Size-controlled green synthesis of silver nanoparticles mediated by gum ghatti (Anogeissus latifolia) and its biological activity
© Kora et al; licensee Springer. 2012
Received: 17 October 2011
Accepted: 9 May 2012
Published: 9 May 2012
Gum ghatti is a proteinaceous edible, exudate tree gum of India and is also used in traditional medicine. A facile and ecofriendly green method has been developed for the synthesis of silver nanoparticles from silver nitrate using gum ghatti (Anogeissus latifolia) as a reducing and stabilizing agent. The influence of concentration of gum and reaction time on the synthesis of nanoparticles was studied. UV–visible spectroscopy, transmission electron microscopy and X-ray diffraction analytical techniques were used to characterize the synthesized nanoparticles.
By optimizing the reaction conditions, we could achieve nearly monodispersed and size controlled spherical nanoparticles of around 5.7 ± 0.2 nm. A possible mechanism involved in the reduction and stabilization of nanoparticles has been investigated using Fourier transform infrared spectroscopy and Raman spectroscopy.
The synthesized silver nanoparticles had significant antibacterial action on both the Gram classes of bacteria. As the silver nanoparticles are encapsulated with functional group rich gum, they can be easily integrated for various biological applications.
KeywordsAntibacterial Autoclaving Gum ghatti Silver nanoparticles Surface-Enhanced Raman Scattering (SERS)
A survey of earlier literature suggests that various natural polymers such as starch , chitosan , and tannic acid  have been reported as reducing agents for the synthesis of silver and gold nanoparticles. It has been demonstrated that the plant-based exudate gums such as gum Acacia  and gum kondagogu  can be utilized as reducing and stabilizing agents for the silver nanoparticle biosynthesis. Gum gellan, a microbial heteropolysaccharide, was employed for similar purpose in the case of gold nanoparticles . Gum ghatti is a naturally occurring water soluble, complex polysaccharide derived as an exudate from the bark of Anogeissus latifolia (Combretaceae family), a native tree of the Indian sub-continent. The name gum ghatti has originated from its transportation through mountain passes or ghats. This native Indian gum is collected from the forests by the tribals and marketed through government organizations such as Girijan Co-operative Corporation Ltd., Visakhapatnam, India. The world production of gum ghatti is about 1,000–1,500 MT/year [7, 8]. This biopolymer is an arabinogalactan type of natural gum and its morphological, structural, physico-chemical, compositional, solution, thermal, rheological, and emulsifying properties have been well documented and studied [9–17]. This biopolymer is a high-arabinose, protein rich, acidic heteropolysaccharide, occurring in nature as mixed calcium, magnesium, potassium, and sodium salt [12–14, 16]. The primary structure of this gum is composed of sugars such as, l-arabinose, d-galactose, d-mannose, d-xylose, and d-glucuronic acid in a molar ratio of 48:29:10:5:10 and < 1% of rhamnose, which is present as non-reducing end-groups. The gum contains alternating 4-O- substituted and 2-O-substituted α d-mannopyranose units and chains of 1 → 6 linked β d-galactopyranose units with side chains of l-arabinofuranose residues. Six percent of rhamnose in the polysaccharide is linked to the galactose backbone as α-Rhap-(1 → 4) β-galactopyranose side chain. It has a molecular weight of 8.94 × 107 g/mol [12, 13, 15, 16].
The gum ghatti with a CAS number 9000-28-6 is recognized as “generally recognized as safe” (GRAS) and approved as a food ingredient (Code 184.1333) by the Food and Drug Administration, USA, under the function of emulsifier and emulsifier salt. Its use in food is also approved in Japan, China, South Korea, Singapore, Russia, Australia, South Africa, Iran, Saudi Arabia, Latin America, and other countries. But, it is not approved as a food additive in European Union and not been accorded a European food safety E number. It is considered as a food grade additives of food by the Bureau of Indian Standards, India under Indian Standard IS 7239:1974 [13, 15, 16]. In India, the application of this hydrocolloid in traditional medicine and food preparations is well known for centuries. The gum is fed to the lactating mothers in the form of laddu to enhance the nutrients in milk as well as to prevent the post-delivery backache . The gum laddu is also eaten as a heating agent during winter season [18, 19]. The gum ghatti is comprised of around 80% soluble dietary fiber and acts a prebiotic by supplying the matrix required to sustain the bacterial flora of the human colon. This hydrocolloid is resistant to gastrointestinal enzymes and known to be degraded enzymatically only by the specific microflora of the colon such as Bifidobacterium longum, thereby aiding in bifidus fermentation [20–22]. This gum is also given for the treatment of diarrhea and diabetes . Earlier studies on gum ghatti fed white leghorn cockerels and albino rats have established the hypolipidemic activity of gum ghatti [24, 25]. Recent studies have established that gum ghatti has a potential application as a release modifier for controlled drug delivery . Gum ghatti has long been used in non-food applications, such as, calico printing, explosives, varnishes, car polishes, ceramics, cosmetics; and in pharmaceutical, textile, paper, petroleum, and mining industries. Also, this biopolymer aids in various photoelectric determinations [7, 8, 13, 16, 23].
The attractive features of gum ghatti prompted us to use this biopolymer for the synthesis and stabilization of silver nanoparticles due to its (i) edible nature and GRAS ; (ii) natural availability and low cost ; (iii) intermediate viscosity between gum arabic and gum karaya [14, 15]; (iv) greater stability to pH acidification, electrolyte addition, and high-pressure treatment [15, 17]; (v) higher emulsification ability and superior emulsion storage stability at lower concentrations , and (vi) exceptional interfacial characteristics with faster kinetics . The green synthesis of inherently safer silver nanoparticles depends on the adoption of the basic requirements of green chemistry; the solvent medium, the benign reducing agent, and the non-hazardous stabilizing agent [1, 27]. In this context, we have explored and developed a facile and green synthetic route for the production of silver nanoparticles using a proteinaceous, edible, renewable natural plant polymer, gum ghatti as both the reducing and stabilizing agents. Being a natural polymer, gum ghatti is amenable for biodegradation. The synthesis was carried out in aqueous medium by autoclaving, without the addition of any external chemical reducing agent. In this study, autoclaving was adopted as a synthetic route to produce sterile silver nanoparticles that are completely free from bacteria, viruses, and spores, which would suit biological applications. The focus of this study was on (i) the synthesis, (ii) characterization, and (iii) capping and stabilization of silver nanoparticles. In addition, we have also demonstrated the antibacterial activity of the prepared nanoparticles on Gram-positive and Gram-negative bacteria for finding out the potential of the generated nanoparticles for various environmental and biomedical applications.
Characterization of synthesized silver nanoparticles
In order to study the formation of silver nanoparticles, the UV–Visible absorption spectra of the prepared colloidal solutions were recorded using an Elico SL 196 spectrophotometer (Hyderabad, India), from 250 to 800 nm, against autoclaved gum blank. The absorption spectra of gum before and after autoclaving were also recorded against ultra pure water blank. The size and shape of the nanoparticles were obtained with Hitachi H 7500 (Tokyo, Japan) and JEOL 3010 (Tokyo, Japan) transmission electron microscopes (TEM), operating at 80 and 200 kV, respectively. Samples were prepared by depositing a drop of colloidal solution on a carbon-coated copper grid and drying at room temperature. The X-ray diffraction (XRD) analysis was conducted with a Rigaku, Ultima IV diffractometer (Tokyo, Japan) using monochromatic Cu Kα radiation (λ = 1.5406 Å) running at 40 kV and 30 mA. The intensity data for the nanoparticle solution deposited on a glass slide were collected over a 2θ range of 35–85° with a scan rate of 1°/min. The nanoparticles were recovered from the synthesized solutions by centrifugation and made into powders using a FTS Systems, Dura-DryTM MP freeze dryer (New York, USA). The IR spectra of the lyophilized samples were recorded using a Bruker Optics, TENSOR 27 FT-IR spectrometer (Ettlingen, Germany); over a spectral range of 400–4000 cm–1. The Raman spectrum of the synthesized nanoparticles was recorded at room temperature using the 532-nm line from a SUWTECH, G-SLM diode laser (Shanghai, China). The scattered light was collected and detected using a CCD-based monochromator, covering a spectral range of 150–1700 cm–1. The sample solution was taken in a standard 1 cm × 1 cm cuvette and placed in the path of the laser beam.
Results and discussion
Synthesis of silver nanoparticles
The present experimental investigation reports the green synthesis of silver nanoparticles using gum ghatti by autoclaving. This method utilizes a proteinaceous, edible, renewable, and water soluble biopolymer; gum ghatti which functions as both reducing and stabilizing agents during synthesis. By virtue of being a natural polymer, this gum is also amenable for biodegradation. The process of autoclaving makes the silver nanoparticles intrinsically safe and sterile, in environmentally benign solvent water. Moreover, generation of gum–silver nanoparticles by autoclaving is a prerequisite for biological applications. Thus, the adopted method is meeting the requirements of green chemistry principles.
Proposed mechanism of reduction
During autoclaving at 121°C under the influence of temperature and pressure (103 kPa), this biopolymer expands and becomes more accessible for the silver ions to interact with the available functional groups on the gum as observed earlier for starch . The gum has been categorized under arabinogalactan due to the abundance of arabinose and galactose. This acidic heteropolysaccharide is known to be rich in uronic acid content and shows a pH of 4.5–5.5 [8, 14–17]. The presence of hydroxyl and carboxylic groups on this biopolymer  facilitates the complexation of silver ions. Subsequently, these silver ions oxidize the hydroxyl groups to carbonyl groups, during which the silver ions are reduced to elemental silver. In addition to this inherent oxidation, the dissolved air may also causes oxidation of the existing hydroxyl groups to carbonyl groups such as aldehydes and carboxylates. In turn, these powerful reducing aldehyde groups along with the other existing carbonyl groups reduce more and more of silver ions to elemental silver. Further, these nanoparticles are probably capped and stabilized by the polysaccharides along with the proteins present in the gum. As these carbohydrate polymers are very complex, it is most likely that more than one mechanism is involved in the complexation and subsequent reduction of silver ions by gum ghatti during autoclaving. Silver ion complexation by hydroxyl groups and its subsequent reduction by aldehyde groups are reported for starch, in which silver nanoparticles were produced by autoclaving . Silver nanoparticles produced using gum Acacia, carboxylate groups involving complexation of silver ions and its subsequent reduction by hydroxyl groups were reported .
The reduction of silver ions by this gum even at room temperature was observed. But, the formed nanoparticles were not stable and aggregated due to lack of stabilization of the synthesized nanoparticles. It was noticed that the autoclaving at 121°C and 103 kPa of pressure, increased the extent of synthesis and stabilization of the nanoparticles. It is known that elevated temperature and pressure accelerate the synthesis of nanoparticles . Besides, this process complexly eliminates the microbial contamination possibly acquired during gum secretion, collection, handling, and transportation.
Characterization of synthesized silver nanoparticles
Transmission electron microscopy
Fourier transform infrared spectroscopy (FTIR)
Inhibition zones (mm) observed with different bacterial culture plates loaded with silver nanoparticles and silver nitrate at a silver concentration of 5 μg
S. aureus 25923
E. coli 25922
E. coli 35218
P. aeruginosa 27853
Synthesis of silver nanoparticles
Silver nitrate (AgNO3) (E. Merck, Mumbai, India) of analytical reagent grade was used for the synthesis. “Gum ghatti” grade-1 was purchased from Girijan Co-operative Corporation Ltd., Hyderabad, India. All the solutions were prepared in ultra pure water. Gum ghatti was powdered in a Prestige high-speed mechanical blender (Bengaluru, India) and sieved to obtain a mean particle size of 38 μm. Then, 0.5% (w/v) of homogenous gum stock solution was prepared by adding this powder to reagent bottle containing ultra pure water and stirring overnight at room temperature. Then this solution was centrifuged to remove the insoluble materials and the supernatant was used for all the experiments. The protein concentration in the gum solution was quantified by Lowry’s method using a Bangalore GeneiTM protein estimation kit, Cat No 105560 (Bengaluru, India). The silver nanoparticles were synthesized by autoclaving the silver nitrate solutions containing various concentrations of gum ghatti at 121°C and 103 kPa of pressure for different durations of time, under dark conditions. The effect of concentration of gum and reaction time on nanoparticle synthesis was studied.
The well-diffusion method was used to study the antibacterial activity of the synthesized silver nanoparticles. All the glassware, media, and reagents used were sterilized in an autoclave at 121°C, 103 kPa of pressure for 20 min. Staphylococcus aureus (ATCC 25923); and Escherichia coli (ATCC 25922), E. coli (ATCC 35218), and Pseudomonas aeruginosa (ATCC 27853) were used as model test strains for Gram-positive and Gram-negative bacteria, respectively. Bacterial suspension was prepared by growing a single colony overnight in nutrient broth and by adjusting the turbidity to 0.5 McFarland standard . Mueller Hinton agar plates were inoculated with this bacterial suspension and 5 μg of silver nanoparticles was added to the center well with a diameter of 6 mm. The nanoparticles used were prepared with 0.1% gum solution containing 1 mM AgNO3, autoclaved for 30 min. Negative control plates were maintained with autoclaved gum-loaded wells. The culture plates loaded with silver nitrate at a silver concentration of 5 μg were included as positive controls. These plates were incubated at 37°C for 24 h in a bacteriological incubator and the zone of inhibition (ZOI) was measured by subtracting the well diameter from the total inhibition zone diameter. Three independent experiments were carried out with each bacterial strain.
This study reports the facile synthesis of silver nanoparticles from silver nitrate using gum ghatti. The adopted method is compatible with green chemistry principles as the gum serves as a dual functional reductant and stabilizer for the synthesis of nanoparticles. At a given gum concentration, the efficiency of nanoparticle synthesis increases with reaction time, a property attributable to the large reduction capacity of the gum. As the particle size of the nanoparticles can be controlled, this method can be implemented for the large-scale production of monodispersed and spherical nanoparticles of around 5.7 nm due to the availability of low-cost plant-derived biopolymer. The hydroxyl and carboxylate groups of the gum facilitate the complexation of silver ions during autoclaving. Subsequently, these silver ions are reduced to elemental silver possibly by in situ oxidation of hydroxyl groups; and by the intrinsic carbonyl groups in addition to those produced by the air oxidation. This proposed mechanism is also substantiated by the FTIR data. Further, the formed silver nanoparticles had significant antibacterial action on both the Gram classes of bacteria. The surface reactivity provided by capping enables these functionalized nanoparticles as promising candidates for various pharmaceutical, biomedical, and environmental applications. Notably, the selective enhancement of Raman bands of the organic capping agents bound to the silver colloids allows these nanoparticles as suitable substrates for SERS. In view of this, further studies are envisaged to explore the other potential applications of this gum-based nanoparticles.
We thank Dr. S. V. Narasimhan, Associate Director and Dr. Tulsi Mukherjee, Director, Chemistry Group, BARC, for their constant support and encouragement for this study. The support rendered for high-resolution TEM measurements by the DST unit on Nanoscience, Sophisticated Analytical Instrument Facility (SAIF) at IIT-Madras, Chennai, is gratefully acknowledged.
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