The antimicrobial and antioxidants properties of nanoemulsions synthesized with Moringa oleifera essential oil in laboratory conditions

Moringa can serve as a natural plant growth enhancer, as its leaves are rich in zeatin, a plant hormone belonging to the cytokinin group. Leaf extract can stimulate plant growth and increase crop yields [1, 2].

 

It has been demonstrated that several moringa parts—fruits, leaves, flowers, and stems—help prevent cancer, a fatal illness. Moringa's isolated thiocarbamate and isothiocyanate chemicals suppress tumor cell growth [3]. It was discovered that the

 

dichloromethane fraction was cytotoxic to MCF7 breast cancer cells [4]. In chemical carcinogenesis, niazimincin has been proposed as an effective chemopreventive agent [5].  Reactive oxygen spices have been studied using bioactive substances from Moringa pods, including flavonoids [9], thiocarbamates [8], glycosylates [6], isothiocyanates [7], and several other chemicals. It has been demonstrated that the aqueous extract is a strong scavenger of free radicals [10]. Previous studies suggest that the antioxidant potential might be due to kaempferol, which is mainly found in plant leaves [ 11]. 

 

Research has highlighted the potent antimicrobial properties of Moringa oleifera seed extract, which can be utilized in the treatment of fungal diseases [12]. Antimicrobial peptides present in the extract lead to the separation of two microbial membranes (outer and inner), causing water to penetrate the cell, increase cell volume, and result in cell death [13 ].

 

Currently, the use of natural enzyme inhibitors, such as those found in essential oils, is gaining attention due to minimal side effects on the human body [14]. Additionally, nanoemulsions have numerous other advantages, such as minimizing the sensory impact on the taste of food products and enhancing bioactivity due to better size distribution and dispersion. Oil-in-water (O/W) nanoemulsions consist of oil droplets with an average size ranging from 20 to 200 nanometers, dispersed in an aqueous medium and stabilized by an emulsifier layer. Surfactants suitable for use in food (poly sorbates, sugar esters, natural gums, modified vegetable proteins, and even some fractionated alkali-hydrolyzed guar gums) serve as emulsifying agents, and in some cases, they contribute to the unique surface behavior of certain nanoemulsions. Nanoemulsions exhibit electrostatic forces, spatial repulsion, rheology, ons can be formulated with food components to have distinctive and unique properties [15,16]. Despite the enough information about the antimicrobial activity of  oil M. oleifera there is a limited information about the the antimicrobial activity of oil nanoemulsion M. oleifera,  the hypothesis of the current study is the oil nanoemulsion of Moringa will acquired  new chemical and physical properties that will enhance its capacity as an antimicrobial .

 

The aim of this study was to evaluate the antimicrobial and antioxidant activities of Moringa oleifera oil nanoemulsion compared with the crude essential oil as well as study the chemical  and physical characterization of the nanoemulsion . This comprehensive evaluation was conducted to provide a deeper understanding of the functional advantages of converting essential oil into a nanoemulsion system.

Microorganism used in the study

Staphylococcus aureus bacteria (ATCC 29213, PTCC 1917) and Escherichia coli (ATCC 25922, PTCC 1399) and the ability to load and respond to environmental stresses. Depending on the size, nanoemulsiwere obtained from the microbial collection of the Iran Institute of Scientific and Industrial Research.

Extraction of Moringa oleifera Essence

The 150 grams of purchased leaf powder, along with water, were placed inside an ultrasonic bath (Elmap30h, Germany) for 40 minutes. Subsequently, the essence was extracted for 3 hours using the water distillation method in a Clevenger apparatus. The separated essence was then dried with sodium sulfate, and the essences were stored in sealed and dark containers in the refrigerator at 4 degrees Celsius until analysis [17].

 

Preparation of Moringa oleifera Essential Oil Nanoemulsion

To prepare the nanoemulsion, Tween 80 (30% by weight of the essential oil), distilled water, and M.oleifera essential oil (30% w/w) were used. The surfactant, along with the essential oil, was continuously and uniformly added drop by drop to distilled water and homogenized at a speed of 3000 rpm for 15 min . Then, the obtained emulsion solution was transferred to a sonicator probe device (OPTIMA, XL 100 K, 20 KHz, Germany).  The 15 mm probe of the device was immersed in a 25 mm depth of the solution, and the nanoemulsion preparation process was carried out for 5 minutes at 200 watts and a temperature of 25 degrees Celsius [18].

 

Chatatrization of  Moringa oleifera Essential Oil Nanoemulsion

1-Fourier-Transform Infrared Spectroscopy (FTIR)

The qualitative examination of the material was conducted using FTIR spectroscopy, a method commonly employed for identifying the structural features of biopolymers [ 19]. FTIR spectra of the samples were obtained in the range of 400–4000 cm-1 utilising a Thermo Scientific Nicolet iS10 Spectrometer with a Smart iTX attachment (Thermo Scientific, Inc., United States) at a spectral resolution of 4 cm-1 and 32 scans. FTIR spectra were acquired in reflection mode via the integrated diamond attenuated total reflectance (ATR) sampling method. The OMNIC Software facilitated the collecting, processing, analysis, and management of FTIR data inside a graphical interface.

 

2- Determining the morphological properties

Scanning electron microscopy was used to study the surface morphology of nanoparticles. After a 6-hour reaction, the colloidal solution was centrifuged for 4 minutes at 14,000 rpm to create the sample. After being re-dispersed in deionised water, the pellet was centrifuged once more. After three iterations of the procedure, acetone was used to wash the material. A drop of the solution was put on the carbon-coated copper grid after the purified nanoparticles had been sonicated for ten minutes to create the suspension. The sample was exposed to a lamp until it was totally dry. The prepared sample was examined using a scanning electron microscope[ 20].

 

Determination Minimum Inhibitory Concentration MIC  of Moringa Nanoemulsion

Using the standard method recommended by Hudzicki [21], the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Moringa nanoemulsion were determined for two bacteria, S. aureus and E. coli .The concentration was  prepared  from 0.004 to 2.5 mg/ml of both M. oleifera nanoemulsion and essential oil )Initially, 13 sterile tubes containing 1 ml of Mueller-Hinton agar medium were prepared.

Then, 1 mL of Moringa nanoemulsion at a concentration of 5 mg/mL was added to each sterile tube containing 1 mL of Mueller-Hinton agar medium. The first tube was poured, and after shaking, 1 mL from the first tube was added to the second tube, and so on, until tube 13. Finally, 1 mL from tube 13 was poured out. Each bacterial suspension was separately added to the tubes at a concentration of 1.0 mL (CFU/mL 1.5 × 10^8), and the process continued from tube 1 to 12 . One tube was considered as a positive control (medium and bacterial suspension), and one tube was considered as a negative control (medium and essential oil without bacteria). The tubes were incubated at 37 degrees Celsius for 24 hr. After the incubation period, the first tube in which no turbidity was observed was considered as the MIC for each of the samples

 

Evaluation of Antioxidant Activity of Moringa oleifera Essential Oil and Nanoemulsion by DPPH Method

The antioxidant activity of Moringa oleifera nanoemulsion was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method figure 2. For this purpose, various concentrations of essential oil and nanoemulsion (5, 10, 20, 25, and 30 mg/ml in methanol) were prepared. Then, 200 microliters of each sample were added to 1.0 millimolar DPPH solution in 8.2 milliliters, and the mixture was incubated in the dark at 25 degrees Celsius for 30 minutes. The absorbance of the solution at 517 nanometers was measured using a spectrophotometer. Distilled water was used as a control in this test. The percentage of DPPH free radical inhibition was calculated using the formula:

SC (%) = 100 × (A blank –A sample)/A blank)

Then, the antioxidant activity of the essential oil and nanoemulsion was determined as the inhibitory concentration at 50% (IC50) using linear regression analysis of the SC values for various sample concentrations. All experiments were conducted in a completely randomized design with at least three repetitions [22].

 

 Statistical Analysis

The results obtained from three repetitions was performed using version 9.1.0 of Graphpad Prism software. Two-way ANOVA was used for analysis, followed by the Tukey test to examine the significance level of the results. A significance level of 0.05 was considered (P < 0.05)

 

In recent years, with the increasing awareness of consumers, the use of herbal medicines and natural products, in general, has significantly increased [23]. Moringa is a tropical tree from the Moringaceae family, which includes fourteen species. This fast-growing deciduous tree has adapted well to arid conditions, with drum-shaped pods containing seeds. The tree can reach a height of up to 12 meters and produces seeds in its first year of growth. Moringa has been used for centuries for nutritional and medicinal purposes. This plant contains vitamin C, vitamin A, calcium, potassium, and all essential amino acids [24, 25]. Essential oils are natural and plant-derived secondary metabolites that, due to their good functional properties such as antimicrobial and antioxidant properties, have been proposed as alternatives to chemical preservatives in food studies [26].

 

The essential oil of the Moringa plant is one of the few essential oils with industrial, health, food, and medicinal applications [27]. The antioxidant compounds present in this plant help the human body reduce oxidative damage. Due to concerns related to food safety and health, food industry specialists are seeking to replace synthetic antioxidants with various natural antioxidants [28, 29]. Despite the high potential of essential oils, their direct use in food products and aqueous solutions is accompanied by limitations such as volatility, low solubility, and changes in sensory properties. On the other hand, essential oils are susceptible to oxidation when exposed to temperature, light, and oxygen, reducing their effectiveness.

 

According to a study by  Almuttairi and Abdulla, 2023 [30].  transforming essential oils into emulsion forms can enhance their physical stability, solubility, and biological activity. Emulsions can generally be classified into two particle size groups: microemulsions and nanoemulsions. Nanoemulsions, produced through various high-energy methods (such as ultrasonication) or low-energy methods (such as phase inversion), have particle sizes in the range of 1-100 nanometers. They consist of an oil phase dispersed in an aqueous phase, surrounded by a thin layer of surfactant or amphiphilic molecules, contributing to the stability of the nanoemulsion [30].

 

In the results of microbial analysis, the nanoemulsion of Moringa essential oil showed better effectiveness against the Gram-positive bacterium Staphylococcus aureus compared to the essential oil alone. In other studies, [31,32] were evaluated the antibacterial effect of Moringa oleifera extract and pomegranate extract on Porphyromonas gingivalis.. [33] investigated the antioxidant and antibacterial activities of  the oil extract of Moringa oleifera showed inhibitory properties against all tested bacteria except E. coli and K.pneumoniae at a concentration of 125 microliters per milliliter. The maximum antibacterial activity at a concentration of 500 microliters per milliliter was observed against the tested bacteria, measuring 18.5, 14.16, 11.83, and 11.33 mm, respectively. The MIC of the oil extract for Staphylococcus aureus was reported to be 64 micrograms per milliliter. It was 128 microliters per milliliter for Bacillus cereus and 256 microliters per milliliter for both Escherichia coli and Klebsiella pneumoniae.

 

Accrording to Akintelu et al,(2021) [34] studied the antimicrobial activity of Moringa oleifera seed extract against some Gram-positive (S. aureus and Streptococcus pneumoniae) and Gram-negative bacteria (E. coli, Pseudomonas aeruginosa, and K. pneumoniae). Phytochemical analysis of the seed extract revealed the presence of alkaloids, tannins, saponins, phenols, and flavonoids as secondary metabolites. The antibacterial study demonstrated inhibitory zones for all tested bacterial strains. The study highlighted the effectiveness of Moringa oleifera seeds in combating certain bacterial infections.Based on the studies, essential oil extracts, especially in the form of nanoemulsions, may affect Gram-negative bacteria by preventing the production of adenosine triphosphate (ATP) from dextrose and disrupting the cell membrane. Additionally, the hydrophobic property of essential oil nanoemulsions may contribute to their distribution in the structures of Gram-positive bacteria [35].

 

Various factors such as the extraction method, inoculum size, growth phase, growth medium, pH of the environment, duration, and temperature of incubation can influence the results of experiments, leading to reported differences [36]. Moreover, in a study by [30], investigating the antibacterial activity of micro and nanoemulsions of rosemary essential oil, the highest MIC and MBC for micro and nanoemulsions against Gram-negative bacteria Salmonella enteritidis and E. coli were observed at a concentration of 3.6 mg/ml. Staphylococcus aureus was the most sensitive bacterium, and the MIC and MBC of micro and nanoemulsions of rosemary essential oil against it were 0.9 and 1.8 mg/ml

respectively. In general, nanoemulsions can render bacteria inactive or cause their death through different mechanisms.

 

The mode of action of essential oils involves disrupting and destabilizing the structure of phospholipid membrane layers, membrane disruption, interaction with membrane enzymes and proteins as proton carriers, and reduction of pH across the membrane [37]. For instance, a nanoemulsion can release ions that react with thiol groups in bacterial cell surface proteins. As a result, proteins become inactivated, and cell membrane permeability is reduced, ultimately leading to bacterial death. Accumulation of nanoparticles in the bacterial cytoplasm and on its external membrane can be another mechanism involved in preventing bacterial growth and survival. Nanoemulsions produced by high-intensity and high-pressure homogenization methods exhibit higher antimicrobial properties compared to pure essential oils  in additional  the role of nanoparctical  recorded in vivo [38, 39].

The current study demonstrated that the Moringa oleifera essential oil nanoemulsion exhibited markedly stronger antimicrobial activity than the crude essential oil. For S. aureus, the MIC of the nanoemulsion was 1.25 mg/ml compared to 156.0 mg/ml for the essential oil. Similarly, for E. coli, the MIC values were 1.25 mg/ml for the nanoemulsion and 2.5 mg/ml for the essential oil. The antioxidant assessment further showed that the nanoemulsion had a significantly higher free-radical inhibition percentage than the essential oil (p < 0.05), with IC₅₀ values of 17.78 mg/ml and 23.97 mg/ml, respectively. These findings indicate that the essential oil nanoemulsion possesses enhanced antibacterial and antioxidant properties and may serve as a promising approach for use alone or alongside conventional methods in food preservation and in the management of bacterial infections.

 

Data availability

 All data generated or analyzed during this study are available from the authors.

 

Acknowledgments

The authors are grateful to all person who helped improve the quality of this work.

 

Funding

The authors reported there is no funding associated with the work featured in this article.

 

Declarations of competing interests

The authors have declared no conflict of interest.

1.     Daghaghele S, Kiasat AR, safiodin ardebili SM, Mirzajani R  (2022) Evaluation of different extraction methods of phytochemical and antioxidant compounds of Moringa oleifera leaf extract. FSCT. 18 (121) :163-172.  https://doi.org/10.52547/fsct.18.121.13

2.     Kumar V, Pandey N (2011) Antibacterial & antioxidant activity of different extract of moringa oleifera leaves - an in-vitro study. Int. J. Pharm. Sci. Rev. Res. 12(1):89-94.

3.     Vimala, G., & Gricilda Shoba, F. (2014). A review on antiulcer activity of few Indian medicinal plants. International journal of microbiology, 2014(1), 519590.‏ https://doi.org/10.1155/2014/519590

4.     Mohd Fisall, U. F., Ismail, N. Z., Adebayo, I. A., & Arsad, H. (2021). Dichloromethane fraction of Moringa oleifera leaf methanolic extract selectively inhibits breast cancer cells (MCF7) by induction of apoptosis via upregulation of Bax, p53 and caspase 8 expressions. Molecular biology reports, 48(5), 4465-4475. https://doi.org/10.1007/s11033-021-06466-y

5.     Upadhyay, P., Yadav, M. K., Mishra, S., Sharma, P., & Purohit, S. (2015). Moringa oleifera: A review of the medical evidence for its nutritional and pharmacological properties. International Journal of Research in Pharmacy & Science, (2).‏

6.     Nunthanawanich, P., Sompong, W., Sirikwanpong, S., Mäkynen, K., Adisakwattana, S., Dahlan, W., & Ngamukote, S. (2016). Moringa oleifera aqueous leaf extract inhibits reducing monosaccharide-induced protein glycation and oxidation of bovine serum albumin. Springerplus, 5, 1-7.‏ https://doi.org/10.1186/s40064-016-2759-3

7.     Borgonovo, G., De Petrocellis, L., Schiano Moriello, A., Bertoli, S., Leone, A., Battezzati, A., ... & Bassoli, A. (2020). Moringin, a stable isothiocyanate from moringa oleifera, activates the somatosensory and pain receptor TRPA1 channel in vitro. Molecules, 25(4), 976.‏ https://doi.org/10.3390/molecules25040976

8.     Singh, A., & Navneet, X. (2018). Ethnomedicinal, pharmacological and antimicrobial aspects of Moringa oleifera Lam. A review. The Journal of Phytopharmacology, 7(1), 45-50.‏ https://doi.org/10.31254/phyto.2018.7110

9.     Vergara-Jimenez M., Almatrafi M.M., Fernandez M.L. Bioactive Components in Moringa oleifera Leaves Protect against Chronic Disease. Antioxidant. 2017;6:91. https://doi.org/10.3390/antiox6040091

10.   Singh A., Navneet. Ethnomedicinal, pharmacological and antimicrobial aspects of Moringa oleifera lam.: A review. J. Phytopharmacol. 2018;7:45–50. https://doi.org/10.31254/phyto.2018.7110

11.   Mishra, G., Singh, P., Verma, R., Kumar, S., Srivastav, S., Jha, K. K., & Khosa, R. L. (2011). Traditional uses, phytochemistry and pharmacological properties of Moringa oleifera plant: An overview. Der Pharmacia Lettre, 3(2), 141-164.‏

12.   Jawad AM, Allawi AK, Ewadh HM (2018). Essential oils of rosemary as antimicrobial agent against three types of bacteria. Medical journal of Babylon, 15(1), 53-56.‏ https://doi.org/10.4103/MJBL.MJBL_14_18

13.   Saini RK, Sivanesan I, Keum YS (2016) Phytochemicals of Moringa oleifera: a review of their nutritional, therapeutic and industrial significance. 3 Biotech. 6(2): 203. https://doi.org/10.1007/s13205-016-0526-3

14.   Chuang P-H, Lee C-W, Chou J-Y, Murugan M, Shieh B-J, Chen H-M (2007) Anti-fungal activity of crude extracts and essential oil of Moringa oleifera Lam. Bioresource Technology. 98(1):232-6. https://doi.org/10.1016/j.biortech.2005.11.003

15.   Fathiazad F, Ahmadi-Ashtiani H, Rezazadeh S, Jamshidi M, Mazandarani M, Khaki A (2010) Study on phenolics and antioxidant activity of some selected plant of Mazandaran Province. Journal of Medicinal Plants. 9(34).

16.   Najafi V.M Najaf,  Kadkhodaee R (2011) Microencapsulation of Limonene by freeze drying: Effect of type and concentration of wall material. IFSTRJ . 7(3): 210-217. https://doi.org/10.22067/ifstrj.v7i3.10127 

17.   Jafari A, Ghanavi Z (2021) Chemical Properties of Essential Oils Extracted from Seed and Leaf of Moringa Peregrina by Clevenger Method with Ultrasound Pretreatment. JFST (Iran). 18(113): 173-185.

18.   Noori S, Zeynali F, Almasi H (2018) Antimicrobial and antioxidant efficiency of nanoemulsion-based edible coating containing ginger (Zingiber officinale) essential oil and its effect on safety and quality attributes of chicken breast fillets. Food Control. 84: 312-320. https://doi.org/10.1016/j.foodcont.2017.08.015

19.   Ramesan, M. T., & Surya, K. (2017). Fabrication and characterization of biopolymer nanocomposites from natural resource materials. Polymer Composites, 38, E66-E73. https://doi.org/10.1002/pc.23978 ‏

20.   Alkammash, N. M. (2017). Synthesis of silver nanoparticles from Artemisia Sieberi and Calotropis Procera medical plant extracts and their characterization using SEM analysis. Biosciences Biotechnology Research Asia, 14(2), 521-526.‏ DOI : http://dx.doi.org/10.13005/bbra/2474

21.   Hudzicki, J. (2009). Kirby Bauer disk diffusion susceptibility test protocol. Washington, DC: American Society for Microbiology. Retrieved from https://www.asm.org/Protocols/Kirby Bauer Disk Diffusion Susceptibility Test Protocol

22.   Molyneux P. (2004) The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. sci. technol.; 26: 211-219.

23.   Al-Attar MM, Al-Awadi SJ, Abdulfattah SY (2023) Gene Expression and Methylation Levels of PCSK9 Gene in Iraqi Patients with Coronary Artery Disease. Baghdad Sci. J. 20(6): 2078-8665. https://doi.org/10.21123/bsj.2023.7696

24.   Borrin TR, GEOrges EL, Moraes ICF,  Pinho SC (2016) Curcumin loaded nanoemulsions produced by the emulsion inversion point (EIP) method: An evaluation of process parameters and physicochemical stability. J Food Eng. 169: 1-9. https://doi.org/10.1016/j.jfoodeng.2015.08.012

25.   Aviram M, Dornfeld L, Kaplan M (2000) Pomegranate juice flavonoids inhibit low–density lipoprotein oxidation and cardiovascular diseases: studies in atherosclerotic mice and in humans. Drugs Exp. Clin. Res. 28: 49-62.

26.   Carabias-Martínez R, Rodríguez-Gonzalo E, Revilla-Ruiz P, Hernández-Méndez J (2005)Pressurized liquid extraction in the analysis of food and biological samples. J. Chromatogr. A. 1089(1-2):1-17. https://doi.org/10.1016/j.chroma.2005.06.072

27.   Bhatia S, Othman Z, Ahmad AL (2007) Pretreatment of palm oil mill effluent (POME) using Moringa oleifera seeds as natural coagulant. J. Hazard. Mater. 145(1-2):120-6. https://doi.org/10.1016/j.jhazmat.2006.11.003

28.   Chelliah R,  Ramakrishnan SR,  Antony U (2017) Nutritional quality of Moringa oleifera for its bioactivity and antibacterial properties. Int. Food Res. J. 24(2):825-833.

29.   Borrin TR, GEOrges EL, Moraes ICF,  Pinho SC (2016) Curcumin loaded nanoemulsions produced by the emulsion inversion point (EIP) method: An evaluation of process parameters and physicochemical stability. J Food Eng. 169: 1-9. https://doi.org/10.1016/j.jfoodeng.2015.08.012

30.   Almuttairi AA, and Abdulla AA (2023). Occurrence of class 1, 2, and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Babylon Province, Iraq. Medical Journal of Babylon, 20(1), 181-187.‏ https://doi.org/10.4103/MJBL.MJBL_329_22

31.   Faisal Madhloom A, Bashir Hashim Al-Taweel F, Sha AM, Raad Abdulbaqi H (2022) Antimicrobial Effect of Moringa Oleifera L. and Red Pomegranate against Clinically Isolated Porphyromonas gingivalis: in vitro Study. Arch Razi Inst. 77(4):1405-1419. https://doi.org/10.22092/ARI.2022.357513.2051

32.   Gulhane PA, Junghare MP, Dhakate NA (2014) Antimicrobial Activity of Indian Medicinal Plants: Moringa oleifera and Saraca indica. Int.J.Curr.Microbiol.App.Sci. 3(6): 161-169.

33.   Farhan SR, AL-Azawi AH, Salih WY, Abdulhassan AA (2021) The antibacterial and antioxidant activity of Moringa oleifera seed oil extract against some foodborne pathogensIndian J. Forensic Med. Toxicol. 15(4): 2529-2538. https://doi.org/10.37506/ijfmt.v15i4.17085

34.   Akintelu SA, Folorunso AS, Oyebamiji AK (2021) Phytochemical and antibacterial investigation of Moringa oleifera seed: experimental and computational approaches. Eclética Química. 46(2): 17-25. https://doi.org/10.26850/1678 4618eqj.v46.2.2021.p17 25

35.   Fateh MV, Ahmed S, Ali M, Bandyopadhyay S (2013) A review on the medicinal importance of pomegranate. J Pharm sci. 3(4):23- 25.

36.   Al-Quhli SQT, Ibrahim OS, Khashan AA (2024). Antibacterial Activity and Phytochemical Analysis of Moringa oleifera Extract against Staphylococcus aureus Identified by Routine and Molecular Methods. Medical Journal of Babylon, 21(3), 724-728.‏ https://doi.org/10.4103/MJBL.MJBL_1765_23

37.   Shahrampour D, Razavi SMA (2022) Evaluation of stability antimicrobial and antioxidant activity of macromulsion and nanoemulsion of rosemary essential oil. IFSET. 9(3): 195-207.

38.   Holley RA, Patel D (2005) Improvement in shelf life and safety of perishable foods . https://doi.org/10.1016/j.fm.2004.08.006

39.   Abbood, R. S., & Luaibi, N. M.(2023). Subchronic intraperitoneal toxicity of Sio2NPs on body weight and thyroid gland hormones in female Rats.‏