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Biosynthesis of Functionalized Silver Nanomaterials Utilizing Seeds of Black Rosehip (Rosa pimpinellifolia L.) and its Remarkable Antioxidative Capacity

Hamit Emre KIZIL1,*
  • 0000-0001-6193-3734
1Department of Medical Services and Techniques, Vocational School of Health Services, Bayburt University, Bayburt, Türkiye.

  • Submitted01-04-2025|

  • Accepted09-05-2025|

  • First Online 25-06-2025|

  • doi 10.18805/LRF-871

ABSTRACT

Background: Silver nanoparticles (AgNPs) have gained significant attention due to their unique antimicrobial, catalytic and optical properties. This study focuses on the green synthesis of silver nanoparticles using Black rosehip (Rosa pimpinellifolia L.) seeds (Rp-AgNPs) and evaluates their biological activities.

Methods: Rp-AgNPs were synthesized using R. pimpinellifolia seed extract and characterized using multiple analytical techniques including UV-Vis spectroscopy, XRD, FTIR, TEM, SEM, EDAX, zeta potential analysis, DSC, SERS and XPS. The biological activities were assessed through DPPH free radical scavenging assay, cytotoxicity testing against H460 lung cancer cells and antimicrobial activity evaluation against various pathogens.

Result: The synthesized Rp-AgNPs exhibited spherical morphology with an average size of 51.27 nm and demonstrated stability with a zeta potential of -18.9. The nanoparticles showed enhanced antioxidant activity (IC50 = 30.89 μg/mL) compared to the seed extract (IC50 = 136.4 μg/mL). Cytotoxicity studies revealed significant effects against H460 cells with an IC50 of 65.8 μg/mL.Antimicrobial testing demonstrated effectiveness against both Gram-positive and Gram-negative bacteria (MIC 50-100 μg/mL) and Candida strains (MIC 100-200 μg/mL).

INTRODUCTION

Silver nanoparticles (AgNPs) are easy to produce by biological methods, that is low-cost and environmentally friendly (Garg et al., 2020). AgNPs exhibit cellular interactions by binding to cell membrane proteins due to their small size (Kumar et al., 2020). This size-dependent behavior affects both their cellular distribution and biological activity, with smaller AgNPs generally showing greater cytotoxicity (Avalos et al., 2014). AgNPs have anticarcinogenic (Veeragoni et al., 2022), anti-inflammatory (Haider et al., 2020) and antimicrobial (Mousavi et al., 2018) effects. In a complementary investigation, Rengarajan and colleagues achieved successful AgNP synthesis using Calotropis gigantea leaf extract. Their methodological approach incorporated multiple advanced characterization techniques, including UV-visible and FT-IR spectroscopy, electron microscopy (SEM and TEM), XRD and EDX analysis. The resulting nanoparticles showed varied morphologies (spherical to cubic) while retaining key bioactive compounds from the original extract, such as tannins, alkaloids, flavonoids and polyphenols. These results support the wide therapeutic potential of plant-derived AgNP composites in biological and pharmaceutical applications (Rengarajan et al., 2024).
       
Medicinal and aromatic plants effect health and disease processes, especially due to presence of their phytochemical compounds and high antioxidant content that impart anticarcinogenic, antidiabetic (Taşci Çelikel et al., 2024; Orkun Erkılıç and Bayraktar, 2025a; 2025b) and antimicrobial effects (Bayraktar and Tekce, 2018; Bayraktar et al., 2023; Ozcan et al., 2024). Black rose is the fruit of a shrubby plant belonging to the Rosa genus of the Rosaceae family that sheds its leaves in winter. It contains tannins, phenolic compounds such as quercetin, kaempferol and catechin (Mármol et al., 2017); carotenoids (such as beta-carotene, lycopene, lutein and zeaxanthin) (Andersson et  al., 2011) as well as antioxidant (El Wafa, et al., 2025) anti-inflammatory (Rajabi-Moghadam et al., 2025), antimicrobial and anticancer effects (Mallick et al., 2024; Rajabi-Moghadam et al., 2025).The Rosa genus, especially Rosa pimpinellifolia, is an interesting candidate for green synthesis with its rich phytochemical content (Strålsjö et al., 2003). Recent studies have reported that it has the potential to treat non-small cell lung cancer (NSCLC) (Mármol et al. 2017; Demir et al., 2021; Kizil, 2023). Therefore, this study aims to investigate the green synthesis of silver nanoparticles using Black rose seeds and evaluate their biological activities.

MATERIALS AND METHODS

Preparation of the plant extract
 
Plant specimens of Rosa pimpinellifolia L. were collected from Gümüşsu Village, Bayburt Province, Türkiye (N:40o 13' 29.0064''; E: 40o 18' 11.8620'', 1,817 m elevation) Rosehip species identification was confirmed by a botanist from the Department of Organic Agriculture, Faculty of Applied Sciences, Bayburt University.
 
Green synthesis of silver nanoparticles (Rp AgNPs)
 
For the green synthesis of Rp-AgNPs, ground seeds were extracted in distilled water (100 g in 400 mL) at 45oC for 2 hours. The filtered extract was slowly added to silver nitrate solution (0.037 mM, 400 mL) and kept at 55oC for 2 hours until the color changed to brown, indicating nanoparticle formation. The synthesized Rp-AgNPs were purified by three centrifugation cycles (12,000 rpm, 20 minutes) and stored at 4oC (Gecer, 2021).
 
Characterization of Rp AgNPs
 
Synthesized Rp-AgNPs were comprehensively characterized using several techniques. 1UV-Vis spectrophotometry was performed with a Thermo Scientific Multiskan Go. Crystal structure was determined by XRD (Bruker D8 Discover, 2θ = 5o-80o). Average crystalline size (D) was calculated using the Debye–Scherrer formula:
 

Where,
λ = 0.154056 nm (CuKα).
β = FWHM in radians.
θ = diffraction angle.
K = 0.9.
       
Structural characterization included FTIR (Perkin Elmer spectrum device, 4000-450 cm-1) to examine bonds and functional groups (Büyüksirit and Kuleaşan, 2014). Morphological analysis used TEM (FEI Talos-F200S, 200 kV) and SEM (FEI-Quanta FEG 450). TEM samples were prepared on carbon-coated copper grids (200 mesh) with solvent evaporation. Composition was analyzed by EDAX and EDX. Surface charge was determined using a Malvern Instruments Zetasizer Nano ZSP. Thermal properties were investigated by DSC (Perkin Elmer 8000, 5 mg samples, 10oC/min to 500oC) (Niraimathi et al., 2013). Surface plasmon resonance of AgNPs (typically ~400 nm for spheres), influenced by various factors, was examined for its role in SERS (Stamplecoskie et al., 2011). SERS analysis (WITech Alpha 300R) provided insights into biomedical molecule properties (Bindhu et al., 2015). XPS (SpecsGroup Flexmod-Germany) was used for atomic/molecular structure analysis, quantifying seed extract-AgNP binding and elemental composition of Rp-AgNPs (Ag and O).
 
Antioxidant activity analysis
 
The antioxidant capacity of functionalized silver nanomaterials and R. pimpinellifolia seed aqueous extracts was evaluated using a DPPH. (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay with a 0.26 mM DPPH solution (SIGMA). Samples at various concentrations were incubated for 30 minutes at room temperature in the dark and absorbance was measured at 517 nm using UV-Vis spectroscopy (Fig 1). The DPPH scavenging effect was calculated using the equation:

 
Where,
A = Absorbance of the blank.
B = Sample absorbance at 517 nm. (Erenler et al., 2017).
 

Fig 1: DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay.



Cytotoxicity assessment of Rp-AgNPs
 
Cytotoxicity was evaluated using the H460 non-small cell lung cancer cell line (ATCC, USA). Cells were cultured in RPMI medium (Gibco) with 10% FBS (Gibco) in 25 ml flasks and maintained at 37oC in 5% CO2 for 24-48 hours, with PBS (Sigma) washing and trypsin-EDTA (Gibco) for detachment. Cell viability was checked with trypan blue staining (Sigma) and cell counting was done using a Thoma slide after centrifugation (Gecer, 2021). Cell viability was quantified using the WST-8 assay with a CVDK-8 kit (Ecotech Biotechnology®, Türkiye). The procedure included: 1. Seeding H460 cells in 96-well plates for 24 hours. 2. Treating with plant extract (200-12.5 μg/mL in PBS). 3. Incubating for another 24 hours. 4. Assessing viability after 72 hours per kit instructions. 5. Adding 10 μl CVDK-8 reagent and incubating for 3 hours. 6. Measuring absorbance at 450 nm (MultiSkan Go, Thermo Scientific). Statistical significance was set at p<0.05 (Kizil et al., 2023).
 
Antimicrobial activity analysis
 
Disc diffusion assay
 
Rp-AgNPs’ antimicrobial efficacy was assessed against a panel of pathogens (3 Gram-positive, 3 Gram-negative bacteria, 4 yeast-like fungi) using disc diffusion and broth microdilution. Cultures revived on selective media were incubated, pure colonies transferred to liquid media and incubated and standardized suspensions were spread on fresh media. Sterile discs with Rp-AgNPs were placed on inoculated surfaces, incubated and inhibition zones measured (Boztaş et al., 2024).
 
Broth microdilution method
 
The minimum inhibitory concentration (MIC) of Rp-AgNPs against selected pathogens was determined using broth microdilution in 96-well plates. Wells were prepared with 95 µL liquid medium (Mueller Hinton broth for bacteria, Saboraud Dextrose broth for fungi) and 5 µL standardized inoculum (100 µL total). A two-fold serial dilution of Rp-AgNPs (100 µL added to first wells, 100 µL transferred down to the eighth well) was performed. Microplates were incubated at 37oC with shaking (40 rpm) for 24 hours. Then, 5 µL aliquots from each well were transferred to agar plates and incubated for another 24 hours at 37oC. The MIC was the highest concentration showing no microbial growth (Keskin et al., 2023).
 
Statistical analysis
 
All experiments were performed in triplicate and the results were analyzed using GraphPad Prism software (version 8.0.1, United States). The DPPH free radical scavenging activity was evaluated through one-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean±standard deviation, with statistical significance set at p<0.05. Cytotoxicity data, also obtained from triplicate experiments, were analyzed using Student’s t-test and the results are expressed as mean±standard deviation, with statistical significance similarly defined at p<0.05.

RESULTS AND DISCUSSION

The pulverized Black rosehip seeds (100 g) were ground using a coffee grinder and heated with distilled water (400 mL) at 45oC for 2 hours.. The mixture was filtered through Whatman filter paper and a silver nitrate solution (0.04 mM, 400 mL) was gradually added to the extract. The reaction mixture was further heated at 60oC for 2 hours, during which the color changed from orange to dark brown. After the reaction, Rp-AgNPs were collected by repeated centrifugation at 12,000 rpm for 20 minutes and washed thoroughly with distilled water. Finally, the Rp-AgNPs were dried via lyophilization (Fig 2).

Fig 2: Aqueous solutions of Rosa pimpinellifolia L. seeds (A) and Rp-AgNPs (B).


       
The successful synthesis of silver nanoparticles was confirmed by UV–Vis spectroscopy, with a maximum absorption peak at 476 nm, consistent with the typical range of 400–480 nm reported in the literatüre (Fig 3),(Sreeram et al., 2008).

Fig 3: UV-Visible absorption spectra of Rp-AgNPs (A) and Aqueous solutions of Rosa pimpinellifolia L. seeds (B).


       
The XRD analysis of Rp-AgNPs (Fig 4) revealed prominent peaks at 38o, 44o, 64o and 77o, corresponding to the fcc planes (111, 200, 220 and 311) of AgNPs (Rahimi-Nasrabadi et al., 2014). Additional peaks likely indicate metabolites coating the nanoparticles. The calculated crystallite size was 51.93 nm (Rahimi-Nasrabadi et al., 2014).

Fig 4: XRD spectrum of Rp-AgNPs.


       
FTIR analysis was performed to identify functional groups in the R. pimpinellifolia seed extract involved in the synthesis and stabilization of silver nanoparticles. The spectra of the extract and Rp-AgNPs (Fig 5) revealed peaks at 3304.33 cm-1 and 2926.6 cm-1 (O-H stretching), 2855.12 cm-1 and 2111.63 cm-1 (N-H and CºC stretching), 1743.53 cm-1 (C=O stretching), 1637.05 cm-1 (C=C stretching), 1548.06 cm-1 (N-O stretching), 1450.16 cm-1 (C-H bending), 1317.96 cm-1 (C-F stretching and O-H bending), 1242.21 cm-1 (C-O and C-N stretching), 1063.63 cm-1 (S=O and C-O stretching), 969.28 cm-1 and 896.13 cm-1 (C=C bending) and 586.43 cm-1 (C-I stretching).

Fig 5: FTIR spectra of Rp-AgNPs (A) and Aqueous solutions of Rosa pimpinellifolia L. seeds (B).


       
The morphology and size distribution of nanoparticles produced through biological synthesis were examined using TEM, a robust analytical method. The TEM image reveals individual silver particles and illustrates the polydispersity of the nanoparticles. Notably, the silver nanoparticles exhibit an almost spherical shape (Fig 6).

Fig 6: TEM image of Rp-AgNPs nanoparticles. Image scale bar.


       
The surface morphology and dimensions of silver nanoparticles produced under optimal conditions were assessed through SEM, as illustrated (Fig 7).

Fig 7: SEM images of Rp-AgNPs magnified at 25,000×, 50,000×, 100,000× and 200,000×.


       
The surface morphology was also identified to possess a spherical nature, with an average size measuring 51.27 nm (Fig 8).

Fig 8: SEM image of nanoparticles from 16 points.


       
In the EDAX analysis, a distinct peak of silver metal was observed at 3 kV, confirming the presence of silver in the complex. Additionally, the peaks corresponding to other elements in the EDAX spectrum are illustrated (Fig 9).

Fig 9: EDAX spectrum of silver nanoparticles synthesized utilizing black rosehip seeds.


       
Zeta potential measures the electrical potential at the solid-liquid interface, influencing nanoparticle stability. Rp-AgNPs showed a zeta potential of -18.9, indicating stability and particle repulsion (Fig 10), (Gecer, 2021).

Fig 10: Zeta potential distribution of Rp-AgNPs.


       
Differential scanning calorimetry (DSC) was used to analyze the thermal stability of Rp-AgNPs. The DSC curve showed endothermic and exothermic processes, with an exothermic peak at 92°C indicating desorption of organic components from the nanoparticle surface (Fig 11).

Fig 11: Differential scanning calorimetry (DSC) of Rp-AgNPs.


       
Surface-enhanced Raman scattering (SERS) is a highly sensitive technique for detecting and characterizing metal nanoparticles (Bindhu et al., 2015). The Raman spectra of silver nanoparticles (AgNPs) showed vibrational modes at 238 cm-1 (Ag-O stretching)  (Linic et al., 2015). 613 cm-1 (C-S-C) and 1301 cm-1 (anti-symmetric C=O stretching) (Joshi et al., 2018) (Fig 12).

Fig 12: RAMAN scattering spectra of the Rp-AgNPs.


       
X-ray Photoelectron Spectroscopy (XPS) was used to analyze the surface composition and chemical state of Rp-AgNPs. The XPS spectrum (Fig 13) revealed Ag 3d 5/2 peaks at binding energies of 365.1 and 369.0 eV, corresponding to oxidized silver (Ag-O) (Maiti et al., 2016).

Fig 13: XPS spectra of Ag 3d 5/2, O 1s and C 1s peak of the Ag NPs functionalized with seed aqueous extract.


       
DPPH free radical scavenging activity showed that ascorbic acid (IC50: 11.53 μg/mL) had the highest activity, followed by Rp-AgNPs (IC50: 30.89 μg/mL) and R. pimpinellifolia seed extract (IC50: 136.4 μg/mL). This is the first study on silver nanoparticles functionalized with R. pimpinellifolia seeds. The cytotoxicity of Rp-AgNPs on H460 lung cancer cells was evaluated after 72 hours, showing significant cytotoxicity (p<0.05) at concentrations ≥25 μg/mL, with an IC50 of 65.8 μg/mL (Table 1).

Table 1: Effects of Rp-AgNP concentration-dependent cytotoxicity on H460 lung cancer cells on survival after 72 hours.


       
The antimicrobial activity of Rp-AgNPs synthesized using Rosa pimpinellifolia seed extract was evaluated against various pathogenic microorganisms. The disk diffusion method and broth microdilution technique were employed to assess in vitro activity, with results summarized in Table 2.

Table 2: In vitro antimicrobial activities of silver nanoparticles (Rp-AgNPs) prepared using Rosa pimpinellifolia seed extract.


       
The increasing issue of antibiotic resistance (Nikolich and Filippov, 2020) has driven interest in alternative antimicrobial strategies, such as nanoparticle-based approaches (Murugaiyan et al., 2022). R. pimpinellifolia has a high polyphenol content (121.38 mg GAE/100 g fresh weight) and shows tolerance to various stresses (Fattahi et al., 2012). Rp-AgNPs at 100 µg/mL showed inhibition zones of 10-14 mm for bacteria and 7-8 mm for Candida strains, with MIC values of 50-100 µg/mL for bacteria and 100-200 µg/mL for Candida. Rp-AgNPs demonstrated in vitro antimicrobial activity against all tested pathogens, consistent with previous findings (Fattahi et al., 2012).
               
Hormones are biomolecules that are involved in many physiological processes that affect the growth and development of cancer cells (Bayraktar and Bayraktar, 2019; Bayraktar, 2020). Plant-synthesized AgNPs have a promising potential in anticancer treatments because they can affect the development of some types of cancer by affecting the endocrine system. Juarez-Moreno et al., (2017) demonstrated effective cytotoxicity across multiple cancer cell lines without DNA damage, suggesting potential applications in combination therapy. Studies with various plant-derived AgNPs have reported significant anticancer activity, with IC50 values ranging from 28.125 μg/mL for lung cancer to 62.5 μg/mL for breast cancer cells (Vijayan et al., 2018; Valsalam et al., 2019). Green-synthesized AgNPs are particularly promising due to their ability to generate ROS and penetrate cells (Jabeen et al., 2021). Furthermore, silver nanoparticles synthesized using black rosehip seed water extract maintained their antibacterial activity for over 8 weeks when stored at 4oC.

CONCLUSION

In our current study, Rp-AgNPs showed strong antimicrobial activity against bacterial and fungal pathogens and remained stable for over 8 weeks at 4oC. It is thought that Rp-AgNPs will be beneficial and contribute to the treatment of wound infections using environmentally friendly methods thanks to their potential in combating antibiotic resistance. Future research should address these gaps to better explore the biomedical potential of these flavonoid-enriched AgNPs, which also show promise for food and pharmaceutical applications.

ACKNOWLEDGEMENT

I would like to thank Associate Prof. Dr. Mehmet Semih Bingöl from Atatürk University, Associate Prof. Dr. Sinan Bayram and  Associate Prof. Dr. Bülent Bayraktar from Bayburt University for their valuable contributions and support.
 
Preprint statement
 
This paper has been uploaded to Authorea as a preprint: DOI: 10.22541/au.172653476.63929696/v1.
 
Disclaimers
 
The authors are solely responsible for the content and its accuracy, which does not necessarily reflect their institutions’ views. They accept no liability for any losses from its use.
 
Informed consent
 
This study was approved by the Ethics Committee of Bayburt University (Decision No: 37/04, Date: 04/30/2024) in accordance with the Declaration of Helsinki principles. All methods were carried out in accordance with relevant guidelines and regulations and all experimental protocols were approved by the Ethics Committee of Bayburt University. The study utilized the NCI-H460 lung cancer cell line obtained from American Type Culture Collection (ATCC | USA). As this research was conducted using commercially available cell lines and did not involve direct human participants, human material, or human data collection, it did not require clinical trial registration, participant consent, or consent to publish.

CONFLICT OF INTEREST

The authors declare no conflicts of interest and the study was not influenced by funding or sponsorship.

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