The silver nitrate (AgNO₃) with ≥99.0% purity was purchased from Sigma Aldrich. The Nutrient Broth and Nutrient Agar (HiMedia) were purchased from commercial sources and used without further purification. The sheep’s tail was purchased from the local market of Ulaanbaatar, Mongolia. The ointment was prepared according to the traditional method; in brief, the sheep’s tail was cut into small pieces and melted at a low temperature to obtain the absorption-based ointment. Afterward, the oil was filtered for further use in the study. 0.5% AgNP ointment was prepared by adding dry fine powder form of AgNP into sheep’s tail cream. All the other relevant reagents are up to standard.

The roots of Rhodiola rosea were washed with tap water to remove the adhering dust and soil particles, followed by washing with distilled water. The plant’s roots were cut into fine pieces for further study. Plant extracts were prepared by adding 100 ml of distilled water to 5 g of roots from Rhodiola rosea. Afterward, it was boiled for 15 min and cooled at room temperature, then centrifuged twice at 10,000 rpm/20 min. Finally, the plant extract is ready for the subsequent study.

The plant extract of Rhodiola rosea was added to the 10⁻³M AgNO₃ solution with a ratio of 1:32. The reaction took place at ambient temperature with direct exposure to sunlight. To obtain the dry powder form of AgNP, the nanoparticles were centrifuged three times at 18,000 rpm for 20 min and washed with deionized water. Afterward, pellets were kept inside the oven at a temperature of 400°C for 3 h.

The color change from pale yellow to colloidal dark brown as a function of time indicates the presence of AgNP. The UV-Vis spectrophotometer (Shimadzu UV-2500PC Series) records the absorption spectra; the maximum absorbance of spectra shows the size and yield of the synthesized AgNP. The colloidal AgNP was measured at different time intervals from 5 min to 4 h within 300–700 nm wavelength. The biomolecules of the roots of Rhodiola rosea responsible for reducing silver ions and stabilizing AgNP were determined via FTIR spectrophotometer (Prestige-21, Shimadzu, Japan) operating within the range of 500–4000 cm⁻¹ through the potassium bromide powder method. The Zeta Potential (ZetaCompact, CAD Instruments, France) was used to determine the surface charge of the synthesized AgNP. The size and shape of the synthesized silver nanoparticles were measured with Scanning Electron Microscope (ZEISS, Germany). Elemental analysis of the synthesized AgNP was performed on an Energy Dispersive X-ray spectroscope instrument (TM-10000 with EDX). The XRD analysis was employed to determine the average crystallite size of the synthesized AgNP. XRD (Shimadzu, Maxima-X-7000) with CuKα radiation and operating at 40 kV with a current of 30 mA in the range of 20°–90°. The average crystallite size of the synthesized AgNP was determined with the Scherrer equation. The particle size distribution of the biosynthesized AgNP was studied using Photon Cross-Correlation Spectrum (PCCS) on the NANOPHOX NX0137 instrument (Sympatec GmbH, Germany).

The antibacterial activity of the synthesized AgNP with Rhodiola rosea (300 and 450 μg/ml) was assessed on Staphylococcus aureus and Pseudomonas aeruginosa using the agar well diffusion method. Standard antibiotics (Biolab ltd., Budapest, Hungary) including erythromycin 15 mcg/disc (P. aeruginosa) and penicillin G 6 mcg/disc (S. aureus), plant extract and HEPES buffer were selected as the positive and negative control group of the study. First, the bacteria were cultured in the liquid broth medium at 37°C until it reached OD₆₀₀ = 0.6. Afterward, 100 µl of the respective bacterial suspension was spread on the agar plates. The samples and control groups were transferred and filled into the well on the agar plates, followed by incubation at 37°C for 24 h. Finally, the diameter of the inhibition zone around the wells was measured and analyzed.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of National University of Mongolia. BALB/c mice (n = 10) were housed in two separate cages with free access to water and standard rodent chow. The environment was controlled at 24–26°C under 12 h light/dark cycle. All mice were anesthetized with an intraperitoneal injection of ketamine (13 mg/kg) and xylazine (8 mg/kg) (Gomathi et al., 2017). Afterward, the dorsum area of the mice was swabbed with ethanol 70% solution before shaving.

Before inducing burn injury, BALB/c mice were randomly divided into two groups (n = 5 each): a control group and AgNP-ointment treatment group. Burn injury was induced by applying 38% HCl solution to the dorsum area of the mice for 1 min. AgNP-ointment was used twice a day in the treatment group.

The closure of the burn injury was recorded using a digital camera on different days, and the surface area of the burn injury was measured with ImageJ software. The healing rate of burn injury was expressed as the change in a wound area relative to the original wound area. The mice were sacrificed at 1 and 4 days; their skin tissue was collected from the wound site and fixed in 4% buffered formaldehyde. Afterward, the skin tissue samples were embedded in paraffin, and 10 µm thick sections were obtained using a rotary microtome. Sections were stained by Hematoxylin and Eosin (H&E) and Toluidine Blue staining methods.

After inflicting burn injury, the mice were euthanized by cervical dislocation on day 4. Tissue samples were collected from the dorsum area of the mice to isolate RNA for PCR analysis. Each tissue sample was homogenized in 1 ml of Trizol (Sigma Aldrich) reagent with 0.2 ml of chloroform (Chen et al., 1007). After centrifugation at 13,000 rpm at 4°C for 20 min, the supernatant containing RNA was transferred into a new vial and the RNA was precipitated with isopropanol. After an incubation period of 10 min and another centrifugation step of 15 min at 13,000 rpm, the supernatant was discarded. The pellet was washed with 1 ml of 70% ethanol and centrifuged for 15 min at 13,000 rpm. The supernatant was then discarded and the pellet dried. After adding 50 μl diethylpryocarbonate (DEPC)-treated water, the pellet was allowed to dissolve on ice for 10 min. The amount and purity of total RNA were determined using 260 and 280 nm absorbance measured by a spectrophotometer. cDNA library was developed with an RNA template using M-MLV RTase. Primer sequences used for PCR are listed in Table 1. The expression level of each gene was normalized with GAPDH. The ImageJ software (National Institutes of Health, Bethesda, MD) was used to conduct a quantitative analysis of PCR amplicons on digitized gel images.

All data expressed as the mean ± standard deviation (SD) and statistical significance were analyzed with a Student t-test, and a value of p < 0.05 was considered significant.

The presence of AgNP was first identified by color change as a function of time. The mixture turned brown immediately after adding plant extract to the AgNO₃ solution and later changed to a darker shade (Figure 1A). The reaction mixture was observed from 5 min to 4 h. The change in color of the colloidal solution confirms the bioreduction of silver ions due to the plant extracts. The dark brown color of the colloidal solution occurs as a result of light absorbance in the visible region and collective oscillation of free electrons of AgNP, referred to as surface plasmon resonance (SPR). The color change from pale yellow to dark brown for the synthesized AgNP is in line with other published reports (Javed and Mashwani, 2020; UP et al., 2020; Vorobyova et al., 2020). UV-Vis spectroscopy analysis further confirmed the formation of the AgNP synthesized with Rhodiola rosea. The method is utilized in the preliminary stages to determine the size of AgNP, synthesis rate, yield, and stability by measuring the optical absorbance spectra of nanoparticles. The measurement results revealed the maximum absorbance at around 430 nm with a single narrow SPR peak, confirming the presence of spherical and monodisperse AgNPs (Figure 1B). An appearance of an SPR peak between 400 and 500 nm indicates the presence of synthesized silver nanoparticles (Sherin et al., 2020). The SPR peak value or wavelength provides approximate size information. Characterization on UV-Vis spectrophotometer showed a stable absorbance as a function of time and absence of peak shift that correlates to high yield and stability of AgNP. The flavonoids and phenolic compounds in the plant extract may be the main reducing and capping agents for forming AgNPs. Several studies reported that the flavonoid is the main compound responsible for reducing and capping AgNP (Alomar et al., 2020).

The surface chemistry, functional groups, and residues adhered to the nanoparticles can be studied using the FTIR technique (Nayaka et al., 2020). AgNPs were synthesized with Rhodiola rosea. The synthesized AgNP was analyzed by FTIR spectroscopy to study the functional groups responsible for reducing and capping nanoparticles (Figure 2). FTIR analysis results further prove the crucial role of biologically active compounds in the plant extract in reducing and capping the AgNP. The functional groups detected in the AgNP is as follows, the bands were identified at 532.38, 670.29, 833.28, 987.60, 1021.35, 1068.61, 1125.51, 1177.59, 1320.33, 1426.42, 1553.73, 1642.46, 1695.50, 2361.94, 2931.93, 3419.94, and 3740.13 cm⁻¹. The broadband at 3419.94 cm⁻¹ is related to O-H stretching, which may refer to phenols, alcohols, or the NH functional group of amides or amine (Hafeez et al., 2021). Other bands found in AgNP, such as 1426.42, 1553.73, 1642.46, 1695.50, 2361.94, and 2931.93 cm⁻¹ are assigned to S=H sulfur ester, C–O stretch, C=C stretch, C=O stretch, P–H phosphine and C–H stretch respectively.

Zeta Potential is an essential parameter for understanding the synthesized nanoparticles’ stability and surface charge in the colloid system. Nanoparticles can be considered stable if the zeta potential values are above +30 mV or below −30 mV; this is due to the electrostatic repulsion between nanoparticles in suspension (Kumar et al., 2019). The silver nanoparticle synthesized with the medicinal plant Rhodiola rosea revealed a zeta potential of −68.38 ± 3.4 mV, indicating strong stability and a low tendency to aggregation (Table 2). The negative zeta potential implies that the synthesized silver nanoparticle is capped and stabilized by an electronegative compound, which may be the biomolecules within the Rhodiola rosea plant.

The crystalline nature of the synthesized AgNP with Rhodiola rosea was studied and carried out by X-Ray crystallography. The XRD pattern of the nanoparticles was analyzed with an XRD instrument and shown in Figure 3. Bragg reflection of the 2θ peaks was observed at 20° to 90° and corresponded to (111), (200), (220), (311), and (222) plane lattice, which can be indexed to the face-centered cubic crystal nature of the silver with crystallite size of 23 nm. The result matches the reported reference value of ICSD No. 064994. PCCS is a technique that measures the average nanoparticle size (grain size) based on the Brownian motion. In Figure 4, the average particle size of AgNP was approximately X₉₀ = 67.5 nm, SMD = 57.7 nm, and VMD = 58.4 nm. The difference between PCCS and XRD analysis lies in the measurement method of the particle. Scanning Electron Microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDX) characterized the morphological and elemental analysis. EDX analysis revealed the elemental composition of the synthesized AgNP (Figure 5), which included 81.9% silver, 2.6% chlorine, 13.8% phosphorus, and 1.7% potassium elements (Table 2). In addition, the nitrogen element was not detected in the sample, implying no presence of trace ions from AgNO₃. And the SEM imaging has confirmed the presence and spherical shape of the synthesized AgNP and monodisperse size distribution of ∼23 nm (Figure 6).

The antibacterial activity of AgNP (300 μg/ml and 450 μg/ml) was evaluated against P. aeruginosa and S. aureus bacteria. The erythromycin and penicillin G were selected as the positive control, and the plant extract and HEPES buffer as the negative control group of the study. Figure 7A shows the diameter of the inhibition zone of AgNP, the results reveal antibacterial activity against both P. aeruginosa and S. aureus bacteria. The plant extract and HEPES buffer did not show antibacterial activity on the studied bacteria. In addition, P. aeruginosa bacteria were more susceptible to the synthesized AgNP compared to S. aureus bacteria (Figure 7B), this may be due to the difference in bacterial cell wall structures (Vijayan et al., 2018). However, the AgNP synthesized with Rhodiola rosea exhibited an effective activity against both pathogenic bacteria. Currently, the antibacterial effect of AgNP is not clearly understood yet. It has been postulated that the antibacterial mechanism of action is a result of a simultaneous process (Hambardzumyan et al., 2020): 1) penetration of AgNP in the cell wall of bacteria leading to cellular content leakage (Rajesh et al., 2020); 2) silver ion release (Vorobyova et al., 2020); reaction with sulfhydryl groups inside the cytosol (Mickymaray, 2019).

After post-burn injury of the murine model, the TNF-α, IL-1β, and IL-6 expression levels in the treatment group were significantly less than in the control group (Figure 9). On the other hand, there was an elevation of IL-10 expression in the treatment group. The anti-inflammatory cytokine IL-10 prevents excessive inflammation during the wound healing phase by inhibiting the synthesis of pro-inflammatory cytokines, including IL-6, IL-1, and TNF-α. The down-regulation of pro-inflammatory cytokine IL-6 leads to less macrophage and neutrophil recruitment on the wound site, thus fewer cytokine releases that may prolong the inflammatory phase, thereby delaying the wound healing. Additionally, TNF-α is considered the first responder to inflammation, and the cytokine activates keratinocytes and macrophages to release ROS, iNOS, and IL-1β. Overall, biosynthesized AgNP revealed that it could suppress pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, and promote the expression of anti-inflammatory cytokine IL-10 on burn injury.

The inflammatory response is an essential part of the wound healing process and one of the significant causes of burn complications. The effect of AgNP-ointment on burn wound healing was studied using macroscopic evaluation of burn wound surface area and changes in spleen length using digital photographs (Figure 10). The burn wound healing is shown in Figure 10; there was a significant difference in the closure of the burn wound surface area between the control and treatment groups by 61 ± 9 mm² and 5 ± 4 mm² respectively (Figure 10E). All tested mice in the control group developed scabs on the wound site, while mice in the treatment exhibited newborn hair follicles on the wound site (Figures 10A–D). Overall, the macroscopic appearance and closure of the burn wound area suggest accelerated wound healing with the application of AgNP-ointment. There were no significant changes in spleen length between the control and treatment groups which signifies that AgNP-ointment did not exert adverse systemic toxicity in the spleen (Figure 10F). The regeneration of the epithelial layer and mast cell migration of the control and treatment groups was investigated using H&E, Toluidine Blue staining (Figure 11). The re-epithelization process involves the thickening to thinning of the epithelial layer, where cells evolve from proliferation to differentiation until skin thickness is normalized (Wang et al., 2020). In the control group, a thick epithelial layer is observed after inflicting burn injury; in contrast, the AgNP-treatment group exhibited thinning of the epithelial layer (Figures 11A–D). Subsequently, Toluidine Blue staining also revealed pronounced mast cell migration in the control group; in comparison, mice treated with AgNP-ointment were infiltrated with fewer mast cells on the wound site (Figures 11E–H).