Bovine mastitis is a destructive disease of cattle that causes significant economic losses in the dairy industry (1). Staphylococcus aureus is a common cause of bovine mastitis. The disease is linked to oxidative stress from bacterial invasion, as indicated by changes in the oxidative stress parameters in the blood (2, 3). During inflammation, phagocytes produce reactive oxygen species (ROS) that destroy the bacteria (4). Excessive ROS production can overwhelm the antioxidant system and adversely affect the immune system of cows (5). ROS can oxidize macromolecules, such as proteins, lipids, and deoxyribose nucleic acid (DNA), causing oxidative cell damage and altering metabolic pathways (6). Oxidative stress can enhance the adherence of active neutrophils to mammary endothelial cells, worsening inflammation (7). Clinical and subclinical mastitis leads to the release of free radicals and a reduction in the total antioxidant capacity (8). Severe mastitis results in antioxidant imbalance due to excessive peroxynitrite production (9). Evaluating peroxidative damage products (TBARS) and antioxidants, such as glutathione and enzymes (SOD, GPx, and catalase), may serve as markers of oxidative stress and antioxidant status (10). Mastitis alters redox potential, increases oxidative free radicals, and decreases protective antioxidant enzymes (10). In addition to oxidative stress, bacterial infections in mastitis are difficult to combat because of the ability of bacteria to evade the host immune response through biofilms, exotoxins, proteases and bacterial superantigens, and by adhering to mammary epithelial cells (11). Staphylococcus aureus induced mastitis poses a significant challenge in the dairy industry because of the ability of bacteria to survive in phagocytes and epithelial cells, rendering antibiotic treatment ineffective (12). Therefore, alternative treatment options are needed. Studies indicate that adequate antioxidant intake in dairy cows enhances immunological functions such as phagocytosis, bacterial killing, and neutrophil oxidative metabolism (13). Recent studies have highlighted that inorganic nanoparticles effectively scavenge reactive oxygen species (14, 15).

Nanomedicine is an emerging field that involves the fabrication of nanoparticles for therapeutic applications (16, 17). Nanoparticles exhibit unique physicochemical properties (17). Various materials, including metals, metal oxides, and silicates, have been used to create nanoparticles (18). Noble metals like copper (Cu), silver (Ag), gold (Au), and titanium (Ti) are commonly used for nanoparticle fabrication (19).

While AgNPs can induce oxidative stress in disease-causing organisms, which indirectly reduces free radical generation, their free radical scavenging activity is attributed to the functional groups present on their surfaces (20). Zinc oxide nanoparticles have demonstrated antioxidant properties in both intracellular and extracellular environments (21). By activating antioxidant enzymes, ZnO nanoparticles reduce the quantity of free radicals intracellularly, whereas their use of electron transfer reduces free radicals in the extracellular environment to perform their free radical scavenging action (22). However, green-synthesized nanoparticles have been found to have higher antioxidant properties, which is attributed to the capping and stabilizing properties of various phytochemicals involved in their production (23).

The production of large quantities of nanoparticles often involves physical techniques that can yield highly pure nanoparticles; however, these techniques typically require expensive equipment, high pressures and temperatures (24, 25), as well as a significant amount of energy. Alternatively, chemical processes such as chemical reduction and electrochemical and sol–gel processes can also be used to create nanoparticles, but these methods may produce hazardous or polluting waste due to the inclusion of toxic reagents or solvents (26, 27).

The synthesis of nanoparticles using green methods primarily involves the incorporation of cell extracts, such as those derived from plants, microorganisms, algae, and fungi, into a substrate without the use of harmful chemicals. The aerial parts of plants, such as the leaves and flowers, are frequently utilized in green synthesis. Numerous researchers have found that proteins and secondary metabolites present in plant extracts serve as reducing and capping agents that promote the production of nanoparticles (28, 29). Phytochemicals, such as vitamins, amino acids, polysaccharides, terpenoids, alkaloids, and other compounds extracted from plants, help in the effective bio-reduction of metal ions during the synthesis of nanoparticles, which exhibit stability and variability in their structure and dimension. Plant components, ranging from leaves to roots, are widely used to produce metal oxide nanoparticles.

Thespesia populnea of the Malvaceae family, commonly known as the Indian tulip tree, is widely distributed in the southeastern and coastal forests of India. The bark, blossoms, and leaves of this tree, also known as the portia tree, possess medicinal benefits that can be used to treat skin infections (30). Research has shown that T. populnea leaves contain flavonoids, tannins, saponins, terpenoids, polyphenols, glycosides, alkaloids, quercetin, phytosterols, lupeol, and rutin (31, 32). The phytochemicals found in T. populnea have been shown to possess anti-inflammatory, anti-diarrheal, antibacterial, antifungal, and haemostatic properties so it is used in traditional medicinal systems like Sidha and Ayurveda especially the bark and leaves are often used in decoctions or poultices, while the fruits and seeds are be used in oil preparations (33, 34). However, studies examining the effectiveness of T. populnea herbal extract in eliminating oxidative stress related to bacterial mastitis using metal nanoparticles are limited. Considering this, the current study aimed to investigate the green synthesis and characterization of Ag and ZnO nanoparticles from T. populnea leaf extract, as well as the antioxidant activity of these nanoparticles in the treatment of mastitis in a murine model.

This study focuses on the synthesis and characterization of silver (AgNPs), ZnO nanoparticles derived from Thespesia populnea extract using green synthesis approach. The main objective is to evaluate the antimicrobial activity of the nanoparticles against Staphylococcus aureus induced mouse mastitis model, assess their antioxidant properties in vivo, and investigate their potential for reducing oxidative stress in mastitis model. The study did not include long-term toxicity assessments and the plant extract was sourced during a single season, which may limit the seasonal variability of its bioactive compounds.

The experiment was conducted at the Department of Veterinary Biochemistry, College of Veterinary Science, Rajendranagar, Telangana, India, using T. populnea leaves collected from Andhra Pradesh, India which were harvested during the flowering season (February to March). Higher concentrations of bioactive compounds were found during this period in Thespesia populnea.

Synthesis and Characterization of Nanoparticles UV–VIS Analysis: Figures 1A,B display the UV–visible absorption spectra of the TPNS and TPNZ particles, respectively. TPNS particles exhibited a maximum absorbance peak at 421 nm, confirming the bioreduction of Ag⁺ to Ag (0). The absorption spectrum of the TPNZ particles, recorded between 200 and 800 nm, showed a peak at 260 nm, indicating the formation and stability of ZnO nanoparticles.

Green synthesis of nanoparticles, leveraging various phytochemicals in plant extracts, is biocompatible and environmentally friendly, making it efficient for large-scale biomedical applications (44). In this study silver and ZnO nanoparticles were synthesized using T. populnea methanolic leaf extract and characterized by UV–VIS analysis, SEM, DLS, and Zeta potential measurements. The addition of 1 mM silver nitrate and zinc acetate to T. populnea leaf extract resulted in a color change, confirming the production of TPNS and TPNZ (45, 46). UV–VIS spectroscopy indicated peaks at 421 nm and 260 nm for TPNS and TPNZ, respectively, suggesting bioreduction of aqueous silver ions (Ag+) upon exposure to plant extracts. Phytochemicals in T. populnea leaf extracts facilitate the transformation of silver ions into metallic nanoforms (47). Previous studies have shown peaks around 420 nm for T. populnea-synthesized silver nanoparticles (48) and 295 nm for TPNZ (49), while ZnO nanoparticles from Deverra tortuosa and the aqueous extract exhibited peaks in the 200–800 nm range (50), which is consistent with the findings of this studyTPNS and TPNZ particles were further characterized using SEM to examine their morphologies and structures. The SEM image analysis in this study revealed the formation of elliptical to spherical agglomerated TPNS, consistent with the findings of Bhuyar et al. (51) and Widatalla et al. (52) using Padina sp. and green tea leaf extracts. SEM images of TPNZ showed uniformly distributed spherical particles, aligning with results from Yedurkar et al. (53) and Muhammad et al. (54), who synthesized spherical ZnO nanoparticles using Ixora coccinea and Papaver somniferum leaf extracts, respectively. DLS, a technique for measuring particle size through laser beam analysis of Brownian motion in suspension, revealed sizes of 99 nm for TPNS and 87.7 nm for TPNZ. Similar diameters were reported for TPNS synthesized from Rizophora apiculata (99 nm) (55). Comparable sizes of 70 and 100 nm have reported for TPE-mediated silver nanoparticles (48) and M. oleifera seed extract-mediated silver nanoparticles (56). Sundrarajan et al. (57) reported a size of 100 nm for Pongamia pinnata leaf extract-mediated nano ZnO particles via DLS, while Shukla et al. (58) showed a size range of 76.2 to 183.8 nm for Zinc oxide nanoparticles synthesized from Aspergillus niger.

The zeta potential method is crucial for estimating the surface charge of nanoparticles, which is essential for their characterization and understanding the physical stability of nanosuspensions (19). Studies (59) have indicated that stable particles have a zeta potential of ≥ + 30 mV or ≤ −30 mV. A positive charge value of +37.4 mv of zeta potential for silver nanoparticles synthesized using Morus alba leaf extract were reported by Das et al. (60).

TPNS displays higher zeta potential than TPNZ, suggesting that TPE can effectively mediate nano-silver compared to nano ZnO particles.

Reactive oxygen species (ROS) are the natural byproducts of cellular metabolism. Oxidative stress occurs when ROS production exceeds the antioxidant defense capacity (61). In dairy cattle, both clinical and subclinical mastitis increase free radical production, increase total oxidant capacity, and reduce total antioxidant capacity (8). Lipid peroxidation products, particularly polyunsaturated fatty acids susceptible to free radical attack, are commonly used as oxidative stress markers, with TBARS being a widely recognized indicator (62). The elevated TBARS levels in Group II indicated oxidative stress. Among the treated groups, higher restoration of TBARS values was observed in group IV, followed by VI, V, and III, suggesting that TPNS had a stronger antioxidant effect than ceftriaxone, TPNZ, and TPE alone. Siddique and Al-Samman (63) observed a similar decrease in TBARS with Delphinium denudatum wall. Root extract-mediated AgNPs in mice with nephrotoxicity. However, aloin-mediated nano-silver and 11-α-keto boswellic acid-mediated nano-silver did not effectively exert antioxidant effects (64, 65). Kiyani et al. (66) reported a significant (p < 0.01) reduction in TBARS, approaching control values, in gout-affected mice treated orally with nano ZnO (Figure 5).

Endogenous antioxidants include enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reductase, play a crucial role in mitigating oxidative stress. The activities of these enzymes increase during oxidative stress to neutralize excess free radicals. During mastitis, PMNs are activated and generate reactive oxygen species (ROS) such as H₂O₂, superoxide anions, hydroxyl radicals, and halogen reactive species, partially reducing O₂ and lowering antioxidant enzyme levels (67). In this study, the activities of SOD, CAT, GSH, GPx, and GST significantly decreased (p < 0.01) in the blood of mastitis-induced mice, indicating oxidative stress, which is consistent with the findings of Chinchali and Kaliwal (68). The reduced enzyme activities in the mastitis-affected group were normalized more effectively in group IV, followed by groups VI, V, and III, suggesting a higher antioxidant activity of TPNS, likely due to its increased free radical scavenging capacity. GPx activity was nearly restored in mice treated with Rhizophora apiculata-derived AgNPs in hepatotoxin-induced liver damage (69). Yadav et al. (70) reported a significant (p < 0.001) increase in SOD, catalase, and GPx activity in the granulation tissue of rats treated with T. portulacastrum-mediated nano ZnO compared to untreated rats in an induced wound model.

Suresh et al. (71) reported the antioxidant activity of Cassia fistula-mediated nano ZnO in in vitro assays. Ilavarasan et al. (72) and Pandanaboina et al. (73) demonstrated the antioxidant activity of TPE in rats with carbon tetrachloride-induced liver injury and alcohol-induced hepato-renal injury, respectively. Chaitanya et al. (64) observed improved glutathione levels with aloin-mediated silver nanoparticles in mastitis-induced mice. Jacob and Rajiv (74) showed that Curcuma longa-mediated nano ZnO particles possess free radical scavenging abilities through in vitro assays. ZnO nanoparticles enhance antioxidant enzyme activities, reduce free radical levels (OH., O₂., H₂O₂), and scavenge free radicals by electron transfer. Silver nanoparticles synthesized via plant extract phytochemicals efficiently reduce reactive oxygen species (ROS) and protecting biomolecules (75). Biosynthesized AgNPs exhibit superior antioxidant activity compared to extracts alone because of their large surface area, which enhances bioactive chemical adsorption (76). Our findings align with recent literature suggesting that AgNPs, particularly when interacting with antioxidants or phytochemicals, may offer therapeutic benefits. Specifically, studies have demonstrated that AgNPs act as catalysts in antioxidant reactions or facilitate cellular repair under controlled conditions, such as low doses or in combination with herbal compounds (77). TPE can be mediated with nano silver and nano ZnO particles, exerting more effective antioxidant effects than the methanolic extract alone.

This study explores the green synthesis and characterization of nanoparticles, specifically silver and ZnO nanoparticles, using TPE-mediated leaf extract. In the present study, it was shown that the overall antioxidant activity of TPNS was higher than that of ceftriaxone, TPNZ, and TPE indicating that biologically synthesized nanoparticles are more potent than the TPE extract alone, likely due to the combined antioxidant effect of phytochemicals and nanoparticles. Further safety studies are necessary for the upscaling and potential parental use of TPE-mediated nanoparticles as effective antioxidant agents.