# Green Synthesis, Characterization of Gold Nanomaterials usingGundelia tournefortii Leaf Extract, and Determination of Their Nanomedicinal (Antibacterial, Antifungal, and Cytotoxic) Potential

Authors: Cumali Keskin; Ayşe Baran; Mehmet Fırat Baran; Abdulkerim Hatipoğlu; Mehmet Tevfik Adican; Mehmet Nuri Atalar; Irada Huseynova; Rovshan Khalilov; Elham Ahmadian; Ömer Yavuz; Sevgi İrtegün Kandemir; Aziz Eftekhari
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Introduction. Fighting against cancer and antibiotic resistance are important challenges of healthcare systems, and developing new treatment methods has become the most concentrated area of researchers. Method and Materials. Green synthesis, characterization, and some biological activities of gold nanomaterials (AuNPs) obtained with Gundelia tournefortii (kenger) leaf extract were investigated in this study. Fourier scanning electron microscope, UV-visible spectrophotometer, Fourier transform ınfrared spectroscopy, energy-dispersive X-ray spectrophotometer, X-ray diffraction diffractometer, transmission electron microscope, and Zetasizer instrument data were used to elucidate the structures of nanoparticles. Results. The maximum surface plasmon resonance was observed at 532.15 nm after 1 hour. With the powder XRD model, the mean cubic crystallite size was determined as 23.53 nm. It was observed that the shapes of the obtained AuNPs were spherical, and the dimensions were 5-40 nm and hexagonal. Surface charges (-27 mV) and average size (365.3 nm) of gold nanoparticles were measured with a zeta analyzer. Conclusion. The suppressive effects of AuNPs on the growth of pathogenic microorganisms and healthy and cancer cell lines were determined using the MIC and MTT methods, respectively.


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## Body

## 1. Introduction

Nanobiotechnology is a new promising field seeing the design, manipulation, and practice of nanoparticles (NPs) to produce new solutions needed in many fields [1]. The unique properties of nanoparticles rely on monodispersed size and surface morphology; various shapes and sizes of nanomaterials can be obtained by changing the synthesis stage [2, 3]. Nanoparticles are widely produced in the industry by physical and chemical methods [4]. Nowadays, instead of conventional methods, it is more preferred to produce nanoparticles with fast and low-cost green synthesis procedures that do not use toxic solvents do not pollute the environment [5]. In this context, scientific studies have focused on synthesizing these nanomaterials from biological sources such as plants, algae, seaweeds, viruses, bacteria, and fungi [6, 7].In nanoparticle studies, zinc [8], gold [9], silver [10], nickel [11], iron [12], platinum [13], and selenium [14] salts are metals that have been extensively studied in nanoparticle synthesis. Especially, AuNPs are widely preferred in biomedical and imaging applications. One of the important reasons of AuNPs is preferred in biological applications that can be easily synthesized and have the least toxicity compared to other metals [1].Due to the unaware use of drugs and the increase of antibiotic resistance in pathogenic microorganisms, incidence of infectious diseases and drug consumption has increased significantly worldwide [15]. Researchers have reported that metallic nanoparticles can be an alternative to conventionally used antimicrobial agents [16–20].On the other hand, today, fighting cancer with different varieties, which is very difficult to treat and often insufficient, and developing new treatment methods have become the most concentrated area of researchers [15]. In this context, researches are carried out on the anticancer properties of metal nanoparticles [14]. While metal nanoparticles are being evaluated as antimicrobial and anticancer agents, they are also being evaluated in diagnostic, cell labelling, biomarker, drug delivery, cancer treatment, and water treatment applications [21–24].Gundelia tournefortii (GT), a member of the Asteraceae family, is an artichoke-like medicinal plant that grows in countries such as Iran, Cyprus, Egypt, Jordan, Turkey, Azerbaijan, and Turkmenistan [25, 26]. All parts of GT have been used both as a food source and as a medicinal plant since ancient times [27]. It has been reported that it was used in the treatment of many diseases in traditional folk medicine [25–27]. The size of AuNPs is smaller compared to that of nanoparticles synthesized from metals such as copper, titanium, zinc, iron, and silver. These properties facilitate their passage through cell membranes. For this reason, they show stronger antimicrobial, cytotoxic, and anti-inflammatory activity. This is the first study to show that the AuNPs were synthesized by an environmentally friendly way using GT leaf extract. In this study, the synthesis, characterization, and anticancer and antimicrobial potential of AuNPs were evaluated in detail. The obtained results will make a significant contribution to the literature.


## 2. Material and Method

### 2.1. Material

#### 2.1.1. Herbal Material

GT used in the study was collected from Diyarbakır (Karacadağ/Yiğityolu village) in March-April. It is located at 37°52′19″ north latitude and 39°52′28″ east longitude at an altitude of 962 masl. Taxonomically identified specimens (Gundelia tournefortii L.) were stored in the Herbarium of Mardin Artuklu University (2021-6-MAU), Mardin, Turkey.


#### 2.1.2. Chemicals and Reactive

Tetrachloroauric acid (HAuCl4·3H2O) (Alpha Aesar), Mc Farland solution (0.5), RPMI medium, Mueller Hinton Broth, fluconazole, vancomycin, and colistin antibiotics (Sigma Aldrich, USA) used in the study were commercially available.


#### 2.1.3. Microorganisms and Cell Lines Used in Biological Assay

Gram-positive strains (B. subtilis ATCC 11774; S. aureus ATCC 29213), gram-negative (E. coli ATCC 25922; P. aeruginosa ATCC27853) strains, and Candida albicans yeast were supplied from Mardin Artuklu University Microbiology Laboratory. Cancerous (ovarian sarcoma (Skov-3); human colorectal adenocarcinoma (Caco-2); glioblastoma (U118)) and healthy (human dermal fibroblast (HDF)) cell lines were supplied from Dicle University Central Research Cell Laboratory.


### 2.2. Method

#### 2.2.1. Preparing the Plant Extract ofGundelia tournefortii

The green leaves of GT, which were collected beforehand for the experimental study, were flushed and dried. 10 grams of dried green leaves and 100 mL of distilled water were left to boil. Afterwards, it was filtered and the obtained extract was left to cool at room conditions.


#### 2.2.2. Biosynthesis of the Gold Nanoparticles (AuNPs)

For synthesizing of AuNPs, 3 mM aqueous Au solution was prepared from the commercially purchased solid form of tetrachloroauric acid (HAuCl4·3H2O). 50 mL of GT leaf extract and 75 mL of 3 mM HAuCl4·3H2O were left to react in a flask at 45°C. The color change was seen after one hour, and the reaction was complete. Then, the solution (dark black colored) was centrifuged (10000 rpm), and the solid part was separated. The separated solid part was washed several times. The residue was left in the oven at 55°C for 48 hours to dry completely. The dried part was ground for further use.


#### 2.2.3. Determination of Characteristic Properties of Biologically Synthesized AuNPs

A UV-vis spectrophotometer (Agilent CARY 60) detected the presence of the AuNPs by scanning the reaction mixture (300-800 nm). Shape, nanosize, crystalline structure, and surface plasmon distribution of AuNPs were characterized by FE-SEM-TEM (Quanta), EDX (Quanta FEG 250), XRD (Ultima IV, Rigaku, CBO), and Zetasizer (Malvern Panalytical). The average crystalline size of the gold nanoparticles was calculated by the following formula:D=Kλ/βcosθ [28, 29]. FT-IR (P.E. Spect. 100) was used to determine the functional groups.


#### 2.2.4. Antimicrobial Activities of AuNPs on Pathogen Microorganisms

The antimicrobial activities of the synthesized nanoparticles on the gram (-) and gram (+) bacteria strains and yeast were determined by the microdilution method. The lowest concentration at which growth was observed after incubation was determined as the MIC value [30, 31]. Also, commercial antibiotics fluconazole, vancomycin, and colistin and 1 mM HAuCl4·3H2O solution were used to compare the antimicrobial effects of plant-based synthesized gold nanoparticles on pathogen microorganisms.


#### 2.2.5. Evaluation of Cytotoxic Activities of AuNPs on Cancerous and Healthy Cell Lines by the MTT Method

RPMI 1640 medium was used in culture medium to generate Skov-3 cells, and DMEM was used for other cell types. Conditions for cell cultures were determined according to the literature [32]. Cell lines grown in culture medium were exposed to AuNPs for 48 hours in the concentration range of 25-200 μg/mL. At the end of the incubation period, MTT solution was added to the microplate wells and incubated for 3 hours. Finally, DMSO was added to the culture medium and kept at 25±2°C for 15 min. The absorbance of the microplates is measured spectrophotometrically (MultiScan Go, Thermo) at 540 nm. The % viability was calculated using the formula below [32].
(1)%viability=UC∗100,whereU is the absorbance of cell lines treated with AuNPs and C is the absorbance of control cells.


## 2.1. Material

### 2.1.1. Herbal Material

GT used in the study was collected from Diyarbakır (Karacadağ/Yiğityolu village) in March-April. It is located at 37°52′19″ north latitude and 39°52′28″ east longitude at an altitude of 962 masl. Taxonomically identified specimens (Gundelia tournefortii L.) were stored in the Herbarium of Mardin Artuklu University (2021-6-MAU), Mardin, Turkey.


### 2.1.2. Chemicals and Reactive

Tetrachloroauric acid (HAuCl4·3H2O) (Alpha Aesar), Mc Farland solution (0.5), RPMI medium, Mueller Hinton Broth, fluconazole, vancomycin, and colistin antibiotics (Sigma Aldrich, USA) used in the study were commercially available.


### 2.1.3. Microorganisms and Cell Lines Used in Biological Assay

Gram-positive strains (B. subtilis ATCC 11774; S. aureus ATCC 29213), gram-negative (E. coli ATCC 25922; P. aeruginosa ATCC27853) strains, and Candida albicans yeast were supplied from Mardin Artuklu University Microbiology Laboratory. Cancerous (ovarian sarcoma (Skov-3); human colorectal adenocarcinoma (Caco-2); glioblastoma (U118)) and healthy (human dermal fibroblast (HDF)) cell lines were supplied from Dicle University Central Research Cell Laboratory.


## 2.1.1. Herbal Material

GT used in the study was collected from Diyarbakır (Karacadağ/Yiğityolu village) in March-April. It is located at 37°52′19″ north latitude and 39°52′28″ east longitude at an altitude of 962 masl. Taxonomically identified specimens (Gundelia tournefortii L.) were stored in the Herbarium of Mardin Artuklu University (2021-6-MAU), Mardin, Turkey.


## 2.1.2. Chemicals and Reactive

Tetrachloroauric acid (HAuCl4·3H2O) (Alpha Aesar), Mc Farland solution (0.5), RPMI medium, Mueller Hinton Broth, fluconazole, vancomycin, and colistin antibiotics (Sigma Aldrich, USA) used in the study were commercially available.


## 2.1.3. Microorganisms and Cell Lines Used in Biological Assay

Gram-positive strains (B. subtilis ATCC 11774; S. aureus ATCC 29213), gram-negative (E. coli ATCC 25922; P. aeruginosa ATCC27853) strains, and Candida albicans yeast were supplied from Mardin Artuklu University Microbiology Laboratory. Cancerous (ovarian sarcoma (Skov-3); human colorectal adenocarcinoma (Caco-2); glioblastoma (U118)) and healthy (human dermal fibroblast (HDF)) cell lines were supplied from Dicle University Central Research Cell Laboratory.


## 2.2. Method

### 2.2.1. Preparing the Plant Extract ofGundelia tournefortii

The green leaves of GT, which were collected beforehand for the experimental study, were flushed and dried. 10 grams of dried green leaves and 100 mL of distilled water were left to boil. Afterwards, it was filtered and the obtained extract was left to cool at room conditions.


### 2.2.2. Biosynthesis of the Gold Nanoparticles (AuNPs)

For synthesizing of AuNPs, 3 mM aqueous Au solution was prepared from the commercially purchased solid form of tetrachloroauric acid (HAuCl4·3H2O). 50 mL of GT leaf extract and 75 mL of 3 mM HAuCl4·3H2O were left to react in a flask at 45°C. The color change was seen after one hour, and the reaction was complete. Then, the solution (dark black colored) was centrifuged (10000 rpm), and the solid part was separated. The separated solid part was washed several times. The residue was left in the oven at 55°C for 48 hours to dry completely. The dried part was ground for further use.


### 2.2.3. Determination of Characteristic Properties of Biologically Synthesized AuNPs

A UV-vis spectrophotometer (Agilent CARY 60) detected the presence of the AuNPs by scanning the reaction mixture (300-800 nm). Shape, nanosize, crystalline structure, and surface plasmon distribution of AuNPs were characterized by FE-SEM-TEM (Quanta), EDX (Quanta FEG 250), XRD (Ultima IV, Rigaku, CBO), and Zetasizer (Malvern Panalytical). The average crystalline size of the gold nanoparticles was calculated by the following formula:D=Kλ/βcosθ [28, 29]. FT-IR (P.E. Spect. 100) was used to determine the functional groups.


### 2.2.4. Antimicrobial Activities of AuNPs on Pathogen Microorganisms

The antimicrobial activities of the synthesized nanoparticles on the gram (-) and gram (+) bacteria strains and yeast were determined by the microdilution method. The lowest concentration at which growth was observed after incubation was determined as the MIC value [30, 31]. Also, commercial antibiotics fluconazole, vancomycin, and colistin and 1 mM HAuCl4·3H2O solution were used to compare the antimicrobial effects of plant-based synthesized gold nanoparticles on pathogen microorganisms.


### 2.2.5. Evaluation of Cytotoxic Activities of AuNPs on Cancerous and Healthy Cell Lines by the MTT Method

RPMI 1640 medium was used in culture medium to generate Skov-3 cells, and DMEM was used for other cell types. Conditions for cell cultures were determined according to the literature [32]. Cell lines grown in culture medium were exposed to AuNPs for 48 hours in the concentration range of 25-200 μg/mL. At the end of the incubation period, MTT solution was added to the microplate wells and incubated for 3 hours. Finally, DMSO was added to the culture medium and kept at 25±2°C for 15 min. The absorbance of the microplates is measured spectrophotometrically (MultiScan Go, Thermo) at 540 nm. The % viability was calculated using the formula below [32].
(1)%viability=UC∗100,whereU is the absorbance of cell lines treated with AuNPs and C is the absorbance of control cells.


## 2.2.1. Preparing the Plant Extract ofGundelia tournefortii

The green leaves of GT, which were collected beforehand for the experimental study, were flushed and dried. 10 grams of dried green leaves and 100 mL of distilled water were left to boil. Afterwards, it was filtered and the obtained extract was left to cool at room conditions.


## 2.2.2. Biosynthesis of the Gold Nanoparticles (AuNPs)

For synthesizing of AuNPs, 3 mM aqueous Au solution was prepared from the commercially purchased solid form of tetrachloroauric acid (HAuCl4·3H2O). 50 mL of GT leaf extract and 75 mL of 3 mM HAuCl4·3H2O were left to react in a flask at 45°C. The color change was seen after one hour, and the reaction was complete. Then, the solution (dark black colored) was centrifuged (10000 rpm), and the solid part was separated. The separated solid part was washed several times. The residue was left in the oven at 55°C for 48 hours to dry completely. The dried part was ground for further use.


## 2.2.3. Determination of Characteristic Properties of Biologically Synthesized AuNPs

A UV-vis spectrophotometer (Agilent CARY 60) detected the presence of the AuNPs by scanning the reaction mixture (300-800 nm). Shape, nanosize, crystalline structure, and surface plasmon distribution of AuNPs were characterized by FE-SEM-TEM (Quanta), EDX (Quanta FEG 250), XRD (Ultima IV, Rigaku, CBO), and Zetasizer (Malvern Panalytical). The average crystalline size of the gold nanoparticles was calculated by the following formula:D=Kλ/βcosθ [28, 29]. FT-IR (P.E. Spect. 100) was used to determine the functional groups.


## 2.2.4. Antimicrobial Activities of AuNPs on Pathogen Microorganisms

The antimicrobial activities of the synthesized nanoparticles on the gram (-) and gram (+) bacteria strains and yeast were determined by the microdilution method. The lowest concentration at which growth was observed after incubation was determined as the MIC value [30, 31]. Also, commercial antibiotics fluconazole, vancomycin, and colistin and 1 mM HAuCl4·3H2O solution were used to compare the antimicrobial effects of plant-based synthesized gold nanoparticles on pathogen microorganisms.


## 2.2.5. Evaluation of Cytotoxic Activities of AuNPs on Cancerous and Healthy Cell Lines by the MTT Method

RPMI 1640 medium was used in culture medium to generate Skov-3 cells, and DMEM was used for other cell types. Conditions for cell cultures were determined according to the literature [32]. Cell lines grown in culture medium were exposed to AuNPs for 48 hours in the concentration range of 25-200 μg/mL. At the end of the incubation period, MTT solution was added to the microplate wells and incubated for 3 hours. Finally, DMSO was added to the culture medium and kept at 25±2°C for 15 min. The absorbance of the microplates is measured spectrophotometrically (MultiScan Go, Thermo) at 540 nm. The % viability was calculated using the formula below [32].
(1)%viability=UC∗100,whereU is the absorbance of cell lines treated with AuNPs and C is the absorbance of control cells.


## 3. Results

### 3.1. Spectroscopic Analysis Data

One hour after the GT green leaves and 1 mM gold solution used in the study were left in the mixture, the formation of AuNPs is seen in Figure1, with a distinct peak at 532.82 nm due to the color change in the UV-vis spectrum.Figure 1
Maximum absorbance value with UV-vis spectrophotometer as a result of interaction ofGundelia tournefortii plant extract and HAuCl4·3H2O solution.


### 3.2. FE-SEM and TEM Data of Plant-Based AuNPS

Shape and nanosize distribution of plant-based synthesized AuNPs were investigated in detail using FE-SEM and TEM images (Figures2 and 3).Figure 2
FE-SEM image of AuNPs obtained fromGundelia tournefortii plant extract at 1 μm.Figure 3
TEM image of AuNPs obtained fromGundelia tournefortii plant extract at 1 μm (a) and 200 nm (b).
(a)(b)


### 3.3. FTIR

FTIR data were used to identify functional groups possessed by biologically synthesized GT-AuNPs in the 600-4000 cm-1 spectrum range before and after synthesis (Figures 4(a) and 4(b)). Figure 4(b) shows the bands observed on the FTIR spectrum of GT-AuNPs at 3993, 3851, 3350, 2289, 2109, 2001, 1636, and 1363 cm-1.Figure 4
(a) FT-IR spectroscopy diagram ofGundelia tournefortii plant extract; (b) FT-IR spectroscopy diagram of the liquid taken after color change (GT-AuNPs).
(a)(b)


### 3.4. Formation Data of Gold Nanoparticles (EDX Diagram)

The elemental combination of plant-based synthesized AuNPs was determined by EDX diagrams. The absorption of MNPs (gold) crystallised was characterized by strong EDX signals at 2.2 and 9.4 keV (Figure5).Figure 5
Image of elemental composition as a result of EDX analysis of the presence of AuNPs obtained from Gundelia tournefortii plant extract.


### 3.5. XRD Analysis Data of GT-AuNPs

The X-ray diffraction pattern of the synthesized GT-AuNPs is shown in Figure6. The average size of the GT-AuNP nanoparticles was calculated as 23.53 nm [29].Figure 6
X-ray diffraction diagram of the crystal morphology of biocompatible synthesis GT-AuNPs.


### 3.6. Zeta Potential (ZP) and Size Distribution by Intensity

The ZP distribution of the GT-AuNPs was found as -9.96 mV (Figure7). Also, the size distribution of the synthesized AuNPs was found as 146.0 nanometers in the measurements made with Zetasizer (Figure 8).Figure 7
Zeta potential distribution of biocompatible synthesis GT-AuNPs.Figure 8
Zetasizer distribution of biocompatible synthesis GT-AuNPs.


### 3.7. Evaluation of Antimicrobial Activities of AuNPs

The inhibitory effects of synthesized gold nanoparticles on the growth of pathogenic microorganisms (gram (+, -); yeast) were compared using standard antibiotics (vancomycin, colistin, and fluconazole: 128 mg/mL) (Table1).Table 1
The minimum inhibition concentrations values of GT-AuNPs, AuCI4·3H2O, and standard antimicrobial agents (mg/mL).
Pathogen microorganismsGT-AuNPsAuCI4·3H2OStandard antibiotics∗S. aureus ATCC 292130. 250.252B. subtilis ATCC117740.1250.51E. coli ATCC259221.01.02P. aeruginosa ATCC278530.55.01C. albicans0.50.1252∗S. aureus, B. subtilis (vancomycin 128 mg/mL); E. coli, P. aeruginosa (colistin 128 mg/mL); C. albicans (fluconazole 128 mg/mL).


### 3.8. Cytotoxic Activities of AuNPs on Cancerous Cell Lines

AuNPs obtained by biosynthesis of GT leaf extract was applied to the healthy cell (Human Dermal Fibroblasts/HDF), and three different cancerous cell lines (Skov-3, CaCo-2, and U118) and their anticancer effects were determined by the MTT method. Figure9 shows the data obtained as a result of this application.Figure 9
Evaluation of the percent viability rates as a result of the cytotoxic effect of AuNPs two days after combining with cell lines.


## 3.1. Spectroscopic Analysis Data

One hour after the GT green leaves and 1 mM gold solution used in the study were left in the mixture, the formation of AuNPs is seen in Figure1, with a distinct peak at 532.82 nm due to the color change in the UV-vis spectrum.Figure 1
Maximum absorbance value with UV-vis spectrophotometer as a result of interaction ofGundelia tournefortii plant extract and HAuCl4·3H2O solution.


## 3.2. FE-SEM and TEM Data of Plant-Based AuNPS

Shape and nanosize distribution of plant-based synthesized AuNPs were investigated in detail using FE-SEM and TEM images (Figures2 and 3).Figure 2
FE-SEM image of AuNPs obtained fromGundelia tournefortii plant extract at 1 μm.Figure 3
TEM image of AuNPs obtained fromGundelia tournefortii plant extract at 1 μm (a) and 200 nm (b).
(a)(b)


## 3.3. FTIR

FTIR data were used to identify functional groups possessed by biologically synthesized GT-AuNPs in the 600-4000 cm-1 spectrum range before and after synthesis (Figures 4(a) and 4(b)). Figure 4(b) shows the bands observed on the FTIR spectrum of GT-AuNPs at 3993, 3851, 3350, 2289, 2109, 2001, 1636, and 1363 cm-1.Figure 4
(a) FT-IR spectroscopy diagram ofGundelia tournefortii plant extract; (b) FT-IR spectroscopy diagram of the liquid taken after color change (GT-AuNPs).
(a)(b)


## 3.4. Formation Data of Gold Nanoparticles (EDX Diagram)

The elemental combination of plant-based synthesized AuNPs was determined by EDX diagrams. The absorption of MNPs (gold) crystallised was characterized by strong EDX signals at 2.2 and 9.4 keV (Figure5).Figure 5
Image of elemental composition as a result of EDX analysis of the presence of AuNPs obtained from Gundelia tournefortii plant extract.


## 3.5. XRD Analysis Data of GT-AuNPs

The X-ray diffraction pattern of the synthesized GT-AuNPs is shown in Figure6. The average size of the GT-AuNP nanoparticles was calculated as 23.53 nm [29].Figure 6
X-ray diffraction diagram of the crystal morphology of biocompatible synthesis GT-AuNPs.


## 3.6. Zeta Potential (ZP) and Size Distribution by Intensity

The ZP distribution of the GT-AuNPs was found as -9.96 mV (Figure7). Also, the size distribution of the synthesized AuNPs was found as 146.0 nanometers in the measurements made with Zetasizer (Figure 8).Figure 7
Zeta potential distribution of biocompatible synthesis GT-AuNPs.Figure 8
Zetasizer distribution of biocompatible synthesis GT-AuNPs.


## 3.7. Evaluation of Antimicrobial Activities of AuNPs

The inhibitory effects of synthesized gold nanoparticles on the growth of pathogenic microorganisms (gram (+, -); yeast) were compared using standard antibiotics (vancomycin, colistin, and fluconazole: 128 mg/mL) (Table1).Table 1
The minimum inhibition concentrations values of GT-AuNPs, AuCI4·3H2O, and standard antimicrobial agents (mg/mL).
Pathogen microorganismsGT-AuNPsAuCI4·3H2OStandard antibiotics∗S. aureus ATCC 292130. 250.252B. subtilis ATCC117740.1250.51E. coli ATCC259221.01.02P. aeruginosa ATCC278530.55.01C. albicans0.50.1252∗S. aureus, B. subtilis (vancomycin 128 mg/mL); E. coli, P. aeruginosa (colistin 128 mg/mL); C. albicans (fluconazole 128 mg/mL).


## 3.8. Cytotoxic Activities of AuNPs on Cancerous Cell Lines

AuNPs obtained by biosynthesis of GT leaf extract was applied to the healthy cell (Human Dermal Fibroblasts/HDF), and three different cancerous cell lines (Skov-3, CaCo-2, and U118) and their anticancer effects were determined by the MTT method. Figure9 shows the data obtained as a result of this application.Figure 9
Evaluation of the percent viability rates as a result of the cytotoxic effect of AuNPs two days after combining with cell lines.


## 4. Discussion

Colloidal solutions of AuNPs show intense color due to SPR caused by bulk oscillation of free conduction electron [30]. For this reason, UV-vis spectroscopy should be used to reveal the formation and stability of AuNPs in an aqueous solution by color change [31]. Recent studies have reported that the absorption SPR peak value of metal nanoparticles can be measured in the wavelength range of 200-800 nm in metallic nanoparticles [32]. It has also been reported that the SPR value in AuNPs is typically in the wavelength range of 500-550 nm. The maximum absorbance value determined at 532.82 nm in our study is compatible with the reference values and shows the stability of the synthesized gold nanoparticles [19, 30, 32–35].FESEM and TEM analyzes data were used to determine the dimensions of the nanoparticles. Analysis data show that biologically synthesized gold nanoparticles coexist in stable small groups owing to their functional groups and surface charges [31]. These data confirm that AuNPs obtained by plant-based synthesis are stable and nanosized. The FESEM and the TEM images show that the gold particles are predominantly spherical and hexagonal shaped (Figure 3). Many researchers have also reported spherical-shaped AuNPs in their studies [35–37]. Nanoparticles with these shapes can easily pass through the cell membrane. Therefore, it can damage target cell organelles and enzymatic systems and the damaged cells are forced into apoptosis [35].In order to determine the presence of specific functional groups responsible for the reduction reactions of gold nanoparticles from gold salt using biological material, the FTIR measurements obtained before (Figure4(a)) and after synthesis (Figure 4(b)) were compared and evaluated. Examining the biomolecules involved in reduction during the formation of AuNPs (Figures 4(a) and 4(b)), the absorption peak at 1636 cm-1 may be due to the reduction in carbonyl (C=O) stretch vibrations. It can be said that the absorption peak at 3350 cm-1 belongs to the -OH and -NH stretches, and the absorption peak at 2109 cm-1 belongs to the -C=C groups. Probably, these specific groups are liable for the degradation of gold ions [38]. EDX data clearly reveals that the biosynthesized metallic nanosized crystals contain gold in their content and have an elemental structure. Moreover, other prominent nonmetal peaks (C, Cl, and O) seen in the EDX profile can be speculated to be due to the structural content of the GT plant [38].ZP analysis measures the surface charge of the synthesized metallic nanoparticle. A negative measurement of the ZP value indicates that it has the potential to bind with molecules. This is another indication that AuNP has a stable structure. The average ZP values of AuNPs synthesized from different plants were reported to be between -12 mV and -37 mV. On the other hand, nanoparticles with a significantly lower negative charge can more easily enter the cell and interact with cellular structures [39–44]. This creates an important advantage for studies with pathogenic microorganisms and cancerous cells. The strong antimicrobial and anticancer activity of synthesized gold nanoparticles is highly dependent on their surface charge [35].It is known that pathogenic microorganisms develop various resistance mechanisms against traditionally used antibiotics over time. This situation encourages researchers to find alternative ways. One of the reasons why researchers have recently increased interest in nanoparticles is that they can be used as potential antimicrobial agents [34, 45]. The antimicrobial mechanism of AuNPs is due to the fact that they have functional groups, especially thiol, that cause cell death by causing damage to the bacterial cell membrane, bacterial cell wall, ribosome, and mitochondria [1]. Although AuNPs significantly suppress the proliferation of bacteria [44], it was reported that they are generally more resistant to some fungal species than bacteria [45]. The results obtained in the study confirm the findings of previous studies. It was determined that the synthesized GT-AuNPs were 8, 8, 4, 2, and 2 times more effective on the growth of S. aureus, B. subtilis, C. albicans, E. coli, and P. aeruginosa compared to the antibiotics used as a standard, respectively.The effect of several concentrations (25, 50, 100, and 200μg mL−1) of GT-AuNPs on the U118, CaCO-2, SK-OV-3, and HDF cell line has been examined using an in vitro MTT assay. The obtained results show that the biosynthesized AuNPs have been prominent cytotoxic activity against the cancerous cell lines. When the data are analyzed after 48 hours, it is seen that there is no toxic effect in HDF with a survival rate of 76% at 25 μg/mL concentration. It was observed that 25 μg/mL concentration suppressed viability by 14-59% in cancer cell lines. Despite the increase in the concentration of AuNPs in cancerous cells, the enhancement in the percentage of viability is due to the proliferative properties of AuNPs for these cells [46].It is known that NPs show strong oxidative properties [47]. NPs tend to settle in biomolecules such as cell membranes and nuclei. After localization, they exert a toxic effect by stimulating apoptosis with an increase in ROS [46]. Exposure time, charge, degree of accumulation, concentration, the chemistry of surface composition, shape, and size be an able significant effect on the toxicity of AuNPs [48]. It was reported that the concentrations above 3.75 μg/mL and 5 μg/mL of NPs showed toxic effects on Caco-2 cells [49]. In other studies, suppressor concentrations in Skov-3 cells were reported as 9.4 μg/mL [50] and 29.36 μg/mL [51].


## 5. Conclusion

Most of the spherical nanoparticle and stable structure gold colloids were synthesized using an aqueous GT leaf extract. This preferred method is environmentally friendly, simple, economical, and efficient. UV-vis absorption, XRD, and EDX analysis confirmed the synthesis of AuNPs. TEM and FE-SEM structure analyzes revealed that AuNPs were predominantly spherical with an average size of approximately 23.53 nm. It was observed that the obtained nanoparticles showed strong antibacterial and anticandidal effects even at low concentrations.The cytotoxic effects of GT-AuNPs in their use were evaluated by the MTT method. For healthy cell HDF, 25μg/mL concentrations did not show any toxic effect. In cancer cell lines, 25 μg/mL concentrations of GT-AuNPs suppressed viability by 14-59%. The cytotoxic effect was studied for the first time with this application to the U118 glioblastoma cancer cell line. Different applications can be made on the parameters that determine the properties of GT-AuNPs, contributing to medical applications for the search for antimicrobial and anticancer agents. The most important advantage in the biological synthesis of gold nanoparticles is the rich secondary metabolite content of plants. This provides that the surface of the gold particles obtained in nanosize is covered by a negative charge. Charged nanoparticles can bond more easily with biomolecules in cells. Because of these properties, they can be used as novel anticancer and antimicrobial materials.



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