Green synthesis of silver nanomaterials using Ganoderma Lucidum extract as reducing agent and stabilizer with ultrasonic assistance and application as an antibacterial agent

. Synthesis of silver nanoparticles (AgNPs) using plant extracts extracted from Ganoderma lucidum in the buffer zone of Bach Ma National Park, Vietnam is a simple, convenient, economical, and environmentally friendly method. This study describes the biosynthesis of silver nanoparticles in both cases with and without ultrasonic assistance using Ganoderma lucidum extract as a reducing and protective agent. Transmission electron microscopy, scanning electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the as-synthesized AgNPs. Compared to the heating reflux method, the proposed ultrasonic wave assisted heating reflux method produced AgNPs had higher efficiency, smaller and more uniform particle size 6.08  1.80 nm of nm in a short synthesis time period. The antibacterial and antifungal properties of ultrasonically synthesized silver nanomaterials (US-AgNPs) were also investigated. US-AgNPs are important nanomaterials that can find many good applications in practice.


Introduction
Today, along with the development of nanotechnology, nano-metallic materials (NPs) have been receiving special attention from domestic and foreign scientists because of their superior properties, such as optical properties, electrical properties, magnetic properties, mechanical properties, and catalytic properties [1].
These properties have opened up many new applications and research opportunities.Precious metal nanomaterials such as Au, Ag, and Pt with particles from 1 to 100 nanometers in size have many unique chemical and optical properties due to surface plasmon resonance (SPR) [2][3][4].In particular, silver nanomaterials (AgNPs) have attracted attention because of their potential applications in many different fields, namely biomedical science [5], wastewater treatment [6], agriculture [7], antimicrobial agents and biosensor [8,9], etc.The mechanisms of antibacterial activity of silver nanoparticles (AgNPs) are explained by the fact that AgNPs with small size and large surface area can adhere to the surface of microbial cell membranes, which causes the osmotic and respiratory malfunctions of cells [10].AgNPs not only interact with the surface of the membranes but can also penetrate into the bacteria [11].When Ag + ions interact with the membrane of pathogenic bacteria, they will react with the sulfhydryl group (-SH) of the oxygen-transport enzyme molecules and disable this enzyme, causing the inhibition of cellular respiration, which thereby inactivating the bacteria [12].These interactions block DNA replication, thereby killing the bacteria [13].
Currently, silver nanomaterials are synthesized by various physical and chemical methods, such as photochemical reduction [14], chemical reduction [15], using gamma radiation [16], electrochemical [17], hydrothermal [18], microwave [19], ultrasonic waves [20].However, these methods require modern and expensive equipment, not to mention their harmful effects on the environment and human health.Therefore, the synthetic method, which is called the Green Synthetic Method, uses extracts from plants, bacteria, and fungi [21,22] as both a reducing agent and surface stabilizer without adding other chemicals.
Ganoderma Lucidum (G.lucidum) is a rare and valuable herb used in traditional medicine.G. lucidum contains active ingredients, the most important of which are polysaccharides and triterpenoids [23] with immunomodulatory, freeradicals scavenging, anti-allergic, antiinflammatory, antiviral, anti-cancer, anti-radiation damage to DNA abilities [24,25].
Many studies have shown that the speed of silver nanomaterial synthesis can be increased using assisted techniques such as microwaves, gamma waves, or ultrasonic waves.In particular, the ultrasonic waves method draws much attention since it requires simple machines and synthesis processes but achieves high efficiency.
The ultrasonic wave method is a method that uses ultrasonic waves with wavelengths from 10 cm to 10 -3 cm.This wavelength does not generate enough energy to interact directly with chemical bonds.
The ultrasonic wave technique was developed based on the acoustic cavitation phenomenon, which produces air bubbles that form, grow, and collapse in a liquid solution.This collapse generates heat and creates high pressure at the breaking point with extremely high temperature (about 5000), extremely high pressure (about 20 Mpa), and extremely high-temperature rise/fall rate (about 10 10 K.s -1 ) [26].This process acts as a mediator to receive energy and support the synthesis of silver nanomaterials.
In the scope of this study, silver nanomaterials (AgNPs) were synthesized by the green method using extracts from Lingzhi mushroom (Ganoderma Lucidum), which acts as both a reducing agent and surface stabilizer.
Moreover, the advancement of ultrasonic wave assisted synthesis compared to the heating reflux method for prepared AgNPs was successfully demonstrated; furthermore the optical and morphological properties, crystal phase and size distribution of AgNPs were examnined.Then, the synthesized AgNPs material was used to evaluate the antibacterial and antifungal activity.

Materials
The fruiting bodies of Ganoderma Lucidum were harvested in the buffer zone of Bach Ma National Park, Nam Dong district, Thua Thien Hue province.They were then dried and ground into powder.

Preparation of aqueous extract from fruiting bodies of Ganoderma Lucidum
Aqueous extraction of Ganoderma lucidum was prepared by the previously reported procedure

Biosynthesis of silver nanoparticles
AgNPs were synthesized as per two processes using the heating reflux method (CH-AgNPs) and ultrasonic-assisted heating reflux method (US-AgNPs).

Synthesis of silver nanomaterials by heating reflux method
CH-AgNPs were synthesized with optimal synthesis conditions based on published research [27].The synthesis process is summarized as follows: The dried extract of G. Lucidum extract was re-dissolved in water to obtain a G. Lucidum extract with a concentration of 1 g/mL.90 mL of G.
Lucidum extract was mixed with 10 mL of 1 mM AgNO3 solution.The mixture was adjusted to pH = 9 with NH3 solution (25%).The reduction time was 6 hours, and the reaction temperature was 85°C.The result was the colloidal silver solution, which was then precipitated with ethanol, and centrifuged.The solid fraction was collected and dried at 90°C for 24 hours to obtain CH-AgNPs.

Synthesis of silver nanomaterials by ultrasonicassisted heating reflux method
US-AgNPs were synthesized with an Ultrasound frequency: 42,000 Hz, Heating power: 100 W, Ultrasonic Power: 120 W and were presented as follows: 90 mL of Ganoderma Lucidum extract with 1 g/mL concentration was mixed with 10 mL of 1 mM AgNO3 solution, and the mixture was adjusted to pH = 9 with NH3 solution (25%).The mixture was placed in the reaction system, as shown in

Material Characterization Techniques
UV-Vis spectra of AgNPs/GL colloidal solutions were measured on a UV-Vis spectrometer Jasco-V630, Japan.X-ray diffraction was performed on a D8 ADVANCE system (Bruker, Germany).

Dilution of the test sample
The initial sample was diluted in 2 steps in 100% DMSO and sterilized distilled water into a range of

Characterization of the obtained materials
In this study, the influence of ultrasonic waves on the synthesis of silver nanomaterials was studied by comparing some characteristics of silver nanomaterials synthesized by two methods: the heating reflux method (CH-AgNPs) and ultrasonic wave method (US-AgNPs).
Before the reaction, the mixture, including the G. luciudm aqueous extract and silver nitrate was yellow and after the reaction, the color of the mixture became dark brown color.This alternation of color could be contributed to the confirmation of the formation silver nanoparticles.Moreover, the UV-Vis spectrometer was utilized to study the optical properties of both samples: CH-AgNPs and US-AgNPs.From that, it can be predicted that the synthesis of silver nanomaterials by the ultrasonic method has higher efficiency, and the obtained nanoparticles have a more small and uniform size [28].
TEM images reveal the morphology and size distribution of CH-AgNPs and US-AgNPs (Fig. 3).
The results show that the morphology of both samples is spherical nanoparticles (Fig. 3a, 3c).
Through TEM images and the ImageJ software, In which:  is the width at half peak height;  is the diffraction angle (in radians);  is the wavelength of the X-ray (0.154 nm); K is the Scherrer constant (0.9).XRD diagrams were recorded on a D8-Advanced Bruker instrument (Germany) with Cu anode, recording interval 2θ = 30 -90°, step angle 0.01°.The results showed that the average crystal size of the US-AgNPs sample (3.17 nm) was smaller than that of the CH-AgNPs sample (18.65).The results of the average crystal size obtained from XRD spectra had the same pattern as the average particles size gained from TEM images of CH-AgNPs and US-AgNPs.This observation could be explained by comparing the heating mechanism between two methods: the heating reflux method and the ultrasonic-assisted heating reflux method.
The difference in the size of the two samples US-AgNPs and CH-AgNPs is explained based on the effect of ultrasonic waves with their unique properties.Samples of CH-AgNPs were synthesized in a liquid medium and dispersed by mechanical agitation.This solution only increases the stretching of the C-N group [31].The spectra of US-AgNPs are similar to the spectra of G. lucidum, leading to the that there were certain functional groups on the surface of silver nanoparticles.
The elemental composition of US-AgNPs was examined by using the EDX technique; furthermore, the EDX elemental mapping was observed in Fig. 7.The component elements, including C, O, and Ag accounted for 9.59%: 5.71%: 84.70%, respectively, by mass (Table 1).The   From the above results, it can be seen that the effect of ultrasonic waves makes the reaction time shorter, the synthesis efficiency high, and the material particle and crystal size smaller.The samples of silver nanoparticles synthesized with the assistance of ultrasonic waves are further used to conduct test on the antibacterial and antifungal properties.

Antibacterial and anti-fungal ability
The inhibitory effect on tested microorganisms is shown in Table 2.The results showed that silver

Conclusion
In this study, we presented a process to synthesize silver nanomaterials by the "green method" by both the heating reflux method and the ultrasonic wave assisted heating reflux method using extracts from Ganoderma Lucidum as a reducing and protective agent.The results show that the synthesis of silver nanomaterials by ultrasonicassisted heating reflux method has higher efficiency, a shorter reaction time, and a smaller and more uniform size of obtained nanoparticles compared to the heating reflux method.
The synthesized silver nanoparticles have effective antibacterial and antifungal properties, opening up many potential applications in medicine and many other fields.

[
35].Briefly, 2.0 g of Ganoderma Lucidum powder was added to 200 mL of distilled water.The mixture was refluxed at 85 o C for 4 h under stirring conditions.Then, the mixture was centrifuged at 4300 rpm for 20 min to remove the underlying residue and collect the yellowish extract.The yellowish extract was concentrated to 50 mL and then precipitated with ethanol 96% (v/v) at a volume ratio of extract: ethanol of 1: 4. The mixture was kept in the refrigerator for 24 hours, and then centrifuged at 4300 rpm for 20 minutes.The precipitate was washed with acetone and dried at 75 °C for 6 h.The dried aqueous extraction was stored for further experiments.

Figure 1 .Fig. 1 .
Figure 1.The reaction was carried out for 1.5 h and the temperature was kept at 60 °C.The resulting colloidal silver solution was then precipitated with ethanol and centrifuged.The solid fraction was collected and dried at 75 °C for 24 h to obtain US-AgNPs.
in the test were 256 µg/mL with the extract and 128 µg/mL with the clean substance.Test the activities and process the resultsTesting microorganisms were kept at -80 0 C. Before the experiment, the testing microorganisms were activated in a culture medium so that the concentration of bacteria reached 5x105 CFU/ml, and fungal concentration reached 1x10 3 CFU/ml.Then, 10 µl of sample solution at different concentrations was placed into a 96-well PCR plate, and 190 µl of the above activated bacterial and fungal solution was added and incubated at 37 o C/16 -24 hours.The MIC (Minimum Inhibitor Concentration) value was determined at the well with the lowest reagent concentration that completely inhibited the growth of microorganisms.The IC50 (50% Inhibitor Concentration) value was determined through the percentage value of inhibiting microorganisms from growing and the computer software Raw Data.% =  (+) −    (+) −  (−) .%   =   − ( % − ).(  − ow  )  % − ow % (In which, HighConc/LowConc: reagent at high concentration/low reagent at low concentration; HighInh%/LowInh%: percentage inhibition at high concentration/ percentage inhibition at low concentration and OD: optical density).

Figure 2
Figure 2 presents the results of UV-Vis spectra of colloidal silver solution synthesized by two methods: the heating reflux method and the ultrasonic-assisted heating reflux method.From the UV-Vis results, it can be seen that both the two materials samples CH-AgNPs and US-AgNPs have absorption peaks at 425 nm.However, the US-AgNPs sample has higher peak intensity and sharper peak shape than the CH-AgNPs sample.

Origin 8 . 5 . 1 ,
the particle size distributions corresponding CH-AgNPs and US-AgNPs were built (Fig. 3b, 3d).The results of particle size calculation showed that the average particle sizes of CH-AgNPs and US-AgNPs samples were 10.72  2.95 nm and 6.08  1.80 nm, respectively.This shows that samples synthesized with the assistance of ultrasonic waves have smaller particle sizes and more uniform distribution than samples synthesized by the reflux method.XRD patterns are used to demonstrate the crystal structure of both samples CH-AgNPs and US-AgNPs (Fig. 4).It can be seen that both material samples have four sharp diffraction peaks and completely coincide with the standard spectrum of silver (JCPDS: 03-065-2871) at positions 37.96, 44.12, 64.26 and 77.25 for corresponding sides (111), (200), (220) and (311).On the other hand, the crystal size (D) is calculated according to the Sherer equation:

Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .
Fig. 3. UV-Vis TEM images and the diagrams of the particle size distribution of samples CH-AgNPs (a, b) and US-AgNPs (c, d) distribution of elements C, O, and Ag in the AgNPs material sample is presented in Figure 7. b, c, and d.It can be seen that elemental silver is evenly distributed throughout the US-AgNPs sample.

Fig. 7 .
Fig. 7. EDX spectrum and SEM-Mapping images of the elemental distribution map of US-AgNPs material samples nanomaterials have inhibitory ability against seven strains of bacteria and fungi, namely Gram (+): S. aureus, B. subtilis, L. fermentum; Gram (-): S. enterica, E. coli, P.aeruginosa and C.albicans fungus at concentrations from 1 µg/mL to 256 µg/mL.In general, samples of US-AgNPs have high antibacterial ability against Gram (+) and Gram (-) strains with IC50 value < 5.0 (µg/mL) (except B.subtilis which is 34.7 µg/mL) and the MIC ranged from 4 to 16 (µg/mL).However, the resistance to C.albican fungus is not good with IC50 and MIC values of 145.84 and >256 µg/mL, respectively.In general, the ability to inhibit all strains of Gram-positive and Gram-negative bacteria of silver nanomaterials is very strong with IC50 values < 5.0 (µg/mL) (except B.subtilis, which is 34.7 µg/ mL) and MICs in the range of 4-16 (µg/mL).This proves that silver nanomaterials have potential in their application as valuable biosafety antibiotics.

Table 1 .
The elemental composition of MW-AuNPs was analyzed using EDX

Table 2 .
Inhibitory effect on seven strains of microorganisms and fungi