Facile bio-inspired fabrication of AgNPs from Salvia elegans leaf extract and determination of their cytotoxic DNA photocleavage potential

1 Introduction

Biomedicine, sensing, catalysis, information technology, electronics, and other fields are only a few of the many applications that nanotechnology has taken up at this time of rapid scientific progress (Tovar-Lopez, 2023, Malik et al., 2023, Hossain et al., 2023, Al-Thaqafy et al., 2023, Shaikh et al., 2022). Various techniques such as electrochemical methods, photochemical reactions, chemical reduction and environmentally friendly approaches, the so-called green chemistry, are used to produce and chemically stabilize metal nanoparticles (Jamkhande et al., 2019, Khan et al., 2019, Sakamoto et al., 2009, Khan and Asiri, 2016). Among these techniques, use of plants for nanoparticle synthesis represents an innovative and environmentally sustainable chemical approach. Biogenically produced silver nanoparticles (AgNPs) are among the inorganic nanomaterials that are most commonly employed because of their special physicochemical characteristics, which come from surface coatings of natural origin in addition to their size and form. This method outperforms traditional chemical and physical techniques in terms of expenditure and environmental impact. Importantly, it is scalable for large-scale production without requiring high pressure, elevated temperatures, or hazardous chemicals (Kharissova et al., 2019, Shankar et al., 2004).

Salvia elegans (S. elegans), a member of the Lamiaceae plant family, possesses caffeic acid groups, making it potentially beneficial for reduction reactions. In a prior study, Albeladi et al. emphasized the efficiency, reliability, and cost-effectiveness of AgNPs synthesized from Salvia officinalis. Phytochemicals present in S. officinalis, particularly rosmarinic acid, are crucial in enhancing the stability of the AgNPS. (Royji Albeladi et al., 2020).

Nanoparticles possess exceptional ability to load drugs and display notable photoluminescent characteristics, which makes them ideal for the specific delivery of chemotheraputic medications and imaging (Sarma and Das, 2021, Bae et al., 2011, Aldabaan et al., 2024, Cheng et al., 2021, Muddapur et al., 2022, Iqbal et al., 2021). This enables the accurate delivery of drug loaded nanomedicines to organs like the brain, which are typically protected by specific barriers. Ongoing advancements in nanomedicine are expected to provide even greater benefits in the future, particularly as researchers focus on enhancing the properties of nanomaterials at the intersection of biology and nanotechnology (Mitchell et al., 2021).

Studies on chemotherapy drugs containing metal compounds have acted as a catalyst for the development of novel cancer drugs. This is primarily attributed to the potential of these drugs to exhibit reduced toxicity while improving antiproliferative properties against malignant tumors (Zhou et al., 2020). Transition metal complexes have gained tremendous research potential in recent decades owing to their remarkable similarity to nucleases in their mode of action. The mechanism behind their activity is driven by the redox properties of the metals, which, when combined with dioxygen, generate reactive oxygen species that lead to DNA damage through base modification or strand breakage (Collin, 2019).

Current experiments in this field have focused on DNA degradation using metal nanoparticles such as platinum and gold nanoparticles. The generation of singlet oxygen using metal nanoparticles is considered advantageous, especially in the company of nanoparticles synthesized from gold and platinum (Jose et al., 2011). The production of singlet oxygen can be enhanced by the presence of metal nanoparticles, as demonstrated in a recent study by Zhang et al (Zhang et al., 2007). Enhanced electromagnetic fields near metal nanoparticles contribute to enhanced penetration. Furthermore, the amount of absorption and the relative augmentation of singlet oxygen generation by photosensitizers can be predicted using a variety of computation techniques (Pucelik et al., 2020). The primary aim of this research was to assess the DNA binding and DNA photocleavage properties of AgNPs synthesized using S. elegans extract. To thoroughly characterize the materials, a range of analytical techniques, including UV–Vis spectrophotometry, TEM, SEM, and XRD were used.

This study supports the trend of harnessing natural resources to create nanomaterials, which is in line with the growing emphasis on the use of nanotechnology in biomedical applications. The synthesis of AgNPs and the assessment of their biological effects place this study at the intersection of materials science and pharmacology. The research aims to develop a facile and eco-friendly approach for synthesizing AgNPs using S. elegans leaf extract while simultaneously assessing their cytotoxic potential through DNA photocleavage assays. This study not only contributes to the field of green nanotechnology but also opens avenues for the development of innovative therapeutic agents against cancer, enhancing our understanding of the interplay between nanomaterials and biological systems.

2 Experimental

2.1

2.1 Identification and Extraction of the plant

Fresh leaves of S. elegans (Tangerine Sage) were collected from the Thurahalli forest area in Bengaluru, Karnataka, India (Fig. 1). The leaves were carefully gathered, washed, and sun-dried, repeating this process four to five times to eliminate any dust and impurities. A total of 25 g of the leaves were cut into small pieces and placed in a 250 mL flask containing demineralized water (100 mL). The mixture was heated at 100 °C for 45 min, it was allowed to cool. The leaf extracts were then filtered using Whatman filter paper, and the resulting extracts were utilized for synthesizing silver nanoparticles.

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Fig. 1

Photograph of S. elegans plant.

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3 Results and Discussion

This study describes a practical method for the synthesis of AgNPs using S. elegans extract. The reduction of silver ions to AgNPs is probably due to the phytochemicals, especially caffeic acid and phenolic compounds. During the synthesis reaction, the color of the solution was found to remain constant, indicating depletion of all silver salt in the solution. The subsequent analysis of the AgNPs confirmed the exceptional efficiency of the product. The effectiveness of the reaction was finally assessed by examining the absorption spectra collected at regular intervals. A recent study successfully illustrated the use of a natural plant extract as a catalyst for the formation of C = N bonds, thereby facilitating an organic process. The data points provided in the text are expressed as a range of numbers, in particular (Vidyavathi et al., 2021, Vidyavathi et al., 2022).

Additionally, an additional research study was conducted using natural catalysts such as caffeic acid (extracted from S. elegans) for the synthesis. This study revealed significant production of AgNPs within a period of 5 to 15 min when a stoichiometric amount of Salvia elegans extract was introduced.

3.1

3.1 UV–vis spectroscopy

The synthesis of metallic nanoparticles by reduction in an aqueous solution was observed using the UV–visible spectroscopy method following interaction with S. elegans extract. Fig. 2 shows the absorption spectra of AgNPs using the UV–Vis method. Throughout the reaction, the AgNPs solution consistently exhibited a surface plasmon band at approximately 424 nm (Pattanayak et al., 2017). The nanoparticles were evenly distributed throughout the aqueous solution, as evidenced by the absence of aggregation in the UV–visible absorption spectra.

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Fig. 2

UV–Vis absorption spectra of AgNPs.

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3.2

3.2 SEM analysis

SEM was employed to estimate the morphology of the material and reveal the presence of nanostructured AgNPs. The nanoparticles exhibit a configuration characterized by both stochasticity and nonuniformity, as shown in Fig. 3. This observation is consistent with findings from previous studies (Mukherji et al., 2019), which found that these nanoparticles typically adopt a spherical shape.

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Fig. 3

SEM image of the AgNPs.

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3.3

3.3 TEM analysis

A detailed examination of the transmission electron microscopy (TEM) images in Fig. 4 predicted the sizes of the AgNPs, which were found to be 60 nm. The characteristic dot pattern observed in the images indicates the presence of single-crystal particles. The use of extracts from the S. elegans plant leads to the synthesis of crystalline nanoparticles characterized by uniform particle size and minimal coagulation. This phenomenon facilitates the homogeneous distribution of the particles and thus ensures a uniform size (Mukherji et al., 2019).

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Fig. 4

TEM image of the AgNPs.

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3.4

3.4 XRD analysis

The AgNPs were characterized using the XRD method, as depicted in Fig. 5. The confirmation of the crystalline structure of the AgNPs occurred subsequent to the identification of five peaks [38.063 (111), 44.1392 (200), 64.4001 (220), 77.3686 (311), and 81.558 (222)], aligning with cubic silver nanoparticles (Fig. 5), and corresponding precisely with data from JCPDS file no. 89–3722. Particle size was determined using the Debye-Scherrer formula, analyzing the width of the first peak.

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Fig. 5

XRD analysis of the AgNPs.

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3.5

3.5 DNA interaction studies

3.5.1

3.5.1 Electronic absorption studies

Studying the interaction between the AgNPs and CT-DNA can be carried out using UV–Vis absorption bands. When AgNPs were introduced into CT-DNA at a steady concentration, a significant reduction in the intensity of their UV–Vis absorption bands, known as hypochromism, was observed. In addition, a slight shift toward longer wavelengths, particularly a red shift of 3 to 4 nm, was evident, as shown in Fig. 6. The data shows that the AgNPs penetrate the DNA effectively.

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Fig. 6

UV–Vis spectrum of the synthesized AgNPs.

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The intrinsic binding constant (K_b) for AgNPs is determined to be 2.4 × 10⁴ M⁻¹. This measure is largely consistent with the results of a previous study (Das et al., 2021), indicating a high level of agreement. The binding affinity values obtained from electronic spectrum analysis suggest that AgNPs have a higher degree of intercalation with nuclear DNA compared to commercially available drugs.

3.6

3.6 DNA photocleavage studies

To analyze the influence of AgNPs on pUC19 DNA, a series of experiments were performed. DNA samples were subjected to sequential treatment with increasing concentrations of AgNPs ranging from 50 to 200 μM. This treatment lasted 100 min at a 37 °C in phosphate buffered saline (PBS) diluted sufficiently to achieve a pH of 7.4. The treated DNA samples were then processed for further analysis. Following incubation, the DNA was was subjected to electrophoresis on an agarose gel. A significant association was found between the amount of DNA degradation caused by AgNPs and the concentration of AgNPs applied (Fig. 7, lanes 5–8).

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Fig. 7

Image of the gel electrophoresis for the control trials with SC DNA (0.5 µg) and additional additives at 365 nm after an hour of exposure. Lane 1 represents DNA Control + supernatants; lane 2 represents DNA + DMSO (4 µl) + AgNPs; lane 3 represents DNA + NaN₃ (38 µM) + AgNPs; lane 4 represents DNA + Tris (14 µl) + AgNPs; lane 5 to 8 represents DNA + 50, 100, 150, 200 µM AgNPs, respectively.

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Throughout the DNA incubation process, control samples were prepared using solutions containing S. elegans extract and the supernatant of the AgNP dispersion coupled with silver nitrate. However, none of these components had the ability to break down DNA. This discovery lends support to the theory that the presence of AgNPs is the primary cause of DNA degradation. Chemical scavengers were used to locate and remove the active species from the reaction mixture in order to more thoroughly understand the molecular mechanism of the degradation process (Vadivel et al., 2021; Vinay Kumar et al., 2021). The molecules used in this study included dimethyl sulfoxide, which acts as a scavenger for hydroxyl free radicals, as well as sodium azide and tris-[hydroxylmethyl]aminomethane, both of which act as scavengers for singlet oxygen (Bin-Jumah et al., 2020).

The experimental data depicted that the inclusion of sodium azide (0.2 M) and tris-[hydroxylmethyl]-aminomethane (0.2 M) resulted in complete suppression of DNA degradation facilitated by AgNPs, as shown in Fig. 7, lanes 3 and 4 shown. Conversely, the effectiveness of dimethyl sulfoxide (0.2 M) in attenuating complete DNA degradation was observed to be limited (Fig. 7, lanes 2). The data obtained from the study provide convincing evidence that DNA degradation caused by AgNPs is facilitated by the involvement of singlet oxygen.

4 Conclusion

This research investigated the synthesis of silver nanoparticles (AgNPs) from Salvia elegans leaf extract and assessed their cytotoxic potential through DNA photocleavage. Characterization revealed that the AgNPs had an average diameter of 60 nm. Notably, these nanoparticles significantly induced DNA degradation at 37 °C in a dose-dependent manner, highlighting the pivotal role of AgNPs in this process. The study also confirmed the involvement of activated oxygen species in DNA damage, supported by previous findings that sodium azide and tris-[hydroxylmethyl]-aminomethane effectively inhibited such damage. These results provide empirical evidence for the capacity of AgNPs to induce DNA damage via singlet oxygen production. Consequently, AgNPs demonstrate considerable promise as cytotoxic agents, with potential applications in targeting cancer cells in therapeutic settings.

Future Implications: The implications of this research extend beyond the immediate findings, offering avenues for developing novel cancer therapies and enhancing our understanding of the interactions between nanomaterials and biological systems. Continued exploration in this field could lead to breakthroughs in cancer treatment and a more sustainable approach to nanotechnology. Several cancer cell lines are particularly relevant for evaluating the cytotoxic effects of AgNPs synthesized from S. elegans. Cervical cancer cells (HeLa cell line), widely used in cancer research, serve as a baseline for studying cytotoxicity. MCF-7 cells, which are estrogen receptor-positive breast cancer cells, are useful for investigating hormone-responsive tumors. A549 cells, a human lung adenocarcinoma line, are relevant for lung cancer therapies, while HCT116 cells aid in assessing the efficacy of AgNPs in gastrointestinal malignancies. Lastly, U87MG cells, representing glioblastoma, are crucial for exploring treatments for aggressive brain tumors.

CRediT authorship contribution statement

Mufarreh Asmari: Data curation, Conceptualization. A.H. Shridhar: Formal analysis. Joy H. Hoskeri: Methodology. B. Vinay Kumar: Writing – review & editing, Software, Project administration, Methodology. Nayef Abdulaziz Aldabaan: Validation, Methodology. Ibrahim Ahmed Shaikh: Writing – original draft, Validation. Abdulaziz Hassan Alhasaniah: Visualization. Mater H. Mahnashi: Visualization, Resources, Funding acquisition. Arun Shettar: Formal analysis. Basheerahmed Abdulaziz Mannasaheb: Validation, Methodology. Aejaz Abdullatif Khan: Validation, Formal analysis. Amal Bahafi: Methodology, Formal analysis. Uday M. Muddapur: Writing – review & editing, Methodology. S.M.Shakeel Iqubal: Writing – review & editing, Writing – original draft.