Optimization of biogenic synthesis of silver nanoparticles from flavonoid-rich Clinacanthus nutans leaf and stem aqueous extracts — TEI-tagged view

Siti Nur Aishah Mat YusufpersName, Che Nurul Azieyan Che MoodpersName, Nor Hazwani AhmadpersName, Doblin SandaipersName, Chee Keong LeepersName, Vuanghao LimpersName (2020date). “Optimization of biogenic synthesis of silver nanoparticles from flavonoid-rich Clinacanthus nutans leaf and stem aqueous extracts.” Royal Society Open Sciencetitle·j.

Object of study — biogenic synthesisterm·key of silver nanoparticlesterm·dom with Clinacanthus nutansterm·key leaf and stem aqueous extracts

Research question — which phytochemicals the extracts carry, and how to synthesise and characterise AgNPsterm·dom with them

Contribution — optimised extract, AgNO3, temperatureterm·dom and time conditions for plant-mediated AgNPterm·dom synthesis

Full text — tagged

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Optimization of biogenic synthesisterm·key of silver nanoparticlesterm·dom from flavonoid-rich Clinacanthus nutansterm·key leaf and stem aqueous extracts Abstract Background : Silver nanoparticlesterm·dom (AgNPsterm·dom) are widely used in food industries, biomedical, dentistry, catalysis, diagnostic biological probes and sensors. The use of plant extractterm·dom for AgNPsterm·dom synthesis eliminates the process of maintaining cell culture and the process could be scaled up under a non-aseptic environment. The purpose of this study is to determine the classes of phytochemicals, to biosynthesize and characterize the AgNPsterm·dom using Clinacanthus nutansterm·key leaf and stem extracts. In this study, AgNPsterm·dom were synthesized from the aqueous extracts of C. nutans leaves and stems through a non-toxic, cost-effective and eco-friendly method. Results : The formation of AgNPsterm·dom was confirmed by UV-Vis spectroscopy, and the size of AgNP-L (leaf) and AgNP-S (stem) were 114.7 and 129.9 nm, respectively. Transmission electron microscopy (TEMterm·mth) analysis showed spherical nanoparticlesterm·dom with AgNP-L and AgNP-S ranging from 10 to 300 nm and 10 to 180 nm, with average of 101.18 and 75.38 nm, respectively. The zeta potentialsterm·mth of AgNP-L and AgNP-S were recorded at −42.8 and −43.9 mV. X-ray diffraction analysis matched the face-centred cubic structure of silver and was capped with bioactive compounds. Fourier transform infrared spectrophotometer analysis revealed the presence of few functional groups of phenolic and flavonoidterm·key compounds. These functional groups act as reducing agents in AgNPsterm·dom synthesis. Conclusion : These results showed that the biogenically synthesized nanoparticlesterm·dom reduced silver ions to silver nanoparticlesterm·dom in aqueous condition and the AgNPsterm·dom formed were stable and less toxic.

Introduction The field of nanotechnology has undergone substantial developments and has attracted considerable interest from researchers owing to its wide range of application. Generally, nanoparticlesterm·dom include 1–100 nm particles [ 1 ]. The physical and chemical properties of nanoparticlesterm·dom are different from those of bulk materials. Nanoparticlesterm·dom have distinct properties because of their high surface-area-to-volume ratios and small sizes [ 2 ]. The biological activities and surface energy of nanoparticlesterm·dom increase with their surface areas [ 3 ]. Nanoparticlesterm·dom such as silver nanoparticlesterm·dom (AgNPsterm·dom) are widely used in food industries, biomedicine, dentistry, catalysis, diagnostic biological probes and sensing [ 4 , 5 ].

AgNPsterm·dom 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 AgNPsterm·dom 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 AgNPsterm·dom [ 10 ]. Nevertheless, the use of plant extractsterm·dom has become a major interest in AgNPsterm·dom synthesis. In this method, plant extractsterm·dom act as reducing and capping agents because of their various bioactive compounds [ 11 , 12 ]. AgNPsterm·dom synthesis using plant extractsterm·dom is a preferable option compared to other methods because it eliminates the process of maintaining cell culture and can be scaled up under a non-aseptic environment [ 13 ].

The plant extractsterm·dom that induce AgNPterm·dom formation contain reducing agents [ 14 ], such as the metabolites of phenolic compoundsterm·key, flavones, alkaloids and sterols [ 15 – 17 ]. Compared with microorganismsterm·dom, plant extractsterm·dom are inexpensive, easily available and suitable for industrial use because they do not require purification or the use of cultures [ 18 ]. Meanwhile, microbes require aseptic conditions and high maintenance. The time required for the formation of AgNPsterm·dom is faster for plant extractsterm·dom compared to microorganismsterm·dom.

Clinacanthus nutansterm·key ( 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, flavonoidsterm·key, diterpenes and phytosterols were present in the methanol extracts of the leaves [ 24 ]. The aim of this study was to biogenically synthesize the AgNPsterm·dom from C. nutans leaves and stem extracts and to optimize the parameters used in this biogenic synthesisterm·key. Figure 1. C. nutans leaves and stems.

Materials and methods Plant collection The dried leaves and stems of C. nutans were purchased from Botani Sdn. Bhd, Manjung, Perak. Clinacanthus nutansterm·key 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 temperatureterm·dom 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 temperatureterm·dom 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 flavonoidsterm·key. 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 experimentsterm·mth. 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 temperatureterm·dom. 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 flavonoidterm·key content Total flavonoidterm·key 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 temperatureterm·dom. Next, 15 µl of AlCl 3 (10% w/v) solution was added into the mixture and left at room temperatureterm·dom 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 nanoparticlesterm·dom The synthesis of AgNPsterm·dom 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 AgNPsterm·dom 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 AgNPsterm·dom such as extract and AgNO 3 concentration, incubation time and temperatureterm·dom were studied. Table 1 shows the parameter conditions used for optimization study. Table 1. Parameter variables used to optimize the synthesis of AgNPsterm·dom. parameter variable extract concentration 5, 10 and 15% v/v AgNO 3 concentration 1, 3, 5 mM temperatureterm·dom 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 nanoparticlesterm·dom The formation of AgNPsterm·dom 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 sizeterm·dom and zeta potentialterm·mth analysis, the dried powder of AgNPsterm·dom was suspended in deionized water at 1 : 10 ratio. The measurements were conducted at room temperatureterm·dom with Zetasizer Nano ZS (Malvern, Worcestershire, UK) [ 12 ]. For the surface morphology study, the dried AgNPsterm·dom 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 (TEMterm·mth; Libra 120; Carl Zeiss, Oberkochen, Germany) was used to identify the shapes, sizes and morphologies of the synthesized AgNPsterm·dom. For TEMterm·mth analysis, the AgNPsterm·dom 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 AgNPsterm·dom. 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 temperatureterm·dom. The percentage yield of the leaf and stem extracts are 25.31% and 13.12%, respectively. Maceration method was selected because plants contain thermolabile compounds and are suitable for large scale. Thermolabile compounds degrade or decompose when heated for a long time. In this study, distilled water was chosen as the extraction solvent, which is easily available and able to produce high percentage yield (polar solvent) [ 30 ].

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, flavonoidsterm·key, 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 + + flavonoidsterm·key Alkaline reagent + + reducing sugar Fehling + + Benedict + + glycosides Keller–Kilani + + + indicates present/positive reaction, − indicates absent/negative reaction.

Plant extractsterm·dom are known to contain large number of phytonutrients. Based on the previous study, saponin, phenolics, flavonoidsterm·key, 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 nanoparticlesterm·dom. 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 nanoparticleterm·dom synthesis. Therefore, plant extractsterm·dom 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 compoundterm·key, and the intensity was measured by microplate reader. The contents of the phenolic compoundsterm·key 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 , the TPC of CNL was higher (75.52 ± 0.905 µg GAE/mg dry extract) than CNS (37.08 ± 0.477 µg GAE/mg dry extract). TFC was obtained through the aluminium chloride (AlCl 3 ) method. TFC in the samples were calculated by using the calibration curve of quercetin ( y = 0.0003x + 0.0318; R 2 = 0.995) and was expressed as µg of quercetin equivalents per mg of dry extract (µg QE/mg dry extract). All the values were expressed as average ± s.d. Based on the result ( figure 2 ), the TFC of CNL was higher (27.97 ± 1.273 µg QE/mg dry extract) than that of CNS (12.42 ± 1.443 µg QE/mg dry extract). Figure 2. ( a ) TFC and ( b ) TPC of CNL and CNS (mean ± s.d., n = 3).

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 compoundsterm·key donate hydrogen in nature, hence the presence of phenolic compoundsterm·key in the plant extractterm·dom 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 compoundsterm·key, flavonoidsterm·key are commonly present in plants and these compounds are beneficial to human health. Previous study reported that the antioxidant activity of flavonoidsterm·key is due to their hydroxyl groups. Besides, the phytochemical compounds from plant sources can act as reducing and stabilizing agent in AgNPsterm·dom synthesis [ 32 ]. This includes the hydroxyl and carbonyl groups from flavonoidsterm·key [ 39 ]. Therefore, the phytochemicals that were previously identified in C. nutans extracts may be responsible for the biosynthesis of AgNPsterm·dom, where they act as reducing agent in AgNPsterm·dom synthesis.

Biosynthesis of silver nanoparticlesterm·dom The reduction of silver ion (Ag + ) to AgNPsterm·dom 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 AgNPsterm·dom due to the conversion of silver ions into AgNPsterm·dom [ 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 AgNPsterm·dom 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 AgNPsterm·dom 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 nanoparticlesterm·dom [ 42 ]. In this regard, C. nutans extracts act as reducing agents which are cheap and easily available without any capping agents. The synthesis of AgNPsterm·dom 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 AgNPsterm·dom could be derived from flavonoidsterm·key and phenolic compoundsterm·key (reducing agent). These compounds act as electron or hydrogen donors [ 44 ]. The ketoform on the backbone of a flavonoidterm·key 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 AgNPsterm·dom.

UV-Vis spectroscopy analysis UV-Vis spectroscopy is an important technique to observe the formation of AgNPsterm·dom by monitoring the optical properties and electronic structure of the synthesized nanoparticlesterm·dom. The electron cloud of a nanoparticleterm·dom can oscillate on the surface of the nanoparticleterm·dom, which absorbs electromagnetic waves at a particular frequency. This phenomenon is called surface plasmon resonanceterm·dom (SPRterm·dom) and recorded as electromagnetic wavelengths by UV-Vis spectrophotometer [ 46 ]. Figure 4 shows the optimization of incubation time during AgNPsterm·dom synthesis. Based on the spectra, UV wavelengths of AgNP-L (AgNPsterm·dom from leaf extract) and AgNP-S (AgNPsterm·dom from stem extract) were taken from 15 min until 24 h of incubation. The peaks intensified as the incubation time increases. The increase in intensity of the wavelength absorbance is due to the increasing number of nanoparticlesterm·dom formed from the reduction of silver ions and biomolecules in the aqueous plant extractterm·dom solution [ 47 , 48 ]. From the result, 24 h was selected as the best incubation time for AgNPsterm·dom synthesis. Besides that, figure 4 shows the wavelength absorbance of both leaves and stem extracts. The higher absorbance intensity from leaves extract showed a higher flavonoidterm·key and its derivatives content compared to stem extract [ 49 ]. This correlates with the results from TPC which showed leaves extract has 27.97 ± 1.273 µg QE/mg of dry extract compared to stem extract at 12.42 ± 1.443 µg QE/mg of dry extract. Figure 4. UV-Vis spectra of ( a ) AgNP-L and ( b ) AgNP-S as a function of time. Reaction of 5% C. nutans extract in 1 mM AgNO 3 at 25°C.

The parameters of extract concentrations, AgNO 3 concentrations and temperaturesterm·dom were conducted to optimize the AgNPsterm·dom 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 AgNPsterm·dom and homogeneous distribution [ 50 ]. Moreover, SPRterm·dom 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 AgNPsterm·dom [ 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 AgNPsterm·dom being synthesized. Therefore, based on the absorption spectrum, 5 mM AgNO 3 showed the highest formation of AgNPsterm·dom for both extracts. Thus, this concentration was used for subsequent experimentsterm·mth [ 51 ]. Figure 6. UV–Vis spectrum of ( a ) AgNP-L and ( b ) AgNP-S at different concentrations of AgNO 3 .

Finally, the effect of temperatureterm·dom on the synthesis of AgNPsterm·dom was also studied. Different temperaturesterm·dom 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 absorbances of AgNP-L and AgNP-S increased with temperatureterm·dom, with the highest absorbance obtained at 60°C ( figure 7 ). Previous study on the synthesis of AgNPsterm·dom using Pulicaria glutinosa extract showed that AgNPsterm·dom production increased proportionally with temperatureterm·dom [ 29 ]. The maximum absorption peaks for both synthesized AgNP-L and AgNP-S were between 421–443 and 423–443 nm, respectively. The temperatureterm·dom of 60°C was selected for both AgNP-L and AgNP-S as the amount of AgNPsterm·dom synthesized is highest with average mean diameter of the nanoparticlesterm·dom. Figure 7. UV–Vis spectra of ( a ) AgNP-L and ( b ) AgNP-S at different temperaturesterm·dom.

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 sizeterm·dom of nanoparticlesterm·dom increases. A narrow peak at short wavelength indicates small particle sizeterm·dom. From these analyses, it was concluded that the optimum conditions for the synthesis of AgNPsterm·dom were 15% v/v of plant extractterm·dom in 5 mM of AgNO 3 solution at the temperatureterm·dom of 60°C and 24 h incubation time.

The SPRterm·dom bands became sharper and shifted to shorter wavelengths when the temperatureterm·dom increases, indicating decrease in particle sizeterm·dom. The reason for the decrease is the increase in reduction rate during the synthesis process. Silver ions are consumed during the process, thus blocking any secondary reduction process on the surface of the AgNPsterm·dom [ 24 ]. The shape of the synthesized particles can be predicted theoretically, with spherical shape of particles appearing at the wavelengths within the range of 400–490 nm, pentagon (500–590 nm) and triangular (600–700 nm) [ 50 , 52 ]. From the UV-Vis spectra, the morphology of the synthesized AgNPsterm·dom were mostly spherical as the maximum absorption occurred at 421 nm (AgNP-L) or 431 nm (AgNP-S).

Particles size and zeta potentialterm·mth measurement The size of the synthesized AgNPsterm·dom was determined through dynamic light scatteringterm·mth (DLSterm·mth) and transmission electron microscopy (TEMterm·mth). In DLSterm·mth measurement, the average size distribution was recorded at 114.7 ± 1.012 nm for AgNP-L and 129.9 ± 1.400 nm for AgNP-S. The polydispersity indices (PDIs) for AgNP-L and AgNP-S were 0.206 ± 0.007 and 0.183 ± 0.004, respectively ( table 3 ). Table 3. Particle sizeterm·dom and zeta potentialterm·mth analysis. samples polydispersity indexterm·mth (PDI) particle sizeterm·dom (nm) zeta potentialterm·mth (mV) AgNP-L 0.206 ± 0.007 114.7 ± 1.012 −42.8 AgNP-S 0.183 ± 0.004 129.9 ± 1.400 −43.6 The TEMterm·mth images showed that AgNP-L was spherical in shape and had a size distribution ranging from 10 to 300 nm (average = 101.18 nm; figure 8 ). AgNP-S was also spherical in shape and had a size distribution range of 10–180 nm (average = 75.38 nm). Figure 8. TEMterm·mth images of ( a ) AgNP-L, ( b ) AgNP-S with magnifications of 63 000 × and size distribution of ( c ) AgNP-L and ( d ) AgNP-S.

The DLSterm·mth measures the average size of nanoparticlesterm·dom in liquid suspension, which requires low volume of sample. This measurement applies the Brownian motionterm·dom theory for particle sizeterm·dom. Brownian motionterm·dom is a random movementterm·dom of particles in a gas or suspension. The average size of the nanoparticlesterm·dom was determined by measuring the dynamic fluctuation from the light scattering intensity and velocity movement of the particles in suspension [ 53 ]. The calculated PDI values of the samples were 0.206 ± 0.007 (AgNP-L) and 0.183 ± 0.004 (AgNP-S), which are within the range of 0–1, where 0 refers to monodisperse and 1 is polydisperse [ 53 ]. This result clearly shows that the synthesized AgNPsterm·dom were in a monodisperse phase, and the particle aggregation is minimal. The size, morphology and stability of the synthesized AgNPsterm·dom are influenced by the experimental conditions, such as reducing and stabilizing agents in synthesis process [ 6 ]. Saxena et al . [ 54 ] and Shameli et al . [ 55 ] suggested that phenolic and other compounds present in the plant extractterm·dom caused the agglomeration of the nanoparticlesterm·dom. However, the average size of synthesized AgNPsterm·dom in DLSterm·mth measurements are larger than those in TEMterm·mth analysis because DLSterm·mth measures the nanoparticlesterm·dom in clusters [ 56 ].

Zeta potentialterm·mth calculates the net charges of a moving particle under the electric field in solution [ 57 ]. Negative zeta potentialterm·mth was observed in AgNP-L and AgNP-S ( table 3 ). The negative value represents the net charges around a particle and not the actual surface charge. The negative charge may be due to the adsorption of bioactive compounds, such as polyphenolic compounds onto the AgNPsterm·dom surface [ 58 ]. Temperatureterm·dom plays an important factor in the stability of the nanoparticlesterm·dom and increases with zeta potentialterm·mth. The stability of the AgNPsterm·dom can be determined by the zeta potentialterm·mth values. High zeta potentialterm·mth causes strong repulsive forceterm·dom among the particles, thus prevent them from aggregating [ 4 ].

Scanning electron microscopy–energy dispersive X-ray analysis The surface morphology and elemental compositions of the synthesized AgNPsterm·dom 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. The EDX spectra showed intense peak at 3 keV, suggesting that Ag is the main element in AgNPsterm·dom ( figure 9 ). The same results were reported by Li et al . [ 59 ], where peak at 3 keV showed weak carbon, oxygen and chlorine. This is due to the binding of biomolecules from the plant extractterm·dom onto the surface of AgNPsterm·dom [ 29 , 60 ]. Figure 9. SEM images of ( a ) AgNP-L, ( b ) AgNP-S with magnifications of 100 000 × and EDX spectra of ( c ) AgNP-L, ( d ) AgNP-S.

X-ray diffraction analysis XRD was used to determine the crystalline phase of the synthesized AgNPsterm·dom. The XRD spectra of AgNPsterm·dom 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). Four peaks were indexed as 111, 200, 220 and 311 on the synthesized AgNPsterm·dom and they matched the face-centred cubic (FCC) structure of silver (JCPDS file no. 04-0783) [ 61 ]. The positions of the peaks were slightly shifted because of the presence of strain in the crystal structure as part of the characteristic of nanocrystalline [ 62 ].

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 extractterm·dom [ 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 AgNPsterm·dom 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 compoundsterm·key that might be involved in the reduction and formation of AgNPsterm·dom [ 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 AgNPsterm·dom to amide groups that enhance the stability of AgNPsterm·dom [ 72 , 73 ]. Ajitha et al . [ 74 ] also suggested that the proteins of plant extractsterm·dom may act as surfactant to stabilize AgNPsterm·dom. The peaks at 1408.57 cm −1 (CNL) and 1410.77 cm −1 (CNS) for both plant extractsterm·dom 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 AgNPsterm·dom 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, flavonoidsterm·key, stigmasterol, β-sitosterol and chlorophyll derivatives [ 77 ]. These compounds were hypothesized to be responsible for the formation of AgNPsterm·dom. C. nutans has been reported to contain high percentage of phenol and flavonoidsterm·key. 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. Although there is no clear evidence on which specific bioactive compound is responsible for the formation AgNPsterm·dom, it is highly accepted that these phytochemical compounds help in the formation of AgNPsterm·dom. Phenolics and flavonoidsterm·key in the plant extractterm·dom may be responsible for the bioreduction of Ag + to AgNPsterm·dom.

Conclusion The biogenic synthesisterm·key of AgNPsterm·dom from C. nutans leaf and stem aqueous extracts was performed. The process was found to be economical, non-toxic and environmentally friendly. The synthesized AgNP-L and AgNP-S were spherical in shape and had average sizes of 114.7 and 129.9 nm, respectively. Tannins, saponins, alkaloids, amino acid, reducing sugar, flavonoidsterm·key and glycosides were detected from the extracts. The optimized parameters for C. nutans leaf and stem aqueous extract-mediated synthesized silver nanoparticlesterm·dom were obtained as follows: 15% (v/v) of extract, 15 mM of AgNO 3 , 60°C and 24 h incubation time. These parameters affect the shape of the nanoparticlesterm·dom, polydispersity indexterm·mth and particle sizeterm·dom distribution. The FTIR analysis showed the presence of functional groups such as -OH, -NH, -C=O and -COOH, which suggests the conjugation of phenolics and flavonoidsterm·key with silver ions in the plant extractsterm·dom.

Supplementary Material