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Abstract
Background : Silver nanoparticles (AgNPs) are widely used in food industries, biomedical, dentistry, catalysis, diagnostic biological probes and sensors. The use of plant extract for AgNPs synthesis eliminates the process of maintaining cell culture and the process could be scaled up under a non-aseptic environment.
Introduction
The field of nanotechnology has undergone substantial developments and has attracted considerable interest from researchers owing to its wide range of application.
AgNPs synthesis has become popular, and many studies on the process have been conducted using various chemical, physical and biological methods [ 6 ]. Sodium borohydride, hydrazine and ethylene glycol are the most common chemicals used in AgNPs synthesis [ 7 , 8 ]. However, the processes using these chemicals are expensive and harmful to living organisms and hence undesirable. Meanwhile, biological synthesis has attracted interest from researchers because it is cost effective, consumes less energy and is non-toxic [ 6 ]. Many studies used natural resources, such as plants [ 9 ], bacteria, fungi and yeast to synthesize AgNPs [ 10 ]. Nevertheless, the use of plant extracts has become a major interest in AgNPs synthesis.
The plant extracts that induce AgNP formation contain reducing agents [ 14 ], such as the metabolites of phenolic compounds, flavones, alkaloids and sterols [ 15 – 17 ].
Clinacanthus nutans ( figure 1 ) is a small shrub belonging to Acanthaceae [ 19 ], and commonly found in tropical Asian countries, mainly Malaysia, Thailand and Indonesia [ 20 ]. C. nutans is commonly known as ‘Belalai gajah' in Malaysia [ 21 ], Phaya yo or Phaya plongtong in Thailand and Dandang gendis in Indonesia [ 20 – 22 ]. Several bioactive compounds from C. nutans extracts have been isolated and studied. Lupeol, isoorientin, orientin, isovitexin, schaftoside, vitexin and β-sitosterol have been isolated from the stem and leaf extracts of C. nutans [ 23 ]. Other bioactive groups isolated from this plant were sulfur-containing glucosides and chlorophyll derivatives. Further to this, saponin, phenolics, flavonoids, diterpenes and phytosterols were present in the methanol extracts of the leaves [ 24 ].
Materials and methods
Plant collection
The dried leaves and stems of C. nutans were purchased from Botani Sdn. Bhd, Manjung, Perak. Clinacanthus nutans was authenticated with voucher specimen No. 11465 and deposited at Herbarium Unit, School of Biological Sciences, Universiti Sains Malaysia. The dried leaves and stems were pulverized into fine powder with a grinder (Ultra Centrifugal Mill ZM200, Retsch, Haan, Germany). The powder was then sealed in a glass bottle and kept at room temperature until further use.
Plant extraction
The extraction method was adapted from previous study with some modifications [ 25 ]. Briefly, 50 g of fine leaf powder of C. nutans (CNL) was macerated in 500 ml of distilled water (dH 2 O) in an orbital shaker (Orbitron, Bottmingen, Switzerland) at room temperature for 24 h at 150 r.p.m. The aqueous extract was then filtered and lyophilized (EYELA FDU 1200, Fisher Scientific, Loughborough, UK). The extract was labelled, weighed and kept in desiccator until further use. The percentage yield of the aqueous extract was calculated according to equation (2.1). These procedures were repeated for powdered stem of C. nutans (CNS). 2.1 Percentage yield = w 2 − w 1 w 0 × 100 % , where w 0 is the weight of the initial dried sample, w 1 is the weight of the container and w 2 is the weight of the dried extract and container.
Preparation of aqueous extract for qualitative analysis
The qualitative phytochemical analysis was performed for tannins, saponins, alkaloids, proteins, carbohydrates and flavonoids. Each test was expressed as negative (−) or positive (+) reactions. Briefly, 1 g of CNL extract was dissolved in 10 ml of dH 2 O [ 26 ]. The sample was filtered, and the filtrate was used for further experiments. This procedure was repeated for CNS extract. All screening tests were conducted according to the standard methods [ 27 ].
Total phenolic content
The Folin–Ciocalteu method was adapted from previous study with modification [ 26 ]. Briefly, the sample of the extract was prepared at concentration of 1 mg ml −1 with dH 2 O. Then, 30 µl of the sample was mixed with 150 µl of 10% (v/v) Folin–Ciocalteu's phenol reagent (diluted with dH 2 O) in 96-well plate (FalconTM, Fisher Scientific, Loughborough, UK) and the mixture was left for 5 min at room temperature. Then, 120 µl of Na 2 CO 3 (7.5% w/v) was added and the mixture was incubated for 30 min in the dark. The absorbance was measured at 765 nm with FLUOstar Omega microplate reader (Offenbury, Germany). The amount of total phenolic content (TPC) was calculated in term of µg of gallic acid equivalent (GAE) per mg extract based on the gallic acid standard curve.
Total flavonoid content
Total flavonoid content (TFC) was determined by using aluminium chloride method described by Mishra and co-workers with some modifications [ 28 ]. Briefly, the extract was prepared by dissolving 1 mg of CNL or CNS in 1 ml of dH 2 O. Then, 25 µl of each extract was mixed with 125 µl of dH 2 O and 7.5 µl of NaNO 3 (5% w/v). The mixture was incubated for 5 min at room temperature. Next, 15 µl of AlCl 3 (10% w/v) solution was added into the mixture and left at room temperature for 6 min before 50 µl of NaOH (1 M) and 27.5 µl of dH 2 O were added into the mixture. The mixture was incubated in the dark for 20 min. The absorbance was measured at 510 nm using microplate reader. The TFC was expressed as µg quercetin equivalents (QE) per mg extract using quercetin as the standard curve.
Biosynthesis of silver nanoparticles
The synthesis of AgNPs began with the addition of 95 ml of 0.001 M silver nitrate (AgNO 3 ) to 5 ml of CNL extract (1 mg ml –1 in dH 2 O). The mixture was allowed to stir for 24 h. The formation of AgNPs was indicated by the color changes from yellow to dark brown. After 24 h, the mixture was centrifuged (6000 r.p.m., 30 min) and washed with dH 2 O three times. The pellets were dried overnight in an oven at 40°C for further characterization [ 12 ]. The same procedure was repeated for the CNS extract. Different parameters that affect the synthesis of AgNPs such as extract and AgNO 3 concentration, incubation time and temperature were studied. Table 1 shows the parameter conditions used for optimization study. Table 1. Parameter variables used to optimize the synthesis of AgNPs. parameter variable extract concentration 5, 10 and 15% v/v AgNO 3 concentration 1, 3, 5 mM temperature 25°C, 40°C and 60°C time 15 and 30 min, 1, 2, 4, 8, 10, 12, 20, 22 and 24 h
Characterization of silver nanoparticles
The formation of AgNPs was monitored by measuring the reduction of silver ions with a Perkin Elmer double beam UV-Vis spectroscope (Lambda-25). The sample absorbance was measured between 300 and 600 nm after incubation. The sample was diluted with dH 2 O at 1 : 1 volume ratio before the measurement [ 29 ]. For particle size and zeta potential analysis, the dried powder of AgNPs was suspended in deionized water at 1 : 10 ratio. The measurements were conducted at room temperature with Zetasizer Nano ZS (Malvern, Worcestershire, UK) [ 12 ]. For the surface morphology study, the dried AgNPs were placed on the stud and analysed with scanning electron microscope (SEM; Quanta Feg 650) attached with X-Max 50 energy dispersive X-ray spectrometer (EDX; Oxford Instrument, Abingdon, UK). Transmission electron microscope (TEM; Libra 120; Carl Zeiss, Oberkochen, Germany) was used to identify the shapes, sizes and morphologies of the synthesized AgNPs. For TEM analysis, the AgNPs were dispersed in dH 2 O and sonicated for 15 min. The sample was dropped on a copper grid coated with carbon (400 mesh, 305 mm diameter) and observed [ 12 ]. X-ray diffractometer (D8 advance, Bruker, Berlin, Germany) was used to identify the phase and crystallinity of the synthesized AgNPs. All X-ray diffraction (XRD) data were collected in the angular range of 35° ≤ 2 θ ≤ 80° at 40 kV and 30 mA with Cu K α radiation (1.5405 Å). The presence of functional groups that were responsible for the reduction of silver ions was identified with Fourier transform infrared spectrophotometer (FTIR) using KBr disc (Avatar 670; Nicolet, Wisconsin, USA). The absorbance was taken from 4000 to 500 cm −1 [ 29 ].
Results and discussions
Plant extraction
The leaf and stem extracts of C. nutans were obtained through the maceration method at room temperature.
Qualitative phytochemical analysis
The CNL and CNS were quantitatively analyzed for the presence of bioactive groups. The phytochemical study of CNL and CNS showed that both extracts contain tannins, saponins, alkaloids, amino acid, flavonoids, reducing sugars and glycosides ( table 2 ). Table 2. Phytochemical screenings of CNL and CNS. phytochemical group test crude extract CNL CNS tannins FeCl 3 + + saponins Frothing + + alkaloids Dragendroff's + + Mayer's + + amino acid Ninhydrin + + flavonoids Alkaline reagent + + reducing sugar Fehling + + Benedict + + glycosides Keller–Kilani + + + indicates present/positive reaction, − indicates absent/negative reaction.
Plant extracts are known to contain large number of phytonutrients. Based on the previous study, saponin, phenolics, flavonoids, diterpenes and phytosterols were present in the methanol extracts of C. nutans leaves [ 31 ]. The biomacromolecules and secondary metabolites of plant have been widely explored for the ex vivo synthesis of metal nanoparticles. According to Mohamad et al . [ 32 ], the hydroxyl and carbonyl groups of bioactive compounds act as natural reducing agent in formation, capping and stabilizing the nanoparticle synthesis. Therefore, plant extracts are one of the potential sources of natural reducing agents for green synthesis strategies, as this method is easy, efficient, economical and feasible to conduct [ 33 ].
Quantitative phytochemical analysis
TPC was obtained by using Folin–Ciocalteau method. The blue colour complex formed from the reduction of phosphotungstic–phosphomolybdic complex by the phenolic compound, and the intensity was measured by microplate reader. The contents of the phenolic compounds were confirmed according to the TPCs of CNL and CNS. The calibration curve of gallic acid (y = 0.0032x + 0.0407; R 2 = 0.998) was constructed, where the TPC was obtained and expressed as µg of gallic acid equivalents per mg of dry extract (µg GAE/mg dry extract). All the values were expressed as the average ± standard deviation (s.d.). As shown in figure 2 ,
Based on the results, CNL had higher TPC and TFC compared to CNS. However, in a previous study, the TPC of aqueous leaf extract of C. nutans was measured at 30.15 ± 3.09 mg g −1 GAE, which was higher than the TPC in this extract [ 34 ]. The antioxidant activity may be promoted by the presence of carboxyl group of phenolic which is capable of reducing activity [ 32 ]. Phenolic compounds donate hydrogen in nature, hence the presence of phenolic compounds in the plant extract can prevent the oxidative stress-related damage. The variation in phenolic content among the parts of a plant is due to differences in gene expression, which affect the biological properties of the extracts [ 35 , 36 ]. The amount of TPC can vary at the sub-cellular level and within a plant tissue. Moreover, TPC in a plant may vary according to the solvents used, type of extraction methods, age of plant and extraction time [ 37 , 38 ].
Apart from phenolic compounds, flavonoids are commonly present in plants and these compounds are beneficial to human health. Previous study reported that the antioxidant activity of flavonoids is due to their hydroxyl groups. Besides, the phytochemical compounds from plant sources can act as reducing and stabilizing agent in AgNPs synthesis [ 32 ]. This includes the hydroxyl and carbonyl groups from flavonoids [ 39 ]. Therefore, the phytochemicals that were previously identified in C. nutans extracts may be responsible for the biosynthesis of AgNPs, where they act as reducing agent in AgNPs synthesis.
Biosynthesis of silver nanoparticles
The reduction of silver ion (Ag + ) to AgNPs after the addition of CNL or CNS into the AgNO 3 solution was observed after 24 h of incubation. Upon addition of extract into AgNO 3 solution, the color of the mixture gradually changed to dark brown ( figure 3 ) indicating the presence of AgNPs due to the conversion of silver ions into AgNPs [ 40 ]. Previous study has also reported the same where formation of dark red solution from bright yellow resulted after incubation process [ 41 ]. Figure 3. Biosynthesis of AgNPs from ( a ) CNL mixed with AgNO 3 solution at 0 h, ( b ) after 24 h, ( c ) CNS mixed with AgNO 3 solution at 0 h, ( d ) after 24 h.
The green synthesis of AgNPs requires the following conditions: (i) types of solvents used for synthesis, (ii) types of reducing agents, and (iii) non-toxic material used for stabilizing the nanoparticles [ 42 ]. In this regard, C. nutans extracts act as reducing agents which are cheap and easily available without any capping agents. The synthesis of AgNPs using AgNO 3 as the precursor and water (solvent) provides high chemical stability and is inexpensive [ 43 ]. From the TPC and TFC results, the mechanism of chemical reaction for the formation of AgNPs could be derived from flavonoids and phenolic compounds (reducing agent). These compounds act as electron or hydrogen donors [ 44 ]. The ketoform on the backbone of a flavonoid compound reduces from Ag + to Ag 0 [ 45 ]. FTIR analysis was conducted to confirm the presence of the functional groups present in the extract that may be responsible in the formation of AgNPs.
UV-Vis spectroscopy analysis
UV-Vis spectroscopy is an important technique to observe the formation of AgNPs by monitoring the optical properties and electronic structure of the synthesized nanoparticles. The electron cloud of a nanoparticle can oscillate on the surface of the nanoparticle, which absorbs electromagnetic waves at a particular frequency.
The parameters of extract concentrations, AgNO 3 concentrations and temperatures were conducted to optimize the AgNPs synthesis. The analysis for extract concentration was performed at 25°C for 24 h with extract concentrations at 5%, 10% and 15% (v/v). The synthesized AgNP-L peaks were observed between 440 and 454 nm, whereas 441–447 nm for AgNP-S ( figure 5 ). As the concentrations of CNL and CNS increase, the sharpness of the absorption peaks was also increased. The sharpness of the peak indicates the formation of spherical shape of synthesized AgNPs and homogeneous distribution [ 50 ]. Moreover, SPR shifts were also observed along the increasing concentration of extract concentration for both leaves and stem synthesis. These shifts showed reduction in size of the synthesized AgNPs [ 50 ]. Based on the results, 15% (v/v) of both extracts were selected for the optimization of next parameter. Figure 5. UV–Vis spectra of ( a ) AgNP-L and ( b ) AgNP-S for different concentrations of C. nutans extract.
The effect of AgNO 3 solution was then studied. Different concentrations of AgNO 3 (1, 3 and 5 mM) were tested with 15% (v/v) of C. nutans extract at 25°C and 24 h incubation time. The absorption peaks of the synthesized AgNP-L and AgNP-S were found between 442–455 nm and 445–450 nm, respectively ( figure 6 ). The highest concentration of AgNO 3 yielded the highest absorption with slightly lower wavelength which indicates the highest amount of AgNPs being synthesized. Therefore, based on the absorption spectrum, 5 mM AgNO 3 showed the highest formation of AgNPs for both extracts. Thus, this concentration was used for subsequent experiments [ 51 ]. Figure 6. UV–Vis spectrum of ( a ) AgNP-L and ( b ) AgNP-S at different concentrations of AgNO 3 .
Finally, the effect of temperature on the synthesis of AgNPs was also studied. Different temperatures tested, i.e. 25°C, 40°C and 60°C with 15% (v/v) of C. nutans extracts and 5 mM of AgNO 3 . The reaction mixture was incubated for 24 h.
The peak for 60°C is narrower compared to the peaks at 25°C and 40°C. According to Khan et al . [ 29 ], the absorbance peak is broad at a high wavelength when the particle size of nanoparticles increases. A narrow peak at short wavelength indicates small particle size. From these analyses,
Particles size and zeta potential measurement
The DLS measures the average size of nanoparticles in liquid suspension, which requires low volume of sample. This measurement applies the Brownian motion theory for particle size.
Scanning electron microscopy–energy dispersive X-ray analysis
The surface morphology and elemental compositions of the synthesized AgNPs were determined by scanning electron microscopy–energy dispersive X-ray (SEM-EDX). Based on the SEM images, the AgNP-L and AgNP-S had rough surfaces and were spherical in shape.
X-ray diffraction analysis
XRD was used to determine the crystalline phase of the synthesized AgNPs. The XRD spectra of AgNPs obtained ranged from 20° to 80°. The peaks at 2 θ values for AgNP-L were 38.42° (111), 46.43° (200), 65.31° (220) and 76.89° (311). Figure 10 shows the peaks for AgNP-S at 38.32° (111), 46.35° (200), 64.56° (220) and 76.85° (311). The unassigned peaks with (*) symbol were recorded at 27.94°, 32.31°, 54.97° and 67.74° (AgNP-L); 27.94°, 32.33° and 54.89° (AgNP-S). Figure 10. XRD spectra of AgNP-L and AgNP-S.
The observed peaks were compared with the silver database from the Joint Committee on Powder Diffraction Standard (JCPDS) or International Centre for Diffraction Data (ICDD).
The unassigned peaks with (*) symbol recorded at 27.94°, 32.31° and 54.97° for AgNP-L, whereas 27.94°, 32.33° and 54.89° for AgNP-S, were similar to the reported unassigned peaks in studies that used cow's milk, palm oil mill effluent (POME), Annona squamosa peel and Murraya koenigii extracts [ 48 , 63 – 65 ]. In addition, the peaks recorded at 32.31° and 54.97° were similar to those reported as silver oxide [ 66 ]. The peaks with (*) are related to organic compound in plant extract [ 67 , 68 ].
Fourier transform infrared spectrophotometer spectroscopy analysis
FTIR was conducted to determine the possible functional groups responsible for the reduction of Ag + . The comparison was made between C. nutans extract and synthesized AgNPs at wavenumbers from 4000 to 500 cm −1 . The FTIR spectra in figure 11 show the peaks at 3415.97, 1623.42, 1408.57 and 1093.41 cm −1 in CNL and 3415.08, 1616.26, 1410.77 and 1043.45 cm −1 in CNS. AgNP-L exhibited peaks at 3414.10, 1622.25 and 1382.47 cm −1 while 3416.83, 1624.68 and 1383.51 cm −1 for AgNP-S. Figure 11. FTIR spectra of ( a ) CNL, ( b ) AgNP-L, ( c ) CNS and (d) AgNP-S.
The peaks at 3415.97 and 3415.08 cm −1 for CNL and CNS are represented by the -OH or -NH group [ 55 ]. Based on phytochemical analysis, both CNS and CNL extracts contain phenolic compounds that might be involved in the reduction and formation of AgNPs [ 69 ]. The -OH group in the C. nutans extract may be responsible for the oxidation of alcohol to aldehyde during the reduction of Ag + to Ag 0 [ 70 ]. The peaks at 1623.42 cm −1 (CNL) and 1616.26 cm −1 (CNS) shifted to 1622.25 cm −1 (AgNP-L) and 1624.68 cm −1 (AgNP-S) because of the carbonyl stretch in amide [ 71 ]. These peaks are related to the binding of AgNPs to amide groups that enhance the stability of AgNPs [ 72 , 73 ]. Ajitha et al . [ 74 ] also suggested that the proteins of plant extracts may act as surfactant to stabilize AgNPs. The peaks at 1408.57 cm −1 (CNL) and 1410.77 cm −1 (CNS) for both plant extracts shifted to 1382.47 cm −1 (AgNP-L) and 1383.51 cm −1 (AgNP-S). This corresponds to the C-O-H bending (in-plane) of carboxylic acids vibrations [ 75 ]. The peaks of both C. nutans extracts at 1093.41 and 1043.45 cm −1 show the major reductions on the AgNPs FTIR spectra. The peaks also indicate the presence of C-N of aliphatic amines [ 76 ].
C. nutans aqueous extract has been reported to contain various bioactive compounds, including phenolics, flavonoids, stigmasterol, β-sitosterol and chlorophyll derivatives [ 77 ]. These compounds were hypothesized to be responsible for the formation of AgNPs. C. nutans has been reported to contain high percentage of phenol and flavonoids. High-performance liquid chromatography analysis conducted by Sarega et al . [ 78 ] on the aqueous leaf extract of C. nutans showed the presence of four major compounds, namely, protocatechuic acid, chlorogenic acid, caffeic acid and ferulic acid.
Conclusion
The biogenic synthesis of AgNPs from C. nutans leaf and stem aqueous extracts was performed.
Supplementary Material