Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2024 Dec 11;14:30445. doi: 10.1038/s41598-024-81157-9

Effect of ozonation on the phytochemicals of black seed oil and its anti-microbial, anti-oxidant, anti-inflammatory, and anti-neoplastic activities in vitro

Aisha M H Al-Rajhi 1, Tarek M Abdelghany 2,✉, Mohammed S Almuhayawi 3, Mohammed H Alruhaili 3,4, Amna A Saddiq 5, Afra M Baghdadi 5, Soad K Al Jaouni 6, Hibah M Albasri 7, Moayad S Waznah 7, Faisal A Alraddadi 7, Samy Selim 8,✉
PMCID: PMC11634964  PMID: 39663384

Abstract

Black seed has been applied for several decades to cure an extensive variety of illnesses and ailments. In this report, the chemical profile of both crude and ozonized black seed oil was assessed after the oil was exposed to 0 to 5 L/minute of ozone for four hours. The in vitro effects of black seed oil following being exposed to ozone including antimicrobial properties versus Bacillus cereus (ATCC11778), Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC8739), Salmonella typhi (ATCC 6539), and Klebsiella pneumoniae (ATCC13883), Candida albicans (ATCC10221), and Aspergillus niger (ATCC16888). Besides, antioxidant effects, anti-inflammatory capacity, and antineoplastic function versus HCT cells were assessed. The chemical profile of ozonized black seed oil showed elevation of essential molecules of oil as well as presence of some characteristic molecules to both forms of oil. Besides, it could be noticed that exposing of oil to ozone improves its antimicrobial activity towards all tested microbes except for C. albicans. Both forms of oil showed no activity towards A. niger. Black seed oil exposed to ozone showed a promising antioxidant capacity with IC50 of 2.93 ± 0.2 µg/ml. A dramatic improvement in anti-inflammatory impact of ozonized oil as well as its antitumor capacity towards HCT cells could be seen in the laboratory outcomes. The current findings point to a novel method for enhancing some of the in vitro medicinal uses of black seed oil by exposing it to ozone.

Keywords: Black seed oil, Ozone, Gas chromatography, Antimicrobial, Anti-inflammatory, Anti-oxidant, Antineoplastic

Subject terms: Biotechnology, Microbiology, Plant sciences

Introduction

Plant-based diets fulfill the body’s basic dietary requirements, maintain bodily wellness, and strengthen the body’s defenses to a range of diseases1,2. The idea of “functioning foodstuff” has gained popularity in the last several decades within people who are concerned about their health because a balanced diet and a normal lifespan are closely related3. Small but essential substances like herbs and spices, which primarily serve as flavoring additions and additives for preservation, are rich in therapeutic molecules throughout the range of utilized food substances4,5. Despite being mostly used in food preparation, the majority of these edible herbs and spices are also well-known for their nutritious qualities because of their immense capacity to improve health6,7.

Nigella sativa (Black cumin) is becoming known as a miraculous herb with a rich historic and religious history with several studies demonstrating its extensive range of medicinal prospects8,9. The term “black seed” refers to N. sativa which it is grown in many nations worldwide, including the Middle Eastern Mediterranean region10. During history, people have utilized the oil and seeds of N. sativa to treat a wide range of illnesses. In the conventional medical system of India, it is also a significant drug11. N.sativa seeds are used medicinally in many traditional herbal fields for a variety of ailments, such as various respiratory disorders, paralysis, hypertension, diabetes, digestive tract issues, inflammation, and pains like chronic headaches and back pain. Additionally, it has been administered topically to swollen joints, orchitis, eczema, blisters, and nasal abscesses6,9. Because black seed is said to be the cure for all ailments in a prophetic hadith, it is regarded as one of the best types of medicinal treatment accessible. It is also advised to be used frequently in prophesy healthcare12.

The spread of food-borne and human pathogenic microorganisms, in addition to their resistance to many antibiotics, has become a global problem13. This problem requires the collaboration of scientists to search for safer and more effective alternatives or to develop the utilized compounds to inhibit these microbes14. Cancer is one of the main causes of death, and in many cases death is certain as a result of infection by this disease15. It is even considered the second cause of death after heart diseases. The third most common cancer worldwide is colorectal cancer, with rising incidence rates in developing economies, even as incidence rates among individuals with average age start have fallen in high-income economies16. Advances in research during the last 10 years have improved overall survival rates and treatment alternatives. One of the advantages of our current study is that it deals with safe oil and is developing it through the ozonization process. It can be added to foodstuffs or used orally to combat microbes, whether present in foodstuffs or pathogenic to humans. Indeed, there are some active substances in the oil used that have been proven effective against microbes.

Black cumin’s botanical molecules changes according to the cultivation area, development phase, preparation procedures, and separation procedures17. Black cumin has health advantages for practically every biological system in human body, including the neurological, gastrointestinal systems18. The majority of this plant’s medicinal qualities are a result of thymoquinone, a significant active ingredient in the oil that is used for medicine19. Thymoquinone was applied medicinally for suppress several multidrug resistance bacteria including Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus besides Candida albicans and Aspergillus niger20,21. Due to their extremely low toxicity, black seeds are also employed as flavoring additives in bread and pickles22.

The emphasis on reducing hazardous disinfection byproducts (DBPs) in any product has increased due to growing worries about safety of environment and public health. Even though conventional techniques like electrochlorination, ultraviolet (UV) irradiation, and chlorination are efficient for disinfection, they frequently result in the DBPs formation, which are dangerous23. Ozone’s strong oxidative properties and capacity to be applied at various phases of the treatment process make it an extremely effective and adaptable disinfectant. Comparing ozone DBPs to those produced by chlorination, few research have been done to identify, measure, and assess their toxicity24. In the food companies, ozonation is probably one of the greatest effective techniques for decontaminating newly produced products25. Because of its ability to break down oxygen quickly and leave no trace behind, ozone also appears to be a promising substitute for disinfection26. Besides, because ozone is a strong oxidizer, using it for extended periods of time or at high quantities may cause essential oils to break down. When it comes to spice plants that are used to enhance flavor and scent27. Study on the impact of ozonation on the composition of essential oils has been conducted; however, it is important to note that ozone does not alter the chemical formulas of essential oils28. Moreover, certain commercial products that contain essential oils but are derived from vegetal oils do not provide evidence of biomedical effects29. In the present work, the Gas chromatography-Mass spectrometry (GC-MS) examination was applied to test the ingredients of the black seed oils before and after administration of ozone. The bioactivities of the crude and ozonized black seed oils including anti-microbial, anti-oxidant, anti-inflammatory impacts, and anti-neoplastic impact towards human colorectal carcinoma (HCT) cell line was tested.

Materials and methods

Chemicals and test microorganisms

Since black seeds are frequently utilized in Egyptian culture, the oil used in the current work was bought from a local market in Nasr City, Cairo, Egypt. Every chemical utilized in this investigation was bought from Sigma, Egypt. The examined microbial isolated were obtained from the culture collection of Mycology Center , Al-Azhar University, Egypt.

Exposing the black seeds oil to ozone

Ozone gas was produced by an electric shock ionization generator. A 1.0 L drechsel tank with 0.5 mL of black seed oil was placed near the plasma reactor’s output in a – 6 °C cooling bath. For four hours, at a flow rate of 0–6 L/min, the ozone formed bubbles in the coconut oil, producing in a partially solid state. Once the black seed oil was ozonized, it was taken out of the drechsel vessel placed in an empty packaging, quantified, and stored at 3 °C30,31.

Gas chromatography-mass spectrometry (GC-MS) for the crude and ozonized oil

The Rt-560 column (99.0 m × 0.28 mm × 0.22 μm; SCION, UK), auto-sampler (570-SCION, UK), and flame-ionization detection were used for the GC assessment. A chromatography equipment was used to extract information from the FID (Compass CDS collecting data and programs, SCION, USA). As a transfer gas, helium was administered by rip (98:1). The pre-run lasts for nine minutes. It takes 0.6 min to reach equilibrium. Two ramping were completed: ramp 1.0 at a rate of 4 °C/min, resulting in an end temperature of 205 °C, and ramp 2 at a value of 5 °C/min. The temperature reached a maximum of 285 °C after reaching 46 °C initially. Systemized heat mode was used for the evaluations, starting at 100–285 °C and ending at homogeneity for 16.0 min. A 1.6 mL/min transport flow was applied32.

Antimicrobial action

To determine the black seeds oil and ozonized oil’s in vitro anti-microbial impact versus a variety of test microbes of bacteria and fungi, the well agar diffusion technique was utilized. The plates made of Mueller Hinton agar were used for the bacteria, and malt extract medium was utilized for the fungi. An overall volume of 100 µl of various suspended bacterial cells (1.7 × 106 colony forming units/mL) was employed for the bacteria. Applying a disinfected cork borer edge, the tested oils were then added to the well that has been created in medium. DMSO was used as the control substance and gentamicin (0.06 mg/mL) and fluconazole (0.26 µg/mL) as the norm. The inhibitory zone was assessed following 72 h of development at 36 °C for bacterial cells, 5–7 days, and 29 °C for mold33.

Determination for minimal inhibitory level (MIC) and minimal bactericidal value (MBC)

Using nutritious broth for microbes, the micro-dilution broth technique was used to determine the samples’ minimum inhibitory concentration (MIC). Each of the samples under investigation had been diluted twice to determine the ultimate levels, which varied from 0.98 to 1000 µg/mL. To prepare the 96-well micro-titrate plate, 200 µL of the samples dilutions under evaluation in broth media has been placed to each hole. After utilizing new microbial cultures that satisfied the turbidity requirements of the 1.0 McFarland standard to create the inoculum, 2.0 µL of sterile 0.9% NaCl was added to each well to attain a 3.0 × 106 CFU/mL level. After that, the microbes were cultured at 36 °C for 72 h for bacteria and 35 °C for 5–7 days for fungi. All of the samples were optically measured to determine MIC that occurred when the growth of the reference strain was totally suppressed. There was a negative reference (estimated samples without an inoculum) and a positive reference (an inoculum lacking the studied samples) on each microplate34. In each well, 100 milliliters of the microbial culture were sub-cultured onto plates containing medium with 100% growth suppression, from the development control, and from the final positive sample. This allowed for the determination of minimum bactericidal concentration (MBC). The MBC was discovered to have the lowest value of specimens that did not support microbial growth during incubation period at proper temperature.

Anti-oxidant impact

To assess the evaluated samples’ antioxidant capacity. The ethanol solution was used, which contained 0.1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH). Three milliliters of this solution were mixed with 1 milliliter of different concentrations of the specimens in ethanol, varying between (3.9–1000 µg/mL). The mixture was given a thorough shake before being allowed to stand at room temperature for half an hour. The absorption at 517 nm being measured for the resultant color35.

Anti-inflammatory action

The membrane stabilization testing was carried out to determine the anti-inflammatory characteristics of both specimens. Multiple amounts of specimens between 100 and 1000 µg/mL were prepared to do. The obtained specimens were incorporated with hypotonic liquid. Both distilled water and indomethacin were employed as negative and positive standards. The fresh erythrocyte suspension (3.0%) in 0.8 mL saline was incorporated with 500 µL of the samples, and the combination underwent incubation for two hours at 36 °C. The combination was subsequently spun at 13,000 × g for 24 min at 6 °C. The absorbance of samples was detected at 570 nm36.

Anti-neoplastic action

The cytopathic effect on HCT cells was observed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) (MTT) technique for ozonized and crude oil after dissolved in DMSO. The outcome, when measured by traditional phases, is a blue tint whose values are directly related to the number of living cells. A computerized microplate analyzer (BMG, LABTECH, AUSTRILLIA) was used to determine the absorbance at 560 nm. Following a 24-hour period of adhesion till combining, specimens ranging from 1000 to 31.25 µg/mL were introduced, and the cells were then kept at 35 °C for an additional 24 h. When the fresh media was added, 100 µL of MTT solution (5.0 mg/mL) was added, and it remained at 35 °C for a period of 4 h. Using a microscope (Accu-Scope INC, USA) and a CCD camera, the cells were seen37.

Statistical testing

Every test was conducted thrice, and the results are displayed as the average ± SD. The Graph Pad Prism V5 (San Diego, CA, USA) program was used for determining the distinction between means using the t-test and one way ANNOVA. Results indicating p < 0.05 were labeled as significant changes.

Results

Determination of variations in compounds in crude black seed oil and after exposure to ozone using GC-MS

The testing of crude black seed oil by GC-MS chromatography revealed the presence of 28 compounds which consisted 12 major compounds with about 5 different classes of (fatty acids, terpenoid, phytosterols, hydrocarbon, and phenols) of including: Phenol, 2-methyl-5-(1-methylethyl)-; 1,2-Benzenedicarboxylic acid, dimethyl ester; t-Butylhydroquinon; 2,4-Decadienal; ç-Sitosterol; Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl)-; Oleic Acid; Phenol, 2,2’-methylenebis[6-(1,1-dimethylethyl)-4- methyl; 9,12-Octadecadienoic acid (Z, Z)-; 2,3-dihydroxypropyl ester; o-Cymene; n-Hexadecanoic acid; citronellal, consecutively, as shown in (Fig. 1a; Table 1). Besides, examination ozonized black seed oil via GC-mass chromatography showed the presence of 34 compounds with 11 major molecules of six different classes of (Fatty acids, hydrocarbon, terpenes, flavonoid, phytosterol and Sesquiterpene) including: o-Cymene; Thymoquinone; Oleic Acid; Citronellal; Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl); 9,12-Octadecadienoic acid (Z, Z)-; Globulol; 9,12-Octadecadienoic acid (Z, Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester; E,E, Z-1,3,12-Nonadecatriene-5,14-diol; n-Hexadecanoic acid and ç-Sitosterol. Some compounds in both forms of oil could be seen in minor levels. Four compounds could be seen only in crude oil including:1,2-Benzenedicarboxylic acid, dimethyl ester; t-Butylhydroquinon; 1,2–15,16-Diepoxyhexadecane and Androst-5-en-4-one. Besides, about 10 molecules could be seen only in ozonized oil including: Linalool, methyl ether; 1-Butene, 2,3,3-trimethyl-; (+)-cis-Verbenol, acetate; Tricyclo[5.4.0.0(2,8)]undec-9-ene, 2,6,6,9-tetramethyl-, (1R,2 S,7R,8R)-; Dimethyl phthalate; Isopulegol; Phthalic acid, 2,7-dimethyloct-7-en-5-yn-4-yl isobutyl ester; 2,3-Dihydro farnesyl acetate; E,E, Z-1,3,12-Nonadecatriene-5,14-diol and Squalene as depicted in (Fig. 1b; Table 1).

Fig. 1.

Fig. 1

Open in a new tab

GC-mass chromatogram showing of different molecules in (A) crude black seed oil, (B) ozonized black seed oil.

Table 1.

Various separated molecules and their classes in crude and ozonized black seed oils.

RT
(Crude)		 Peak name Molecular weight Molecular formula Peak area % RT (O3) Peak name Molecular weight Molecular formula Peak area %
7.76 Pentane, 2,2-dimethyl- (Neoheptane) 100 C7H16 1.2 7.95 Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl)- (Bicyclic monoterpenoids) 136 C10H16 7.02
7.94 Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl)- (Bicyclic monoterpenoids) 136 C10H16 2.54 8.36 Linalool, methyl ether (Monoterpenoid) 168 C11H20O 0.70
8.35 1,5-Heptadien-4-ol, 3,3,6-trimethyl- (Monoterpene alcohol) 154 C10H18O 0.7 9.96 1-Butene, 2,3,3-trimethyl- (Alkene) 98 C7H14 1.49
11.59 o-Cymene (Aromatic hydrocarbon) 134 C10H14 1.75 11.64 o-Cymene (Aromatic hydrocarbon) 134 C10H14 17.99
11.83 D-Limonene (Monoterpene) 136 C10H16 1.06 11.76 (+)-cis-Verbenol, acetate (Bicyclic montropene alchol) 194 C12H18O2 0.39
15.70 Nerol, methyl ether (Noncyclic sesquiterpene alkene alcohol) 168 C11H20O 0.62 11.85 D-Limonene (Monoterpene) 136 C10H16 0.73
21.07 Thymoquinone (Flavonoid) 164 C10H12O2 0.6 15.71 Nerol, methyl ether (Noncyclic sesquiterpene alkene alcohol) 168 C11H20O 2.01
23.9 2,4-Decadienal, (E, E)- (Medium chain aldehyde) 152 C10H16O 2.98 21.23 Thymoquinone (Flavonoid) 164 C10H12O2 14.71
24.63 Phenol, 2-methyl-5-(1-methylethyl)- (Monoterpenic phenol) 150 C10H14O 10.12 21.91 Furan, 2-(2-furanylmethyl)-5-methyl (heteraromatic methyl compound) 162 C10H10O2 0.45
28.14 Longifolene (Tricyclic sesquiterpene) 204 C15H24 0.7 24.01 2,4-Decadienal, (E, E)- (Medium chain aldehyde) 152 C10H16O 1.57
29.17 1,2-Benzenedicarboxylic acid, dimethyl ester (Dieter of carobxylic acid) 194 C10H10O4 3.89 24.66 Phenol, 2-methyl-5-(1-methylethyl)- (Monoterpenic phenol) 150 C10H14O 1.27
35.34 t-Butylhydroquinon (phenol, derviative of quinol) 166 C10H14O2 3.58 25.98 Tricyclo[5.4.0.0(2,8)]undec-9-ene, 2,6,6,9-tetramethyl-, (1R,2 S,7R,8R)- (Sesquiterpene) 204 C15H24 0.44
40.43 1,2–15,16-Diepoxyhexadecane (Hydrocarbon) 254 C16H30O2 0.57 28.16 Longifolene (Tricyclic sesquiterpene) 204 C15H24 1.66
47.55 Androst-5-en-4-one (Steroid hormone) 272 C19H28O 0.7 29.21 Dimethyl phthalate (organic ester) 194 C10H10O4 0.58
48.38 1,7-Hexadecadiene (Alkane hydrocarbon) 222 C16H30 1.08 35.36 p-Cymene-2,5-diol (Terpenes) 166 C10H14O2 1.17
49.53 n-Hexadecanoic acid (fatty acid) 256 C16H32O2 1.73 35.38 Hexadecane (Alkane hydrocarbon) 226 C16H34 0.44
52.52 Citronellal (Monoterpenoid) 154 C10H18O 1.5 40.46 Isopulegol 158 C10H18O 1.02
53.02 Villosin (Sesquiterpene lactone) 300 C20H28O2 0.5 47.55 Phthalic acid, 2,7-dimethyloct-7-en-5-yn-4-yl isobutyl ester 356 C22H28O4 0.88
55.30 9,12-Octadecadienoic acid (Z, Z)- (Fatty acid) 280 C18H32O2 0.72 48.4 Tetradecane (Alkane hydrocarbon) 198 C14H30 0.88
55.51 Oleic Acid (Fatty acid) 282 C18H34O2 2.56 48.58 2,3-Dihydro farnesyl acetate (p-menthane monoterpenoid) 266 C17H30O2 0.29
58.40 Globulol (Sesquiterpene) 222 C15H26O 1.2 49.84 n-Hexadecanoic acid (Fatty acid) 256 C16H32O2 2.94
62.84 Phenol, 2,2’-methylenebis[6-(1,1-dimethylethyl)-4- methyl (Methyl phenol) 340 C23H32O2 2.54 52.64 Citronellal (Monoterpenoid) 154 C10H18O 7.45
65.70 Glycerol 1-palmitate (Monoacylglycerols) 330 C19H38O4 0.7 53.08 Villosin (Sesquiterpene lactone) 300 C20H28O2 0.73
70.31 9,12-Octadecadienoic acid (Z, Z)-, 2,3-dihydroxypropyl ester (fatty acid ester) 354 C21H38O4 1.75 54.39 Linoleic acid ethyl ester (Fatty acid ester) 308 C20H36O2 0.73
70.49 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester ( fatty acid ester) 356 C21H40O4 1.06 56.10 9,12-Octadecadienoic acid (Z, Z)- (Fatty acid) 280 C18H32O2 5.99
80.76 Olean-13(18)-ene (Triterpenes) 410 C30H50 0.62 54.27 Oleic Acid (Fatty acid) 282 C18H34O2 8.24
84.15 Stigmasterol (Tetracyclic triterpenes) 412 C29H48O 0.6 60.39 E, E,Z-1,3,12-Nonadecatriene-5,14-diol (Terpenes) 294 C19H34O2 3.27
87.2 ç-Sitosterol (Phytosterols) 414 C29H50O 2.98 64.19 Glycerol 1-palmitate (Monoacylglycerols) 330 C19H38O4 1.08
68.96 Globulol (Sesquiterpene) 222 C15H26O 5.88
70.46 9,12-Octadecadienoic acid (Z, Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester (Fatty acid ester) 354 C21H38O4 4.23
73.96 Squalene 410 C30H50 0.39
81.96 Olean-13(18)-en (Tetracyclic triterpenes) 410 C30H50 0.54
84.94 Stigmasterol (Tetracyclic triterpenes) 412 C29H48O 0.67
88.70 ç-Sitosterol (Phytosterols) 414 C29H50O 2.17
Open in a new tab

Determination of the effect of ozone on antimicrobial action of black seed oil

There is a notable enhancement of the antibacterial action towards Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC8739), Salmonella typhi (ATCC 6539), and Klebsiella pneumoniae (ATCC13883) of black seed oil upon exposing it to ozone compared to antibacterial role of crude oil as well as standard drugs. On the other hand, exposing of black seed oil to ozone has no impact of antimicrobial activity towards Candida albicans (ATCC10221). Lastly, it could be noticed that either crude or ozonized black seed oils has no impacts towards Aspergillus niger (ATCC16888) as depicted in Table 2; Fig. 2.

Table 2.

Illustration of antimicrobial action of crude black seed oil and after exposing it to ozone relative to standard drugs (gentamicin (0.06 mg/ml) for bacteria and fluconazole (0.26 µg/ml) for fungi), (the outcomes were tabulated as means (mm) ± SD).

Microbes Standard drug Crude black seed oil Ozonized black seed oil
B. cereus  (ATCC 11778) 26.2 ± 0.2 25.0 ± 0.1 30.0 ± 0.1
S.aureus (ATCC 6538) 23.0 ± 0.2 21.0 ± 0.1 25.0 ± 0.2
E. coli (ATCC 8739 25.0 ± 0.2 25.0 ± 0.1 25.0 ± 0.2
S. typhi (ATCC 6539) 22.0 ± 0.2 24.0 ± 0.2 22.0 ± 0.2
K. pneumoniae (ATCC13883) 18.0 ± 0.1 20.0 ± 0.2 18.0 ± 0.1
A. niger (ATCC16888) 25.0 ± 0.2 NA NA
C. albicans (ATCC10221) 31.0 ± 0.2 30.0 ± 0.2 30.0 ± 0.1
Open in a new tab

Fig. 2.

Fig. 2

Open in a new tab

Antimicrobial activity of various tested microbes (1) Blank, (2) Control (standard drug), (3) crude black seed oil, and (4) crude black seed oil exposed to ozone (outcome are sown as means ± SD).

Detection of MIC and MBC of crude and ozonized black seed oil

There is a notable reduction (P ≤ 0.05) in both minimal inhibitory levels and minimal bactericidal values of ozonized black seed oil compared to crude black seed oil towards B. cereus (ATCC 11778), S. aureus (ATCC6538), E. coli (ATCC8739), S. typhi (ATCC 6539), and K. pneumoniae (ATCC13883). On the other hand, there is no reduction in MIC and MBC towards minimal bactericidal values in both oil forms as illustrated in Table 3.

Table 3.

Evaluation of the MIC and MBC outcomes (µg/ml) of crude and ozonized black seed oil, (the data were recorded as means ± SD) (* P ≤ 0.05).

Microorganisms Crude black seed oil Ozonized black seed oil
MIC MBC MIC MBC
B. cereus ( ATCC11778) 15.62 ± 0.6 31.25 ± 0.7 7.8 ± 0.7* 7.8 ± 0.6*
S.aureus (ATCC 6538) 62.5 ± 0.5 125.0 ± 0.8 15.62 ± 0.9* 31.25 ± 0.3*
E. coli (ATCC 8739 31.25 ± 0.4 125.0 ± 0.7 15.62 ± 0.7* 31.25 ± 0.4*
S. typhi (ATCC 6539) 15.62 ± 0.8 62.5 ± 0.6 15.62 ± 0.8* 31.25 ± 0.5*
K. pneumoniae ( ATCC13883) 62.5 ± 0.9 125.0 ± 0.4 15.62 ± 0.4* 31.25 ± 0.6*
C. albicans (ATCC10221) 15.62 ± 0.1 31.25 ± 0.3 15.62 ± 0.1* 15.62 ± 0.2*
Open in a new tab

Assessment of the effect of ozone on anti-oxidant action of black seed oil

In this work, antioxidant action of both crude black seed oil as well as ozonized oil was compared relative to ascorbic acid as antioxidant standard. The antioxidant value (IC50) of ascorbic was 2.22 ± 0.1 µg/mL. A notable enhancement (P ≤ 0.05) of the antioxidant action of crude black seed oil upon exposing it to ozone was observed, where IC50 for crude and ozonized oils were 3.95 ± 0.1 and 2.93 ± 0.2 µg/mL, consecutively as depicted in Fig. 3.

Fig. 3.

Fig. 3

Open in a new tab

Anti-oxidant action (%) of crude and ozonized black seed oils using DPPH assay (results are illustrated as means ± SD).

Detection of the effect of ozone on anti-inflammatory activity

The lysis of erythrocytes was evaluated upon using various levels of crude and ozonized black seed oil was compared to the positive control (indomethacin). The anti-inflammatory potential of crude and ozonized black seed oil was assessed via inhibition of hemolysis as showed in Fig. 4A and B, respectively. It’s cleared that hemolysis inhibition was lower using crude black seed oil than that ozonized particularly at low concentrations 3.9, 7.8, 15.62, and 31.25 µg/mL as illustrated in Eppendorf tubes No 2, 3, 4, and 5, respectively compared to untreated sample as in Eppendorf tubes No 1 or Eppendorf tubes No 10 containing sample treated by indomethacin. The estimated hemolysis inhibition was documented in Fig. 4C, where there was dramatic difference (P ≤ 0.05) of impact via using various levels of ozonized black seed oil relative to activity of crude oil.

Fig. 4.

Fig. 4

Open in a new tab

(A) Anti-inflammatory action various levels of (A) crude black seed oil, (B) ozonized black seed oil and (C) hemolysis inhibition at different concentrations. 0, untreated; 2–9, treated by 3.9–1000 µg/ml oil; 10, treated by indomethacin. Statistical illustrations of various levels of crude and ozonized black seed oil relative to indomethacin (Outcomes are drawn as means ± SD).

Detection of the impact of ozone on anti-cancer action

The toxic roles of crude and ozonized black seed oil were examined by MTT procedure via HCT cells and it could be seen that upon treatment by various values from 1000 to 31.25 of the oil to ozone a dramatic elevation (P ≤ 0.05) of its cytotoxic impact where IC50 was 56.93 ± 0.6 and 102.54 ± 1.23 µg/mL for crude and ozonized oil consecutively as shown in Table 4; Fig. 5.

Table 4.

Evaluation of cytopathic impact of various levels of black seed oil and prior to exposure to O3 on HCT cells (results are recorded as means ± SD).

Sample Conc. (µg/mL) Mean of A560 Viability (%) Toxicity (%) SE
HCT cell line None 0.719 100 0.00 0.002
Oil + O3 1000 0.020 2.77 97.23 0.001
500 0.024 3.37 96.63 0.001
250 0.025 3.51 96.49 0.002
125 0.216 29.91 70.09 0.006
62.5 0.716 99.35 0.65 0.003
31.25 0.719 99.77 0.23 0.002
IC50 102.54 ± 1.23 µg/mL
Crude oil 1000 0.018 2.50 97.50 0.0
500 0.019 2.68 97.32 0.001
250 0.020 2.73 97.27 0.001
125 0.041 5.73 94.27 0.008
62.5 0.284 39.34 60.66 0.008
31.25 0.714 99.08 0.92 0.003
IC50 56.93 ± 0.6 µg/mL
Open in a new tab

SE, standard error.

Fig. 5.

Fig. 5

Open in a new tab

(A) A normal monolayer HCT control cells, (B) HCT cells exposed to different values of crude black seed oil and, (C) HCT cells treated by various levels of ozonized black seed oil, (D) Assessment of role of crude black seed oil and via exposure to O3 on viability % versus HCT cells at various concentrations of oils. (E) Assessment of role of crude black seed oil and via exposure to O3 on toxicity % versus HCT cells at various concentrations of oils.

Discussion

Numerous Semitic civilizations have long utilized black seed oil as a therapeutic remedy38. The research field has provided information on the use of different devices related to biotechnology to enhance the pharmacological characteristics, agronomical features, and tolerance of black seed oil with the goal of boosting its medicinal qualities39.

The raw black seed oil in the research study was processed via this technique, which involved ozonizing the unrefined oil using a specified system to provide an ozone delivery rate of 0 to 6 L/minute. Ozone generators are used to create ozonized oils, and the effectiveness of ozonized products can vary depending on a number of factors. The effectiveness and quality of ozone producers; ozonation circumstances such as duration, level of ozone and supply, temperature, and reaction mixture movement; kind and the amount of vegetal oil; and the incorporation of water or another catalyst are some of the factors that affect ozonation processes40. Additionally, it might be able to prevent the creation of possibly harmful nitrated by-products by using pharmaceutical-quality oxygen41. Ozonized content is a crucial indicator of an oil’s pharmacological ability to supply active oxygen and other medicinal compounds that can be employed to treat skin conditions42. Therefore, understanding the formation of by-products aids in determining the level of ozonation necessary to provide the intended medicinal properties. Monitoring biochemical characteristics43 is one way to evaluate the grade of the ozonized oils that are formed; these parameters are crucial for detection and characterization.

In the current investigation, treatment of crude black seed oil to ozone changed the oil’s chemical profile and increased the amount of several components e.g.: Bicyclo[3.1.0]hex-2-ene, 4-methyl-1-(1-methylethyl)-, o-Cymene, Thymoquinone, Longifolene, Oleic Acid and n-Hexadecanoic acid in ozonated oil. Besides, the ozation of balck seed oil led to reduce the concnetration of some compounds including: D-Limonene, Nerol, methyl ether, 2,4-Decadienal, (E, E)-, Phenol,2-methyl-5-(1-methylethyl)-. On the other hand, it could be noticed that the amount of molecules improved due to exposing of raw oil to ozone. Numerous volatile substances, including as thymoquinone, p-cymene, limonene, and citronellol, have been found in N. sativa seed oil by earlier research44. There are still a lot of unidentified volatile substances that need to be characterized, though, based on the area in which the seeds were grown45. According to Bourgou et al.46, the volatile oil of N. sativa seeds from Tunisia has a unique form of p-cymene that is not present in seeds from other sources.

In this work exposure of black seed oil substatially boost the antibacterial impact of the oil versus B. cereus, S. aureus, E. coli, S. typhi, and K. pneumoniae. Certain components of essential oils have antimicrobial properties that can partially block bacterial cells, ultimately causing the bacterium to collapse47. Typically, plants that chemically comprise several families of chemical compounds are used for obtaining essential oils48. Phenols and oxygen-substituted phenolic ring structures were responsible for antibacterial phytochemicals found in several herbs and spices49. The –OH groups in the phenolic molecules are responsible for the molecules’ inhibitory effects. Other researchers suggested that the vast usage of black seed oil in therapeutics was attributable to the presence of thymoquinone components50.

Ozonation of black seed oil improve the antiooxidant potential of the oil as seen in the results in this report. Black seed oils’ remarkable anti-oxidant properties have been used in both conventional and complementary medicine51. Products made from meat have been kept from oxidizing by using black seed’s antioxidant properties. While black seed oils’ radical scavenging capability was significantly lower, their antioxidant capacity was considerable in the amino acid and fat fractions52,53. Black cumin’s thymoquinone has a significant antioxidant capability and reduces peroxidation of lipids54,55.

Cells prone to stress from oxidation include erythrocytes. Their high concentration of polyunsaturated fatty acids in their membrane lipid structure, frequent contact with oxygen, and the generation of reactive oxygen species by stimulated inflammatory cells are all linked to their sensitivity56. Furthermore, red blood cells are very susceptible to auto-oxidation when there is an abundance of hemoglobin present57. Hemolysis is therefore frequently used as a significant marker of damage caused by free radicals influencing the erythrocyte membrane58. Conversely, it is well recognized that antioxidant chemicals may neutralize free radicals and shield cells in opposition to oxidative stress-induced hemolysis. The capacity of ozonized black seed oil to shield red blood cells from damage caused by oxidation was evaluated in the present research. A substantial improvement in anti-inflammatory activity prior to ozonation could be detected.

The MTT test, a superior-throughput cell-based test, is used to assess the cytotoxic response of the HCT cell line to varying doses of either crude or ozonized black seed oil. MTT is a common colorimetric test that assesses color alterations which reflect the cellular proliferation. According to Mosmann59, numerous advantages were associated to MTT assay such as speed, quantitation, and assaying of several samples. Employment of this protocol to chemosensitivity examination appears to be appreciated and beneficial. The action mechanism of MTT test incudes cell respiration measures and the produced quantity of formazan is relative to the amount of living cells60. A rise or decline in the number tested cells is associated to the formed amount of formazan, demonstrating the level of cytotoxicity of oil. IC50 is the dose of the of either crude or ozonized black seed oil which cause 50% death of the HCT cells and can be predictive of the degree of cytotoxic effect. Numerous studies demonstrated the well-known anticancer properties of black seed oil33,37,61. In a prior investigation, the anticancer properties of thymoquinone and thymohydroquinone presnet in black seed oil have been contrasted and it was discovered that while both ingredients exhibited dose-related activity, thymohydroquinone was less efficient versus cancer cells at low concentrations than thymoquinone62. The presnt results showed that ozonation improve anticcncnr impact of black seed oil. It has been proposed that the act of ozonation leads to chemical alterations like oxidation, which alter and convert molecules and accumulate more potent ones. The toxic roles of crude and ozonized black seed oil were examined by MTT procedure via HCT cells and it could be seen that upon treatment by various values from 1000 to 31.25 of the oil to ozone a dramatic elevation (P ≤ 0.05) of its cytotoxic impact. The high level of cytotoxic is represented by low quantity of IC50, therefore in our study the ozonized oil possess higher anticancer than crude oil as indicated by IC50 values of 56.93 ± 0.6 and 102.54 ± 1.23 µg/mL, respectively.

Conclusions and future perspectives

The current findings imply that ozone applied to black seed oil at a flow rate of 0–5 L/minute for four hours alters the relative concentration of bioactive molecules, particularly thymoquinone. This may be responsible for variations in the molecules’ antimicrobial, antioxidant, and anti-inflammatory properties as well as their destructive influence on HCT. From the obtained results, numerous future perspectives will take in our consideration in further studies such as separation of active compounds under the effect on ozonization, silico studies on the interaction of active compounds with target proteins in cancer cells to understand the different pathways of signaling as well as recognizing their related to biomarkers of carcinogenesis. Moreover, in our study the extracts were experimented on one type of cancer, subsequently their activity should be including at least four kinds of cancer. In overall, there is still potential for in live animal models and additional clinical investigations in the scientific examination of ozonized black seed oil for application of this approach.

Acknowledgements

The authors would like to acknowledge Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Author contributions

Conceptualization, methodology, formal analysis A.M.H.A., T.M.A., M.S.A., M.H.A.; investigation, A.A.S., A.M.B., S.K.A., H.M.A.; writing—original draft preparation, writing—review and editing M.S.W., F.A.A., S.S. 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 (PNURSP2024R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Data availability

The results from the present investigation are available from the corresponding author upon reasonable appeal.

Competing interests

The authors declare no competing interests.

Footnotes

The original online version of this Article was revised: In the original version of this Article, Samy Selim was omitted as a corresponding author. Correspondence and requests for materials should also be addressed to sabdulsalam@ju.edu.sa. Furthermore, an incorrect email address for the corresponding author Tarek M. Abdelghany was quoted. Correspondence and requests for materials should be addressed to tabdelghany.201@azhar.edu.eg

Publisher’s note

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

Change history

1/30/2025

A Correction to this paper has been published: 10.1038/s41598-025-88223-w

Contributor Information

Tarek M. Abdelghany, Email: tabdelghany.201@azhar.edu.eg

Samy Selim, Email: sabdulsalam@ju.edu.sa.

References

  • 1.Al-Rajhi, A. M. H. et al. Anti-Helicobacter Pylori, antioxidant, antidiabetic, and anti-alzheimer’s activities of laurel leaf extract treated by moist heat and molecular docking of its flavonoid constituent, naringenin, against acetylcholinesterase and butyrylcholinesterase. Life13(7), 1512. 10.3390/life13071512 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bakri, M. M. et al. Impact of moist heat on phytochemical constituents, anti-Helicobacter pylori, antioxidant, anti-diabetic, hemolytic and healing properties of rosemary plant extract in vitro. Waste Biomass Valor15, 4965–4979. 10.1007/s12649-024-02490-8 (2024). [Google Scholar]
  • 3.Qanash, H. et al. Anticancer, antioxidant, antiviral and antimicrobial activities of Kei Apple (Dovyalis caffra) fruit. Sci.Rep.12, 5914. 10.1038/s41598-022-09993-1 (2022). [DOI] [PMC free article] [PubMed]
  • 4.Alsalamah, S. A., Alghonaim, M. I., Jusstaniah, M. & Abdelghany, T. M. Anti-yeasts antioxidant and healing properties of Henna pre-treated by moist heat and molecular docking of its major constituents, chlorogenic and ellagic acids, with Candida albicans and Geotrichum candidum proteins. Life13(9), 1839. 10.3390/life13091839 (2023). [DOI] [PMC free article] [PubMed]
  • 5.Al-Rajhi, A. M. et al. Anticancer, anticoagulant, antioxidant and antimicrobial activities of Thevetia peruvianalatex with molecular docking of antimicrobial and anticancer activities. Molecules27(10), 3165. 10.3390/molecules27103165 (2022). [DOI] [PMC free article] [PubMed]
  • 6.Dabeer, S. et al. History and traditional uses of black seeds (Nigella sativa). In Black Seeds (Nigella sativa) 1–28 (Elsevier, 2022).
  • 7.Hossain, M. S., Saha, K. C., Islam, M. M., Noor, M. Z. & Choudhury, D. A. Assessment of nutritional composition, bioactive compounds and antioxidant activity of Chia seeds grown in Bangladesh. Bangladesh J. Agric. Res.48 (2), 259–270 (2023). [Google Scholar]
  • 8.Tariq, M. Nigella sativa sfolklorelklore treatment in modern day medicine. Saudi J. Gastroenterol.14, 105–106. 10.4103/1319-3767.41725 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Shoeb, S., Huzaifa, M. & Saman, B. A comprehensive review of black cumin. Int. J. Res. Pharm. Allied Sci.3(3), 146–153 (2024). [Google Scholar]
  • 10.Khare, C. P. Encyclopedia of Indian Medicinal Plants (Springes, 2004).
  • 11.Goreja, W. G. Black Seed: Nature’s Miracle Remedy (Amazing Herbs, 2003).
  • 12.Al-Bukhari, M. I. The collection of authentic sayings of prophet Mohammad (peace be upon him), division 71 on medicine. 2nd ed (ed. Al-Bukhari, S.) (Hilal Yayinlari, 1976).
  • 13.Khalid, A. & Ahmad, S. S. Antibacterial activity of Nigella sativa against multi-drug resistant bacteria. Int. J. Pathol.22(2), 45–95. 10.59736/IJP.22.02.899 (2024). [Google Scholar]
  • 14.Mahros, M. M., Abd-Elghany, S. M., Sayed-Ahmed, M. Z., Alqahtani, S. S. & Sallam, K. I. Improving the microbiological quality, health benefits, and storage time of cold-stored ground mutton supplemented with black seed. Lwt138, 110673. 10.1016/j.lwt.2020.110673 (2021). [Google Scholar]
  • 15.Constantinou, V. & Constantinou, C. Focusing on colorectal cancer in young adults. Mol. Clin. Oncol.20(1), 1–10. 10.3892/mco.2023.2706 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Sheikhnia, F., Rashidi, V., Maghsoudi, H. & Majidinia, M. Potential anticancer properties and mechanisms of thymoquinone in colorectal cancer. Cancer Cell Int.23(1), 320. 10.1186/s12935-023-03174-4 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Akram, K. M. & Afzal, M. Chemical composition of Nigella sativa Linn: part 2 recent advances. Inflammopharmacology24, 67–79. 10.1007/s10787-016-0262-7 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Srinivasan, K. Cumin (Cuminum cyminum) and black cumin (Nigella sativa) seeds: traditional uses, chemical constituents, and nutraceutical effects. Food Qual. Saf.2, 1–16. 10.1093/fqsafe/fyx031 (2018). [Google Scholar]
  • 19.Ardiana, M., Pikir, B. S., Santoso, A., Hermawan, H. O. & Al-Farabi, M. J. Effect of Nigella sativa supplementation on oxidative stress and antioxidant parameters: a meta-analysis of randomized controlled trials. Sci. World J.2020, 45. 10.1155/2020/2390706 (2020). [DOI] [PMC free article] [PubMed]
  • 20.Rahman, A. U., Abdullah, A., Faisal, S., Mansour, B. & Yahya, G. Unlocking the therapeutic potential of Nigella sativa extract: phytochemical analysis and revealing antimicrobial and antioxidant marvels. BMC Complement. Med. Ther.24(1), 266. 10.1186/s12906-024-04470-w (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jin, H. W. & Eom, Y. B. Antibacterial and anti-biofilm effects of Thymoquinone against Carbapenem-resistant uropathogenic Escherichia coli. Indian J. Microbiol.2024, 1–10. 10.1007/s12088-024-01231-8 (2024). [DOI] [PMC free article] [PubMed]
  • 22.Aumeeruddy, M. Z. & Mahomoodally, M. F. Combating breast cancer using combination therapy with 3 phytochemicals: piperine, sulforaphane, and thymoquinone. Cancer125(10), 1600–1611. 10.1002/cncr.32022 (2019). [DOI] [PubMed] [Google Scholar]
  • 23.Pulicharla, R., Proulx, F., Behmel, S., Sérodes, J. B. & Rodriguez, M. J. Trends in ozonation disinfection by-products—occurrence, analysis and toxicity of carboxylic acids. Water12, 756 (2020). [Google Scholar]
  • 24.Pérez-Barragán, J. et al. Iron (magnetite) nanoparticle-assisted dark fermentation process for continuous hydrogen production from Rice Straw Hydrolysate. Appl. Sci.14(21), 9660. 10.3390/app14219660 (2024). [Google Scholar]
  • 25.Ghasemi, F., Nili-Ahmadabadi, A., Omidifar, N. & Nili-Ahmadabadi, M. Protective potential of thymoquinone against cadmium, arsenic, and lead toxicity: a short review with emphasis on oxidative pathways. J. Appl. Toxicol.43(12), 1764–1777. 10.1002/jat.4459 (2023). [DOI] [PubMed] [Google Scholar]
  • 26.Ölmez, H. & Kretzschmar, U. Potential alternative disinfection methods for organic fresh-cut industry for minimizing water consumption and environmental impact. LWT-Food Sci. Technol.42, 686–693 (2009). [Google Scholar]
  • 27.Gabler, F. M., Smilanick, J. L., Mansour, M. F. & Karaca, H. Influence of fumigation with high concentrations of ozone gas on postharvest gray mold and fungicide residues on table grapes. Postharvest Biol. Technol.55, 85–90 (2010). [Google Scholar]
  • 28.Kazi, M., Chatzopoulou, P. & Christos, L. Effect of ozonation on the essential oil composition of dried aromatic plants. In Proceedings of the 8th International Conference on Information and Communication Technologies in Agriculture, Food and Environment; Chania, Greece (2017).
  • 29.Karlsson, M. F. et al. Plant odor analysis of Potato: response of Guatemalan Moth to above- and Belowground Potato volatiles. J. Agric. Food Chem.57, 5903–5909. 10.1021/jf803730h (2009). [DOI] [PubMed] [Google Scholar]
  • 30.Pietrocola, G., Ceci, M., Preda, F., Poggio, C. & Colombo, M. Evaluation of the antibacterial activity of a new ozonized olive oil against oral and periodontal pathogens. J. Clin. Exp. Dent.10, e1103–e1108. 10.4317/jced.54929 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Elvis, A. M. & Ekta, J. S. Ozone therapy: a clinical review. J. Nat. Sci. Biol. Med.2, 66–70. 10.4103/0976-9668.82319 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kabir, Y., Akasaka-Hashimoto, Y., Kubota, K. & Komai, M. Volatile compounds of black cumin (Nigella sativa L.) seeds cultivated in Bangladesh and India. Heliyon6(10), e05343. 10.1016/j.heliyon.2020.e05343 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Mohammed, S. J. et al. Structural characterization, antimicrobial activity, and in vitro cytotoxicity effect of black seed oil. Evid. Based Complement. Alternat Med.2019, 6515671. 10.1155/2019/6515671 (2019). [DOI] [PMC free article] [PubMed]
  • 34.Nazemi, S. B. et al. The comparison of antimicrobial effect of Nigella sativa nanoparticle and chlorhexidine emulsion on the most common dental cariogenicic bacteria. Med. J. Islam Repub. Iran.35, 149. 10.47176/mjiri.35 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Gueffai, A. et al. Phenolics from defatted black cumin seeds (Nigella sativa L.): ultrasound-assisted extraction optimization, comparison, and antioxidant activity. Biomolecules12 9), 1311. 10.3390/biom12091311 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Amina, M., Arumugham, M. I., Ramalingam, K. & Rajeshkumar, S. Evaluation of the anti-inflammatory, antimicrobial, antioxidant, and cytotoxic effects of Chitosan Thiocolchicoside-Lauric Acid Nanogel. Cureus15(9), e46003. 10.7759/cureus.46003 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Alsanosi, S. et al. The potential role of Nigella sativa seed oil as epigenetic therapy of Cancer. Molecules27, 2779. 10.3390/molecules27092779 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Bhatti, I., Zubair, H., Bakhsh, M. S. & Akhlaq, M. The scientific importance of Nigella sativa (Kalonji) and honey in accordance with Tib-E-Nabvi. Gomal Univ. J. Res.29, 27–30 (2013). [Google Scholar]
  • 39.Rahim, M. A. et al. A narrative review on various oil extraction methods, encapsulation processes, fatty acid profiles, oxidative stability, and medicinal properties of black seed (Nigella sativa). Foods11, 2826. 10.3390/foods11182826 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Guerra, B. P., Poznyak, T., Chairez, I. & Brito, A. M. Correlation of structural characterization and viscosity measurements with total unsaturation: an effective method for controlling ozonation in the preparation of ozonated grape seed and sunflower oils. Eur. J. Lipid Sci. Technol.117, 988–998. 10.1002/ejlt.201400292 (2015). [Google Scholar]
  • 41.Travagli, V., Zanardi, I., Valacchi, G. & Bocci, V. Ozone and ozonated oils in skin diseases: a review. Mediat Inflamm.2010, 610418. 10.1155/2010/610418 (2010). [DOI] [PMC free article] [PubMed]
  • 42.Song, M. et al. The antibacterial effect of topical ozone on the treatment of MRSA skin infection. Mol. Med. Rep.17, 2449–2455. 10.3892/mmr.2017.8148 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Sadowska, J. et al. Characterization of ozonated vegetable oils by spectroscopic and chromatographic methods. Chem. Phys. Lipids. 151, 85–91. 10.1016/j.chemphyslip.2007.10.004 (2008). [DOI] [PubMed] [Google Scholar]
  • 44.Solati, Z., Baharin, B. S. & Bagheri, H. Antioxidant property, thymoquinone content and chemical characteristics of different extracts from Nigella sativa L. seeds. J. Am. Oil Chem. Soc.91(2), 295–300 (2014). [Google Scholar]
  • 45.Ahmad, N. et al. Ultra-high-performance liquid chromatography-based identification and quantification of thymoquinone in Nigella sativa extract from different geographical regions. Phcog Mag. 14(57), 471–480 (2018). [Google Scholar]
  • 46.Bourgou, S., Ksouri, R., Skandrani, I., Chekir-Ghedira, L. & Marzouk, B. Antioxidant and antimutagenic activities of the essential oil and methanol extract from Tunisia Nigella sativa. Ital. J. Food Sci.20(2), 191–202 (2008). [Google Scholar]
  • 47.Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R. & De Feo, V. Effect of essential oils on pathogenic bacteria. Pharmaceuticals6(12), 1451–1474. 10.3390/ph6121451 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Al-Rajhi, A. M. & Abdelghany, T. A. Nanoemulsions of some edible oils and their antimicrobial, antioxidant, and anti-hemolytic activities. BioResources18(1), 1465–1481. 10.15376/biores.18.1.1465-1481 (2023). [Google Scholar]
  • 49.Bazaka, K., Jacob, M. V., Chrzanowski, W. & Ostrikov, K. Anti-bacterial surfaces: natural agents, mechanisms of action, and plasma surface modification. RSC Adv.5(60), 48739–48759. 10.1039/c4ra17244b (2015). [Google Scholar]
  • 50.Cardoso, T., Galhano, C. I. C., Ferreira Marques, M. F. & Moreira Da Silva, A. Thymoquinone β-cyclodextrin nanoparticles system: a preliminary study. Spectroscopy: Int. J.27(5–6), 329–336. 10.1155/2012/902486 (2012).
  • 51.Okur, Ö. D. Determination of antioxidant activity and total phenolic contents in Yogurt added with black cumin (Nigella sativa) Honey. Ovidius Univ. Ann. Chem.32, 1–5 (2021). [Google Scholar]
  • 52.Lutterodt, H. et al. Fatty acid profile, thymoquinone content, oxidative stability and antioxidant properties of cold-pressed black cumin seed oils. LWT Food Sci. Technol.43, 1409–1413 (2010). [Google Scholar]
  • 53.Muzolf-Panek, M. et al. A chemometric approach to oxidative stability and physicochemical quality of raw ground chicken meat affected by black seed and other spice extracts. Antioxidants9, 903 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Bordoni, L. et al. Antioxidant and anti-inflammatory properties of Nigella sativa Oil in Human pre-adipocytes. Antioxidants8, 51 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Dalli, M., Azizi, S. E., Kandsi, F. & Gseyra, N. Evaluation of the in vitro antioxidant activity of different extracts of Nigella sativa seeds, and the quantification of their bioactive compounds. Mater. Today Proc.45, 7259–7263 (2021). [Google Scholar]
  • 56.Welbourn, E. M., Wilson, M. T., Yusof, A., Metodiev, M. V. & Cooper, C. E. The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane. Free Radic Biol. Med.103, 95–106 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Liao, W. et al. Protective effects of kaempferol against reactive oxygen species-induced hemolysis and its antiproliferative activity on human cancer cells. Eur. J. Med. Chem.114, 24–32 (2016). [DOI] [PubMed] [Google Scholar]
  • 58.Al-Rajhi, A. M., Yahya, R., Bakri, M. M., Yahya, R. & Abdelghany, T. M. In situ green synthesis of Cu-doped ZnO based polymers nanocomposite with studying antimicrobial, antioxidant and anti-inflammatory activities. Appl. Biol. Chem.65(1), 35. 10.1186/s13765-022-00702-0 (2022). [Google Scholar]
  • 59.Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55–63. 10.1016/0022-1759(83)90303-4 (1983). [DOI] [PubMed] [Google Scholar]
  • 60.Selim, S. et al. Estimation and action mechanisms of cinnamon bark via oxidative enzymes and ultrastructures as antimicrobial, anti-biofilm, antioxidant, anti-diabetic, and anticancer agents. BioResources19(4), 7019–7041. 10.15376/biores.19.4.7019-7041 (2024). [Google Scholar]
  • 61.Agbaria, R., Gabarin, A., Dahan, A. & Ben-Shabat, S. Anticancer activity of Nigella sativa (black seed) and its relationship with the thermal processing and quinone composition of the seed. Drug Des. Devel Ther.9, 3119–3124. 10.2147/DDDT.S82938 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Jafri, S. H. et al. Thymoquinone and cisplatin as a therapeutic combination in lung cancer: in vitro and in vivo. J. Exp. Clin. Cancer Res.29, 29–87 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The results from the present investigation are available from the corresponding author upon reasonable appeal.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES