# Mycosynthesis of Zinc Oxide Nanoparticles Coated with Silver usingGanoderma lucidum (Curtis) P. Karst and Its Evaluation of In Vitro Antidiabetic and Anticancer Potential

Authors: D. S. Ranjith Santhosh Kumar; N. Elango; Gayathri Devi Selvaraju; Paul A. Matthew; Senthilkumar Palanisamy; Hayoung Cho; Fatimah S. Al Khattaf; Ashraf Atef Hatamleh; Amit Dutta Roy
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Nanotechnology is an evolving interdisciplinary field of research interspersing material science and nanobiotechnology. Nanoparticles are studied extensively for their specific catalytic, magnetic, electronic, optical, antimicrobial, theranostic, diagnosis, wound healing, and anti-inflammatory properties. ZnO nanoparticles (NPs) have many applications owing to their unique characteristics, which include low cost, nontoxicity, abundance in nature, and the ability to prepare compounds with varying morphologies having different properties. The main aim of the study is to biosynthesis of ZnO nanoparticles coated with silver from the aqueous extract ofGanoderma lucidum (Curtis) P. Karst and to evaluate its antidiabetic potential by performing alpha-glucosidase inhibition and alpha-amylase inhibition assays and to evaluate the anticancer potential by cytotoxicity (MTT) assay against human breast cancer MDA-MB 231 cell lines. The biosynthesis of ZnO nanoparticles coated with Ag was characterized by UV–vis spectroscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray analysis, scanning electron microscope, and transmission electron microscopy. An increasing concentration in the biosynthesized ZnO nanoparticles coated with Ag produces strong antidiabetic activity through enzyme inhibition effect and anticancer activity through the reduction of cell viability. The present study recommended that the “Biological” method of biological nanoparticle production is a promising approach that allows synthesis in aqueous conditions, with low energy requirements and low costs. In the future, the mycosynthesized nanoparticles might be used in the medical arena to treat and prevent diseases.


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## Body

## 1. Introduction

In the sphere of research, nanotechnology has become increasingly essential. Nanotechnology is a field of science and technology that works with tiny molecules and is commonly employed for therapeutic purposes in underdeveloped nations. Nanoparticles are defined as particles with a diameter of fewer than 100 nm [1].ZnO, also known as zincite, is a versatile material with a wide range of applications in technology, including electromagnetic shielding, LEDs, and other light-emitting devices [2]. Many research investigations have focused on ZnO NPs, and they have a wide range of industrial uses, including textiles, cosmetics, water treatment, and UV emitting devices; ZnO NPs play a vital role in therapeutic as well as pharmaceutical areas because of having fewer side effects. ZnO NPs have a huge surface area, which allows them to have an effective target action on cancer cells [3].The coating is a process in which the metals such as gold, silver, chromium, etc., are doped with the main synthesized nanoparticles to stabilize the particles and also avoid agglomeration. The coating can be used to expand the advantages of any metal nanoparticles [4]. The three most common ways of creating nanoparticles are physical, chemical, and biological. Among all the methods of nanoparticle synthesis, we chose to proceed with the green synthesis because of its rapid, cost-effective, eco-friendly procedures, and avoiding the production of undesirable or harmful byproducts [5].Ganoderma lucidum (Curtis) P. Karst is a medicinal mushroom belonging to the family of Ganodermataceae. G. lucidum is also known as Lingzhi in China, and it has been dubbed the “mushroom of immortality.” In Asian countries, it was traditionally used to enhance the immune power and treat various diseases [6]. There is a huge number of bioactive components including triterpenes, polysaccharides, sterols, and peptides found in the G. lucidum. Those identified compounds have numerous health benefits and are also used to treat the diseases, such as cancer, diabetes, asthma, arthritis, etc. [7, 8].There are no studies on the mycosynthesis of ZnO nanoparticles coated with Ag fromG. lucidum that we are aware of. Therefore, we aimed to synthesize the silver-coated ZnO nanoparticles and evaluate their antidiabetic and anticancer potential through some respected assays [9].


## 2. Materials and Methods

### 2.1. Sample Collection

G. lucidum was gathered from the Maruthamalai foothills (11°2′46″ N, 76°51′7″ E) in the Western Ghats, Coimbatore, Tamil Nadu, India. The Mycology Division of the Indian Forest Genetics and Tree Breeding Institute, Coimbatore, validated G. lucidum, and the voucher specimen (RT-25406/9-1-2016) was kept in our laboratory for future reference.


### 2.2. Preparation of Aqueous Extract

Deionized water was used to completely clean the mushroom sample, removing dirt and other contaminants. It was then air-dried for 5 days in the shade at room temperature. The dried mushroom sample was crushed into a fine powder after being cut into small pieces. In an Erlenmeyer flask, 3 g of mushroom powder was mixed with 300 ml deionized water and heated in a mantle for 2 hr at 80°C, then cooled and filtered using Whatman no. 1 filter paper. As a reducing agent, the filtrate aqueous extract is employed [10].


### 2.3. Synthesis of Silver-Coated Zinc Oxide Nanoparticles

To make silver-coated zinc oxide nanoparticles (Ag–ZnO NPs), 450 ml ofG. lucidum aqueous extract was mixed with 45 ml of 1 mM zinc nitrate solution in a 1,000 ml Erlenmeyer flask. The precipitate was separated from the reaction solution by centrifugation at 10,000 rpm for 15 min and the pellet was collected; the centrifugation process was then done twice more and the remaining pellet was collected. Pellets were dried in a hot air oven until all liquid ingredients were evaporated, then kept in airtight bottles for future research [11].


### 2.4. Silver Coating

Five milliliters of 10 mM silver nitrate was added drop wisely with the mixture of aqueous extract ofG. lucidum and 1 mM zinc nitrate solution under constant stirring. The mixture was stirred for 2–3 hr with the use of a magnetic stirrer. Then the mixture of the aqueous solution was placed in the open shaker to improve the synthesis of Ag–ZnO NPs [12, 13].


### 2.5. Characterization of Biosynthesized Ag-Coated ZnO NPs

The obtained Ag–ZnO NPs fromG. lucidum were characterized by using UV–vis spectrometry, FTIR analysis, EDX, SEM, and TEM analysis.UV/VIS 3000+ double beam UV visible ratio–recording scanning spectrophotometer from Lab India (SKU : 174-0020) with dimensions of (WDH)/weight = 540440390 mm/36 kg was used to study the optical absorption spectra of Ag–ZnO NPs. FTIR is a qualitative analysis and it shows the presence of different functional groups, which give rise to the well-known signatures in the IR region of the electromagnetic spectrum. The presence of Ag-coated ZnO nanoparticles was confirmed by an energy-dispersive analysis X-ray (EDX) spectrum using an X-ray microanalyzer (Oxford Instruments, UK). The structural characterization of the ZnO nanoparticles coated with Ag was carried out by SEM (JEOL JEM 2100) and TEM (JEOL JEM 2100). The sample was prepared by air-drying drops of diluted solutions of the preparations on carbon films supported by copper grids.


### 2.6. Antidiabetic Activity of Silver-Coated Zinc Nanoparticles

#### 2.6.1. In Vitro Alpha-Glucosidase Inhibition Assay

Matsui et al. [14] used an adapted approach to investigate the effect of ZnO NPs coated with Ag on alpha-glucosidase inhibition. The stock solution of alpha-glucosidase (0.5 U/ml) was also prepared in 20 mmol/l sodium phosphate buffer (pH 6.9) in 96 well plates. Alpha-glucosidase has been prepared by varying concentrations of Ag–ZnO NPs and acarbose standard drug (1–5 μg/ml), respectively. The mixture was incubated at 37°C for 15 min, and 100 µl of 4-nitrophenyl-β-D-glucopyranoside (PNPG) was added further. The reaction mixture was incubated for 10 min at 37°C. To stop the reaction, 750 ml of Na2CO3 (0.1 M) was added, and the OD values of spectrophotometer (UV-100 Cyber Lab, USA) readings were taken at 405 nm in triplicate. Acarbose alone was used as reference and controls for assay contain only 4-nitrophenyl-β-D-glucopyranoside (PNPG). The percentage of alpha-glucosidase inhibition was measured as follows:(1)%Activityofsample=A405ofsample−A405ofcontrolsA405ofcontrol×100,To prevent the action of the alpha-glucosidase by 50% (IC50), the inhibitory concentration of the gold nanoparticles was calculated graphically [10, 14].


#### 2.6.2. In Vitro Alpha-Amylase Enzyme Inhibition Assay

The approach was used to determine the activity of alpha-amylase (Hansawasdi et al. [15]). In each tube, 2 mg of starch azure was suspended in 0.2 ml of 0.5 M Tris–HCl buffer (pH 6.9) and 0.01 M CaCl2. The tubes holding the substrate solution were boiled for 5 min before being incubated for 5 min at 37°C. Ag–ZnO NPs were taken in each tube containing different concentrations of dimethyl sulfoxide (10, 20, 40, 60, 80, and 100 g/ml) [15].PPA from the porcine pancreas was dissolved in Tris–HCl buffer to a concentration of 2 units/ml, and 0.1 ml of this enzyme solution was added to each of the tubes stated above. The absorbance of the resulting supernatant was measured at 595 nm using a spectrophotometer (UV–vis spectrophotometer UV-2450, Shimadzu). The alpha-amylase inhibitory activity was calculated as follows:(2)Ac+–Ac−–As−AbAc+–Ac−×100,where Ac+, Ac−, As, and Ab are defined as the absorbance of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme activity), a test sample (with enzyme), and a blank (a test sample without enzyme), respectively.


### 2.7. Anticancer Activity of Silver-Coated Zinc Nanoparticles

#### 2.7.1. MTT Assay

The cytotoxic effects of Ag–ZnO NPs against MDA-MB 231 cell lines were determined using the MTT test. The cell lines were seeded in 96-well microtiter plates, which were then filled with the appropriate amounts of Ag–ZnO NPs stock solutions and cultured at 37°C for 48 hr. As a control, cells that had not been treated were utilized. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, tetrazole) colorimetric assay was used to test the cultivated cell. MTT (5 mg/ml, 20l) was added to each group of cells, and the plates were incubated for another 4 hr. The medium was then withdrawn, and DMSO (200L, Sigma–Aldrich, USA) was added to dissolve the formazan crystals formed by metabolically active cells reducing the tetrazolium salt [16].MTT is reduced into metabolically active cells to yield an insoluble purple formazan product. The cell suspensions were dispensed (100μl) in triplicate into 96-well culture plates at optimized concentrations of 1 × 105 cells/well for each cell line, after a 24 hr recovery period. Assay plates were read using a spectrophotometer at 560 nm. The absorbance of the samples was measured using a microplate (ELISA) reader [17].Trypan blue assay was performed to quantify the dead cells following the treatment of cytotoxic stimuli using trypan blue dye.


## 2.1. Sample Collection

G. lucidum was gathered from the Maruthamalai foothills (11°2′46″ N, 76°51′7″ E) in the Western Ghats, Coimbatore, Tamil Nadu, India. The Mycology Division of the Indian Forest Genetics and Tree Breeding Institute, Coimbatore, validated G. lucidum, and the voucher specimen (RT-25406/9-1-2016) was kept in our laboratory for future reference.


## 2.2. Preparation of Aqueous Extract

Deionized water was used to completely clean the mushroom sample, removing dirt and other contaminants. It was then air-dried for 5 days in the shade at room temperature. The dried mushroom sample was crushed into a fine powder after being cut into small pieces. In an Erlenmeyer flask, 3 g of mushroom powder was mixed with 300 ml deionized water and heated in a mantle for 2 hr at 80°C, then cooled and filtered using Whatman no. 1 filter paper. As a reducing agent, the filtrate aqueous extract is employed [10].


## 2.3. Synthesis of Silver-Coated Zinc Oxide Nanoparticles

To make silver-coated zinc oxide nanoparticles (Ag–ZnO NPs), 450 ml ofG. lucidum aqueous extract was mixed with 45 ml of 1 mM zinc nitrate solution in a 1,000 ml Erlenmeyer flask. The precipitate was separated from the reaction solution by centrifugation at 10,000 rpm for 15 min and the pellet was collected; the centrifugation process was then done twice more and the remaining pellet was collected. Pellets were dried in a hot air oven until all liquid ingredients were evaporated, then kept in airtight bottles for future research [11].


## 2.4. Silver Coating

Five milliliters of 10 mM silver nitrate was added drop wisely with the mixture of aqueous extract ofG. lucidum and 1 mM zinc nitrate solution under constant stirring. The mixture was stirred for 2–3 hr with the use of a magnetic stirrer. Then the mixture of the aqueous solution was placed in the open shaker to improve the synthesis of Ag–ZnO NPs [12, 13].


## 2.5. Characterization of Biosynthesized Ag-Coated ZnO NPs

The obtained Ag–ZnO NPs fromG. lucidum were characterized by using UV–vis spectrometry, FTIR analysis, EDX, SEM, and TEM analysis.UV/VIS 3000+ double beam UV visible ratio–recording scanning spectrophotometer from Lab India (SKU : 174-0020) with dimensions of (WDH)/weight = 540440390 mm/36 kg was used to study the optical absorption spectra of Ag–ZnO NPs. FTIR is a qualitative analysis and it shows the presence of different functional groups, which give rise to the well-known signatures in the IR region of the electromagnetic spectrum. The presence of Ag-coated ZnO nanoparticles was confirmed by an energy-dispersive analysis X-ray (EDX) spectrum using an X-ray microanalyzer (Oxford Instruments, UK). The structural characterization of the ZnO nanoparticles coated with Ag was carried out by SEM (JEOL JEM 2100) and TEM (JEOL JEM 2100). The sample was prepared by air-drying drops of diluted solutions of the preparations on carbon films supported by copper grids.


## 2.6. Antidiabetic Activity of Silver-Coated Zinc Nanoparticles

### 2.6.1. In Vitro Alpha-Glucosidase Inhibition Assay

Matsui et al. [14] used an adapted approach to investigate the effect of ZnO NPs coated with Ag on alpha-glucosidase inhibition. The stock solution of alpha-glucosidase (0.5 U/ml) was also prepared in 20 mmol/l sodium phosphate buffer (pH 6.9) in 96 well plates. Alpha-glucosidase has been prepared by varying concentrations of Ag–ZnO NPs and acarbose standard drug (1–5 μg/ml), respectively. The mixture was incubated at 37°C for 15 min, and 100 µl of 4-nitrophenyl-β-D-glucopyranoside (PNPG) was added further. The reaction mixture was incubated for 10 min at 37°C. To stop the reaction, 750 ml of Na2CO3 (0.1 M) was added, and the OD values of spectrophotometer (UV-100 Cyber Lab, USA) readings were taken at 405 nm in triplicate. Acarbose alone was used as reference and controls for assay contain only 4-nitrophenyl-β-D-glucopyranoside (PNPG). The percentage of alpha-glucosidase inhibition was measured as follows:(1)%Activityofsample=A405ofsample−A405ofcontrolsA405ofcontrol×100,To prevent the action of the alpha-glucosidase by 50% (IC50), the inhibitory concentration of the gold nanoparticles was calculated graphically [10, 14].


### 2.6.2. In Vitro Alpha-Amylase Enzyme Inhibition Assay

The approach was used to determine the activity of alpha-amylase (Hansawasdi et al. [15]). In each tube, 2 mg of starch azure was suspended in 0.2 ml of 0.5 M Tris–HCl buffer (pH 6.9) and 0.01 M CaCl2. The tubes holding the substrate solution were boiled for 5 min before being incubated for 5 min at 37°C. Ag–ZnO NPs were taken in each tube containing different concentrations of dimethyl sulfoxide (10, 20, 40, 60, 80, and 100 g/ml) [15].PPA from the porcine pancreas was dissolved in Tris–HCl buffer to a concentration of 2 units/ml, and 0.1 ml of this enzyme solution was added to each of the tubes stated above. The absorbance of the resulting supernatant was measured at 595 nm using a spectrophotometer (UV–vis spectrophotometer UV-2450, Shimadzu). The alpha-amylase inhibitory activity was calculated as follows:(2)Ac+–Ac−–As−AbAc+–Ac−×100,where Ac+, Ac−, As, and Ab are defined as the absorbance of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme activity), a test sample (with enzyme), and a blank (a test sample without enzyme), respectively.


## 2.6.1. In Vitro Alpha-Glucosidase Inhibition Assay

Matsui et al. [14] used an adapted approach to investigate the effect of ZnO NPs coated with Ag on alpha-glucosidase inhibition. The stock solution of alpha-glucosidase (0.5 U/ml) was also prepared in 20 mmol/l sodium phosphate buffer (pH 6.9) in 96 well plates. Alpha-glucosidase has been prepared by varying concentrations of Ag–ZnO NPs and acarbose standard drug (1–5 μg/ml), respectively. The mixture was incubated at 37°C for 15 min, and 100 µl of 4-nitrophenyl-β-D-glucopyranoside (PNPG) was added further. The reaction mixture was incubated for 10 min at 37°C. To stop the reaction, 750 ml of Na2CO3 (0.1 M) was added, and the OD values of spectrophotometer (UV-100 Cyber Lab, USA) readings were taken at 405 nm in triplicate. Acarbose alone was used as reference and controls for assay contain only 4-nitrophenyl-β-D-glucopyranoside (PNPG). The percentage of alpha-glucosidase inhibition was measured as follows:(1)%Activityofsample=A405ofsample−A405ofcontrolsA405ofcontrol×100,To prevent the action of the alpha-glucosidase by 50% (IC50), the inhibitory concentration of the gold nanoparticles was calculated graphically [10, 14].


## 2.6.2. In Vitro Alpha-Amylase Enzyme Inhibition Assay

The approach was used to determine the activity of alpha-amylase (Hansawasdi et al. [15]). In each tube, 2 mg of starch azure was suspended in 0.2 ml of 0.5 M Tris–HCl buffer (pH 6.9) and 0.01 M CaCl2. The tubes holding the substrate solution were boiled for 5 min before being incubated for 5 min at 37°C. Ag–ZnO NPs were taken in each tube containing different concentrations of dimethyl sulfoxide (10, 20, 40, 60, 80, and 100 g/ml) [15].PPA from the porcine pancreas was dissolved in Tris–HCl buffer to a concentration of 2 units/ml, and 0.1 ml of this enzyme solution was added to each of the tubes stated above. The absorbance of the resulting supernatant was measured at 595 nm using a spectrophotometer (UV–vis spectrophotometer UV-2450, Shimadzu). The alpha-amylase inhibitory activity was calculated as follows:(2)Ac+–Ac−–As−AbAc+–Ac−×100,where Ac+, Ac−, As, and Ab are defined as the absorbance of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme activity), a test sample (with enzyme), and a blank (a test sample without enzyme), respectively.


## 2.7. Anticancer Activity of Silver-Coated Zinc Nanoparticles

### 2.7.1. MTT Assay

The cytotoxic effects of Ag–ZnO NPs against MDA-MB 231 cell lines were determined using the MTT test. The cell lines were seeded in 96-well microtiter plates, which were then filled with the appropriate amounts of Ag–ZnO NPs stock solutions and cultured at 37°C for 48 hr. As a control, cells that had not been treated were utilized. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, tetrazole) colorimetric assay was used to test the cultivated cell. MTT (5 mg/ml, 20l) was added to each group of cells, and the plates were incubated for another 4 hr. The medium was then withdrawn, and DMSO (200L, Sigma–Aldrich, USA) was added to dissolve the formazan crystals formed by metabolically active cells reducing the tetrazolium salt [16].MTT is reduced into metabolically active cells to yield an insoluble purple formazan product. The cell suspensions were dispensed (100μl) in triplicate into 96-well culture plates at optimized concentrations of 1 × 105 cells/well for each cell line, after a 24 hr recovery period. Assay plates were read using a spectrophotometer at 560 nm. The absorbance of the samples was measured using a microplate (ELISA) reader [17].Trypan blue assay was performed to quantify the dead cells following the treatment of cytotoxic stimuli using trypan blue dye.


## 2.7.1. MTT Assay

The cytotoxic effects of Ag–ZnO NPs against MDA-MB 231 cell lines were determined using the MTT test. The cell lines were seeded in 96-well microtiter plates, which were then filled with the appropriate amounts of Ag–ZnO NPs stock solutions and cultured at 37°C for 48 hr. As a control, cells that had not been treated were utilized. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, tetrazole) colorimetric assay was used to test the cultivated cell. MTT (5 mg/ml, 20l) was added to each group of cells, and the plates were incubated for another 4 hr. The medium was then withdrawn, and DMSO (200L, Sigma–Aldrich, USA) was added to dissolve the formazan crystals formed by metabolically active cells reducing the tetrazolium salt [16].MTT is reduced into metabolically active cells to yield an insoluble purple formazan product. The cell suspensions were dispensed (100μl) in triplicate into 96-well culture plates at optimized concentrations of 1 × 105 cells/well for each cell line, after a 24 hr recovery period. Assay plates were read using a spectrophotometer at 560 nm. The absorbance of the samples was measured using a microplate (ELISA) reader [17].Trypan blue assay was performed to quantify the dead cells following the treatment of cytotoxic stimuli using trypan blue dye.


## 3. Results and Discussion

### 3.1. Visual Observation

Analysis of ZnO nanoparticles was made by coating Ag particles, the aqueous solution ofG. lucidum was then mixed with the solution of zinc nitrate. Later, the silver nitrate slowly started to change its nature which resulted in the color change. This color transition served as evidence for the synthesis completion [18]. The final brownish color change is shown in Figure 1, which exhibited the reduction of zinc nitrate to zinc oxide and it occurs due to the excitation of the surface plasmon resonance effect [11]. The observation was alike to the green synthesis of silver nanoparticles from an aqueous extract of brown seaweed of Padina boergesenii, which was completed by Chikkanna et al. [19].Figure 1
Mycosynthesis of Ag-coated ZnO NPs fromG. lucidum: (a) initial and final color of Ag-coated ZnO NPs; (b) Ag coated with the mixture of G. lucidum extract and zinc nitrate solution.
(a)(b)


### 3.2. UV–Visible Spectral Analysis

UV–vis spectroscopy was used to confirm the formation of synthesized nanoparticles in the initial stage. The dark brownish sample of ZnO NPs was coated with Ag nanoparticles, which were synthesized from the extract ofG. lucidum. The synthesized ZnO NPs were scanned by using UV–vis spectroscopy under the range between 300 and 600 nm [20]. The result obtained from UV–vis spectral analysis showed (Figure 2) a high-absorption peak at 370 nm, which corresponds to synthesized ZnO NPs. Also, this intense response of ZnO NPs could be due to the surface plasmon resonance effect. The synthesized product is confirmed to claim ZnO NPs coated with Ag because no other peaks were observed in the UV spectrum region [21]. A similar observation was also reported earlier by Siva Vijayakumar et al. [22] in the green synthesis of zinc oxide nanoparticles from the extracts of leaf, stem, and in vitro grown callus of Mussaenda frondosa L.: characterization and their applications.Figure 2
UV–vis absorption spectra of Ag-coated ZnO NPs synthesized fromG. lucidum.


### 3.3. Fourier-Transformed Infrared (FTIR) Spectral Analysis

The FTIR absorption spectra of the water-soluble extract before and after reduction of Ag-coated ZnO NPs showed the capping ligand of the silver-coated zinc oxide nanoparticles which may be due to the linkage of an alkenes, alkyl sulfides groups, carboxyl groups, primary amides, and nitro groups as described in Table1 and Figure 3. FTIR analysis of the synthesized Ag–ZnO NPs exposes strong bands at 1,991, 1,644, 1,515, 1,392, and 958 cm−1. We confirmed that the strong peak was focused at 1,515 cm−1, which corresponds to the carboxyl groups from amino acid residues, and that proteins had the highest ability to bind metals, based on the FTIR results. This intercalation might explain why proteins create metal oxide nanoparticles (i.e., capping of silver-coated zinc nanoparticles) to avoid agglomeration and maintain the medium. It has already been reported in the green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract, that the same functional groups of phytochemicals induce the nanoparticles were synthesized using amines, alkanes, and carboxyl ions that are widely seen in secondary metabolites such as terpenoids, flavonoids, and alkaloids.Figure 3
FTIR spectrum of Ag-coated ZnO NPs synthesized fromG. lucidum: (a) control water-soluble extract (G. lucidum); (b) Ag-coated ZnO NPs with water-soluble extract of G. lucidum.
(a)(b)Table 1
FTIR analysis of Ag-coated ZnO NPs.
Sr. No.Wavenumber (cm−1)StretchFunctional groups11,991NH2Primary amides21,644N=ONitrogen group31,515CO2Carbonyl group41,392C=CAlkenes5958C–SAlkyl sulfides


### 3.4. Scanning Electron Microscope Analysis

The silver-coated zinc nanoparticles synthesized with the aid ofG. lucidum extract were scanned using SEM in order to determine the morphology, polydispersed shape, and size of the synthesized nanoparticle. SEM images (Figure 4) showed that the particles formed were spherical, hexagonal, and triangular. The nanoparticles formed were in the range of 20–100 nm in size. The obtained SEM results were similar to the synthesis of ZnO NPs from the leaves of Passiflora caerulea L. (Passifloraceae), which was reported earlier by Santhoshkumar et al. [23].Figure 4
SEM analysis of Ag-coated ZnO NPs synthesized fromG. lucidum: (a) synthesized nanoparticles; (b) enlarged portion of Ag-coated ZnO NPs.
(a)(b)


### 3.5. Energy Dispersive Diffraction Analysis

The elemental composition of mycosynthesized ZnO NPs coated with Ag fromG. lucidum was investigated using EDX to confirm the presence of Zn in the nanoparticle solution that was purified using the ultracentrifugation process. The EDX findings (Figure 5) revealed a significant peak of zinc and silver in the suspension, confirming its existence [24]. The presence of silver alongside zinc shows that biomolecules were involved in the creation of ZnO nanoparticles coated with Ag and that they may have also functioned as stabilizing molecules to ensure the nanoparticles’ stability. The obtained result from EDX was similar to the study of Santhoshkumar et al. [23]. In his study, the synthesis of zinc oxide nanoparticles was using the plant leaf extract of Passiflora caerulea L. (Passifloraceae), in which Zn was elevated in the nanoparticles suspension which was visible in the result of EDX analysis.Figure 5
EDAX spectrum of Ag-coated ZnO NPs synthesized fromG. lucidum.


### 3.6. Transmission Electron Microscopy (TEM) Analysis

TEM analysis was carried out for further confirmation of the synthesized ZnO NPs coated with Ag fromG. lucidum. The findings of the TEM are shown in Figure 6, which showed that the produced particles were mostly polydistributed and were spherical, hexagonal, and triangular. The selected area electron diffraction (SAED) pattern also reveals the crystalline structure of the ZnO nanoparticles coated with Ag. The particle sizes varied from 10 to 20 nm [25, 26]. In previous kinds of literature, it was observed that nanoparticles ranging from 2 to 20 nm are assumed to be good characteristics of a nanoparticle. So, the resultant product also has the same mentioned range in size, which was enough to conclude the good formation of synthesized ZnO NPs coated with Ag. A similar observation has been reported in the green synthesis of zinc oxide nanoparticles using flower extract of Nyctanthes arbor-tristis by Jamdagni et al. [27].Figure 6
TEM analysis of Ag-coated ZnO NPs synthesized fromG. lucidum.


### 3.7. Alpha-Amylase Inhibition Assay and Alpha-Glycosidase Assay

Antidiabetic activity of synthesized ZnO NPs coated with Ag was evaluated by the inhibition assays of alpha-glucosidase and alpha-amylase enzymes. The results obtained from both enzyme inhibition assays shown in Tables2 and 3 exhibited the concentration of both Ag–ZnO NPs and standard drug (acarbose) on their respective enzymes. It reveals the carbohydrate digestive enzyme inhibition effect of synthesized Ag–ZnO NPs from G. lucidum. So, the results exhibited the significant antidiabetic potential of Ag–ZnO NPs. With an increasing concentration of Ag–ZnO NPs, the enzyme inhibition level was automatically elevated remarkably, so it might be used to prepare effective antidiabetic drugs without any harmful actions. Thus, the Ag–ZnO NPs seem to be a promising and effective antidiabetic agent that can induce a significant reduction of enzymes. A similar carbohydrate digestive enzyme inhibition effect was reported earlier in the antidiabetic activity of Ag NPs from green synthesis using Lonica japonica leaf extract by Balan et al. [28].Table 2
Alpha amylase inhibition assay effect of Ag-coated ZnO NPs synthesized fromG. lucidum.
Sr. No.Concentration (µg/ml)Percent (%) inhibition of alpha-glucosidase activityStandard (acarbose)1.1032.33 ± 0.0113.06 ± 0.012.2046.52 ± 0.1020.09 ± 0.013.4058.90 ± 0.0139.30 ± 0.014.6075.32 ± 0.0153.59 ± 0.035.8082.21 ± 0.0670.78 ± 0.016.10094.37 ± 0.0685.34 ± 0.027.IC 50 (µg/ml)2439Values are mean ± SD (n = 6), values not sharing a common letter differ significantly at <0.05 by DMRT.Table 3
Alpha-glucosidase inhibition effect of Ag-coated ZnO NPs synthesized fromG. lucidum.
Sr. No.Concentration (µg/ml)Percent (%) inhibition of alpha-amylase activityStandard (acarbose)1.1014.01 ± 0.0118.17 ± 0.012.2028.67 ± 0.0121.34 ± 0.013.4037.25 ± 0.0131.21 ± 0.024.6045.34 ± 0.0242.78 ± 0.015.8068.12 ± 0.0160.34 ± 0.036.10079.24 ± 0.0174.71 ± 0.017.IC 50 (µg/ml)4750Values are mean ± SD (n = 6), values not sharing a common letter differ significantly at <0.05 by DMRT.


### 3.8. Cytotoxicity–MTT Assay

MTT assay was performed on chemoresistance breast cancer MDA-MB 231 cell lines to investigate the anticancer activity of Ag–ZnO NPs synthesized fromG. lucidum. The attained results shown in Figure 7 exhibits that the cell viability was significantly decreased when increasing the concentration of both ZnO NPs coated with Ag synthesized from G. lucidum and crude extract of G. lucidum on cell lines. Trypan blue assay resulted in maximum quantity of more than 90% of dead cells stained by trypan blue assay counted using a hemocytometer on a basic upright microscope. Nanoparticles are a good therapeutic agent in anticancer therapy. G. lucidum is a good anticancer medicine since ancient days [29]; so, nanoparticle and G. lucidum will be a good combination in treating breast cancer. Our synthesized Ag–ZnO NPs might be an alternative treatment and therapeutic approach against cancer because other treatments include chemotherapy, radiotherapy, and drugs are potentially cause severe side effects like pain, strain, etc. In the future, the production of drugs by using natural sources instead of toxic chemicals will play a major role in pharmaceutical industries [30]. Currently, several studies are going on with the combination of nanoparticles and other therapeutic techniques like infrared laser light for treating the various types of cancers. In those researches, the positive results are appearing in the animal models which were proved by many researchers. Hence, more clinical trials are needed to successfully approve nanoparticle-related treatments for testing on humans [31]. In this study, Ag–ZnO NPs and crude extract of mushroom were treated on human chemoresistance breast cancer MDA-MB 231 cell lines.Figure 7
Percentage of cell viability for crude extract and synthesized ZnO NPs coated with Ag based on the MTT assay.


## 3.1. Visual Observation

Analysis of ZnO nanoparticles was made by coating Ag particles, the aqueous solution ofG. lucidum was then mixed with the solution of zinc nitrate. Later, the silver nitrate slowly started to change its nature which resulted in the color change. This color transition served as evidence for the synthesis completion [18]. The final brownish color change is shown in Figure 1, which exhibited the reduction of zinc nitrate to zinc oxide and it occurs due to the excitation of the surface plasmon resonance effect [11]. The observation was alike to the green synthesis of silver nanoparticles from an aqueous extract of brown seaweed of Padina boergesenii, which was completed by Chikkanna et al. [19].Figure 1
Mycosynthesis of Ag-coated ZnO NPs fromG. lucidum: (a) initial and final color of Ag-coated ZnO NPs; (b) Ag coated with the mixture of G. lucidum extract and zinc nitrate solution.
(a)(b)


## 3.2. UV–Visible Spectral Analysis

UV–vis spectroscopy was used to confirm the formation of synthesized nanoparticles in the initial stage. The dark brownish sample of ZnO NPs was coated with Ag nanoparticles, which were synthesized from the extract ofG. lucidum. The synthesized ZnO NPs were scanned by using UV–vis spectroscopy under the range between 300 and 600 nm [20]. The result obtained from UV–vis spectral analysis showed (Figure 2) a high-absorption peak at 370 nm, which corresponds to synthesized ZnO NPs. Also, this intense response of ZnO NPs could be due to the surface plasmon resonance effect. The synthesized product is confirmed to claim ZnO NPs coated with Ag because no other peaks were observed in the UV spectrum region [21]. A similar observation was also reported earlier by Siva Vijayakumar et al. [22] in the green synthesis of zinc oxide nanoparticles from the extracts of leaf, stem, and in vitro grown callus of Mussaenda frondosa L.: characterization and their applications.Figure 2
UV–vis absorption spectra of Ag-coated ZnO NPs synthesized fromG. lucidum.


## 3.3. Fourier-Transformed Infrared (FTIR) Spectral Analysis

The FTIR absorption spectra of the water-soluble extract before and after reduction of Ag-coated ZnO NPs showed the capping ligand of the silver-coated zinc oxide nanoparticles which may be due to the linkage of an alkenes, alkyl sulfides groups, carboxyl groups, primary amides, and nitro groups as described in Table1 and Figure 3. FTIR analysis of the synthesized Ag–ZnO NPs exposes strong bands at 1,991, 1,644, 1,515, 1,392, and 958 cm−1. We confirmed that the strong peak was focused at 1,515 cm−1, which corresponds to the carboxyl groups from amino acid residues, and that proteins had the highest ability to bind metals, based on the FTIR results. This intercalation might explain why proteins create metal oxide nanoparticles (i.e., capping of silver-coated zinc nanoparticles) to avoid agglomeration and maintain the medium. It has already been reported in the green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract, that the same functional groups of phytochemicals induce the nanoparticles were synthesized using amines, alkanes, and carboxyl ions that are widely seen in secondary metabolites such as terpenoids, flavonoids, and alkaloids.Figure 3
FTIR spectrum of Ag-coated ZnO NPs synthesized fromG. lucidum: (a) control water-soluble extract (G. lucidum); (b) Ag-coated ZnO NPs with water-soluble extract of G. lucidum.
(a)(b)Table 1
FTIR analysis of Ag-coated ZnO NPs.
Sr. No.Wavenumber (cm−1)StretchFunctional groups11,991NH2Primary amides21,644N=ONitrogen group31,515CO2Carbonyl group41,392C=CAlkenes5958C–SAlkyl sulfides


## 3.4. Scanning Electron Microscope Analysis

The silver-coated zinc nanoparticles synthesized with the aid ofG. lucidum extract were scanned using SEM in order to determine the morphology, polydispersed shape, and size of the synthesized nanoparticle. SEM images (Figure 4) showed that the particles formed were spherical, hexagonal, and triangular. The nanoparticles formed were in the range of 20–100 nm in size. The obtained SEM results were similar to the synthesis of ZnO NPs from the leaves of Passiflora caerulea L. (Passifloraceae), which was reported earlier by Santhoshkumar et al. [23].Figure 4
SEM analysis of Ag-coated ZnO NPs synthesized fromG. lucidum: (a) synthesized nanoparticles; (b) enlarged portion of Ag-coated ZnO NPs.
(a)(b)


## 3.5. Energy Dispersive Diffraction Analysis

The elemental composition of mycosynthesized ZnO NPs coated with Ag fromG. lucidum was investigated using EDX to confirm the presence of Zn in the nanoparticle solution that was purified using the ultracentrifugation process. The EDX findings (Figure 5) revealed a significant peak of zinc and silver in the suspension, confirming its existence [24]. The presence of silver alongside zinc shows that biomolecules were involved in the creation of ZnO nanoparticles coated with Ag and that they may have also functioned as stabilizing molecules to ensure the nanoparticles’ stability. The obtained result from EDX was similar to the study of Santhoshkumar et al. [23]. In his study, the synthesis of zinc oxide nanoparticles was using the plant leaf extract of Passiflora caerulea L. (Passifloraceae), in which Zn was elevated in the nanoparticles suspension which was visible in the result of EDX analysis.Figure 5
EDAX spectrum of Ag-coated ZnO NPs synthesized fromG. lucidum.


## 3.6. Transmission Electron Microscopy (TEM) Analysis

TEM analysis was carried out for further confirmation of the synthesized ZnO NPs coated with Ag fromG. lucidum. The findings of the TEM are shown in Figure 6, which showed that the produced particles were mostly polydistributed and were spherical, hexagonal, and triangular. The selected area electron diffraction (SAED) pattern also reveals the crystalline structure of the ZnO nanoparticles coated with Ag. The particle sizes varied from 10 to 20 nm [25, 26]. In previous kinds of literature, it was observed that nanoparticles ranging from 2 to 20 nm are assumed to be good characteristics of a nanoparticle. So, the resultant product also has the same mentioned range in size, which was enough to conclude the good formation of synthesized ZnO NPs coated with Ag. A similar observation has been reported in the green synthesis of zinc oxide nanoparticles using flower extract of Nyctanthes arbor-tristis by Jamdagni et al. [27].Figure 6
TEM analysis of Ag-coated ZnO NPs synthesized fromG. lucidum.


## 3.7. Alpha-Amylase Inhibition Assay and Alpha-Glycosidase Assay

Antidiabetic activity of synthesized ZnO NPs coated with Ag was evaluated by the inhibition assays of alpha-glucosidase and alpha-amylase enzymes. The results obtained from both enzyme inhibition assays shown in Tables2 and 3 exhibited the concentration of both Ag–ZnO NPs and standard drug (acarbose) on their respective enzymes. It reveals the carbohydrate digestive enzyme inhibition effect of synthesized Ag–ZnO NPs from G. lucidum. So, the results exhibited the significant antidiabetic potential of Ag–ZnO NPs. With an increasing concentration of Ag–ZnO NPs, the enzyme inhibition level was automatically elevated remarkably, so it might be used to prepare effective antidiabetic drugs without any harmful actions. Thus, the Ag–ZnO NPs seem to be a promising and effective antidiabetic agent that can induce a significant reduction of enzymes. A similar carbohydrate digestive enzyme inhibition effect was reported earlier in the antidiabetic activity of Ag NPs from green synthesis using Lonica japonica leaf extract by Balan et al. [28].Table 2
Alpha amylase inhibition assay effect of Ag-coated ZnO NPs synthesized fromG. lucidum.
Sr. No.Concentration (µg/ml)Percent (%) inhibition of alpha-glucosidase activityStandard (acarbose)1.1032.33 ± 0.0113.06 ± 0.012.2046.52 ± 0.1020.09 ± 0.013.4058.90 ± 0.0139.30 ± 0.014.6075.32 ± 0.0153.59 ± 0.035.8082.21 ± 0.0670.78 ± 0.016.10094.37 ± 0.0685.34 ± 0.027.IC 50 (µg/ml)2439Values are mean ± SD (n = 6), values not sharing a common letter differ significantly at <0.05 by DMRT.Table 3
Alpha-glucosidase inhibition effect of Ag-coated ZnO NPs synthesized fromG. lucidum.
Sr. No.Concentration (µg/ml)Percent (%) inhibition of alpha-amylase activityStandard (acarbose)1.1014.01 ± 0.0118.17 ± 0.012.2028.67 ± 0.0121.34 ± 0.013.4037.25 ± 0.0131.21 ± 0.024.6045.34 ± 0.0242.78 ± 0.015.8068.12 ± 0.0160.34 ± 0.036.10079.24 ± 0.0174.71 ± 0.017.IC 50 (µg/ml)4750Values are mean ± SD (n = 6), values not sharing a common letter differ significantly at <0.05 by DMRT.


## 3.8. Cytotoxicity–MTT Assay

MTT assay was performed on chemoresistance breast cancer MDA-MB 231 cell lines to investigate the anticancer activity of Ag–ZnO NPs synthesized fromG. lucidum. The attained results shown in Figure 7 exhibits that the cell viability was significantly decreased when increasing the concentration of both ZnO NPs coated with Ag synthesized from G. lucidum and crude extract of G. lucidum on cell lines. Trypan blue assay resulted in maximum quantity of more than 90% of dead cells stained by trypan blue assay counted using a hemocytometer on a basic upright microscope. Nanoparticles are a good therapeutic agent in anticancer therapy. G. lucidum is a good anticancer medicine since ancient days [29]; so, nanoparticle and G. lucidum will be a good combination in treating breast cancer. Our synthesized Ag–ZnO NPs might be an alternative treatment and therapeutic approach against cancer because other treatments include chemotherapy, radiotherapy, and drugs are potentially cause severe side effects like pain, strain, etc. In the future, the production of drugs by using natural sources instead of toxic chemicals will play a major role in pharmaceutical industries [30]. Currently, several studies are going on with the combination of nanoparticles and other therapeutic techniques like infrared laser light for treating the various types of cancers. In those researches, the positive results are appearing in the animal models which were proved by many researchers. Hence, more clinical trials are needed to successfully approve nanoparticle-related treatments for testing on humans [31]. In this study, Ag–ZnO NPs and crude extract of mushroom were treated on human chemoresistance breast cancer MDA-MB 231 cell lines.Figure 7
Percentage of cell viability for crude extract and synthesized ZnO NPs coated with Ag based on the MTT assay.


## 4. Conclusion

The biosynthesis method used in this study exhibited a simple, eco-friendly meanwhile budget-friendly process when compared to other methods. Thus, the results of the present study successfully proved the antidiabetic and anticancer potential of our mycosynthesized Ag–ZnO NPs. The effect of Ag–ZnO NPs on chemoresistance breast cancer MDA-MB 231 cell lines revealed promising cytotoxicity. Due to nontoxic and less usage of chemicals, these kinds of biosynthesized Ag–ZnO NPs will predominantly take their place in the pharmaceutical field.



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*Source: 2798532-2022-12-23.xml*