Rapid biological synthesis of silver nanoparticles using plant leaf extracts

Original Paper

DOI: 10.1007/s00449-008-0224-6

Cite this article as:
Song, J.Y. & Kim, B.S. Bioprocess Biosyst Eng (2009) 32: 79. doi:10.1007/s00449-008-0224-6


Five plant leaf extracts (Pine, Persimmon, Ginkgo, Magnolia and Platanus) were used and compared for their extracellular synthesis of metallic silver nanoparticles. Stable silver nanoparticles were formed by treating aqueous solution of AgNO3 with the plant leaf extracts as reducing agent of Ag+ to Ag0. UV-visible spectroscopy was used to monitor the quantitative formation of silver nanoparticles. Magnolia leaf broth was the best reducing agent in terms of synthesis rate and conversion to silver nanoparticles. Only 11 min was required for more than 90% conversion at the reaction temperature of 95 °C using Magnolia leaf broth. The synthesized silver nanoparticles were characterized with inductively coupled plasma spectrometry (ICP), energy dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and particle analyzer. The average particle size ranged from 15 to 500 nm. The particle size could be controlled by changing the reaction temperature, leaf broth concentration and AgNO3 concentration. This environmentally friendly method of biological silver nanoparticles production provides rates of synthesis faster or comparable to those of chemical methods and can potentially be used in various human contacting areas such as cosmetics, foods and medical applications.


Biological synthesisNanoparticlesSilverPlant extractsSize control


Nanoparticles usually referred as particles with a size up to 100 nm [1, 2]. Nanoparticles exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology, if compared with larger particles of the bulk material they are made of. Nanoparticles present a higher surface to volume ratio with decreasing size of nanoparticles. Specific surface area is relevant for catalytic reactivity and other related properties such as antimicrobial activity in silver nanoparticles. As specific surface area of nanoparticles is increased, their biological effectiveness can increase due to the increase in surface energy [1].

Silver has long been recognized as having an inhibitory effect toward many bacterial strains and microorganisms commonly present in medical and industrial processes [3]. The most widely used and known applications of silver and silver nanoparticles are in the medical industry. These include topical ointments and creams containing silver to prevent infection of burns and open wounds [4]. Another widely used applications are medical devices and implants prepared with silver-impregnated polymers [5]. In addition, silver-containing consumer products such as colloidal silver gel and silver-embedded fabrics are now used in sporting equipment.

Production of nanoparticles can be achieved through different methods. Chemical approaches are the most popular methods for the production of nanoparticles. However, some chemical methods cannot avoid the use of toxic chemicals in the synthesis protocol. Since noble metal nanoparticles such as gold, silver and platinum nanoparticles are widely applied to human contacting areas, there is a growing need to develop environmentally friendly processes of nanoparticles synthesis that do not use toxic chemicals. Biological methods of nanoparticles synthesis using microorganism [68], enzyme [9], and plant or plant extract [10] have been suggested as possible ecofriendly alternatives to chemical and physical methods. Using plant for nanoparticles synthesis can be advantageous over other biological processes by eliminating the elaborate process of maintaining cell cultures [10]. It can also be suitably scaled up for large-scale synthesis of nanoparticles.

Shankar et al. [10] reported on the synthesis of pure metallic nanoparticles of silver and gold by the reduction of Ag+ and Au3+ ions using Neem (Azadirachta indica) leaf broth. However, little has been carried out about engineering approaches such as rapid nanoparticles synthesis using plant extracts and size control of the synthesized nanoparticles. The times required for more than 90% reduction of Ag+ and Au3+ ions using Neem leaf broth were about 4 and 2 h, respectively. If biological synthesis of nanoparticles can compete with chemical methods, there is a need to achieve faster synthesis rates. In this study, we screened several plant leaf extracts and compared their synthesis of silver nanoparticles by monitoring the conversion using UV-visible spectroscopy. We also investigated the effects of reaction conditions such as reaction temperature, leaf broth concentration and AgNO3 concentration on synthesis rate and particle size of the silver nanoparticles.

Materials and methods

Five plant leaves were collected and dried for 2 days at room temperature. They were Pine (Pinus desiflora), Persimmon (Diopyros kaki), Ginkgo (Ginko biloba), Magnolia (Magnolia kobus) and Platanus (Platanus orientalis). The plant leaf broth solution was prepared by taking 5 g of thoroughly washed and finely cut leaves in a 300 mL Erlenmeyer flask with 100 mL of sterile distilled water and then boiling the mixture for 5 min before finally decanting it. They were stored at 4 °C and used within a week.

Typically, 10 mL of leaf broth was added to 190 mL of 1 mM aqueous AgNO3 solution for reduction of Ag+ ions. The effects of temperature on synthesis rate and particle size of the prepared silver nanoparticles were studied by carrying out the reaction in water bath at 25–95 °C with reflux. The concentrations of AgNO3 solution and leaf broth were also varied at 0.1–2 mM and 5–50% by volume, respectively. The silver nanoparticle solution thus obtained was purified by repeated centrifugation at 15,000 rpm for 20 min followed by redispersion of the pellet in deionized water. UV-vis spectra were recorded as a function of reaction time on a UV-1650CP Shimadzu spectrophotometer operated at resolution of 1 nm. After freeze drying of the purified silver particles, the structure and composition were analyzed by scanning electron microscopy (SEM, Hitachi S-2500C), field emission transmission electron microscopy (FE-TEM, Tecnai F30 S-Twin, FEI), energy dispersive X-ray spectroscopy (EDS, Sigma), and X-ray photoelectron spectroscopy (XPS, ESCALAB 210). Silver concentrations and conversions were determined using inductively coupled plasma spectrometry (ICP, JY38Plus). Average particle size and distribution were measured using particle analyzer (NICOMP™ 380 ZLS).

Results and discussion

Synthesis and characterization of silver nanoparticles

It is well known that silver nanoparticles exhibit yellowish-brown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles [10]. Reduction of the silver ion to silver nanoparticles during exposure to the plant leaf extracts could be followed by color change and thus UV-vis spectroscopy. Figure 1a–c show the UV-vis spectra recorded from the reaction medium as a function of reaction time using Persimmon, Magnolia and Pine leaf broth, respectively. It is observed that the maximum absorbance occurs at ca. 430 nm and steadily increases in intensity as a function of reaction time. The final absorption intensities at 430 nm were more than 1.0 a.u. and increased up to 1.5 a.u. using Magnolia leaf broth which was higher than about 0.5 a.u. reported with Neem leaf broth by Shankar et al. [10], suggesting that the conversion to silver nanoparticles may be higher using the plant leaf extracts in this study. Since the peak wavelength did not shift during the reaction, we could quantitatively monitor the concentrations of silver nanoparticles and thus conversion by measuring the absorbance at 430 nm. The linear relationship was obtained between the silver concentration determined by ICP and the absorbance at 430 nm.
Fig. 1

UV-vis spectra recorded as a function of reaction time of 1 mM AgNO3 solution with plant leaf broth. aDiopyros kaki. bMagnolia kobus. cPinus densiflora. Insets respective plots of absorbance at 430 nm as a function of time

Figure 2 shows the time courses of silver nanoparticles production with different reaction temperatures obtained with Persimmon leaf broth. As the reaction temperature increased, both synthesis rate and final conversion to silver nanoparticles increased. The final conversion at 25 °C was 60% and reached almost 100% at more than 55 °C. Besides Persimmon leaf broth, we investigated the effect of reaction temperature with Magnolia leaf broth and obtained similar results. Rai et al. [11] also mentioned the increase of reduction rate with increasing the reaction temperature for the gold nanotriangles synthesized using lemongrass extract. The average particle sizes with temperature are shown in the inset of Fig. 2. The particle size decreased from 50 nm at 25 °C to 16 nm at 95 °C. Regarding the reason of decrease in particle size with temperature, we can hypothesize as follows. As the reaction temperature increases, the reaction rate increases and thus most silver ions are consumed in the formation of nuclei, stopping the secondary reduction process on the surface of the preformed nuclei. Similar trend was observed with gold nanotriangles synthesized using lemongrass extract [11].
Fig. 2

Time courses of silver nanoparticles formation obtained with 1 mM AgNO3 and 5% Diopyros kaki leaf broth with different reaction temperature. Inset effect of reaction temperature on average silver particle size

Figure 3a and b are SEM and TEM images, respectively, obtained with 5% Persimmon leaf broth and 1 mM AgNO3 solution at 55 °C. It is shown that relatively spherical nanoparticles are formed with average diameter of 32 nm with some deviations. The silver nanoparticles showed Gaussian distributions (the inset of Fig. 3a). The coefficient of variance of the silver nanoparticles, defined as the ratio of standard deviation to average diameter, was 0.672. Other values of the coefficient of variance were in the range of 0.52–0.68. The inset of Fig. 3b shows the selected area electron diffraction (SAED) pattern recorded from the silver nanoparticles. The ring-like diffraction pattern indicates that the particles are crystalline. The diffraction rings could be indexed on the basis of the fcc structure of silver. Four rings arise due to reflections from (111), (200), (220), and (311) lattice planes of fcc silver, respectively. Similar SAED pattern was obtained with silver nanoparticles synthesized using Aloe vera extract by Chandran et al. [12]. EDS and XPS spectra recorded from the silver nanoparticles are shown in Fig. 3c and d, respectively. EDS profile shows strong silver signal along with a weak oxygen and carbon peak, which may originate from the biomolecules that are bound to the surface of the silver nanoparticles. Together with TEM images, Shankar et al. [10] reported that nanoparticles synthesized using plant extracts are surrounded by a thin layer of some capping organic material from plant leaf broth. Our TEM image in Fig. 3b also shows that the silver particles synthesized using plant extracts are surrounded by a thin layer of some capping material and were stable in solution during 4 weeks after their synthesis possibly due to the capping material on the surface of nanoparticles. XPS spectrum shows characteristic silver peaks on the surface of nanoparticles, suggesting that silver nanoparticles are successfully synthesized using plant leaf broth in this study.
Fig. 3

Characterization of silver nanoparticles formed with 1 mM AgNO3 and 5% Diopyros kaki leaf broth at 55 °C. a SEM image. Inset particle size distribution. b TEM image. Inset electron diffraction pattern recorded from the particles shown in b with lattice planes of fcc silver. c Spot profile EDS spectrum. d XPS spectrum

In order to screen plant with high production capability of silver nanoparticles, we compared several plant extracts for their synthesis rate of silver nanoparticles. As shown in Fig. 4, the synthesis rate was highest with Magnolia leaf broth. Only 11 min was required for more than 90% conversion at 95 °C. Although rapid synthesis of silver nanoparticles within 5 min was recently reported using culture supernatants of Enterobacteria [13], the silver nanoparticles synthesized were unstable after 5 min. Using plant extracts for nanoparticles synthesis is another advantage over using bacteria because the nanoparticles are stable for a long time.
Fig. 4

Time courses of silver nanoparticles formation obtained with 1 mM AgNO3 and 5% various plant leaf extracts at 95 °C

Control of reaction rate and particle size

We further investigated the possibility of controlling the reaction rate and particle size by changing the composition of the reaction mixture. Figure 5 shows the time courses of silver nanoparticles formation with different Magnolia leaf broth concentrations at 5–50% and 1 mM AgNO3. The reaction rate was highest at 20% leaf broth concentration, but similar reaction rates were obtained with more than 10% leaf broth concentrations. The inset in Fig. 5 shows that the average particle size increases with increasing the leaf broth concentration. Sub-micro scale particles between 100 and 800 nm were obtained with high concentrations of leaf broth more than 10%, suggesting that too many reducing agents cause aggregation of the silver particles synthesized possibly due to the interactions between capping molecules bound to the surface of particles and secondary reduction process on the surface of the preformed nuclei.
Fig. 5

Time courses of silver nanoparticles formation obtained with 1 mM AgNO3 and various concentrations of Magnolia kobus leaf broth at 95 °C. Inset effect of leaf broth concentration on silver particle size

Figure 6 shows the effect of AgNO3 concentration on conversion and particle size with 5% Magnolia leaf broth. The times required for more than 90% conversion were less than 11 min with 0.1 and 1 mM AgNO3 concentration. With 2 mM AgNO3, about 90 min was required for 90% conversion, but the final conversion reached almost 100%. The average particle size decreased with increasing the AgNO3 concentration. The reason of decrease in particle size with AgNO3 concentration is not clear at this point. It is considered that particle size and shape are dependent on various conditions such as plant type, nanoparticle type, reaction temperature and composition.
Fig. 6

Time courses of silver nanoparticles formation obtained with 5% Magnolia kobus leaf broth and various concentrations of AgNO3 at 95 °C. Inset effect of AgNO3 concentration on silver particle size

Currently, the mechanism of biological nanoparticles synthesis is not fully understood. For gold nanoparticles synthesized extracellularly by the fungus Fusarium oxysporum, it was reported that the reduction occurs due to NADH-dependent reductase released into the solution [14]. With Neem leaf broth, it was reported that terpenoids are believed to be the surface active molecules stabilizing the nanoparticles and reaction of the metal ions is possibly facilitated by reducing sugars and/or terpenoids present in the Neem leaf broth [10]. Recent results with Capsicum annuum L. extract indicated that the proteins which have amine groups played a reducing and controlling role during the formation of silver nanoparticles in the solutions, and that the secondary structure of the proteins changed after reaction with silver ions [15]. More elaborate studies are required to elucidate the mechanism of biological nanoparticles synthesis. In conclusion, an environmentally friendly method using plant extracts was proposed to synthesize silver nanoparticles. Only 11 min was required for over 90% conversion by using Magnolia leaf broth and increasing the reaction temperature to 95 °C, which was faster or comparable to the synthesis rate of chemical methods. The average particle size could be controlled from 15 to 500 nm by changing the reaction temperature, leaf broth concentration and AgNO3 concentration.

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Department of Chemical EngineeringChungbuk National UniversityCheongjuRepublic of Korea