et al., 2012).In this work, we report a simple, nontoxic, and eco-friendly green synthesis of silver nanoparticles using aqueous seed extract of G. kola and no toxic chemicals are used as reducing and stabilizing agent during the synthesis.

METHODOLOGY Materials
G. kola seeds and Silver nitrate (AgNO3) of analytical grade was purchased from Sigma-Aldrich.

Plants and preparation of extract
G. kola seeds were obtained from the local market in Lafia, the capital of Nasarawa State, Nigeria. 10 g of seeds was milled using an ordinary coffee grinder and ground kernel were boiled with 100 mL triply distilled/ deionized water for 10 min.After filtration through Whatman No 1, filter clear seed extract was obtained for further use.All the aqueous solutions were prepared using triply distilled deionized water and stored at 4°C for further use.

Synthesis of silver nanoparticles
10 ml of aqueous seed extract was added to 40 ml of 1 mM aqueous silver nitrate solution; the mixture was heated at 70 o C in time intervals ranging from 2 to 30 min.Reduction of the Ag + ions was monitored by measuring the UV-visible spectrum of the solution on a spectrophotometer (T60 UV-vis spectrophotometer) operating at a resolution of 1 nm.

Characterization
The crystallinity and phases of the Ag nanoparticles were characterized by X-ray diffractometer (XRD-6000, Shimadzu, Japan) with Cukα radiation (λ = 1.5412Å) in the range of 30°-90° with 2°/min scanning rate.The functional groups of Ag nanoparticles were characterized by Fourier-Transform Infrared (FTIR, Perkin Elmer, Spectrum BX) spectroscopy in the range of 4000-350 cm -1 .In addition, time-resolved absorption spectra of prepared Ag nanoparticles was analyzed via UV-visible Spectrophotometer (T60 UV-Vis spectrophotometer).The morphology and size of the prepared Ag nanoparticles was observed by Transmission Electron Microscopy.

Evaluation of antibacterial activity
The antimicrobial screening of the silver nanoparticles was carried out against Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, Rhizopus stolonifer and Candida albican using Agar-well diffusion method.Gentamycin and Tioconazole were used as control for bacteria and fungi respectively.Using a micropipette, 50 μL of nanoparticle solution was poured onto each of the 8mm diameter well made on the prepared agar plates.On incubation at room temperature for 72 h, the diameter of zone inhibition was measured in millimeter; the values are shown in Table 1.

UV-Vis spectra analysis
The color change was noted by visual observation in the Erlenmeyer flask that contains AgNO 3 solution with G. kola extract.The color of the AgNO 3 /seed extract solution changed from light yellow to light brown after 5 min and eventually to dark brown.This color change indicates the formation of Ag nanoparticles in the solution.Seed extract without AgNO 3 did not show any color changes.The formation of Ag nanoparticles was further confirmed by using X-ray diffraction (XRD), Fourier-Transform infrared spectroscopy (FTIR) and Transmission electron microscopy (TEM).
Figure 1 shows the UV-vis absorption spectrum of the synthesized Ag nanoparticles.Silver nanoparticles have free electrons, which give surface plasmon resonance (SPR) absorption band.This is due to the combined vibration of electrons of silver nanoparticles in resonance with light wave.A broad absorption peak was observed at 440 nm, which is a characteristic band for silver nanoparticles.No other peak was observed in the spectrum which confirms that the synthesized products are Ag° only.

XRD analysis
Figure 2 shows XRD patterns for Ag nanoparticles synthesized by G. kola seed extract.Four peaks at 2θ, 38.4, 44.5, 64.8, and 77.7 corresponding to (111), ( 200), ( 220) and ( 311) reflect planes of face-centred cubic (fcc) structure of Ag.These peaks are consistent with the standard data file JCPDS No 04-0783 JCPD.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 (Sheny et al., 2011).The average mean size of AgNPs was 10.40 nm.No peaks from any other phase were observed showing that single phase Ag with cubic structure nanoparticles has been obtained directly.

FTIR spectra analysis
FTIR measurements were carried out on both the extract and the silver nanoparticles from G. kola.This was done to identify the biomolecules for reduction, and efficient stabilization of the metal nanoparticles synthesized by G. kola seed extract.The FTIR spectrum of silver nanoparticles is shown in Figure 3b.The presence of peaks at 3434, 2346, 2096, 1643 and 1028 cm −1 was observed for the crude extract of G. kola (Figure 3a), which indicates that the silver nanoparticles may be surrounded by hydroxyl group, because the peaks indicate -OH symmetric stretching and C-O bonds in aliphatic or carboxylic compounds (Ghosh et al., 2012).The band at 3434 cm -1 corresponds to O-H stretching Hbonded alcohols.The peak at 1643 cm -1 corresponds to C=O of ketone or aldehyde group and was shifted to lower frequency of 1635 cm −1 in the spectra of the silver nanoparticles from G. kola.The peak at 1028 cm -1 was assigned to the stretch of the C−O bond (Renquan et al., 2012).Therefore, the synthesized nanoparticles were surrounded by proteins and metabolites such as terpenoids, biflavonoids having functional groups of alcohols, ketones, aldehydes and carboxylic acids.This suggests that the biological molecules could possibly perform dual functions of formation and stabilization of silver nanoparticles in the aqueous medium (Mukherjee et al., 2001).This shows that the aqueous seed extract of G. kola is a very good and robust bioreductant for the synthesis of silver nanoparticles.

TEM and EDX analysis
The TEM image gives the morphology and size of the synthesized silver nanoparticles.A spherical nanoparticle which conforms with the SPR band in the UV-Vis spectrophotometer and to that of the XRD analysis was obtained with average particle size of 10.40 nm (Figure 4).Table 2 shows the EDX profile of the synthesized silver nanoparticle from G. kola extract.The selected area electron diffraction SAED pattern of G. kola silver nanoparticles was recorded at different scales.The   220) and (311) Plane of Ag°.

Antibacterial assay
The bio-reduced silver nanoparticles from G. kola show a clear and high zone of inhibition as a result of the attachment of the silver ion to the negative charged cell wall.This led to structural changes in the cell wall, permeabilility and subsequently death of the bacteria (Sondi and Salopek-Sondi, 2004) 1).Also due to high zone of inhibition exhibited by S. aureus and A. niger followed by C. albican in Figure 5, the silver nanoparticles from Garcinia kola possess high antimicrobial activity which can be used in household appliances like clothes washer and refrigerator which can kill bacteria and reduce odour

Conclusion
In this paper, we have reported a green method to synthesize silver nanoparticles using the aqueous seed extract of G. kola.The aqueous seed extract of G. kola helped in reducing and stabilizing the silver nanoparticles with remarkable growth onset of 10 min.This makes our green approach superior to conventional methods.The average size was found to be 10.4 nm with high  susceptibility against tested pathogenic bacteria and fungi.
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Figure 1 .
Figure 1.UV-visible spectrum of the silver nanoparticles synthesized with aqueous seed extract of G. kola

Figure 3 .
Figure 3. FTIR spectra of (a) extract of G. kola leave extract (b) silver nanoparticles synthesized using G. kola.

Figure 4 .
Figure 4. (c) TEM micrograph of the Ag nanoparticles synthesized using G. kola (d) EDX graph (e) the particle size distribution of the synthesized silver nanoparticles from G. kola.(f) selected area electron diffraction (SAED) pattern of the synthesized silver nanoparticles from G. kola.

Figure 5 .
Figure 5. Antimicrobial activity of silver nanoparticles synthesized from G. kola.

Table 1 .
The variations in zone of inhibitions (mm).

Table 2 .
EDX elemental analysis of the silver nanoparticles of G. kola.