Green synthesis of AgNPs using plant extract and investigation of its anti-human colorectal cancer application

Yuze Zhai; Benjun Wang; Weiwei Han; Bianfang Yu; Jichen Ci; Fan An

doi:10.1515/chem-2023-0174

Article Open Access

Green synthesis of AgNPs using plant extract and investigation of its anti-human colorectal cancer application

Yuze Zhai , Benjun Wang , Weiwei Han , Bianfang Yu , Jichen Ci and Fan An

Published/Copyright: December 31, 2023

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From the journal Open Chemistry Volume 21 Issue 1

Abstract

Recently, the plant extracts used to synthesize nanoparticles (NPs) have been considered an excellent alternative to physical and chemical ways. The applications of NPs in the fields of agriculture, industry, and medicine are so many and diverse that they cannot be counted. In recent years, silver nanoparticles (AgNPs) have attracted the consideration of several scientists because of their special characteristics and many applications in various fields, including optoelectronic catalysts, biological markers, and pharmaceutical and medical applications. In the current experiment, the cytotoxic potential of the properties of AgNPs green formulation using green tea on human colorectal cancer cells were determined. The NPs characterization was done by field emission-scanning electron microscopes, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. The average diameter of the particles was about 35 nm. The presence of (111), (200), (220), and (311) peaks at the positions of 38°, 44°, 63°, and 77° indicate the presence of AgNPs, which confirms the correct synthesis of AgNPs. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to measure anti-colorectal carcinoma (on HCT-8, HT-29, MDST8, HCA-7 Colony 29, HCT 116, and Ramos.2G6.4C10 cells) properties of AgNPs. The findings indicate that in 3 days, the cancer cell survival percentage in various dilations reduced as much as the NPs concentration increased. The best anticancer effect was reported at 1,000 μg/mL dilation. The IC₅₀ was 141, 46, 149, 125, 125, and 44 µg/mL against HCT-8, HT-29, MDST8, HCA-7 Colony 29, HCT 116, and Ramos.2G6.4C10 colorectal cancer cells, respectively. The results indicated that these NPs could inhibit colorectal cancer cells more strongly than normal cells. After doing the clinical trial studies, the recent AgNPs are a suitable option for colorectal cancer treatment.

Keywords: cancer cell lines; colorectal cancer; MTT assay; silver nanoparticles

1 Introduction

Cancer is a well-known disease that has existed for centuries and has always attracted the attention of scientists and ordinary people and has shown itself as an incurable disease. On the other hand, after the control of infectious diseases, it has a special place in terms of the need for research [1,2,3]. Cancer starts from the cell, which itself is the maker of body tissues, and the tissues make the organs. Naturally, cells grow and divide into newer cells based on the needs of the body [4,5,6]. When cells get old, they die and new cells replace them. Sometimes this natural process has problems. New cells are created when the body does not need them, and old cells that must be destroyed continue to live [3,4,5]. This excessive accumulation of cells creates tissue masses called tumors. Today, several cancer treatments including surgery, chemotherapy, radiation therapy, and palliative care are available [7,8,9,10]. New treatment methods have to be determined to prevent carcinoma [11,12,13]. Recently, the nanotechnology science and related productions have been used in the oncology field for controlling the tumor growth and its removing. Many advantages of metal nanoparticles (NPs) around technology and medical applications are becoming increasingly apparent [11,12,13]. Because of their small size, low volume-to-surface ratio, and limited space, metal NPs have special properties, including optical, electronic, thermal, and catalytic properties. Among metal NPs, silver is known as one of the most common nanomaterials with wide applications [14,15]. AgNPs are formulated in different biological, chemical, and physical ways and have multipurpose medical applications. For example, they have anti-cancer, anti-angiogenic, anti-inflammatory, antiviral, antifungal, and antibacterial properties [16,17,18,19,20]. The production of NPs in conventional chemical and physical ways seems very dangerous and expensive. However, biological silver nanoparticles (AgNPs) have higher efficiency, solubility, and stability. Therefore, biological methods of producing AgNPs are non-toxic, rapid, simple, green, and reliable and can yield well-defined sizes and morphologies under optimal conditions for various purposes. Therefore, the AgNPs green synthesis approach is very promising [15,17].

Previous research has indicated that the cellular toxicity and anticancer mechanisms of AgNPs include disruptions in the mitochondrial electron transfer chain caused by ROS production, ATP synthesis interruption, and DNA damage. Several different mechanisms, especially ROS excessive production, participate in the toxicity caused by AgNPs. Since cancer cells are more sensitive to the raise of ROS levels than normal cells [18,19,20], cell death may be induced in tumor cells due to increased ROS levels in the anticancer drug’s presence. Studies have presented that AgNPs prevent cell proliferation induced by VEGF, migration, and cell survival through the Akt/PI3K-dependent pathway [19,20,21]. It has also been clarified that AgNPs create more toxicity in cancer cells than in healthy cells, because of the difference in their cellular conditions. Recently, in the science of nanotechnology and medicine, the demand for AgNPs has increased due to their very effective nature [18,19,20,21,22]. Despite having several medical applications, the safety issues of AgNPs are not well defined and need to be investigated [19,20].

In the current experiment, the cytotoxic potential of the properties of AgNPs green formulation using green tea with the scientific name of Camellia sinensis on HCT-8, HT-29, MDST8, HCA-7 Colony 29, HCT 116, and Ramos.2G6.4C10 human colorectal cancer cells were determined. Green tea contains polyphenols, phytochemicals, and catechins, which are the main antioxidant compounds of plants. Antioxidants can fight cancer cells. This combination can inhibit the cancer cells and tumor growth [11,12]. The cancers’ prevalence is very low in people who drink green tea. Scientists think that drinking green tea is a factor in preventing cancer as the antioxidants amount in it is 100 times more effective than vitamin C and 25 times more effective than vitamin E [11,12].

2 Experimental method

2.1 Preparation of extract

The heat-soaking method was applied for plant extraction. After weighing, 30 g of green tea was washed at different times with distilled water to omit the contamination of surface. Then, it was added to 0.3 L of boiling water and after 45 min, the gained extract was passed by Whatman filter paper No. 1. The fresh extract was saved for the biological experiments.

2.2 Preparation of NPs

For the AgNPs synthesis, the precipitation method was carried out with the silver ions reduction by the extract. 180 mL of 1 mM AgNO₃ solution was mixed with 20 mL extract and the solution was kept at laboratory temperature for 2 h. After 120 min of the reaction time, the sediment was washed and centrifuged at 13,500 rpm for 25 min. Then, the produced NPs were placed at 70°C for 100 min [17,18].

2.3 Determining the properties of AgNPs

2.3.1 X-ray diffraction (XRD) method

The XRD method proves that the synthesized particles are AgNPs. To prepare for XRD analysis, after the synthesis of AgNPs, the samples were centrifuged with an ultracentrifuge at a speed of 15,000 rpm for 30 min, then the supernatant solution was discarded and the deposited NPs were examined using X-ray analysis. In this investigation, αCu K Å = (2.44λ) beam diffraction device was used for X-ray radiation. For all samples used, the device voltage was 43 kV and its amperage was 93 mA.

2.3.2 Fourier transform infrared spectroscopy (FT-IR)

To evaluate the changes in the functional group because of the process of reduction, FTIR was performed using the NICOLET infrared spectrometer model iS10.

2.3.3 Scanning electron microscopy (SEM)

To determine the morphology and size of the NPs yielded, an electron microscope, Philips SEM machine, model 300-CMC (Netherlands) was used.

2.4 Anticancer property (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MTT)

The formulated AgNPs’ cytotoxic potentials on common colorectal carcinoma cells i.e., HCT-8, HT-29, MDST8, HCA-7 Colony 29, HCT 116, and Ramos.2G6.4C10 were assessed using MTT.

The colorectal carcinoma cells were incubated in DMEM containing high sugar (10% v/v), penicillin, and streptomycin at 37°C, 5% carbon dioxide. 10⁵ cells were added to each well. Then, DME culture medium, 200 µL of bovine fetal serum, and AgNPs dilutions were added to the wells. 30 µL of MTT solution with a concentration equal to 6 mg/mL was added to all the wells and the plates were kept in a CO₂ incubator for 3 h. After removing the medium, 0.2 mL of DMSO was added to the wells and the AgNPs’ absorbance was read at the wavelength of nanometers using an Eliza reader device. Using control cells, the cell viability curve was drawn [22]:

(1) Cell viability ( % ) = Sample A Control A × 100 .

2.5 Statistical analysis

Each experiment was repeated at least three times independently for each substance, in each dilution and in each of the adjacent times, and each time at least in triplicate. In this study, two-way ANOVA analysis was used for repeated data, followed by one-way variance analysis for repeated data and one-way variance analysis with post-test by Duncan. P ≤ 0.01 was considered as a significant level [23].

3 Results and discussion

3.1 Chemical characterization analysis

3.1.1 Field emission-scanning electron microscopes (FE-SEM) analysis

Morphology of the biosynthesized AgNPs was studied by SEM. The fabricated AgNPs were found to be in polymorphic shapes and aggregated which possibly was due to dehydration exerted during the sample preparation for SEM analysis (Figure 1). In addition, the particles average size is about 35 nm.

Figure 1

FE-SEM image of AgNPs synthesized with plant extract.

3.1.2 FT-IR analysis

The reducing biomolecule’s nature in the formation of AgNPs was studied by FTIR analysis of NPs. As seen in Figure 2, the FTIR spectrum of NPs shows bands, each band corresponding to specific functional groups. The broad peak and intense absorption in the range of 3,490 cm⁻¹ correspond to the stretching fluctuations of the hydroxyl functional group (OH) in alcohols and carboxylic acid [22]. Since this region has two bands, it can be attributed to N–H expansion oscillations in amides and amines [24]. Band 2,984 cm⁻¹ can be related to the stretching vibrations of second-type amines (N–H), amides, or C–H stretching vibrations of hydrocarbon chains [22]. The 1,748 cm⁻¹ band corresponds to the amide I band of proteins or H–C stretching modifiers of amino acids in the extract. Band 1,486 cm⁻¹ is related to carbon groups with double bonds in aromatic rings. The 1,376 cm⁻¹ band corresponds to the CH–CH bond in the lipids or sugars of the extract. The 1,248 cm⁻¹ band corresponds to the amide III band of proteins. The 1,004 cm⁻¹ band corresponds to C–O stretch vibrations in C–N or first-type alcohols in aliphatic amines. It can also correspond to the C–CHO bond, aldehydes, and C–CO–C stretching fluctuations of ketones or halogen compounds. The 757 cm⁻¹ band corresponds to the aldehydes C–H band or halogen compounds such as bromides and chlorides [22,24]. The extract after the reduction of silver salt showed the same FTIR spectra as before the reduction of silver salt, but with a lower intensity. This reduction of the peak shows the activity of different types of phenolic compounds, alcohol, sugars, and proteins in the extract. However, the reduction of the band is seen more in the area of 2,000–3,000 cm⁻¹ and 1,550–1,500 cm⁻¹. Also, the new band presence at 1,723 cm⁻¹ in the spectrum after reduction, which corresponds to the stretching fluctuations of the C═O functional group of carboxylic acid or carbonyl compounds, shows that polyols are mainly responsible for the silver ions reduction, causing them to be oxidized to unsaturated carbonyl groups and causing the mentioned peaks [24].

Figure 2

FT-IR pattern of AgNPs synthesized with plant extract.

3.1.3 XRD analysis

XRD is used to study the crystalline material’s structure. In the electromagnetic spectrum, the X-ray region is in the range between gamma rays and ultraviolet rays. By XRD, information can be obtained about the substance structure and the element’s determination. XRD was used to characterize the material’s crystal structure, including average distances measuring between atomic and layer series, single crystal position and atoms composition determining, and investigating the unknown materials crystal structure, by measuring structural properties such as grain shape and size. According to Figure 3, the spectrum obtained from X-ray analysis corresponds to the standard spectrum of AgNPs and confirms the AgNPs presence.

Figure 3

XRD pattern of AgNPs synthesized with plant extract.

The X-ray diffraction method proves that the formulated NPs are AgNPs. In this investigation, all the spectra taken were compared with standard X-ray spectra. The presence of (111), (200), (220), and (311) peaks at the positions of 38°, 44°, 63°, and 77° indicate the presence of AgNPs, which confirms the correct synthesis of AgNPs (Figure 3).

3.2 Anti-colorectal cancer effects

Nanomedicine is a rapidly promising and developing strategy to fight carcinoma by metal NPs. Recent carcinoma treatments, such as radiation therapy and chemotherapy, have limitations because of unexpected drug-related side effects, lack of low drug concentration specificity at the cancer location, and chemoresistance development. NPs-mediated therapy is the best way for the treatment of cancer [22,23,24,25]. NPs can target patient cells or specific tumor tissues through passive or active targeting by encapsulating remedial agents with NPs. However, several NPs-formulated strategies have progressed, the tumor heterogeneity and its stroma make a notable discussion for clinicians and nanotechnologists to progress specific drugs precisely to carcinoma cells [25,26,27,28]. To gain better efficacy, safety, biocompatibility, reduced toxicity, higher specificity, and overcome conventional chemotherapy limitations, the modern NPs used strategies based on a single platform is another discussion in carcinoma treatment [17,18]. However, it needs to determine the limitations and challenges of using NPs for carcinoma treatment [29,30]. These include manufacturing and regulatory issues, NPs variability, enhanced permeability and persistence effect, limited carrying capacity, and physiological barriers [29,30,31,32]. In recent years, AgNPs have been of great interest in diagnostics and research due to their remedial applications as anticancer agents [18,30]. Cancer is a multifactorial and complex disease, whose characteristic feature is the expansion and uncontrolled growth of abnormal cells caused by many factors, including a combination of environmental, internal, external, and genetic factors [31,33]. Therapeutic options are cancer-specific targeting, immunotherapy, radiation therapy, surgery, hormone therapy, and chemotherapy. The main discussion in targeted carcinoma treatment is to identify sensitive, affordable and effective molecules that have the targeting cells characteristics and raise the cancer cells sensitivity. Recently, different studies have provided evidence that AgNPs have anti-cancer and anti-angiogenic effects [30,31,32,33]. Recent research has indicated that chemotherapy has many side effects in patients. So, it is necessary to progress technologies to inhibit general and specific side effects. Several studies are interested in formulating NPs as a suitable alternative to create drugs that can specifically target cancer cells [33,34]. Studies indicate that AgNPs induce apoptosis and sensitize carcinoma cells. AgNPs induce cell morphology changes, reduce metabolic activity and cell lifespan, and raise oxidative stress that causes mitochondrial damage, raises ROS production and ends with DNA damage. AgNPs’ cellular uptake mainly occurs through endocytosis [34,35]. Instead of treating AgNPs directly in cells, scientists use carrier molecules to deliver silver to cancer cells. Along with the medical technologies advancement, there is a significant interest in applying NPs to replace or ameliorate recent treatment ways [28,29,30,31,32]. Current advances in nanobiotechnology are the NPs used in effective and new medical treatments and diagnostics development [31,32,33,34,35].

The findings of our study indicate that the IC₅₀ was reported to be about 141, 46, 149, 125, 125, and 44 μg/mL on HCT-8, HT-29, MDST8, HCA-7 Colony 29, HCT 116, and Ramos.2G6.4C10; while HUVEC cells indicated a higher survival percentage than carcinoma cells (Figures 4–7) (Table 1).

Figure 4

The cytotoxicity (cell viability (%)) potentials of AgNPs in the concentrations of 1–1,000 µg/mL against human umbilical vein endothelial cell.

Figure 5

The anti-colorectal carcinoma (cell viability (%)) potentials of AgNPs in the concentrations of 1–1,000 µg/mL against HCT-8 (a) and HT-29 (b) cells.

Figure 6

The anti-colorectal carcinoma (cell viability (%)) potentials of AgNPs in the concentrations of 1–1,000 µg/mL against MDST8 (a) and HCA-7 Colony 29 (b) cells.

Figure 7

The anti-colorectal carcinoma (cell viability (%)) potentials of AgNPs in the concentrations of 1–1,000 µg/mL against HCT 116 (a) and Ramos.2G6.4C10 (b) cells.

Table 1

IC₅₀ of AgNPs against cancer cells

	AgNPs (µg/mL)
HCT-8	141 ± 5^b
HT-29	46 ± 2^a
MDST8	149 ± 3^b
HCA-7 Colony 29	125 ± 4^b
HCT 116	125 ± 3^b
Ramos.2G6.4C10	44 ± 3^a
HUVEC	—

The numbers indicate the Mean value ± SD.

4 Conclusion

AgNPs are smart magnetic particles that have a large absorption surface with a very small size. Recent advances in the knowledge of nanotechnology have created a huge change in cancer treatment, and on the other hand, AgNPs were the first particles that were used in cancer. It is known that AgNPs are of special importance in drug delivery and cancer treatment. Because of their very small size and wide absorption surface, these particles can be easily directed to the cancer site. Therefore, AgNPs can play a role as carriers in carrying anti-cancer agents. On the other hand, by providing a magnetic field outside the body, anticancer drugs can be concentrated in the cancer site. Generally, anticancer drugs are loaded with AgNPs and used in cancer treatment. In current years, the anti-angiogenic and anti-cancer potentials of AgNPs have been noticed. The outcomes have revealed that AgNPs can be considered as a strong anti-carcinoma agent. In this study, the nanomaterials green formulation was successfully performed by a green formulation approach with an herbal extract. The NPs were recognized by chemical techniques of FT-IR, FE-SEM, and XRD methods. The outcomes of the study indicated the AgNPs benefits in curing colorectal cancer. A dose-dependency of AgNPs was observed for the cancer cell line viability. The IC₅₀ was 141, 46, 149, 125, 125, and 44 µg/mL against HCT-8, HT-29, MDST8, HCA-7 Colony 29, HCT 116, and Ramos.2G6.4C10 colorectal cancer cells, respectively. AgNPs induced cell apoptosis, accompanied by the up-regulation of pro-apoptotic cleaved caspase-8 and Bax markers regulation and anti-apoptotic Bcl-2 marker down-regulation.

Funding information: Qilu Chinese Medicine Advantage Specialist cluster Project (YWC2022ZKJQ0003). Exploring the mechanism of promoting mesenteric vascular repair in the treatment of Crohn’s enteritis by adding flavor to Si-Miao Yong’an Tang based on TGF-β1/Smads signaling pathway (M-2023305).
Author contributions: All authors had the same role in conceptualization, data curation, formal analysis, acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing – original draft, and writing – review and editing.
Conflict of interest: There is no conflict of interest.
Ethical approval: The experiments were performed according to the ethical guidelines of the International Association for the Study of Human.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Received: 2023-07-27

Revised: 2023-11-20

Accepted: 2023-11-29

Published Online: 2023-12-31

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/chem-2023-0174

Keywords for this article

cancer cell lines; colorectal cancer; MTT assay; silver nanoparticles

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