Potential exploration of silver nitrate nanoparticlesloaded on Conyzacanadensis forin vitro and in vivocytotoxic and immunologicalstudies

doi:10.21203/rs.3.rs-4994409/v1

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Potential exploration of silver nitrate nanoparticlesloaded on Conyzacanadensis forin vitro and in vivocytotoxic and immunologicalstudies

https://doi.org/10.21203/rs.3.rs-4994409/v1

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This study aimed to evaluate the biological effects of C. canadensis extract and its silver nanoparticles, focusing on their cytotoxic and anti-inflammatory properties by assessing their effectiveness in cytokine production and wound healing potential in albino male mice. Our research revealed that C. canadensis extracts are rich in flavonoids, phenols, and saponins. Wound healing results indicated that burns treated with an aqueous extract of C. canadensis healed in 24 days, compared to 18 days for those treated with silver nanoparticles. Notably, mice treated with green synthetic nanoparticles recovered after just 13 days.

The study also demonstrated the anti-inflammatory effects of C. canadensis extract and biosynthesized silver nanoparticles on the in vitro release of cytokines (IL-6 and IL-10) from cultured mononuclear cells. These results highlight the potential of C. canadensis as a valuable source of bioactive compounds for developing new drugs, cosmetics, and food products across various industrial sectors. Silver nanoparticles and C. canadensis extracts could be effectively used as wound healing and anti-inflammatory has been performed for the first time in these species.

Green Synthesis

Silver Nanoparticles

Cytotoxic

wound healing

Conyza canadensis

Nanotechnology has emerged as a highly influential technology in recent years [1]. It is a scientific field that employs various synthesis methods and techniques to manipulate particle sizes and configurations, creating particles ranging from one to one hundred nanometers [2]. Nanoscale particles exhibit unique characteristics, including variations in shape and size distribution, which differ significantly from those of larger particles [3].

The bottom-up method uses plant extracts to produce nanoparticles for biological applications [4]. This approach is part of the developing field of "Phyto nanotechnology," which focuses on using nanoparticles that are ecologically friendly and cost-effective [5]. For the first time in scientific research, we used a plant, Conyza canadensis, for this purpose.

Conyza is a genus classified in the Asteraceae family, encompassing approximately 400 species distributed globally [6]. Some species of this genus have been utilized in traditional medicine to treat wounds, facial acne, rheumatism, swelling, arthritis-induced pain, and various pathological conditions such as inflammation and microbial infections, including urinary and respiratory tract infections [7]. Using plants has been associated with various pharmacological activities, such as antibacterial, antioxidant, anticancer, hypolipidemic, cardiovascular, neurological, respiratory, immune system, anti-inflammatory, analgesic, and antipyretic effects [8, 9]. The effects mentioned, which display antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory, and anti-musical properties, are linked to plant secondary metabolites such as monoterpenes, diterpenes, triterpenes, tetraterpenes, sesquiterpenes, saponins, flavonoids, phenols, steroids, and coumarins [10, 11].

Phytochemical investigations have revealed that C. canadensis contains a wide range of chemical components, such as saponins, diterpenoids, terpenoids, glycosides, tannins, anthraquinones, steroids, phenols, and flavonoids [12]. C. canadensis exhibits a variety of biological activities, including antioxidant, antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and mutagenic properties [7]. Previous research has shown that extracts from C. canadensis demonstrate strong cytotoxic effects on many cancer cell lines [13].

This research aimed to explore and expand the potential use of an aqueous extract of C. canadensis for the production of silver nanoparticles. We evaluated their impact on breast cancer cell lines (MCF7), measured the levels of anti-inflammatory cytokines IL-6 and IL-10, examined their ability to promote wound healing, and compared the effects of C. canadensis-loaded AgNPs with traditional AgNPs. However, there is no clear scientific evidence for using Conyza extract to produce silver nanoparticles for wound healing.

This study is the first novel report combining the phytochemical composition of the plant used in traditional medicine, the biosynthesis of silver nanoparticles from C. canadensis, and the ability of its extract to promote wound healing.

Plant Extract Preparation

C. canadensis was prepared following the methodology outlined by Kadhim [14]. The plant was thoroughly cleaned, and then dried in the dark for three days. The dried plant were ground carefully into fine powder. 50 grams of it were immersed in 1 liter of distilled water at 40°C for 2 days. The mixture was continuously stirred using a shaking incubator. Afterward, the mixture was filtered twice through cheesecloth to remove any insoluble particles. Some extracts were dried, while others

were preserved in solution, depending on their intended use. The extracted substances were then stored in a refrigerator at 4°C until needed.

Bioactive Compound Assessment

This study employed the aluminium chloride colorimetric method to spectrophotometrically determine the total flavonoid content of C. canadensis [15]. The total phenolic content (TPC) was measured using the Folin-Ciocalteu method [16]. The total saponin content was assessed spectrophotometrically using glacial acetic acid and sulfuric acid [17].

Synthesis of silver nanoparticles using C.canadensis extract

Synthesis of silver nanoparticles using C. canadensis extract and various concentrationsofAgNO₃ solution (1, 1.5, 2, 2.5 mM) silver nitrate solution in double distilled water was the source of silver. Silver nitrate and extract were mixed together in a ratio of 1:9. The reaction mixture was heated below the boiling point and continuously stirred at 800 rpm using magnetic stirrer. The mixture turned reddish brown in color within 1 h which indicated the formation of silver nanoparticles. The whole reaction was carried out in the dark. The obtained suspension was centrifuged at 15,000 rpm for 45 min. The pellet containing silver nanoparticles was washed 3–4 times with deionized water to remove silver ions and extract residue. The precipitated nanoparticles were lyophilized. Lyophilized nanoparticles were stored in a cool, dry, and dark place [18].

Cytotoxic Activity

The human breast adenocarcinoma MCF-7 and normal human WRL-68 cell lines were provided by the Department of Pharmacology, Faculty of Medicine, Centre for Natural Product Research and Drug Discovery, University of Malaya, Malaysia. The cells were cultured in RPMI-1640 medium and grown in culture flasks for 24 hours in a humidified incubator with 5% CO₂ at 37°C.

The study aimed to evaluate the cytotoxic effects of C. canadensis extract on MCF-7 and WRL-68 cells in a controlled laboratory setting. The MTT colorimetric assay was used to assess cytotoxicity, with the botanical extract applied at concentrations of 12.5, 25, 50, 100, and 200 µg/mL. The assay was performed on 96-well microtiter plates using an MTT kit manufactured by Intron Biotech, Korea. Cells were plated at a density of 1 x 10^4 cells/mL and incubated for 24 hours at 37°C with 5% CO₂ in 200 µL of complete culture medium per well. After incubation, the medium was removed, and sequential dilutions of nanoparticles made using green methods and C. canadensis at concentrations of 12.5, 25, 50, 100, and 200 µg/mL were added to the wells. A control group of untreated cells was also included. The experiment was replicated three times using undamaged microtiter plates for each concentration and control group. The plates were incubated for 48 hours at 37°C with 5% CO₂. Following exposure, 10 µL of MTT solution was added to each well and the plates were incubated for 4 hours at 37°C with 5% CO₂. The medium was then carefully removed, and 100 µL of solubilization solution was added to each well and incubated for 5 minutes. Absorbance was measured using an ELISA reader at 575 nm. Statistical analysis of the optical density data was performed to determine the concentration of chemicals needed to reduce cell viability by 50% (IC50) for each cell line. Cell viability was calculated using the following equation:

𝐕𝐢𝐚𝐛𝐢𝐥𝐢𝐭𝐲 (%) =\(\:\frac{\text{O}\text{p}\text{t}\text{i}\text{c}\text{a}\text{l}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{O}\text{p}\text{t}\text{i}\text{c}\text{a}\text{l}\:\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:}\)× 𝟏𝟎𝟎

In Vivo Wound Healing Model

In this study, mice were used to test the impact of anti-inflammatory properties on burn healing. The experiment followed the mouse skin wound-healing model outlined in a previous study [19]. Male albino mice, weighing between 23 and 27 grams and aged between 8 and 10 weeks, were utilized. The animals were housed in standard laboratory cages and fed a normal pellet diet prepared by factories in Iraq on an ad libitum basis. The Animal Care and Ethics Committee of the Biotechnology Research Center at Al-Nahrain University, Baghdad, Iraq, approved all experimental protocols (Ref. No. E. B. 102), ensuring compliance with the National Institutes of Health Guidelines (NIH Publication No. 86 − 23 revised in 1996). The animals were divided into groups as outlined in the experimental design section, with each group housed in a separate plastic enclosure.

Experimental Design

This experiment evaluated the anti-inflammatory effect and its ability to promote burn healing. The animals were divided into four groups, each with five mice:

Group I: Mice were treated with distilled water (negative controls).
Group II: Mice were treated with commercial cream (Silverin, Switzerland) for burns, supplied by a Baghdad pharmacy.
Group III: Mice were treated with an aqueous extract of C. canadensis.
Group IV: Mice were treated with green synthesized nanoparticles loaded on C. canadensis (1, 1.5, 2, and 2.5 mM). Thirty-five animals comprised the total number of mice in this stage.

Breast Cancer Patients and Controls

The ethical committee at the Biotechnology Research Center of Al-Nahrain University approved this study (Ref. No. C.B 242). The study involved a team of ten patients diagnosed with breast cancer, as well as a control group of ten individuals. The patients received diagnosis and treatment at Baghdad Teaching Hospital, where diagnosis was made through comprehensive clinical examination and laboratory assessments by the hospital's consultant medical personnel. All participants were of Iraqi Arab descent, aged 25 to 40. They were initially diagnosed in January 2022, but treatment did not begin until April of the same year. Additionally, a control group of ten healthy individuals, without any prior medical history or symptoms of breast cancer, was included in the study. These individuals were university personnel and students.

Isolation of Mononuclear Cells

Peripheral blood samples were collected from each participant through venipuncture using a disposable syringe. Approximately 8–10 ml of blood was drawn and subsequently divided into two equal portions. The first portion (4–5 ml) was collected in a regular tube to obtain serum. The second portion was collected in a test tube containing heparin (10 IU/ml) to separate the mononuclear cells. The blood samples were stored refrigerated and transported to the laboratory. In the laboratory, serum was obtained by centrifuging the samples at 2000 revolutions per minute for 10 minutes. Mononuclear cells were separated from the other blood components using a density-gradient centrifugation method as described in reference [20].

Experimental Design

The experimental design for cytokine production is illustrated below:

Culture 1: Patient cultures without treatment (healthy control).
Culture 2: Patient cultures treated with a concentration of C. canadensis loaded on silver NPs (2 mM).
Culture 3: Patient cultures treated with C. canadensis aqueous extract.
Culture 4: Serum of the patients.

Culture Establishment

According to the experimental design, 2 ml of cells, with a concentration of 2–3 x 106 cells per milliliter, were transferred to a test tube. Then, 0.1 ml of nanoparticles loaded onto a plant extract were added to the test tube. The tube was incubated at 37°C for 48 hours. After the incubation period, the tubes were centrifuged at 2000 revolutions per minute for 5 minutes. The liquid portion above the sediment, known as the supernatant, was collected and divided into smaller portions. These portions were then frozen at -20°C until further analysis of cytokines could be conducted [21].

Assessment of Cytokines IL-6 and IL-10 Levels

The sandwich ELISA technique was used to evaluate the levels of two cytokines (IL-6 and IL-10) in the tissue culture supernatant of breast cancer patients and controls, following the manufacturer's instructions (PeproTech, USA).

Statistical Analysis

This study used the Statistical Package for the Social Sciences (SPSS) version 13.2 for statistical analysis. Descriptive statistics, including means and standard deviations, were used to determine the significance of three concurrent assays. The analysis of variance (ANOVA), the key inferential statistic of this study, was employed to identify variations between groups and differences among means. Statistical significance was set at P < 0.05

Chemical analysis

The total flavonoid and phenol content in an aqueous extract of C. canadensis was spectrophotometrically determined, expressed as rutin and gallic acid equivalents, respectively. The extract contained 144.41 ± 1.13 µg/ml of flavonoids and 28.53 ± 2.01 µg/ml of phenols. Additionally, spectrophotometric analysis revealed that the aqueous extract of C. canadensis contained 74.72 ± 1.38 µg/ml of saponins.

Silver nanoparticles Biosynthesis

The Fig. 1 illustrates the process and results of visually observing the green synthesis of nanoparticles over 24 hours. The formation of AgNPs using green methods was confirmed by detecting color changes and performing UV spectroscopy absorption analysis. The reaction mixture initially changed from yellowish after ten minutes to reddish-brown within an hour and eventually to dark brown after one hour (Fig. 1).

Cytotoxic Activity

Two cell lines, WRL-68 and MCF-7, were selected to evaluate the potential effects of C. canadensis extract and C. canadensis loaded on silver nanoparticles. The MTT assay, with concentrations ranging from 12.5 to 200 µg/ml, was used to assess the impact of the extract and the nanoparticle-loaded extract on these cell lines.

The findings in Figs. 2 and 3 indicated a dose-dependent decrease in cell viability in MCF-7 cells (Fig. 2). At a 200 µg/ml concentration, the viability of MCF-7 cells decreased by 40.895 ± 3.74%. The highest cell viability was observed at a concentration of 12.5 µg/ml, with a value of 85.72 ± 3.21%. The highest concentration had the most significant harmful effect on the cells, with an IC50 value of 14.65 µg/ml. In contrast, the IC50 value for the WRL-68 normal cell line was 70.54 µg/ml (Fig. 3). These findings could help identify potential pharmacological targets for cancer cells and aid in developing therapeutic interventions for cell damage caused by burns.

Figure 3 presents the results of the cytotoxic effect of green synthetic nanoparticles at different molarities (1 mM, 1.5 mM, 2 mM, and 2.5 mM) on MCF-7 and WRL-68 cell lines after 24 hours of incubation at 37°C. The results showed a dose-dependent reduction in cell viability across all tested molarities.

At 1 mM, MCF-7 cell viability decreased to 64.20 ± 2.66% at a concentration of 200 µg/ml, while the highest cell viability (94.48 ± 1.40%) was observed at 12.5 µg/ml. At 1.5 mM, MCF-7 cell viability decreased to 54.09 ± 2.57% at 200 µg/ml, with the highest viability (95.95 ± 0.90%) at 12.5 µg/ml. For the 2 mM concentration, MCF-7 cell viability dropped to 48.23 ± 4.57% at 200 µg/ml, and the highest viability (86.19 ± 3.92%) was observed at 12.5 µg/ml. At 2.5 mM, MCF-7 cell viability was reduced to 41.01 ± 4.18% at 200 µg/ml, with the highest viability (83.22 ± 1.33%) at 12.5 µg/ml.

These results indicate that cell survival decreases with increasing concentrations of green synthetic nanoparticles, demonstrating their potential cytotoxic effects on cancer cells.

Our study revealed that the nanoparticles exhibited significant cytotoxic activity at a concentration of 1 mM, with an IC50 value of 72.38 µg/ml. However, when tested on the standard cell line WRL-68, the IC50 value of the nanoparticles was found to be 78.38 µg/ml (Fig. 3a). At 1.5 mM, the most potent cytotoxic activity was observed, with an IC50 value of 24.45 µg/ml. Nonetheless, the effect of the nanoparticles on the WRL-68 cell line resulted in an IC50 value of 90.15 µg/ml (Fig. 3b). Similarly, at 2 mM, the most potent cytotoxic activity was observed, with an IC50 value of 25.39 µg/ml. An IC50 of 61.3 µg/ml was determined for the effect of the nanoparticles on the WRL-68 cell line (Fig. 3c). Additionally, at 2.5 mM, the most potent cytotoxic activity showed an IC50 value of 67.06 µg/ml. In contrast, the effect of the nanoparticles on the WRL-68 cell line resulted in an IC50 value of 109.9 µg/ml (Fig. 3d).

Wound Healing Effect

The wound healing ability of the plant aqueous extract and C. canadensis loaded on silver nanoparticles at different molarities (1 mM, 1.5 mM, 2 mM, and 2.5 mM) was evaluated, along with silver sulfadiazine (positive control) and a negative control, to treat burns on mice's skin. The days required for recovery from burns were recorded, and average wound healing times for the treated groups were determined on days 9, 11, 13, 18, and 24.

The results indicated that an aqueous extract of C. canadensis healed burns in 24 days, compared to silver sulfadiazine, which took 18 days, and the negative control, which also took 24 days. Green-synthesized nanoparticles loaded with C. canadensis healed burns in 9 days at 1 mM, 11 days at 1.5 mM, 11 days at 2 mM, and 13 days at 2.5 mM (Table 1 and Fig. 4).

Table 1

The healing and recovery from burns in mice after treatments
Groups	Treatment	Period of recovery
Negative Control	Mice With any treatment	24 days
Positive Control	Mice Treatment with silverin	18 days
Vehicle Control	Mice Treatment with Conyza canadensis	24 days
Group A	Mice Green synthesis of silver nanoparticle 1Mm	9 days
Group B	Mice Green synthesis of silver nanoparticle 1.5mM	11 days
Group C	Mice Green synthesis of silver nanoparticle 2mM	11 days
Group D	Mice Green synthesis of silver nanoparticle 2.5mM	13 days

Antiinflammatory Analysis

This study investigated the in vitro effects of C. canadensis extract and C. canadensis loaded on silver nanoparticles on the production of two cytokines (IL-6 and IL-10) from cultured mononuclear cells of breast cancer patients. Table 2 presents the impact of the plant extract and C. canadensis loaded on green nanoparticles on IL-6 and IL-10 production.

Table 2

Effect of plant extract and *C. canadensis* loaded on green nanoparticles on Il-6 and IL10 production from cultured mononuclear cells of breast cancer patients.
Groups	IL-6	IL-10
Untreated (NPs + extract)	24.76 ± 9.25	79.44 ± 11.74
Treated (NPs + extract)	8.40 ± 0.70	32.5 ± 8.3
Treated (plant extract only)	14.4 ± 1.5	34.13 ± 2.30
Healthy control	3.71 ± 2.26	48.30 ± 7.98

For IL-6, the serum level in the culture of untreated breast cancer patient cells at 2 mM of green synthesized nanoparticles was 24.76 ± 9.251 pg/ml. In contrast, the treated culture with green synthesized nanoparticles at 2 mM showed a reduction in IL-6 levels to 8.40 ± 0.70 pg/ml. The level of IL-6 in the culture of cells treated with the plant extract was 14.4 ± 1.5 µg/ml, while the level in the culture of healthy patient control cells was 3.71 ± 2.26 pg/ml.

For IL-10, the serum level in the culture of untreated breast cancer patient cells with green synthesized nanoparticles at 2 mM was 79.44 ± 11.74 pg/ml. Compared to the treated culture with green synthesized nanoparticles at 2 mM, the IL-10 level decreased to 32.5 ± 8.3 pg/ml. In the culture of cells treated with the plant extract at 2 mM, the IL-10 level was 34.13 ± 2.30 µg/ml. The control group of healthy patients had an IL-10 level of 48.30 ± 7.98 pg/ml.

Our study aimed to evaluate the phytochemical composition of C. canadensis and produce silver nanoparticles from it. We assessed their impact on breast cancer cell lines (MCF-7), measured the levels of anti-inflammatory cytokines IL-6 and IL-10, and examined their ability to promote wound healing. Additionally, we compared the effects of AgNPs loaded on C. canadensis with traditional silver-based treatments for burns (silver sulfadiazine) and C. canadensis extract on the burn healing process.

Chemical analysis

The primary parameters assessed in the study included the total flavonoids, total phenols, and total saponins in the aqueous extract of C. canadensis. The values for total phenols and total flavonoids are consistent with a study by Abood and Kadhim (2021), which demonstrated that C. canadensis is a rich source of flavonoids [22]. Phenolic compounds significantly impact human health due to their bioactive qualities, including antiviral, antibacterial, anti-inflammatory, cardioprotective, neuroprotective, and anti-aging activities. The aqueous extract of C. canadensis was subjected to spectrophotometric analysis to determine the total saponin content, yielding 74.72 ± 1.38 µg/ml of saponins. Al-Snafi (2017) also found that C. canadensis contains saponins, which is consistent with this observation [8, 9]. Polat et al. (2022) reported that the extract of C. canadensis had a total flavonoid content of 18.91 ± 1.46 µg/ml and a total phenol content of 71.34 ± 0.53 µg/ml [7]. Shelepova et al. (2020) highlighted the presence of flavonoids and phenolics as lipid peroxidation inhibitors and free radical scavengers in plant sources such as C. canadensis [23]. The

phytochemical analysis of C. canadensis aqueous extract has laid the foundation for further research to determine its biological activities.

Silver nanoparticles Biosynthesis

Silver nanoparticles can be generated through physical, chemical, or biological means. Among these methods, the last one is used by researchers owing to its unique virtues, such as the abundance of synthesis materials, easy operation, eco-friendly nature [24].

The active molecules of C. canadensis extract catalyze the conversion of silver metal ions (Ag) into silver nanoparticles, as evidenced by the color change (Fig. 1) the observed change in color is caused by the stimulation with regards to the plasmon resonance property (SPR) [25, 26]. They also differentiated between artificial and organic nanoparticles by detecting color changes from yellow to brown [24]. Similarly, silver nanoparticles can be made through hydrophilic-hydrophobic interactions that generate intermolecular forces. The AgNP solutions in water stimulate surface plasmon oscillations, resulting in a brown color similar to the reddish-brown seen within an hour in this study. In a survey conducted by Said et al. (2024), comparable results were observed following the production of green nanoparticles using Lawsoniainermis [27].

Cytotoxic Activity

The presence of essential oils in plants coated with nanoparticles (NPs) may account for the cytotoxic effects observed in MCF-7 cells upon exposure to NPs, as these oils are known to have a wide variety of biological functions [28, 29]. It has been observed that silver nanoparticles may disrupt the proper functioning of cellular proteins [30], potentially causing changes in cellular chemistry. This disruption can lead to partial unfolding and aggregation of proteins, resulting in cytotoxicity [31]. Previous studies have shown that extracts from C. canadensis exhibit substantial cytotoxic effects on various cancer cell lines [7]. Notably, the root extracts of C. canadensis are more effective than the aerial components, with IC50 values of 94.73 and 84.85 µg/mL on A549 and H1299 cell lines, respectively. Moreover, these extracts significantly decrease the viability of various types of cancer cells. C. canadensis has been proven to possess cytotoxic, antifungal, antibacterial, antiviral, anti-inflammatory, and antioxidant properties [32].

These results demonstrate that the C. canadensis extract, rich in total phenols and flavonoids and possessing potent antioxidant activities [33], successfully inhibited cell proliferation and reduced inflammation. Furthermore, the flavonoid and essential oil constituents present in C. canadensis exhibit anti-mutagenic, anti-tumor, and anti-cancer properties [34, 35]. The antioxidant systems in our bodies often lack sufficient levels, and studies suggest that damage caused by reactive oxygen species (ROS) plays a crucial role in cancer development [36]. ROS can cause DNA damage, and genetic alterations can occur when cells divide without repairing or incorrectly repairing the damaged DNA [37]. Studies have shown that a 75 µg/ml concentration of silver nanoparticles produced using leaf extract from Solanum surattense can result in a 50% death rate in MCF-7 cells [38, 39]. Silver nanoparticles can trigger mitochondrial death in breast cancer cells, making them a powerful cancer treatment. Silver nanoparticles (AgNPs) increase the expression of pro-apoptotic proteins in MCF-7 cells, thereby initiating the process of apoptosis [40, 41]. The study also demonstrated the importance of nanoparticle size in facilitating their accumulation in tumor tissue by exploiting the vascular gap in the tumor capillary. Therefore, these discoveries are vital and could potentially lead to the discovery of new medicinal compounds.

Wound Healing Effect

The results showed that wound healing was faster in the group treated with C. canadensis extract compared to untreated controls and those treated with silver nanoparticles. The groups treated with silver nanoparticles at concentrations of 1 mM, 1.5 mM, 2 mM, and 2.5 mM showed complete wound healing within 9 to 13 days. Nanoparticles are believed to enhance the migration of hemagglutinocyte cells, aligning with their natural properties and supporting wound healing [42]. Silver compounds have long been used as primary treatments for infections, particularly for complex burns and chronic ulcers [43]. The use of products containing silver nanoparticles, with their unique properties, has significantly accelerated the development of medicinal products utilizing silver NP technology [44]. Earlier studies documented that silver nanoparticles can reduce the infiltration of inflammatory cells and suppress the production of inflammatory cytokines [45]. Additionally, numerous reports have highlighted the biological properties of plants, and various studies have aimed to identify the specific biologically active compounds in Conyza canadensis responsible for its healing effects. Our recent study demonstrated that early treatment with C. canadensis extract promotes burn wound healing in mice. Studies of wound healing on C.canadensis have not been found. To the best of our knowledge, this study has been performed for the first time in this species..

AntiinflammatoryAnalysis

This study investigated the in vitro effects of C. canadensis extract and C. canadensis-loaded silver nanoparticles on the production of cytokines IL-6 and IL-10 from cultured mononuclear cells of breast cancer patients. Previous research has shown that silver nanoparticles (AgNPs) can effectively reduce the influx of inflammatory cells, inhibit the production of pro-inflammatory cytokines, and enhance matrix metalloproteinase expression [46]. For instance, administering AgNPs to mice with ovalbumin-induced allergies has been shown to mitigate inflammation by inhibiting the synthesis of vascular endothelial growth factor and mucous glycoprotein [45]. Additionally, AgNPs have been reported to impact anti-inflammatory activity in the postoperative peritoneal adhesion model, with silver-polyvinyl pyrrolidone nanoparticles demonstrating anti-inflammatory characteristics by reducing TNF-α levels [47].

The medicinal properties of C. canadensis, including its anti-inflammatory, anticoagulant, anti-gastric ulcer, anti-diabetic, antioxidant, anti-cancer, and mutagenic activities, have been well-documented [48]. Our results indicate that C. canadensis extract effectively enhanced IL-6 production in culture supernatants, suggesting its immunomodulatory action compared to control groups. The significant increase in IL-6 levels can be attributed to the high concentration of flavonoids in the C. canadensis extract, which has been shown to enhance IL-2 production [9, 49]. Flavonoids can affect the immune system by promoting CD4 + T cells to increase IL-6 production in both laboratory settings and living organisms [50]. They also modulate immunological responses and potentially exhibit anti-inflammatory characteristics by increasing IL-6 secretion [51].

Moreover, the extract from C. canadensis also increased the synthesis of IL-10, highlighting an additional target affected by the extract. Recent research confirms that flavonoids stimulate cells to secrete IL-10 through mechanisms such as gene and protein expression regulation, enzyme activity modulation, and other related factors [52]. The presence of flavonoids in C. canadensis extract plays a crucial role in stimulating immune cells to produce cytokines, explaining the observed increase in IL-10 production.

Cytokines function as agents that either stimulate or suppress inflammation, working together to restore equilibrium. IL-6 acts as a mediator to stimulate inflammation while also regulating anti-inflammatory responses by triggering potent cytokines like IL-10 [53]. Our study demonstrated that C. canadensis extract enhanced the synthesis of crucial cytokines IL-6 and IL-10 in both breast cancer patients and healthy cells. These cytokines boost the cell-mediated immune response (IL-6), modulate the immunological response (IL-10), and may inhibit malignant cell transformation or development. Key T cells, including Th1, Treg, and Th17, generate these specific cytokines. Research has shown that compounds derived from medicinal plants can influence cytokine production [54]. Conyza canadensis extract has demonstrated anti-inflammatory effects both in vitro and in vivo, the methanol extract of Conyza floribunda acts as an anti-inflammatory agent by inhibiting the production of nitric oxide and various pro-inflammatory cytokines [32]. Additionally, a flavonoid glycoside derived from Conyza floribunda has been found to reduce pro-inflammatory cytokines in laboratory settings [55].

Our study explores the environmentally friendly production of silver nanoparticles through biosynthesis using C. canadensis extract. Notably, the plant exhibits a high content of flavonoids, total phenols, and saponins. Silver nanoparticles produced using this green method and the plant extract showed significant cytotoxic effects on MCF-7 and WRL-68 cell lines, as well as anti-inflammatory activities by reducing IL-6 and IL-10 levels. Applying green synthesis to load silver nanoparticles (AgNPs) onto C. canadensis extract has a considerable impact on biological activity, specifically targeting MCF-7 and WRL-68 cell lines. These resukts demonstrate that the combination of plant extract and C. canadensis incorporated into green nanoparticles significantly enhances the production of IL-6 and IL-10 in the cells of breast cancer patients. The silver nanoparticles and C. canadensis exhibit anti-inflammatory, antioxidant, and anti-cancer characteristics.

Authors contributions

Ahlem SOUSSI conceived and conceptualized the study; Ahlem SOUSSI and Ruqaya M. AL-EZZY designed an experiment Safa SALAH SALMAN prepared material and performed the formal analysis; Safa SALAH SALMAN and Ruqaya M. AL-EZZY performed in vitro assay and spectroscopic analysis;

Safa SALAH SALMAN and Ahlem SOUSSI wrote the original draft of the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

None.

Data Availability

All data generated or analyzed during this study are included in this published manuscript.

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. The study was approved by the Ethics Committee at Biotechnology Research Center Al-Nahrain University Iraq approved. (Ethical approval No. E. B. 102) (Ethical approval No. C.B 242).

Conflict of Interest

The authors declare no competing interests

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Editorial decision: Resubmit revised form; Major revisions required
02 Oct, 2024
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You are reading this latest preprint version

Potential exploration of silver nitrate nanoparticlesloaded on Conyzacanadensis forin vitro and in vivocytotoxic and immunologicalstudies

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Plant Extract Preparation

Bioactive Compound Assessment

Cytotoxic Activity

In Vivo Wound Healing Model

Experimental Design

Breast Cancer Patients and Controls

Isolation of Mononuclear Cells

Experimental Design

Culture Establishment

Assessment of Cytokines IL-6 and IL-10 Levels

Statistical Analysis

RESULTS

Chemical analysis

Silver nanoparticles Biosynthesis

Cytotoxic Activity

Wound Healing Effect

Antiinflammatory Analysis

DISCUSSION

Chemical analysis

Silver nanoparticles Biosynthesis

Cytotoxic Activity

Wound Healing Effect

AntiinflammatoryAnalysis

Conclusion

Declarations

References

Status:

Version 1