- Open Access
Alpinia calcarata: potential source for the fabrication of bioactive silver nanoparticles
© The Author(s) 2018
- Received: 7 July 2018
- Accepted: 19 November 2018
- Published: 6 December 2018
- Silver nanoparticles
- Process optimization
- Antibacterial activity
- Antioxidant activity
Fabrication methods for the synthesis of metal nanoparticles using green chemistry principle have gained significant attention in the past decades. This is mainly due to there is no requirement of toxic chemicals for synthesis protocol. The chemical synthesis approaches have drawback that during synthesis process the colloidal solution being contaminated by several by-products as a result of chemical reactions. Thus, to overcome all these problems there is a need to develop alternative green process which does not need any harmful chemicals. Considerable works have been put to develop eco-friendly approaches for the synthesis of nanoparticles. Microbes and plants are currently used for the synthesis of nanoparticles. The use of plants for the synthesis of nanoparticle is rapid, low cost, environmental benign and a single step method. Plant extract contains a variety of phytochemicals compounds such as flavonoids, terpenoids, phenols, alkaloids some plant enzymes like hydrogenases, reductases, etc. which act as a reducing and capping agent in the presence of metal salts .
Recently metal nanoparticles are gaining the interest of scientist for the novel methods of synthesis because they exhibit unique physicochemical properties including optical catalytic, magnetic, electronic and antimicrobial properties . They have a high surface area and a high fraction of atoms. Nanomaterials such as Ag, Au, Cu, Zn, Pt and Pd have been synthesized by various methods, including biological entities using bacteria, fungi, algae and plants . Among them silver (Ag) nanoparticles play a considerable role in field of medical and biological sciences due to its significant physicochemical properties. Ag nanoparticles are reported to exhibit great antibacterial, antifungal, anti-inflammatory, antioxidant and antiviral activities . Nanoparticles play a crucial role in drug delivery, tissue engineering, gene delivery, artificial implants, diagnosis, imaging and sensing . It has been found that a highly reactive metal nanoparticle shows excellent antimicrobial activities against bacteria  Silver nanoparticles have wide applications in medicines including skin creams and ointments containing silver to inhibit infection of burns and wounds and medical devices.
In the present investigation, we discuss the synthesis of bioactive Ag nanoparticles using the leaf extract of Alpinia calcarata as the biomaterial. A. calcarata is also known as Snap Ginger, which is a plant native to India, belongs to the Zingiberaceae family, as it is commonly available medicinal plant, used for synthesis of Ag nanoparticles. A. calcarata is a perennial herb with horizontal root stock and full leafy stem. The leaves of the plant are simple, alternative 25–35 cm in length and are 2.5–5 cm broad . It is reported that the compounds isolated from the Zingiberaceae plants were found to have anticancer activity against several cell lines and also have strong anti-inflammatory and antioxidant activities . Rhizome of the plant also contains various secondary metabolites majorly diterpenoids, some of them are reported as cytotoxic and can induce cell cycle arrest such as Calcarathan D, Calcarathan E etc. They also show excellent antibacterial activities against some pathogenic Gram +ve and Gram −ve bacteria. Additionally, A. calcarata is also used for the treatment of various diseases such as warming digestive tonic, carminative, stomachic, stimulant, expectorant and fungal infections. Plant is also used as tonic, aphrodisiac and diuretic, in the treatment of headache, lumbago diabetes, chest pain, rheumatic pains, bronchitis, dyspepsia, sore throat, impotence and diseases of kidney and liver. It is particularly considered for its efficacy in chest complains. A wide variety of chemical compounds and various bioactivities, including antimicrobial and antioxidant properties were reported in phytochemical studies from this plant. Ag nanoparticles have been synthesized using several natural products like Crocus sativus L. , Azadirachta indica , Alysicarpus monilifer , Camellia sinensis , Glycine max , Cinnamon zeylanian , Syzygium aromaticum  etc. Plants give a better platform for nanoparticle synthesis as they do not contain any toxic chemicals along with this they also provide natural stabilizing agent. Other advantages may include, use of plant extracts reduces the cost of microorganism’s isolation and culture media increases the cost ambitious viability over nanoparticle synthesis through microorganisms .
The aim of the present investigation was to synthesize the silver nanoparticles using leaf extract of A. calcarata. Phytochemical investigation of leaf extract of plant to evaluate the possible biomolecules responsible for the bioreduction and stabilization of synthesized nanoparticles. Optimization of various reaction conditions for nanoparticle synthesis such as the metal ion concentration, pH and incubation period. Furthermore characterization of synthesized silver nanoparticles by UV–Vis spectroscopy, FTIR, DLS, zeta potential measurements, ICP-OES, TEM and XRD analysis and evaluation of their potentiality for antibacterial activity against pathogenic microbial strains and antioxidant activities.
2.1 Sample collection
Fresh leaves of Alpinia calcarata (Snap Ginger) were collected from campus of Department of Botany, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India. Silver nitrate and other analytical grade chemicals were purchased from Hi-media laboratories, Mumbai, India. The bacterial culture of Escherichia coli (MTCC-44), Pseudomonas aeruginosa (MTCC-424) and Staphylococcus aureus (MTCC-96) were obtained from microbial type culture collection (MTCC), Chandigarh, India.
2.2 Preparation of plant leaf extract
Aqueous extract of leaf of A. calcarata were prepared using freshly collected leaves. They were surface sterilized with running tap water followed by distilled water and finally chopped into fine pieces. About 10 g of finally incised leaf was boiled with 100 ml of distilled water for 30 min. The extract was allowed to cool than filtered with Whatman No. 1 filter paper. Filtrate was collected and stored at 4 °C for further experiment.
2.3 Phytochemical investigation
Qualitative phytochemical analysis of A. calcarata was performed using the standard protocols described by Jigna and Sumitra  for determination of presence of several phytoconstituents like flavonoids, alkaloids, glycosides, tannins, resins, phytosterols, saponins, terpenes, quinones and phenolic compounds. The results of these tests were showed qualitatively as positive (+) or negative (−).
2.4 Biosynthesis of silver nanoparticles
For biosynthesis of silver nanoparticles, 1 ml of the leaf extract was added to the 9 ml of 1 mM aqueous silver nitrate solution. The mixture was incubated in dark condition at room temperature. The visual color change in the reaction mixture from yellow to reddish brown was observed after 5 min with reference to control, which indicates the formation of silver nanoparticles. Further confirmation was done by spectrometric analysis.
2.5 Optimization of various experimental parameters
Several experimental parameters like effect of metal ion concentration, pH and different incubation time were evaluated for biosynthesis process using A. calcarata. The absorbance of resultant samples was measured at 420 nm using UV–Vis spectrophotometer.
2.5.1 Effect of metal ion concentration
To study the effect of concentration of silver nitrate solution on nanoparticle synthesis, various concentration of silver nitrate (0.5–3 mM) was used. During the synthesis 9 ml of each concentration (0.5–3 mM) was taken in different test tubes. 1 ml of leaf extract was added to each of test tubes and incubated in dark condition at room temperature. Synthesis of silver nanoparticles was confirmed by UV–Vis spectrometric analysis.
2.5.2 Effect of pH
To study the effect of pH on nanoparticle biosynthesis, synthesis process was carried out at various pH ranges from (4 to 11). Silver nitrate at optimum concentration obtained by previous study was used and all other parameters for biosynthesis process were remaining the same.
2.5.3 Effect of contact time on nanoparticles biosynthesis
To study the effect of incubation period, synthesis of nanoparticle was carried at different time intervals from 0 to 16 h, absorbance of resulting solution was taken at 420 nm at various time intervals. Optimum silver nitrate concentration and pH was used from the previous experiments and other parameters were remaining the same.
2.6 Characterization of silver nanoparticles synthesized under optimum condition
2.6.1 UV–visible spectroscopy
Initial characterization of the Ag nanoparticles was carried out using UV–visible spectroscopy. Reduction of silver ions to the silver nanoparticles was examined by measuring the surface plasmon resonance of the reaction mixture. The spectra were recorded on, Double Beam Spectrophotometer, 2203 at room temperature regulated at a resolution of 1 nm between 300 and 700 nm ranges. Optical density was taken at different wavelength ranging from 300 to 700 nm and plotted the values on a graph. Scanning was performed after reaction time ranging from 5 min to 24 h.
2.6.2 Dynamic light scattering (DLS) and Zeta potential analysis
The particle size range of the nanoparticle with their polydispersity was analyzed using zetasizer instrument (Zetasizer Nano, Malvern UK). The size of the particle was measured by the time dependent fluctuation of scattering of laser light when particles were under gone to Brownian motion. With the help of this we can also analyze the size distribution pattern and the mean size of the particle inside the sample.
For measurements of zeta potential to infer about their stability of the colloidal AgNPs was analyzed via Zeta Sizer instrument (Malvern-Nano ZS 90). The sample was poured into sample holders of the instrument and data recorded.
2.6.3 Fourier transform infrared spectroscopy (FTIR)
Reaction mixture was centrifuged at 12,000 rpm for 20 min to remove the biological biomass residues. Obtained pellet were redispersed in distilled water and then again centrifuge it. This step is repeated for 2–3 times. Finally samples were dried and grinded with KBr pellets and then subjected to FTIR spectroscopy measurements. The measurements were carried out on a Thermo-Nicolet-Avatar 370 instrument in the different reflectance mode at a resolution of 4 cm−1 in KBr pellet.
2.6.4 Inductively coupled plasma-optical emission spectrometry (ICP-OES)
The technique is mainly used for the qualitative and quantitative determination of the metals and metalloids in the biological samples. The working principle of emission spectrometry (Perkin Elmer Optima 5300 DV ICP-OES) is based on the fact that atoms or ions in an excited state tend, to revert back to the ground state and in so doing emit characteristic wavelength and intensity of that light is proportional to the concentration of that particular element in the sample solution.
2.6.5 Transmission electron microscopy (TEM)
The morphology including their shape and size were determined by transmission electron microscopy (TEM CM 200 instrument). For TEM study sample is prepared by sonicated it first for 20 min and then a drop of it was placed on copper grid and it was allowed to dry in vacuum, resulting an image is produced from the introduction of the electron transmitted through the sample.
2.6.6 X-ray diffraction pattern measurement
2.7 Bactericidal activity of synthesized silver nanoparticles
The Ag nanoparticles synthesized by using leaf extract was tested for dose dependent antibacterial activities by agar well diffusion method against pathogenic bacterial strains E. coli, P. aeruginosa (Gram −ve), and S. aureus (Gram +ve). In brief, the pure cultures of bacteria were subcultured on nutrient agar media (NAM). Each strain was swabbed uniformly on the individual plates. Wells of 6 mm diameter were made on nutrient agar plates by using cork borer. Various volumes of Ag nanoparticles (20, 30 and 50 µl) were added to the centre of the well. The streptomycin (1 mg/ml) and plant extract were used as a positive and negative control for the antibacterial assay. Inoculated plates were incubated at 37 °C for 24 h. After incubation, the different levels of zone of inhibition of bacteria were measured and recorded. The standard deviation was calculated using three replicates of experiments. The results of antibacterial activity were compared with control experiment.
2.8 In vitro antioxidant activities of synthesized silver nanoparticles
2.8.1 DPPH free radical scavenging assay
2.8.2 Hydrogen peroxide scavenging assay
The H2O2 scavenging activity was evaluated by the method describes by Bhakya et al. . Different concentration of silver nanoparticles and plant extract were mixed with 50 µl of 5 mM H2O2 solution and incubated at room temperature for 20 min. Ascorbic acid was used as standard. Absorbance was measured at 610 nm. Activity in terms of H2O2 scavenging was calculated using above mentioned (Eq. 1).
2.9 Statistical analysis
All the experiments were performed in triplicates and the results were expressed as mean ± standard deviation. Significant levels were tested at P < 0.05.
In the present investigation we have demonstrated the biosynthesis of silver nanoparticles using leaf extracts of A. calcarata. Qualitative phytochemical analysis was performed to evaluate the phytoconstituents present in the leaf extracts. Effect of various experimental parameters on silver nanoparticle biosynthesis was also studied. Further, silver nanoparticles synthesized under optimum conditions were characterized by different analytical instruments. Bioactivity of synthesized silver nanoparticles were evaluated by antibacterial and antioxidant assay.
3.1 Phytochemical screening
Phytochemical analysis of leaf extract of Alpinia calcarata in different solvents
3.2 Biosynthesis of silver nanoparticles
3.3 Optimization of various experimental parameters
Optimization of experimental conditions is essential in order to achieve the optimum conditions for silver nanoparticles formation. The optimizing factors involved in this study were silver nitrate concentration, pH and incubation period.
3.3.1 Effect of metal ion concentration
3.3.2 Effect of pH
To study the effect of the pH on nanoparticle biosynthesis we have selected a pH range of 4–11. Synthesized silver nanoparticles stability were determined by their corresponding absorbance at 420 nm at pH from 4 to 11 (Fig. 2b). An increase in the absorbance with increase in pH suggested that alkaline reaction condition was more appropriate for silver nanoparticle biosynthesis. However, when we increase the pH from 9 to 11, aggregation of nanoparticles was observed. Previous studies also show that optimal pH for silver nanoparticle biosynthesis has varied by plant species. Spectral responses of individual nanoparticles are related with the size and shape of the nanoparticles. It has been reported that change in pH affects the size and shape of the particles as pH has the ability to change of biomolecules that might affect their reduction as well as stability. Our results were also shows similarity with the findings of Prathna et al. .
3.3.3 Effect of contact time on nanoparticles biosynthesis
Figure 2c shows the effect of various incubation periods (time) on biosynthesis of silver nanoparticles. As we increase the incubation period the absorbance value was also increases up to 12 h, but thereafter they did not show any increase. In another study Pugazhendhi et al.  A. calcarata shows the formation of silver nanoparticles after 24 h of incubation.
3.4 Characterization of synthesized silver nanoparticles
3.4.1 UV–Vis spectroscopy
UV–Vis spectroscopy analysis is used to determine the formation and stability of silver nanoparticles in the aqueous solution . In aqueous suspension the size and shape of silver nanoparticle is generally recognized with UV–visible spectroscopy. The formation of silver nanoparticles was observed using UV–visible spectroscopy 200–700 nm wavelength range. In our result highest peak was observed at 420 nm (Fig. 1A), which was found to be specific for the biosynthesis of silver nanoparticles. The reaction mixture showed a single SPR band, which confirms the spherical shape of silver nanoparticles , which was further confirmed by TEM micrographs. The UV–Vis spectra also showed that the silver nanoparticles formed rapidly within 10 min and remained stable even after 48 h (Fig. 1B). Intense plasmon band was observed after 5 min between 400 and 430 nm, which indicates its high reducing efficiency. No change in absorbance was recorded after 48 h of incubation, confirming the complete reduction of Ag+ ions to silver nanoparticles. Optical absorbance spectrum of metal nanoparticles is due to SPR (surface plasmon resonance), which leads to a shift towards the red or blue ends depending upon the shape and size of the particle, surrounding medium and state of agglomeration . Stability of the synthesized silver nanoparticles was studied by, measuring its intensity by UV–Vis spectrophotometer over a period of 3 months in same reaction conditions. No significant change in the absorbance was observed, which confirmed its stability over a longer period (data not shown here). Synthesis time of silver nanoparticle is quite shorter as compared to previously reported studies with plant extract [29, 34, 35].
3.4.2 Dynamic light scattering (DLS) and Zeta potential analysis
The surface zeta potential values of AgNPs were measured to be slightly negative and were −19.4 mV (Fig. 3b). Duan et al.  reported that AgNPs mainly exhibit negative charge. The possible cause of the negative surface charges on synthesized AgNPs is may be due to the absorption of free nitrate ions present during the reduction of AgNO3 . The negative charge of AgNPs prevents them from agglomeration and increases their stability, as well as help to enhance their antimicrobial property. Zeta potential is mainly used for the quantification of the magnitude of charge which is a key indicator of the stability of colloidal dispersions. The magnitude of zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in dispersion. When the value for zeta potential is small, attractive force may causes the repulsion and may break and flocculate the dispersion. Nanoparticles have very small diameter and are highly energetic, this makes them highly unstable. So that particles undergo agglomeration/aggregation to stabilize themselves and also develop certain charges on their surface which contributes to their stabilization. Zeta potential has direct relation with the stability of a structure.
3.4.3 Fourier transform infrared spectroscopy (FTIR)
3.4.4 Inductively coupled plasma-optical emission spectrometry (ICP-OES)
The concentration of synthesized silver nanoparticles in the aqueous medium was determined by ICP-OES analysis. In this study we found that leaf extract of A. calcarata produces about 24.81 ppm of silver nanoparticles, per litre of culture filtrate when 1.5 mM silver nitrate was added.
3.4.5 Transmission electron microscopy (TEM)
3.4.6 X-ray diffraction analysis (XRD)
3.5 Antibacterial activity of synthesized silver nanoparticles
Antibacterial activity of synthesized silver nanoparticles
Zone of inhibition (mean ± SD in mm)
AgNO3 (1 mM)
Plant extract (50 µl)
12.17 ± 0.34
24.12 ± 0.61
2.87 ± 0.16
9.13 ± 0.52
22.13 ± 0.54
27.70 ± 0.31
15.06 ± 0.12
19.54 ± 0.73
5.38 ± 0.61
13.41 ± 0.33
17.52 ± 0.37
22.43 ± 0.45
10.11 ± 0.43
27.42 ± 0.57
3.67 ± 0.35
17.09 ± 0.42
21.17 ± 0.63
29.14 ± 0.63
A number of reports available that shows that silver have been permanently utilized to treat or inhibit a wide range of disease caused by both Gram positive and Gram negative bacteria . Results from previous studies also support the antibacterial activity of silver nanoparticles . No zone of inhibition was shown in case of control. Recently nanoparticles has been used as an interesting alternative method to antibiotics and supposed to have a high potential in curing several bacterial diseases in human. In recent years silver nanoparticles have attracted significant attention especially in antimicrobial activity [66, 67].
Similar study was carried out by Paszek et al. , during their study they reported that the high antibacterial activity of silver nanoparticles is due to the release of silver cations from silver nanoparticles that act as reservoirs for the Ag+ bactericidal agent. Silver nanoparticles synthesized from various plant extracts were reported for potent bactericidal activity [69–71].
Results of this study strongly recommended that silver nanoparticle synthesized from A. calcarata exhibit potential antibacterial activity against disease causing pathogenic strains of bacteria and hence can be used as an antibiotic or may be used for the development of nano based antibacterial formulations.
3.6 Antioxidant activities of synthesized silver nanoparticles
The H2O2 scavenging activity of silver nanoparticles was quantitatively analyzed by using spectrophotometer and is shown in (Fig. 8b). As we know that hydroxyl radical is a highly reactive free radical formed in biological system and has been used as a highly damaging species in free radical pathology, having ability to damage a wide range of molecules like proteins, DNA, lipids etc. . Results of present study showed that silver nanoparticles exhibited potential reducing power as compare to ascorbic acid. It has been reported that in the presence of H2O2 silver nanoparticles can hydroxyl radicals (reactive oxygen species). The free radical scavenging activity of silver nanoparticles are might be due to the presence of several phyto-constituents that have ability to donate the hydrogen atom in their OH groups. Results also resembles with earlier reports on antioxidant activity of leaf extract of Erythrina suberosa (Roxb.) . The result obtained from this experiment also recommends the applications of silver nanoparticles as natural antioxidant agent for several health preservation against various oxidative stresses related with degenerative diseases.
The present study revealed the rapid fabrication of silver nanoparticles using leaf extract of A. calcarata. Silver nanoparticles formation was achieved within 10 min, which was confirmed by UV–Vis spectroscopy analysis that showed a sharp peak at 420 nm confirming the formation of silver nanoparticles. The effects of various experimental parameters played a major role in the biosynthesis and size control of the particles. FTIR results suggested that the biomolecules like the secondary metabolites present in the plant leaves are responsible for the reduction and stabilization of nanoparticles. TEM micrograph of synthesized silver nanoparticles showed spherical shaped nanoparticles with average particle size 27 nm. DLS analysis showed that synthesized nanoparticles were well dispersed. XRD analysis concluded that synthesized nanoparticles were highly crystalline in nature. Antibacterial and antioxidant studies revealed that the synthesized Ag nanoparticles have potential bactericidal activities against all the selected pathogenic strains of bacteria and they also show efficient free radical scavenging activity. Hence this green chemistry principle towards the synthesis of silver nanoparticles have several benefits like rapid, convenient, facile and ease to scale up etc. Future prospects may include the formulation of nanomedicines against several human and veterinary pathogens by using such plant extracts and to develop studies in the interface between biology and material structural science.
PK and SKS design and wrote the manuscript. DKS, RKY and LK helped in performing the experiment. All authors read and approved the final manuscript.
The authors are thankful to SAIF IIT Madras for ICP-OES analysis, RGPV, Bhopal for DLS analysis, BIT Mesra for Zeta potential analysis, SAIF IIT Bombay for TEM analysis, STIC SAIF Cochin for FTIR and XRD analysis. The authors also like to acknowledge Head, Department of Botany, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, for providing lab facilities to carry out this work.
The authors declare that they have no competing interests.
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