AAVS_13_8_1756-1763

INTRODUCTION

The term “Nano biotechnology” refers to the study and enhancement of biotechnological processes and their resulting products via the use of nanotechnology (Sumathi et al., 2024). Nano biotechnology is a rapidly developing topic that draws upon many different academic disciplines including those of biology, chemistry, physics and medicine (Tiquia-Arashiro and Rodrigues, 2016). Accordingly, a wider range of plant materials including leaf extracts, fruit, bark, fruit peels, root and callus have recently been added to the green synthesis of nanoparticles employing phytocompounds as bio-reductants (Abedini et al., 2024). Alkaloids, flavonoids and terpenoids were discovered to be the components in plant extracts that reduced and stabilized nanoparticles (Khattak et al., 2024). Green chemistry can be employed to make silver nanoparticles from the extract of Mentha Spicata leaves. This procedure is environmentally friendly, reduces costs and nontoxic (Moosavy et al., 2023). Medicinal plants are an important component of medicinal products and frequently serve as the foundation for indigenous or traditional healing practices that are still employed by the majority of people in many nations (Sharifi-rad et al., 2018). Mentha plants belong to the Lamiaceae family which is extensively cultivated in Europe, Asia, Africa, Australia and North America (Mamadalieva et al., 2017). Extracts from this genus have been used as food for a long time and are very valuable because they contain a lot of antioxidants, such as phenolic bioactive compounds (Salehi et al., 2018).

There are biologically active parts in Mentha species that are used in traditional medicines. Mint species can also be used to treat common health problems like colds, coughs, sinusitis, fever, bronchitis, and nausea (Dhifi et al., 2013). Also, the mint plant has been said to kill insects, fight germs, stop muscle spasms, fight fungi, and protect antioxidant (Öztürk-Yalçın et al., 2024).

Mentha was used in the past in the same way that it is used today in medicinal plants. Common colds, coughs, sinusitis, fevers, bronchitis, nausea, vomiting, indigestion, intestinal colic, and appetite loss can all be treated with mentha. It can also be used to add flavor to chewing gum, toothpaste, confectionery and pharmaceutical preparations (Zamljen et al., 2024). Silver nanoparticles (AgNPs) are known for their diverse biological properties, including antifungal (Deweer et al., 2023), anti-inflammatory (Li et al., 2024), antiviral (Mahendran and Rahman, 2020), anti-angiogenic (Rogers et al., 2008), and antiplatelet activities (Gurunathan et al., 2009). In this study, we synthesized silver nanoparticles by reducing silver ions from silver nitrate using an aqueous extract of Mentha spicata leaves. The biosynthesized nanoparticles were then evaluated for safety and toxicity in female albino rats over 14 days of daily oral administration.

Mentha spicata, a medicinal plant from the Lamiaceae family, is rich in secondary metabolites, particularly essential oils. Traditionally, its aerial parts are used to prepare herbal tea, which has been associated with various therapeutic effects, as documented in ethnopharmacological studies worldwide. Recent experimental studies have confirmed the biological activity of its bioactive compounds. Extracts and essential oils from M. spicata have demonstrated multiple pharmacological benefits, such as antioxidant, anticancer, antiparasitic, antimicrobial, and antidiabetic effects (El Menyiy et al., 2022).

The primary objective of this research was to synthesize AgNPs using Mentha spicata extract and assess their safety and toxicity profile in kidneys albino rats following 14 days of administration.

MATERIALS AND METHODS

Ethical Approval

All experimental procedures were reviewed and approved by the Ethical Committee of the College of Pharmacy, University of Kerbala, Iraq (Approval No.: COPh-6381) that aligns with international guidelines.

Green Synthesis and Characterization of Silver Nanoparticles (AgNPs)

Silver nanoparticles (AgNPs) were synthesized following the method described by Alwan et al. (2021). A 1 mM aqueous solution of silver nitrate (AgNO₃) was prepared and used as the precursor for nanoparticle synthesis. 5 mL of filtered aqueous leaf extract of Mentha spicata was added dropwise to 200 mL of the AgNO₃ solution under ultrasonication for 30 minutes to facilitate the reduction of Ag⁺ ions to Ag⁰.

The reaction mixture was then incubated at room temperature for 20 minutes under vigorous magnetic stirring, during which a rapid color change was observed, indicating the formation of AgNPs. The resulting mixture was allowed to react for 72 hours under gentle stirring to ensure complete nanoparticle formation. Aliquots were collected at predetermined intervals to monitor the synthesis process. The biosynthesized AgNPs were characterized using:

UV-Vis spectroscopy (Shimadzu UV-1600, Japan).

X-ray diffraction (XRD) (Shimadzu, Japan).

Fourier-transform infrared spectroscopy (FTIR) (Shimadzu, Japan).

Scanning electron microscopy (SEM) (CYAN, Belgium).

Atomic force microscopy (AFM) (TOSHIBA, Japan).

All chemicals used were of analytical grade, sourced from Sigma-Aldrich (Germany). Fresh Mentha spicata leaves were obtained from the local market for extract preparation.

Experimental Animals and Study Design

Thirty healthy albino rats (aged 10-12 weeks, weighing 175-200g) were obtained for this investigation. The animals were housed in the animal care facility of the College of Pharmacy, University of Kerbala, Iraq, under controlled environmental conditions with free access to standard rodent chow and water.

The rats were randomly allocated into five experimental groups (n=6 per group):

Control group: Received distilled water only.

T1 group: Administered 6.25 μg/kg AgNPs daily.

T2 group: Administered 12.5 μg/kg AgNPs daily.

T3 group: Administered 25 μg/kg AgNPs daily.

T4 group: Administered 50 μg/kg AgNPs daily.

All treatments were administered orally once daily for 14 consecutive days.

Blood Sample Collection Protocol

Following the 14-day experimental period, blood samples were collected from the albino rats under fasting and anesthesia. The animals were first anesthetized via intramuscular injection of a ketamine (60 mg/kg) and xylazine (12 mg/kg) combination. Once deep anesthesia was confirmed (absence of pedal reflex), cardiac puncture was performed to obtain blood samples (Patlolla et al., 2015), sample were placed in non-heparinized gel tubes and allowed to stand for 30 minutes before being centrifuged (for 15 minutes at 3000 rpm) and stored in firmly sealed tubes for further analysis at -20 C°, to assess the malondialdehyde MDA (Biotech –China), Reduce glutathione (GSH ) (Biosciences –China), urea (Linear- Spain), creatinine (Linear- Spain) and ATPase (Sun long –China).

Statistical Analysis

The experimental data are presented as mean ± standard error (S.E.) in tables. Statistical analyses were performed using SPSS software (version 22). Intergroup comparisons were conducted using one-way analysis of variance (ANOVA), with a p-value ≤ 0.001 considered statistically significant (Snedecor and Cochran, 1980).

RESULTS

AgMSNP characterization the first stage of AgNPs’ creation was distinguished by a noticeable color shift, with the color turning yellowish after 20 minutes. The color then began to alter after an hour to brown then to dark brown after 72 hours, remaining constant at dark brown. The optical absorbance of the synthesized Mentha spicata leaf extract-mediated silver nanoparticles was characterized by UV-Vis spectroscopy across the wavelength range of 200-800 nm, with a spectral resolution of 2 nm. The analysis revealed distinct absorption peaks occurring between 205-665 nm, confirming the formation of nanoparticles, Figure 1. UV-Vis spectroscopy absorbance of AgMSNPs. The FT-IR spectra of dried powdered AgNPs reduced by Mentha Spicata was obtained at various peak ranges ranging from 500 to 4000 cm 1.

Figure 2, FT-IR spectrum of Biogenic AgNPs; showed the FT-IR spectra with distinct peaks bands at band (3404.36, 3390.86, 2956.87, 2927.94, 1620.21, 1516.05, 1357.89, 1062.07, 831.32, 777.31, 615.29, 547.78). XRD analysis showed three distinct diffraction peaks of 20°,36.8° 43°, 58.5°,63.5°,75.5° and 78.5 ° at 2θ values indexed to (110), (111), (200), (220), (300) and (311) peak which were shown in Figure 3. The XRD pattern of biosynthesized AgNPs. SEM of AgMSNPs induced by reduction of AgNO3 with Mentha Spicata leaf extract in the current study showed spherical shape nanoparticles with a dimeter range of 33.5-60.06 nm and 1.526- 2.059 սm (Figure 4). Scanning Electron Microscope image for Biogenic AgNPs). Surface topography analysis was performed using atomic force microscopy (AFM). Figure 5a presents the two-dimensional AFM image of silver nanoparticles synthesized using Mentha spicata aqueous leaf extract (AgMSNPs), revealing aggregated nanoparticle formations. The corresponding three-dimensional representation in Figure 5b demonstrates a homogeneous population of nanoparticles with uniform surface morphology and consistent particle distribution.

Figures 6A, 6B, and 6C demonstrate the dose-dependent effects of Mentha spicata-synthesized silver nanoparticles (AgMSNPs) on renal and metabolic parameters following 14-day administration in adult female rats. Figure 6A, Serum urea concentration variations across treatment groups, Figure 6B, Serum creatinine level alterations, Figure 6C, Changes in ATPase enzyme activity, which showed a significant (p≤ 0.001) increase in serum urea and creatinine with a significant (p≤ 0.05) increase in ATPase concentrations after 14 days of treatment with AgMSNPs. These results were used as a response for estimation of ED as follows: Serum Urea concentration: The results in Figure 6A, showed a positive relationship between serum urea concentration and four successive doses of AgMSNPs. The correlation coefficient was equal (0.9563). From the equation, the estimated ED of AgMSNPs that cause a marked elevation in serum urea concentration was found to be equal to 12.5 µg/Kg B.W. 4.5.2 Serum Creatinine concentration: The results in (Figure 6B) revealed of positive linear relationship between four successive doses of AgMSNPs and serum creatinine concentration. The correlation coefficient was equal to (0.9164) whereas the estimated ED of AgMSNPs was equal to 12.5 µg/Kg B.W. 4.5.3 Serum ATPase concentration: The results in Figure 6C showed a positive relationship between serum ATPase concentration and four successive doses of AgMSNPs. The correlation coefficient was equal (0.9296). From the equation, the dose ED of AgMSNPs in serum concentration was found to be equal to 12.5 µg/Kg B.W.

In Table 1, Effect of different doses of AgMSNPs on serum urea (mg/dl), serum creatinine (mg/dl), showed significant (p≤ 0.001) increase in serum urea in T1, T2, T3 and T4 compare with control, the serum creatinine showed no significant (p> 0.001) different between T2 and T3 compare with control, while a significant (p≤ 0.001) increase in T3 and T4 compare with control, also the serum concentration of ATPase showed significant (p> 0.001) differences between T2, T3 and T4 compare with T1 and control groups. When comparing every population to the control group, there were no appreciable variations in the blood levels of the oxidative stress indicators MDA and GSH, this result showed in Table 2. Effect of different doses of AgMSNPs on MDA and GSH in adult female rats after 14 days of experiment.

Table 1: Effect of different doses of AgMSNPs on serum urea (mg/dl), serum creatinine (mg/dl), in adult female rats after 14 days of experiment.

Groups	Urea mg/dl	Creatinine Mg/dl	ATPase(µmol/g)
Control	31.25±0.25c	0.3115±0.0405 bc	4.2476±0.2162b
T1	32.25±0.337b	0.2855±0.0322 c	4.1975±0.3518b
T2	33.70 ±0.413b	0.31775±0.00297abc	4.3875±0.2891b
T3	33.5±0.042a	0.35225±0.00201ab	4.8875±0.2104a
T4	33.60±0.253a	0.3615±0.00379a	5.7548±0.2308a
P value	< 0.0001	0.0143	0.0143

Table 2: Effect of different doses of AgMSNPs on MDA and GSH in adult female rats after 14 days of experiment.

GSH U/mol	MDA µmol/L	Groups
2.612± 0.756 a	0.135± 0.513 a	Control
2.60± 0.77 a	0.154± 0.25 a	T1
2.672 ± 0.738 a	0.0962± 0.25 a	T2
2.655± 0.102 a	0.148± 0.029 a	T3
2.722± 0.804 a	0.169± 0.139 a	T4

Values are expressed as mean ± SE. n=6 rats / each group. Small letter denote significant differences between groups (p< 0.001).Group 1: Control negative Animals received normal saline. T1: Animals received AgMSNPs (6.25µg/kg B.W) orally. T2: Animals received AgMSNPs (12.5µg/kg B.W) orally.T3: Animals received AgMSNPs (25µg/kg B.W) orally. T4:Animals received AgMSNPs (50 µg/kg B.W) orally.

DISCUSSION

The UV-visible spectra study done for detection of phyto constituents contained in extract of M. Spicata. The average crystallite size of the biosynthesized silver nanoparticles (AgNPs), calculated using the Debye–Scherrer equation, was found to be 36.8 nm. This result aligns with previous findings reported by Reddy et al. (2018). UV-Vis spectroscopy analysis after 72 hours of incubation revealed a distinct peak in the range of 205–665 nm (Figure 1). The pale yellow silver colloids synthesized using Mentha spicata leaf extract exhibited a surface plasmon resonance (SPR) band within this range, suggesting the formation of spherical or nearly spherical AgNPs (Sathishkumar et al., 2009). The appearance of this peak at 205–665 nm confirms the reduction of silver ions, consistent with studies by Awwad et al. (2014) and Nasrollahzadeh et al. (2015). It is important to note that the SPR band position is influenced by factors such as nanoparticle size and shape. The spectra were recorded once the sample color and absorption intensity stabilized, providing preliminary insights into the size distribution of the AgNPs (Sulaiman et al., 2013).

FTIR analysis (Figure 2) indicated shifts in absorption bands, suggesting changes in vibrational frequencies. The spectra confirmed the presence of various functional groups in the Mentha spicata extract, including those associated with phenols, alcohols, alkanes, alkynes, and aldehydes. Similar observations were reported by Jha et al. (2009).

This study aims to evaluate the effective dose of biosynthesized silver nanoparticles (AgNPs) derived from Mentha spicata leaf extract in experimental albino rats. The AgNPs were synthesized through a green synthesis approach, utilizing aqueous M. spicata extract as a reducing agent to convert silver ions (Ag⁺) into metallic silver (Ag⁰). This reduction process is facilitated by the bioactive phytochemicals present in the extract. The color change observed during AgNP formation is attributed to the excitation of surface plasmon vibrations in the nanoparticles (Nawfal et al., 2019; Islam et al., 2024).

The XRD analysis (Figure 3) confirmed the crystalline structure of the synthesized silver nanoparticles (AgNPs), with peak broadening indicating their small particle size (Ali and Khudair, 2019). These findings demonstrate that Mentha spicata leaf extract serves as an efficient and rapid green synthesis route for producing stable AgNPs. SEM and AFM imaging (Figures 4 and 5) revealed spherical AgNPs with smooth surfaces and nanoscale dimensions, corroborating previous reports by Gauthami et al. (2015). SEM analysis further showed well-dispersed nanoparticles (60–80 nm) with minor aggregation, attributed to surface interactions with bioactive cellular compounds acting as capping/reducing agents (Bélteky et al., 2019). Notably, AFM measurements in this study (~60 nm) aligned closely with recent work by Alsamhary (2020).

Throughout the 14-day experimental period, none of the groups treated with varying doses of biosynthesized AgNPs exhibited any mortality (0% mortality rate) or signs of toxicity. This study was specifically designed to evaluate the effective dose of Mentha spicata-derived AgNPs in albino rats, with the aim of assessing their safety and toxicological profile through multiple exposures (Makarov et al., 2014). In an ideal effective dose study, adverse effects typically manifest within 14 days of administration (Reddy et al., 2018); however, no toxicity indicators—including mortality, tremors, convulsions, writhing, analgesia, increased motor activity, cyanosis, or anesthesia—were observed at any administered dose (6.25, 12.5, 25, or 50 µg/kg).

Kim and Ryu (2013) suggest that the toxicity of AgNPs cannot be attributed solely to the presence of Ag⁺ ions in the nanoparticle solution. The safety of these AgNPs may instead be linked to the bioactive compounds in the M. spicata methanol extract, which serve as reducing agents (converting Ag⁺ to Ag⁰), as well as to factors such as nanoparticle size and administered dose. Additionally, the kidneys—being primary target organs for numerous substances due to their vital roles in detoxification and excretion (Mahmoodi et al., 2018; Mohammed et al., 2021)—showed no evidence of toxicity in this study.

The ATPase enzyme functions as a primary transporter, actively pumping protons out of the cell. The notable rise in ATPase activity observed in rats treated with spearmint (Mentha spicata)-derived silver nanoparticles (AgNPs) may result from multiple biochemical and physiological processes. ATPases, including Na+/K+-ATPase and Ca²⁺-ATPase, play a vital role in maintaining ion balance and cellular homeostasis. One possible explanation is that AgNPs stimulate ion transport by interacting with cell membranes (Mikhailova, 2020). Additionally, low concentrations of AgNPs might modulate oxidative stress responses by boosting antioxidant enzymes, thereby protecting ATPase function (Ahamed et al., 2010). Mentha spicata contains polyphenols and flavonoids, which could help reduce oxidative damage to ATPases (Ferrentino et al., 2020). Furthermore, the biogenic synthesis of AgNPs using spearmint may improve their biocompatibility, preventing severe oxidative inhibition of ATPases (Mittal et al., 2013). Interestingly, low doses of AgNPs might enhance ATPase activity by activating cellular repair mechanisms, whereas higher concentrations could suppress it due to excessive reactive oxygen species (ROS) production (Muhamad et al., 2022).

Given the critical role of oxidative stress in organ toxicity—marked by the production of malondialdehyde (MDA), 4-hydroxynonenal, and altered antioxidant enzyme activity (Alwan et al., 2021; Nawfal and Al-Okaily, 2022)—this study evaluated the biochemical impact of M. spicata-derived AgNPs. Notably, no significant changes in MDA or glutathione (GSH) levels were observed compared to controls, aligning with findings by Nasir et al. (2014) and Sood and Khudiar (2018), who reported stable oxidative stress biomarkers after 14 days of AgNP exposure.

Renal function, assessed via urea and creatinine levels, remained unaffected across all groups (control G1 and treated T1–T4) during the 14-day observation period. No mortality or behavioral abnormalities (e.g., lethargy, tremors) were recorded, and biochemical parameters in T1–T3 (6.25–25 µg/kg) showed no significant divergence from controls. These results, consistent with Alwan et al. (2021), suggest that biogenic AgNPs are safe for oral administration in albino rats at tested doses.

However, the high-dose group (T4; 50 µg/kg) exhibited significant biochemical alterations, corroborating reports by Patlolla et al. (2015) and Muhamad et al. (2022) on AgNP-induced cytotoxicity at elevated concentrations. Based on dose-response proximity to control data, 12.5 µg/kg was identified as the effective dose (ED) for subsequent studies.

CONCLUSIONS AND RECOMMENDATIONS

This study demonstrates that Mentha spicata leaf extract serves as an effective reducing agent for green synthesis of silver nanoparticles (AgNPs). Toxicity evaluation in albino rats revealed no mortality, oxidative stress (stable MDA/GSH levels), or renal dysfunction across tested doses. The effective dose (ED) was determined to be 12.5 μg/kg body weight, as it elicited no adverse effects while maintaining biochemical and physiological stability.

ACKNOWLEDGMENTS

The authors extend their sincere gratitude to the staff of the Department of Physiology, Pharmacology, and Biochemistry at the College of Veterinary Medicine, University of Baghdad, Iraq, for their invaluable support and collaboration throughout this research. Their expertise and assistance were instrumental in the successful completion of this work.

NOVELTY STATEMENTS

The primary objective of this research was to synthesize AgNPs using Mentha spicata extract and assess their safety and toxicity profile in kidneys albino rats following 14 days of administration.

AUTHOR’S CONTRIBUTIONS

Alaa Hashim Ali and Luma Walid Khaleel were responsible for designing and conducting the experiments, analyzing the data, providing reagents, materials, and analysis tools, as well as writing the manuscript.

Conflict of Interest

The authors affirm that they have no actual or potential conflicts of interest, whether financial, personal, or professional, that might be construed as influencing the research or its outcomes.

REFERENCES

Abedini, S, Pourseyedi S, Zolala J, Mohammadi H, Abdolshahi R (2024). Green synthesis of Superparamagnetic Iron Oxide and Silver Nanoparticles in Satureja hortensis Leave Extract: Evaluation of Antifungal Effects on Botryosphaeriaceae Species. Curr. Microbiol., 81(6): 149. https://doi.org/10.1007/s00284-024-03647-3

Ahamed M, Alsalhi MS, Siddiqui, MK (2010). Silver nanoparticle applications and human health. Clinica chimica acta; Int. J. Clin. Chem., 411(23-24): 1841–1848. https://doi.org/10.1016/j.cca.2010.08.016

Ali ZS, Khudair KK (2019). Synthesis, Characterization of Silver Nanoparticles Using Nigella sativa Seeds and Study Their Effects on the Serum Lipid Profile and DNA Damage on the Albino rats’ Blood Treated with Hydrogen Peroxide. Iraqi J. Vet. Med., 43(2): 23-37. https://doi.org/10.30539/iraqijvm.v43i2.5266

Alsamhary KI (2020). Eco-friendly synthesis of silver nanoparticles by Bacillus subtilis and their antibacterial activity. Saudi J. Biol. Sci., 27(8): 2185–2191. https://doi.org/10.1016/j.sjbs.2020.04.026

Alwan S, Al-Saeed M, Abid H (2021). Safety assessment and biochemical evaluation of the effect of biogenic silver nanoparticles (using bark extract of C. zeylanicum) on Rattus norvegicus albino rats’, Baghdad J. Biochem. Appl. Biol. Sci., 2(03): 138–150. https://doi.org/10.47419/bjbabs.v2i03.67

Awad AM, Albiss B, Ahmad AL (2014). Green synthesis, characterization and optical properties of zinc oxide nanosheets using Olea europea leaf extract. Adv. Mater. Lett., 5: 520-524. https://doi.org/10.5185/amlett.2014.5575

Bélteky P, Rónavári A, Igaz N, Szerencsés B, Tóth IY, Pfeiffer I, Kiricsi M, Kónya Z (2019). Silver nanoparticles: aggregation behavior in biorelevant conditions and its impact on biological activity. Int. J. Nanomed., 14: 667–687. https://doi.org/10.2147/IJN.S185965

Deweer C, Sahmer K, Muchembled J (2023). Anti-oomycete activities from essential oils and their major compounds on Phytophthora infestans. Environ. Sci. Pollut. Res. Int., 30(51): 110240–110250. https://doi.org/10.1007/s11356-023-29270-6

Dhifi W, Litaiem M, Jelali N, Hamdi N, Mnif W (2013). Identification of a new chemotye of the plant Mentha aquatica grown in Tunisia: chemical composition, antioxidant and biological activities of its essential oil. J. Essent. Oil Bear. Plants, 14(3): 320-328. https://doi.org/10.1080/0972060X.2011.10643941

El Menyiy N, Mrabti HN, El Omari N, Bakili AE, Bakrim S, Mekkaoui M, Balahbib A, Amiri-Ardekani E, Ullah R, Alqahtani AS, Shahat AA, Bouyahya A (2022). Medicinal Uses, Phytochemistry, Pharmacology, and Toxicology of Mentha spicata. Evid. Based Complement. Altern. Med., eCAM, 2022: 7990508. https://doi.org/10.1155/2022/7990508

Ferrentino G, Morozova K, Horn C, Scampicchio M (2020). Extraction of Essential Oils from Medicinal Plants and their Utilization as Food Antioxidants. Curr. Pharm. Des., 26(5): 519–541. https://doi.org/10.2174/1381612826666200121092018

Gauthami M, Srinivasan N, Goud NM, Boopalan K, Thirumurugan K (2015). Synthesis of silver nanoparticles using Cinnamomum zeylanicum bark extract and its antioxidant activity. Nanosci. Nanotechnol. Asia, 5(1):2–7. https://doi.org/10.2174/221068120501150728103209

Gurunathan S, Lee KJ, Kalishwaralal K, Sheikpranbabu S, Vaidyanathan R, Eom SH (2009). Antiangiogenic properties of silver nanoparticles. Biomaterials, 30(31): 6341–6350. https://doi.org/10.1016/j.biomaterials.2009.08.008

Moosavy MH, de la Guardia M, Mokhtarzadeh A, Khatibi SA, Hosseinzadeh N, Hajipour N (2023). Green synthesis, characterization, and biological evaluation of gold and silver nanoparticles using Mentha spicata essential oil. Sci. Rep., 13(1): 7230. https://doi.org/10.1038/s41598-023-33632-y

Mikhailova EO (2020). Silver Nanoparticles: Mechanism of Action and Probable Bio-Application. J. Funct. Biomater., 11(4): 84. https://doi.org/10.3390/jfb11040084

Jha AK, Prasad K, Kumar V, Prasad K (2009). Biosynthesis of silver nanoparticles using Eclipta leaf. Biotechnol. Prog., 25(5): 1476–1479. https://doi.org/10.1002/btpr.233

Khattak M, Khan TA, Nazish M, Ishaq MS, Hameed H, Kamal A, Elshikh MS, Al Farraj DA, Anees M (2024). Exploration of reducing and stabilizing phytoconstituents in Arisaema dracontium extract for the effective synthesis of Silver nanoparticles and evaluation of their antibacterial and toxicological proprties. Microb. Pathog., 192: 106711. https://doi.org/10.1016/j.micpath.2024.106711

Kim S, Ryu DY (2013). Silver nanoparticle-induced oxidative stress, genotoxicity and apoptosis in cultured cells and animal tissues. J. Appl. Toxicol, JAT., 33(2): 78–89. https://doi.org/10.1002/jat.2792

Islam AKMS, Bhuiyan R, Nihad SAI, Akter R, Khan MAI, Akter S, Islam MR, Khokon MAR, Latif MA (2024). Green synthesis and characterization of silver nanoparticles and its efficacy against Rhizoctonia solani, a fungus causing sheath blight disease in rice. PloS one, 19(6): e0304817. https://doi.org/10.1371/journal.pone.0304817

Li W, Li P, Li X, Hou H, Lin H, Jin M, Liu K, Zhang X, Sheng W (2024). Metabolomics-based profiling of anti-inflammatory compounds from Mentha spicata in shanghe, China. Heliyon, 10(16): e35974. https://doi.org/10.1016/j.heliyon.2024.e35974

Mahendran G, Rahman LU (2020). Ethnomedicinal, phytochemical and pharmacological updates on Peppermint (Mentha × piperita L.)-A review. Phytother. Res., 34(9): 2088–2139. https://doi.org/10.1002/ptr.6664

Mahmoodi Esfanddarani H, Abbasi Kajani A, Bordbar AK (2018). Green synthesis of silver nanoparticles using flower extract of Malva sylvestris and investigation of their antibacterial activity. IET Nanobiotechnol., 12(4): 412-416. https://doi.org/10.1049/iet-nbt.2017.0166

Makarov VV, Makarova SS, Love AJ, Sinitsyna OV, Dudnik AO, Yaminsky IV, Taliansky ME, Kalinina NO (2014). Biosynthesis of stable iron oxide nanoparticles in aqueous extracts of Hordeum vulgare and Rumex acetosa plants. Langmuir - ACS Publications – Am. Chem. Soc., 30(20): 5982- 5988. https://doi.org/10.1021/la5011924

Mamadalieva, N. Z., Akramov, D. K., Ovidi, E., Tiezzi, A., Nahar, L., Azimova, S. S. (2017). Aromatic Medicinal Plants of the Lamiaceae Family Composition , and Biological Activities. Medicines Review, 4(8), 1–12.

Mascoloti Spréa R, Caleja C, Pinela J, Finimundy TC, Calhelha RC, Kostić M, Sokovic M, Prieto MA, Pereira E, Amaral JS, Barros L (2022). Comparative study on the phenolic composition and in vitro bioactivity of medicinal and aromatic plants from the Lamiaceae family. Food Res. Int. (Ottawa, Ont.): 161: 111875. https://doi.org/10.1016/j.foodres.2022.111875

Mittal AK, Chisti Y, Banerjee UC (2013). Synthesis of metallic nanoparticles using plant extracts. Biotechnol Adv. 2013; 31(2):346-356. https://doi:10.1016/j.biotechadv.2013.01.003

Mohammed AA, Aldossary ATY, Nawfal AJ, Obaid AH (2021). Oxidative Stress and Diabetics: Assessment of Therelationship Between Glycosalated Hemoglobin Andnitrosative Biomarker on Type 2 Diabetic Patients. Biochem. Cell. Arch., 21(1): 885–890. https://connectjournals.com/03896.2021.21.000

Muhamad M, Rahim, N, Wan Omar WA, Nik Mohamed Kamal NNS (2022). Cytotoxicity and Genotoxicity of Biogenic Silver Nanoparticles in A549 and BEAS-2B Cell Lines. Bioinorg. Chem. Appl., 2022: 8546079. https://doi.org/10.1155/2022/8546079

Nasrollahzadeh M, Sajadi MM, Babaei F, Maham M (2015). Euphorbia helioscopia Linn as a green source for synthesis of silver nanoparticles and their optical and catalytic properties. J. Colloid Interface Sci., 450: 374–380. https://doi.org/10.1016/j.jcis.2015.03.033

Nasir MU, Hussain S, Jabbar S, Rahid F, Khalid N, Mehmood A. (2014). A review on the nutritional content, functional properties and medicinal potential of dates. Sci Lett 2014; 3(1):Online first 2014044-SL. available on: HYPERLINK “https://www.researchgate.net/publication/271590091 A_review_on_the_nutritional_content_functional_properties_and_medicinal_potential_of_dates” https://www.researchgate.net/publication/271590091

Nawfal AJ, Tafash Haithm T, Mahmood Anmar Shukr (2019). Protective Effect of roket Leaves (Eruca Sativa) Extract against Lead Induced Oxidative Damage in Liver and Kidney of Male rats. Indian J. Public Health Res. Dev., 10(5): 365-370. https://10.5958/0976-5506.2019.01029.5

Nawfal AJ, Al-Okaily BN (2022). Effect of the Sublethal Dose of Lead Acetate on Malondialdehyde, Dopamine, and Neuroglobin Concentrations in Rats. World Vet. J., 12 (3): 311-315. https://dx.doi.org/10.54203/scil.2022.wvj39

Öztürk-Yalçın F, Ürkek B, Şengül M (2024). Evaluation of microbiological, antioxidant, thermal, rheological and sensory properties of ice cream fermented with kefir culture and flavored with mint (Menthaspicata L.). Food Sci. Nutr., 12(10): 7358–7369. https://doi.org/10.1002/fsn3.4355

Panácek A, Kolár M, Vecerová R, Prucek R, Soukupová J, Krystof V, Hamal P, Zboril R, Kvítek L (2009). Antifungal activity of silver nanoparticles against Candida spp. Biomaterials, 30(31): 6333–6340. https://doi.org/10.1016/j.biomaterials.2009.07.065

Patlolla AK, Hackett D, Tchounwou PB (2015). Silver nanoparticle-induced oxidative stress-dependent toxicity in Sprague-Dawley rats. Mol. Cell. Biochem., 399(1-2): 257–268. https://doi.org/10.1007/s11010-014-2252-7

Reddy ST, Soman SS, Yee J (2018). Magnesium balance and measurement. Adv. Chronic. Kidney Dis., 25(3): 224-229. https://doi.org/10.1053/j.ackd.2018.03.002

Rogers JV, Parkinson CV, Choi YW, Speshock JL, Hussain SM (2008). Nanoscale Res. Lett. 3: 129–133. https://doi.org/10.1007/s11671-008-9128-2

Salehi B, Stojanović-Radić Z, Matejić J, Sharopov F, Antolak H, Kręgiel D, Sen S, Sharifi-Rad M, Acharya K, Sharifi-Rad R, Martorell M, Sureda A, Martins N, Sharifi-Rad J (2018). Plants of Genus Mentha: From Farm to Food Factory. Plants (Basel, Switzerland): 7(3): 70. https://doi.org/10.3390/plants7030070

Sathishkumar M, Sneha K, Won SW, Cho CW, Kim S, Yun YS (2009). Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity. Colloids Surf. B. Biointerfaces, 73(2): 332–338. https://doi.org/10.1016/j.colsurfb.2009.06.005

Sharifi-Rad J, Tayeboon GS, Niknam F, Sharifi-Rad M, Mohajeri M, Salehi B, Iriti M, Sharifi-Rad M (2018). Veronica persica Poir. extract - antibacterial, antifungal and scolicidal activities, and inhibitory potential on acetylcholinesterase, tyrosinase, lipoxygenase and xanthine oxidase. Cell. Mol. Biol., (Noisy-le-Grand, France): 64(8): 50–56. https://doi.org/10.14715/cmb/2018.64.8.8

Snedecor GW, Cochran WG (1980). Statistical Methods. 6th ed. the Iowa state University press., 238-248. Available at: https://www.amazon.com/Statistical-Methods-Seventh-isbn-0813815606/dp/B0012S4NIE

Sood MA, Khudiar KK (2018). Role of Salvia officinal’s Silver Nanoparticles in Attenuating Renal Damage in Albino rats Exposed to Methotrexate (Part I). Iraqi J. Vet. Med., 42(2): 7-20. https://doi.org/10.30539/iraqijvm.v42i2.281

Sulaiman GM, Mohammed WH, Marzoog TR, Al-Amiery AA, Kadhum AA, Mohamad AB (2013). Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract. Asian Pac. J. Trop. Biomed., 3(1): 58–63. https://doi.org/10.1016/S2221-1691(13)60024-6

Sumathi Y, Dong CD, Singhania RR, Chen CW, Gurunathan B, Patel AK (2024). Advancements in Nano-Enhanced microalgae bioprocessing. Bioresour. Technol., 401: 130749. https://doi.org/10.1016/j.biortech.2024.130749

Tiquia-Arashiro S, Rodrigues DF (2016). Extremophiles: applications in nanotechnology (p. 193). New York, NY, USA: Springer Int. Publishing. https://link.springer.com/book/10.1007/978-3-319-45215-9

Zamljen T, Grohar MC, Medic A (2024). Mint-Scented Species in Lamiaceae: An Abundant and Varied Reservoir of Phenolic and Volatile Compounds. Foods (Basel, Switzerland): 13(12): 1857. https://doi.org/10.3390/foods13121857