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. 2025 May 14;15:76. doi: 10.1186/s13568-025-01849-x

Phytochemical characterization of peanut oil and its ozonized form to explore biological activities in vitro

Aisha M H Al-Rajhi 1, Tarek M Abdelghany 2,✉, Samy Selim 3, Mohammed S Almuhayawi 4, Mohammed H Alruhaili 4,5, Mohanned T Alharbi 6, Soad K Al Jaouni 7
PMCID: PMC12078917  PMID: 40369380

Abstract

Peanut (Arachis hypogaea) is a perennial leguminous crop grown worldwide. This study aims to investigate the chemical composition and pharmaceutical applications of peanut oil using experimental methods for both crude peanut oil and its ozonized form. The peanut oil was exposed to ozone for five hours at flow rates ranging from 0 to 7 L/min to complete the ozonization process. Gas Chromatography-Mass Spectrometry was employed to analyze the chemical composition of peanut oil. The antimicrobial activity of the two oil forms was evaluated against Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Candida albicans, and Aspergillus niger. The protein denaturation assay was used to assess anti-inflammatory properties, while the DPPH assay was employed to evaluate antioxidant activity. Cytotoxicity was tested using normal human fibroblast cells (WI-38) and colon cancer cells. The results revealed that exposure to ozone altered the chemical composition of the oil, increasing the number of identifiable molecules from 10 in the crude oil to 29 in the ozonized form. A significant enhancement in the antimicrobial activity of the crude oil was observed after ozonization. Moreover, ozonization notably increased the oil’s antioxidant capacity with IC50 13.06 ± 0.6 µg/mL, while crude oil provide IC50 23.37 ± 0.3 µg/mL compared to IC50 standard ascorbic acid (3.08 ± 0.4 µg/mL) as well as its anticancer (IC50 was 7.31 ± 0.21 and 15.09 ± 0.37 µg/mL against colon carcinoma cells, IC50 was 29.49 ± 2.03 and 24.68 ± 1.44 µg/mL against lung fibroblast normal cells employing crude oil and ozonized oil, respectively) anti-inflammatory potential.

Keywords: Peanut oil, Ozone, Antimicrobial, Anti-inflammatory, Antioxidant, Anticancer

Introduction

Medicinal and culinary crops have been essential components of both traditional and contemporary medicine, offering a wealth of bioactive substances with potential therapeutic benefits. Beyond their historical use in treating a wide range of ailments, these plants are increasingly relevant for developing functional foods and discovering new pharmaceuticals. The bioactive compounds in these plants are known to combat oxidative stress and metabolic disorders while promoting overall health (Abdelghany et al. 2019; Yahya et al. 2022). As a natural approach to maintaining health, ongoing research into their bioactive constituents contributes to the prevention and management of various diseases (Abdelghany et al. 2021; Qanash et al. 2022).

Peanuts (Arachis hypogaea) are among the plants of particular interest to researchers in nutrition and medicine due to their rich nutritional content and potential medicinal properties (Djeghim et al. 2024a, b). Peanuts rank as the fourth-largest oil crop and the thirteenth-largest food crop globally (Zhao et al. 2021). The peanut comprises outer shells, skins, and seeds, with the latter two serving as dietary components (Cui et al. 2023). According to Yaw et al. (2008), peanut seeds are rich in minerals and vitamins, contain approximately 20% protein, and have an oil content of around 50%. Historically, peanuts were considered unhealthy due to their high oil content (Hammad et al. 2023). However, peanut skins, often an underutilized by-product in the food industry, are rich in natural antioxidants (Dudek et al. 2017). Recent studies suggest that peanuts and their bioactive compounds may have beneficial effects on metabolic health (Djeghim et al. 2024a, b; Vikal et al. 2024). Fatty acids and phenolic compounds in peanuts have also shown promise in biomedical research (Franco et al. 2023) Despite their potential, research on the therapeutic properties of peanut oil remains limited. Gas Chromatography-Mass Spectrometry (GC-MS), a powerful analytical tool for identifying bioactive molecules such as fatty acids, terpenes, and phenolic compounds, can address this gap by determining the chemical constituents of peanut oil and evaluating its therapeutic properties (Shoaib et al. 2023).

Ozone, an inorganic molecule composed of three oxygen atoms, is a powerful oxidizing agent naturally produced in Earth’s atmosphere and vital to life on the planet (Silva et al. 2020). Ozonized oils are created using ozone generators, and the chemical composition of these derivatives can be influenced by several factors, including the type and quantity of vegetable oil, the presence of water or other catalysts, and ozonation parameters such as time, temperature, ozone concentration, flow rate, and stirring efficiency (Akbas and Ozdemir 2006). Additionally, the quality and efficiency of ozone generators play a significant role in the process (Al-Rajhi et al. 2024a). The use of medical-grade oxygen can help prevent the formation of potentially harmful nitrated byproducts, which are associated with nitrogen content in the air and can reduce the effectiveness of ozonation (Ugazio et al. 2020). Cancer has emerged as a worldwide health issue, making evaluations of cancer-related deaths crucial for efficient public health policy development and appropriate distribution of resources. The resistance of cancer to medications and chemotherapy poses a challenge to public health organizations across the globe (Qanash et al. 2023a, b). The most frequently occurring malignant cancer is the colorectal tumor (CRT) in the digestive tract and ranks as the 3rd most common cancer in men, the 2nd in women, and the 4th leading cause of death globally (Nimer et al. 2025). CRT displays several features, such as a rapid rate of reproduction, heightened energy metabolism, and a tendency to invade surrounding tissues (Tan et al. 2025). Therefore the current study aims to investigate the effects of ozone on the biomedical applications of peanut oil, including its antimicrobial, antioxidant, anti-inflammatory, and antitumor activities.

Materials and methods

Chemicals and test microbes

The peanut oil used in this project was sourced from a local vendor in Jazan, Saudi Arabia. All chemicals used in this study were supplied by Sigma Aldrich in Saudi Arabia. The microbial isolates examined in this research were kindly provided by Prof. Tarek Mohamed from Cairo, Egypt. The utilized microorganisms was Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Klebsiella pneumonia (ATCC 13883), Salmonella typhi (ATCC 6539), Candida albicans (ATCC 10221), and Aspergillus niger (ATCC 16888).

Ozonation process for peanut oil

Ozone gas was generated using an electric shock ionization device at the Centre of Plasma, Ain Shams University. A Drechsel tank (1.0 L) containing 0.6 L of peanut oil was placed near the plasma reactor’s effluent in a cooling bath maintained at -8 °C. The ozone layer was bubbled through the peanut oil for five hours at a flow rate ranging from 0 to 7 L/min, leading to a partially solidified state. After ozonization, the peanut oil was removed from the Drechsel tank, transferred to an empty container, weighed, and stored at 5 °C (Khalifa et al. 2022).

Phytochemical analysis of peanut oil and ozonized oil using gas chromatography-mass spectrometry (GC-MS)

The GC-MS analysis was performed using an Rt-560 column (95.0 m × 0.25 mm × 0.20 μm; PerkinElmer, Germany), an auto-sampler (2400-PerkinElmer, Germany), and flame ionization detection (FID). Data from the FID was processed using chromatography software (Compass CDS Data Collection and Programs, PerkinElmer, Germany). Helium was used as the carrier gas at a ratio of 97:1. The pre-run duration was nine minutes, and the equilibrium was achieved within 0.6 min. Two temperature ramps were applied: the first at a rate of 6 °C/min, reaching a maximum temperature of 210 °C, and the second ramp also at 6 °C/min, increasing to a final temperature of 290 °C. The initial temperature was set at 40 °C. The analyses were conducted in a systemized heating mode, running from 100 °C to 280 °C, and achieved thermal stability after 20 min. The carrier gas flow rate was maintained at 1.0 mL/min (Abdelghany et al. 2020).

Evaluation of antimicrobial impact of examined oil forms

The well agar diffusion method was used to evaluate the in vitro antimicrobial effects of peanut oil and ozonized peanut oil against a range of bacterial and fungal test microorganisms. Mueller-Hinton agar plates were used for bacteria, while malt extract agar was used for fungi. For the bacterial assay, 100 µL of suspended bacterial cells (1.8 × 106 colony-forming units/mL) were spread onto the agar plates. Wells were created in the agar medium using a sterilized cork borer, and the test oils were introduced into these wells. Gentamicin (0.07 mg/mL) and fluconazole (0.24 µg/mL) served as standard reference substances, while Dimethyl sulfoxide (DMSO) (5% ) was used as the control. Bacterial plates were incubated at 37 °C for 48 h, while plates inoculated by fungi were incubated at 27 °C for 5 days. After incubation, the diameter of the inhibitory zones around the wells was measured to assess antimicrobial activity (Alghonaim et al. 2023).

Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

The MIC of the specimens was determined using the microdilution broth method in nutrient-rich broth for microorganisms. Final concentrations, ranging from 0.98 to 1000 µg/mL, were prepared by serial two-fold dilution of the specimens under examination. A volume of 100 µL of each dilution in broth media was added to the wells of a 96-well microtiter plate. Fresh microbial cultures adjusted to match the turbidity of the 1.0 McFarland standard were used to prepare the inoculum. Each received 2.5 µL of sterile 0.9% NaCl to achieve a final microbial concentration of 2.0 × 106 CFU/mL. The plates were incubated for 72 h at 38 °C for bacteria and 5–7 days at 27 °C for fungi. Optical measurements were taken to identify the MIC, defined as the lowest concentration of the test specimen that completely inhibited visible growth of the microbial strain. Each microplate included a positive control (inoculum without the test specimens) and a negative control (test specimens without inoculum) (Al-Rajhi et al. 2024b). The minimum bactericidal concentration (MBC) was determined by sub-culturing 100 µL from wells that showed complete growth inhibition onto fresh agar plates. These included the growth control, the highest positive sample, and media with complete growth suppression. The MBC was identified as having the lowest concentration of the test specimens that prevented microbial growth during the incubation period at the specified temperature (Donato et al. 2024).

Testing antioxidant impact of examined oil forms

The antioxidant capacity of the examined specimens was determined using a 0.10 mM of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) solution in ethanol. One milliliter of sample solutions in ethanol, with concentrations ranging from 3.8 to 1000 µg/mL, was mixed with three milliliters of the DPPH solution. The mixture was thoroughly shaken and allowed to stand at room temperature for 35 min. The resulting color change was measured by recording the absorbance at 510 nm (Selim et al. 2024).

Examination of Anti-inflammatory action of examined oil forms

The anti-inflammatory activity was assessed using the protein denaturation assay. A 450 µL volume of a 1% aqueous solution of bovine serum albumin (BSA) was mixed with 50 µL of the test specimens at varying concentrations ranging from 5 µg/mLto 50 µg/mL. The pH of the solution was adjusted to 6.4 using a small amount of 1.0 N hydrochloric acid. The mixture was incubated at room temperature for 20 min, followed by heating in a water bath at 50 °C for 35 min. After cooling, the absorbance of the samples was measured at 650 nm using a Biosystem 310 Plus spectrophotometer (UK). Diclofenac sodium was used as the reference drug for comparison, and dimethyl sulfoxide (DMSO) served as the control (Ameena et al. 2023).

Elucidation of anticancer and cytotoxicity roles of examined oil forms

The MTT assay was employed to evaluate the cytotoxic effects of ozonized peanut oil and crude peanut oil dissolved in DMSO on HCT-116 cells (human colorectal carcinoma) as representative cancer cells, and WI-38 cells (human fibroblasts) as representative normal cells. This method produces a blue color corresponding to the quantity of viable cells. Absorbance was measured at 540 nm using a computerized microplate analyzer (Agilent, USA). Cells were first allowed to adhere for 24 h, after which samples at concentrations ranging from 1000 to 31.25 µg/mLwere added and incubated for another 24 h at 35 °C. Fresh medium was then introduced, followed by the addition of 100 µL of MTT solution (5.0 mg/mL). The mixture was incubated at 38 °C for 240 min. The resulting cellular activity was observed and recorded using a CCD camera and a microscope (Nikon, Japan) (Al-Rajhi and Abdelghany 2023b).

Statistical evaluations

The data are presented as the mean ± SD for each experiment, which was performed in triplicate. Statistical analysis to compare means was conducted using the t-test and one-way ANOVA. GraphPad Prism V5 (San Diego, CA, USA) software was used for the analysis. Differences were considered statistically significant at p < 0.05.

Results

GC-MS analysis of crude peanut oil revealed the presence of ten compounds from five different classes. These compounds included Thymoquinone (Monoterpene), 2,4-Decadienal (E, Z) and 2,4-Decadienal (both Aldehydes), Dodecanoic acid, Tetradecanoic acid, n-Hexadecanoic acid, and 17-Octadecynoic acid (Fatty acids), as well as 9,12-Octadecadienoic acid (Z, Z) and Oleic acid (Fatty acids). Additionally, 9,12-Octadecadienoyl chloride (Z, Z) was identified as a Pyridinecarboxylic acid. In contrast, the ozonized peanut oil contained 29 different compounds from approximately fifteen classes. These included Cyclohexene, 1-methyl-4-(1-methyl-ethenyl) (Hydrocarbon), Oxalic acid, allyl decyl ester (Carboxylic acid ester), 5,5-Dimethyl-cyclohex-3-en-1-ol (Alicyclic hydrocarbon), and 2-Decenal (E) (Aldehyde). Other compounds included 2,4-Decadienal (E, E) (Aldehyde), (-)-Isopinocampheol, acetate (Monoterpene), Tetradecane (Alkane), Oxalic acid, 6-ethyloct-3-yl hexyl ester (Carboxylic acid ester), and Hexadecane (Alkane). Also identified were 2-Heptadecanol (Fatty alcohol), 1,2-Benzenedicarboxylic acid, butyl octyl ester (Di-carboxylic acid ester), n-Hexadecanoic acid (Fatty acid), Oleic acid (Fatty acid), and 10-Methyldodecan-5-olide (Alkane). Other notable compounds were Phenol, 2,2’-methylenebis[6-(1,1-dimethylethyl)-4-methyl] (Phenol), Butyl 9,12-octadecadienoate (Fatty acid), 6-Octadecenoic acid (Fatty acid), 1-Heptatriacotanol (Alcohol), and Eicosanoic acid, isobutyl ester (Fatty acid ester). The ozonized oil also contained Betulin (Triterpene), Butyl 9,12-octadecadienoate (Fatty acid), 9,12-Octadecadienoic acid (Z, Z) (Fatty acid), Ethyl 9-cis, 11-trans-octadecadienoate (Fatty acid), Erucic acid (Fatty acid), Squalene (Hydrocarbon), α-Tocopheryl methyl ether (Methylated phenol), β-Sitosterol (Phytosterol), and 4 H-1-benzopyran-4-one, 2-(3,4-dihydroxyphenyl)-6,8-di-α-D-glucopyranosyl-5,7-dihydroxy (Coumarins). Four compounds were common to both the crude and ozonized oils: 2,4-Decadienal (E, E), n-Hexadecanoic acid, 9,12-Octadecadienoic acid (Z, Z), and Oleic acid. The peak areas of these compounds were significantly reduced (P ≤ 0.05) in the ozonized oil (Figs. 1 and 2, and Table 1).

Fig. 1.

Fig. 1

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GC-MS examination for the various molecules in crude peanut oil

Fig. 2.

Fig. 2

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GC-MS analysis for the different compounds crude peanut oil exposed to ozone

Table 1.

Illustration of various bioactive compounds in crude peanut oil using GC-MS

*RT (min) Area (%) Compound Class Formula ***MW (g/mol)
20.45 1.96 Thymoquinone Monoterpene C10H12O2 164
22.78 2.11 2,4-Decadienal, (E, Z)- A Aldehyde C10H16O 152
23.79 3.89 2,4-Decadienal Aldehyde C10H16O 152
36.55 3.63 Dodecanoic acid Fatty acid C12H24O2 200
43.18 2.83 Tetradecanoic acid Fatty acid C14H28O2 228
50.05 5.37 n-Hexadecanoic acid B Fatty acid C16H32O2 256
56.96 3.00 17-Octadecynoic acid Fatty acid alkyne C18H32O2 280
57.29 70.25 9,12-Octadecadienoic acid (Z, Z)- C Fatty acid C18H32O2 280
62.95 1.91 9,12-Octadecadienoyl chloride, (Z, Z)- Pyridinecarboxylic acid C18H31ClO 298
63.18 5.05 Oleic Acid D Fatty acid C18H34O2 282
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Capital and small superscript letters refer to common molecules in both types of oil with significant difference P ≤ 0.05

Table 2.

Illustration of various bioactive compounds in crude peanut oil exposure to Ozone using GC-MS

*RT (min) Area (%) Compound Class Formula **MW (g/mol)
11 0.46 1.49 Cyclohexene, 1-methyl-4-(1-met hylethenyl)- Hydrocarbon C10H16 136
19.36 0.85 Oxalic acid, allyl decyl ester Carboxylic acid ester C15H26O4 270
20.05 0.55 5,5-Dimethyl-cyclohex − 3-en-1-ol Alicyclic hydrocarbon C8H14O 126
21.11 0.71 2-Decenal, (E) Aldehyde C10H18O 154
23.41 1.49 2,4-Decadienal, (E, E)- a Aldehyde C10H16O 152
25.01 0.85 (-)-Isopinocampheol, acetate Monoterpene C12H20O2 196
27.95 0.55 Tetradecane Alkane C14H30 198
31.93 0.71 Oxalic acid, 6-ethyloct-3-yl hexyl ester Carboxylic acid ester C18H34O4 314
35.81 24.84 Hexadecane Alkane C16H34 226
38.50 1.47 2-Heptadecanol Fatty alcohol C17H36O 256
46.81 1.35 1,2-Benzenedicarboxylic acid, butyl octyl ester Di-carboxylic acid ester C20H30O4 334
48 0.50 1.83 n-Hexadecanoic acid b Fatty acid C16H32O2 256
54 0.02 1.13 Oleic Acid d Fatty acid C18H34O2 282
59 0.79 1.56 10-Methyldodecan-5-o lide Alkane C13H24O2 212
61.46 10.89 Phenol, 2,2’-methylenebis[6-(1,1-dimethylethyl)-4-methyl Phenol C23H32O2 340
61.95 5.61 Butyl 9,12-octadecadienoate Fatty acid C22H40O2 336
62.21 15.27 6-Octadecenoic acid Fatty acid C18H34O2 282
64 0.07 0.82 1-Heptatriacotanol Alcohol C37H76O 536
64.59 1.17 Eicosanoic acid, isobutyl ester Fatty acid ester C24H48O2 368
68.17 1.21 Betulin Triterpene C30H50O2 442
68.60 1.43 Butyl 9,12-octadecadienoate Fatty acid C22H40O2 336
68 0.80 4.12 9,12-Octadecadienoic acid (Z, Z)-, c Fatty acid C21H38O4 354
69.58 0.79 Ethyl 9.cis.,11.trans.-octadec adienoate Fatty acid C20H36O2 308
71.30 1.51 Erucic acid Fatty acid C22H42O2 338
73.18 11.63 Squalene Hydrocarbon C30H50 410
79.62 2.2 ç-Tocopheryl methyl ether Methylated phenol C29H49DO2 431
83.18 2.5 ç-Sitosterol Phytosterol C29H50O 414
86.04 1.47 4 H-1-benzopyran − 4-one, 2-(3,4-dihydroxyp henyl)-6,8-di-á-dglucopyranosyl − 5,7-dihydroxy Coumarins C27H30O16 610
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Capital and small superscript letters refer to common molecules in both types of oil with significant difference P ≤ 0.05

*RT; Retention time, **MW; Molecular weight

It was observed that peanut oil exhibited significant antimicrobial activity against B. subtilis (ATCC 6633), S. aureus (ATCC 6538), K. pneumonia (ATCC 13883), S. typhi (ATCC 6539), and C. albicans (ATCC 10221) when compared to standard antimicrobial agents. However, when the oil was exposed to ozone, its antimicrobial activity increased, surpassing the efficacy of the standard antimicrobial drugs. Additionally, the ozonized oil demonstrated antifungal activity against A. niger (ATCC 16888), with an inhibition diameter of 15 ± 0.1 mm, as shown in (Fig. 3; Table 3). Exposure of peanut oil to ozone resulted in a significant reduction (P ≤ 0.05) of the MIC for B. subtilis (ATCC 6633), from 31.25 ± 0.4 to 15.62 ± 0.2 µg/mL, and its MBC (P ≤ 0.05), from 62.5 ± 0.1 to 31.25 ± 0.3 µg/mL. Additionally, the MIC for S. aureus (ATCC 6538) decreased from 62.5 ± 0.3 to 15.62 ± 0.1 µg/mL, and its MBC dropped from 125 ± 0.1 to 31.25 ± 0.1 µg/mL. A significant reduction in the MIC and MBC levels was also observed for K. pneumoniae (ATCC 13883), S. typhi (ATCC 6539), C. albicans (ATCC 10221), and A. niger (ATCC 16888) (Table 3).

Fig. 3.

Fig. 3

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Comparative antimicrobial examination for the crude peanut oil, ozonized oil towards different examined tested microbes and both DEMSO and standard drugs (Gentamicin ((0.07 mg/mL) for bacteria, fluconazole (0.24 µg/mL) for fungi) were examined as negative and positive controls respectively

Table 3.

Antimicrobial action (mm) of peanut crude oil and ozonized oil relative to standard drug (Data are reported as means ± SD) with mics and MBCs (µg/mL) detection

Pathogenic microorganism Inhibition zone (mm) MIC MBC
Crude Oil Ozonized oil Control drugs Crude Oil Ozonized oil Crude Oil Ozonized oil
B. subtilis 25 ± 0.1 28 ± 0.2 26 ± 0.2 31.25 ± 0.4 15.62 ± 0.2* 62.5 ± 0.1 31.25 ± 0.3#
S. aureus 20 ± 0.1 26 ± 0.2 24 ± 0.2 62.5 ± 0.3 15.62 ± 0.1* 125 ± 0.1 31.25 ± 0.1#
K. pneumonia 19 ± 0.1 22 ± 0.1 18 ± 0.1 62.5 ± 0.2 31.25 ± 0.4* 125 ± 0.1 62.5 ± 0.7#
S. typhi 20 ± 0.2 23 ± 0.1 20 ± 0.1 62.5 ± 0.2 31.25 ± 0.1* 250 ± 0.1 125 ± 0.6#
C. albicans 27 ± 0.1 29 ± 0.1 26 ± 0.1 31.25 ± 0.6 7.8 ± 0.3* 62.5 ± 0.1 15.62 ± 0.1#
A. niger NA 15 ± 0.1 34 ± 0.2 – 125 ± 0.2* – 500 ± 1.1#
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Results are recorded as means ± SD; * in mics, # in MBCs in different test microbes refer to significant difference P ≤ 0.05

In the current study, the DPPH assay was applied to examine the antioxidant activity of peanut oil, revealing a promising impact with an IC50 of 23.37 ± 0.3 µg/mL, compared to ascorbic acid, which was used as the standard with an IC50 of 3.08 ± 0.4 µg/ml. Additionally, exposure of peanut oil to ozone led to an improvement in its antioxidant activity, with the IC50 reaching 13.06 ± 0.6 µg/mL (Fig. 4). The protein denaturation assay revealed the effective anti-inflammatory impact of peanut oil, with an IC50 of 15.38 ± 0.7 µg/ml, compared to the standard, which had an IC50 of 6.83 ± 0.6 µg/mL. Additionally, exposure of peanut oil to ozone led to a dramatic enhancement (P ≤ 0.05) in the oil’s anti-inflammatory impact, with the IC50 reported as 7.36 ± 0.5 µg/mL (Fig. 5).

Fig. 4.

Fig. 4

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Examination of the antioxidant activity by DPPH assay for the crude peanut oil and ozonized oil relative to ascorbic acid which used as a standard (Data are drawn as means ± SD)

Fig. 5.

Fig. 5

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Anti-inflammatory activity for the crude peanut oil and after exposure of oil to ozone relative to diclofenac sodium which used as a standard by using protein denaturation procedure (Data are drawn as means ± SD)

In this study, the MTT assay was used to examine the inhibitory activity of crude peanut oil at different concentrations against colon carcinoma cells (HCT). The oil demonstrated efficient anti-tumor activity with an IC50 of 15.09 ± 0.37 µg/mL. However, exposure of the oil to ozone layers resulted in a significant boost (P ≤ 0.05) in its anticancer activity, with the IC50 reaching 7.31 ± 0.21 µg/mL, as shown in Table 4.

Table 4.

Antitumor impact of peanut crude oil and ozonized oil towards HCT cells

Conc. (µg/mL) Crude Oil Ozonized oil
Viability % Inhibitory % S.D. (±) Viability % Inhibitory % S.D. (±)
500 2.39 97.61 0.35 1.32 98.68 0.06
250 5.97 94.03 0.21 4.17 95.83 0.15
125 11.46 88.54 0.62 8.09 91.91 0.13
62.5 25.31 74.69 0.73 15.78 84.22 0.37
31.25 38.72 61.28 1.24 25.27 74.73 0.91
15.6 49.14 50.86 1.97 34.65 65.35 0.79
7.8 62.53 37.47 1.28 48.47 51.53 1.64
3.9 75.06 24.94 0.68 60.45 39.55 1.39
0.0 100 0.0 0.0 100 0.0 0.0
IC50 15.09 ± 0.37 µg/mL 7.31 ± 0.21 µg/mL
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Outcome are tabulated as means ± SD

Human lung fibroblast normal cells (WI-38) were used to examine the cytotoxic impact of both forms of oil at different concentrations. It was observed that both types of oil exhibited minimal cytotoxicity towards the normal tested cells. The IC50 values for crude peanut oil and ozonized peanut oil were 24.68 ± 1.44 µg/mL and 29.49 ± 2.03 µg/mL, respectively (Table 5).

Table 5.

Cytotoxicity of peanut crude oil and ozonized oil towards WI-38 cells upon

Conc. (µg/mL) Crude Oil Ozonized oil
Viability % Inhibitory % S.D. (±) Viability % Inhibitory % S.D. (±)
500 3.41 96.59 0.15 4.24 95.76 0.17
250 6.87 93.13 0.39 10.51 89.49 0.43
125 13.28 86.72 0.64 19.42 80.58 0.56
62.5 29.65 70.35 1.03 32.75 67.25 0.97
31.25 44.65 55.35 1.61 47.18 52.82 1.36
15.6 57.46 42.54 1.32 72.91 27.09 2.03
7.8 70.39 29.61 1.25 87.29 12.71 0.71
3.9 78.26 21.74 0.48 95.23 4.77 0.45
0.0 100 0.0 0.0 100 0.0 0.0
IC50 24.68 ± 1.44 µg/mL 29.49 ± 2.03 µg/mL
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Data are tabulated as means ± SD

Discussion

Various dietary oils are produced and consumed in increasing quantities worldwide each year. People’s dietary habits have shifted from being high in plant-derived oils to being high in animal-derived oils (Bai et al. 2021; Qanash et al. 2023b). Most research has focused on assessing the nutritional content of edible oils from a dietary and health-promoting perspective (Al-Rajhi and Abdelghany 2023b; Alsolami et al. 2023; Qanash et al. 2023b). Peanut oil is widely consumed due to its distinct flavor, and its components can be significantly affected by the roasting method, which can alter the flavor of the oil (Yang et al. 2021; Yin et al. 2022). The application of biotechnology to improve the pharmaceutical and therapeutic properties of edible oils and enhance their therapeutic potential has been increasingly explored by the research community (Rahim et al. 2022). In this study, crude peanut oil was ozonized for five hours at a flow rate of 0 to 7 L/min using a designated system. Multiple factors, including exposure time, ozone and oxygen levels, temperature, reaction motion, and the type and quantity of vegetable oil, as well as the addition of water or catalysts, can affect the ozonation process and the effectiveness of ozonized oils produced using ozone generators (Tığlı Aydın and Kazancı 2018). Additionally, using pharmaceutical-grade oxygen may prevent the formation of potentially hazardous nitrated by-products (Travagli et al. 2010).

The chemical composition of crude peanut oil significantly changed upon exposure to ozone. While the crude oil contained ten different compounds, the ozonized oil contained twenty-nine compounds. Both oil forms shared four common compounds: 2,4-Decadienal (E, E), n-Hexadecanoic acid, 9,12-Octadecadienoic acid (Z, Z), and Oleic acid, all of which showed reduced peak areas in the ozonized oil. Oleic acid, present in peanuts, has been shown to increase insulin production in type-II diabetic mice (Vassiliou et al. 2009). Research by Akbas and Ozdemir (2006) found that the proportion of free fatty acids in pistachio oil ozonized at 5.0, 7.0, and 9.0 mg L-1 did not increase after exposure for up to 420 min. Ozone primarily interacts with the double carbon–carbon bonds in unsaturated fatty acids, forming ozonides, aldehydes, and peroxides. The amount and ratio of these chemicals depend on several factors, including the type of reactor, process temperature, and ozonation duration (Díaz et al. 2006; Georgiev et al. 2015).

The ozonized peanut oil exhibited significant antimicrobial activity against B. subtilis, S. aureus, K. pneumonia, S. typhi, C. albicans, and A. niger, indicating that ozonation improves the antimicrobial action. Several studies have shown that ozonized oils at varying degrees of ozonation exhibit promising antimicrobial effects (van de Kassteele et al. 2012; Ledea-Lozano et al. 2019). Ozonized oils have been effectively used in humans to treat a variety of skin disorders and sensitivities, as well as to care for skin after surgical procedures, laser treatments, and sunburn (Silva et al. 2021). The antioxidant capacity of both oil types was evaluated in vitro using common tests, such as the DPPH assay (Rahim et al. 2023). According to Prabasheela et al. (2015), peanut oil has lower antioxidant capacity compared to oils such as sunflower and avocado oil. The presence of various molecules in the ozonized oil may enhance its overall functional properties, providing additional health benefits such as antioxidant and antibacterial activities (Al-Snafi 2020). The process of protein denaturation involves changes to hydrophobic, disulfide, and electrostatic hydrogen bonds (Sen et al. 2015). Inflammatory diseases can result in protein denaturation, leading to the generation of autoantigens. Therefore, preventing protein denaturation may reduce inflammatory activity (Sangeetha and Vidhya 2016). Peanut oil’s anti-inflammatory potential has been attributed to its rich fatty acid content (Zhao et al. 2019; Anavi-Cohen et al. 2023). Exposure of oil to ozone layers increases the number of chemical classes responsible for enhancing anti-inflammatory effects. The MTT assay was used to assess the cytotoxic and anticancer effects of peanut oil on tumor cells (Tsai et al. 2024). Peanut oil demonstrated encouraging anticancer potential against colon cancer cells, and exposure to ozone further enhanced its effects. Additionally, both oils exhibited negligible harmful effects on healthy human fibroblast cells. Researchers have shown that peanut oil’s fatty acid composition is effective in treating various types of cancer (Hammad et al. 2023; Jeeunngoi et al. 2024). Finally, the exposure of Peanut oil to ozone for five hours at a flow rate of 0 to 7 L/min significantly influences the relative concentrations of bioactive compounds, particularly 2,4-Decadienal (E, E)-, n-Hexadecanoic acid, 9,12-Octadecadienoic acid (Z, Z)-, and Oleic acid. Enhancment of antimicrobial, antioxidant, anticancer, and anti-inflammatory activities was attributed to ozonized oil. Overall, this approach provides a promising basis for further scientific investigations into ozonized peanut oil, including studies using animal models and additional clinical research. Also, separation of active compounds afterozonization and test its activity separately aagainst more pathogenic microorganisms and cancer cells as wella as study its action mechanisms on the levels of ultrastructures, enzymes, and target proteins.

Acknowledgements

Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Author contributions

Conceptualization, methodology A.M.H.A. and T.M.A.; formal analysis, investigation S.S., M.S.A., M. T. A. and M.H.A.; writing—original draft preparation, writing—review and editing, M.T.A., S.S. and S.K.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Availability of data and material

All data that support the findings of this study are available within the article.

Declarations

Competing interest

The authors declare no conflicts of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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