β-cyfluthrin (Purity >99%) was acquired from Nanjing Panfeng Chem Ltd. (Nanjing, Jiangsu, China). The rest of the reagents were analytical reagent grade. Fresh basil leaves (Ocimum basilicum L. (Lamiaceae) were obtained from the Faculty of Science, where they were recognized and authenticated as Basil; the common name of Ocimum basilicum L.; Lamiaceae Martinov family, Genus: Ocimum L. by a plant taxonomy specialist at the Department of Botany, Faculty of Science, Alexandria University, Alexandria, Egypt.

Basil dry leaves (100 g) were crushed and hydrodistilated with sterile water (1 L) for 3 h using a Clevenger-type device. The essential oil was extracted and dried over anhydrous sodium sulfate before being kept at 4°C for further use.

A Thermo Scientific GC/MS version (5) 2009 system with TG-5MS column (30mX0.32mmID) was used to identify the chemical elements of the extracted essential oil. At a flow rate of 1 ml/min, helium was used as the carrier gas. Five μl essential oil was diluted to 1 ml with dichloromethane, then 2 μl was injected on splitless mode for 1 min, followed by a 1:10 split flow. GC oven temperature was held at 45°C for 2 min then was programmed from 45°C to 165°C at 4°C/min; from 165°C to 280°C at 15°C/min after which was kept constant at 280°C for 10 min. At 250°C, both the interface and injection temperatures were set. The ionization voltage was 70 eV with a mass range between 40–800 m/z (Adams, 2006). The components of the essential oil were identified using mass fragmentation patterns, which were compared to the mass spectral database (version 2) of the National Institute of Standards and Technology (NIST), and their relative percentages were estimated using GC peak regions.

The total phenolic contents in BEO were determined spectrophotometrically using Folin-Ciocalteau method (Kang et al., 2010). The absorbance was measured at 765 nm against blank. The calibration curve was produced using pyrocatechol as a standard. The total phenolic content of the extract was measured in milligrams of pyrocatechol equivalents (PE) per Gram of extract.

The reducing ability of antioxidants toward the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical was estimated to determine the free radical scavenging activity of the sample extract (Kang et al., 2011). Using butylated hydroxytoluene (BHT) as a positive control, the DPPH radical scavenging activity of each sample was measured at 515 nm and computed as follows: percent inhibition = [(AB-AA)/AB] x 100, where AB and AA are the absorbance values of the control and test samples, respectively. The IC₅₀ value is used to represent the radical-scavenging activity of DPPH.

With slight adjustments, the antioxidant capacity was calculated using the approach reported by Re et al. (1999). A spectrophotometer was used to measure the absorbance at 734 nm. The percent inhibition of ABTS•+ was calculated for each test sample using the formula: (percent inhibition) = [100*(Ac–As/Ac)], where Ac represents the control sample absorbance and As represents the test sample absorbance. The amount of oil required to scavenge 50% of the ABTS radicals (IC₅₀) was estimated. Trolox was utilized as a control substance.

As a measure of “antioxidant power,” a ferric reducing ability (FRAP) assay (Benzie and Strain, 1996) was used to estimate the total antioxidant capacity of the sample. The change in absorbance at 593 nm is measured in this experiment.

Twenty-eight male Wistar rats (150 ± 10 g) were given by the Faculty of Medicine, Alexandria University, Alexandria, Egypt. The local Research Ethical Committee accepted the experimental design, which followed the National Institutes of Health’s guidelines (NIH). The rats were kept in groups and fed rat chow and water ad libitum while being kept at 21–24°C and 40–60% relative humidity with 12-h light-dark cycles. Animals were randomly divided into four groups (n = 7/group) after 2 weeks of acclimatization. The first group was given corn oil (negative control), the second group received basil essential oil (BEO; 3 ml/kg), the third group (positive control) was treated with β-cyfluthrin (β-Cyf; 15 mg/kg/day; 1/25 LD₅₀), and the fourth group-administered BEO an hour before β-Cyf intoxication. Dosages of β-Cyf (Singh et al., 2009) and BEO (Farghali et al., 2014) were given orally for 1 month daily. After the end of the treatment period, rats were anesthetized using isoflurane, euthanized and blood and livers were collected for further analysis. The liver was cut into three pieces: the first was held at 20°C (for biochemical assays), the second was kept at 80°C (for RNA extraction), and the third was used right away (for comet assay and flow cytometry).

The body weights were recorded at the start of the treatment period (starting body weight) and at the end of the treatment period (final body weight). The livers were dissected and weighed after the associated tissues were cut away. In addition, organ relative weight was reported as g/100 g of body weight.

Blood samples were drawn through cardiac puncture and allowed to clot for 30 min at 25°C before being centrifuged for 15 min at 3000 g. Serum was taken and kept at −80°C until it was utilized to determine liver function indicators. Other blood samples were placed in EDTA tubes and analyzed using an automated analyzer for the identification of the complete blood count (CBC) (Sysmex kx-21n Automated Hematology Analyzer; JAPAN CARE CO., LTD.).

After rats’ dissection, the liver of each rat was taken and homogenized in sodium-potassium phosphate buffer (0.01 mol/L, pH 7.4). The homogenates were centrifuged for 20 min at 10,000 g (4°C), and the supernatants were used in various assays.

Thiobarbituric acid-reactive substances (TBARS) and hydrogen peroxide (H₂O₂) were measured in liver homogenate according to the methods of Ohkawa et al. (1979) and Velikova et al. (2000), respectively. The concentrations of lipid hydroperoxides (LOOH) in liver homogenates were assessed using Nourooz-Zadeh et al. (1994) method. The oxidative protein damage was measured spectrophotometrically in the tissue by evaluating the advanced oxidized protein products (AOPP) (Witko-Sarsat et al., 1996) and protein carbonyl (PC) (Reznick and Packer, 1994). The levels of hydroxyproline (HYP) were assessed using the method of Bergman and Loxley (1963).

The content of reduced glutathione (GSH) in liver homogenates was determined using the method of Ellman (1959) after the reaction with 5,5′- dithiobis-(2-nitrobenzoic acid). The activities of glutathione peroxidase (GPx; EC 1.11.1.9) and glutathione reductase (GR; EC 1.6.4.2) were evaluated according to Hafeman et al. (1974) while superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), and glutathione S-transferase (GST; EC 2.5.1.18) were carried out using the methods of Misra and Fridovich (1972), Aebi (1984), and Habig et al. (1974), respectively.

The activities of liver aminotransferases (AST; EC 2.6.1.1 and ALT; EC 2.6.1.2), alkaline phosphatase (ALP; EC 3.1.3.1), and lactate dehydrogenase (LDH; EC 1.1.1.27) activities, as well as total protein, albumin, and total bilirubin, were measured using commercially available kits from Biodiagnostic Company, Egypt.

Real-time PCR was used to assess the relative expression of inflammation-related genes (TNF-α, IL-6) in the liver (qPCR). Total RNA was obtained using a commercial kit (Gene JET RNA Purification Kit), as directed by the manufacturer (Thermo Scientific, #K0731, United States). After evaluation of RNA level by Nanodrop (Quawell, Q3000, United States), cDNA was produced by reverse transcription using a commercial kit (RevertAid H Minus Reverse Transcriptase) as described in the manufacturer’s instruction (Thermo Scientific, # EP0451, United States). The qPCR was performed using StepOnePlus real-time PCR system (Applied Biosystem, United States) and a mixture of cDNA, 2X Maxima SYBR Green Master Mix (Thermo Scientific, #K0221, United States), and gene-specific primers (Table 1). The β-actin gene was utilized as a reference (internal control) to calculate the fold change in target genes. The relative gene expression was calculated using the 2–ΔΔCt method The threshold cycle numbers (Ct) of the target gene were normalized to that of reference (ref.) gene, in both the test groups and the control group by using the following equations:

ΔCt test = Ct (target in test groups)—Ct (ref in test groups)

Alkaline single-cell gel electrophoresis was used to measure DNA damage parameters in liver tissues including tail length, percentage of degraded DNA, and tail moment (comet assay). The DNA fragment movement patterns of 100 cells for each dose level were investigated using GelRed stain (red fluorescence) and a 40× objective on a fluorescent microscope with an excitation filter of 420–490 nm (issue 510 nm). Komet 5 image analysis software was used to accomplish the imaging (Kinetic Imaging, Liverpool, United Kingdom).

Flow cytometry was used to determine the influence of various treatments on the number of cells in each phase of the cell cycle, and it was carried out as previously described by Abdelwahab et al. (2019) with minor changes. Collagenase enzyme (0.1 percent, Invitrogen, United States) was used to lyse freshly dissected liver tissues, which were subsequently mesh filtered (0.7 mm nylon) and centrifuged (4,000 rpm/5 min). The cells were fixed and stained with propidium iodide before being examined with an Attune flow cytometer (Applied Bio-system, United States). Each cell cycle phase’s number of cells was counted and expressed as a percentage of the total number of cells.

Results from all groups were represented as means with standard errors (SEM) and analyzed with SPSS software (version 22, IBM Co., Armonk, NY). ANOVA and Tukey’s post-hoc test were used to compare between groups. The sample size (7 rats/group) used with Cohen’s d of two achieved power of 92.91% via Two- Sample t-test using Effect Size (PASS program version 15). p-values ≤ 0.05 were deemed significant.

The overall content of the basil essential oil (yield = 6.32 mg/g) contained 33 compounds, accounting for 97.0% of the total oil (Figure 1; Table 2), including monoterpenes, sesquiterpenes compounds, and phenylpropanoid derivatives. Estragole was the main component (28.6%). Linalool, the second most significant molecule, had a peak area of 21.7%. Other notable chemicals included (E)-methyl cinnamate (14.3%), α-cadinol (7.1%), eugenol (5.9%), 1,8-cineole (4.0%), methyl eugenol (3.1%), and α-bergamotene (2.2%).

In the present study, O. basilicum leaves essential oil showed relatively high phenolic content with 40.9 mg PE/g. In comparison to the standard, BEO has a comparatively strong DPPH radical scavenging capacity, with an IC₅₀ value of 10.79 mg/ml. BHT acid had a strong IC₅₀ value of 6.80 mg/ml as a control. According to the findings, the BEO appears to have high antiradical activity, which could be ascribed to its high phenolic content, which traps the cation radical ABTS^•+ by supplying H⁺. BEO had an IC₅₀ (ABTS) of 0.69 ± 0.020 mg/ml, while Trolox had an IC₅₀ (ABTS) of 3.35 ± 0.003. Furthermore, the FRAP assay is quick and easy to carry out, and the reaction is repeatable and linearly proportional to the antioxidant content present. It is calculated by comparing the total amount of antioxidants to the sample’s reducing capability. BEO has high reducing power (FRAP) equal (IC₅₀ = 1.378 g/L), but less than the reducing power of butylated hydroxytoluene (BHT) (IC₅₀ = 0.908 g/L) (Table 3).

During the research period, no mortality occurred while burrowing, salivation, tremors, writhing, and convulsions, are only observed as a few clinical indications of poisoning. In rats exposed to β-Cyf, there was a substantial decrease in body weights and an increase in relative liver weights when compared to control rats. In comparison to the β-Cyf exposed group, rats supplemented with BEO then treated with β-Cyf showed considerable alleviation in these parameters. BEO did not generate any substantial changes in rats when given alone (Table 4).

Results of the hematological parameters in rats treated with β-Cyf showed a significant drop in hemoglobin (Hb) concentration, red blood cells (RBC), hematocrit (Ht), and thrombocyte values and an increase in white blood cells (WBC) when compared to the control group. Administration with basil alone showed minor changes in the hematological parameters compared to the control group while administration of BEO before β-Cyf intoxication showed significant restoration in these parameters near the normal level in comparison to the β-Cyf group (Table 5).

To examine the effects of β-Cyf administration on lipid peroxidation, TBARs, H₂O_2, and lipid hydroperoxide (LOOH) levels were determined. Significant induction in TBARs, H₂O_2, and LOOH concentrations after β-Cyf administration was detected as compared to the control group while rats who received BEO plus β-Cyf displayed a considerable decline in TBARS, H₂O₂, and LOOH concentrations as compared to the β-Cyf treated group. In addition, protein carbonyl (PC) and advanced oxidized protein products (AOPP) markers of protein oxidation were markedly increased in the liver of the β-Cyf intoxicated group. The increase in PC and AOPP levels in the liver was reduced by BEO administration. Hepatic hydroxyproline (HYP) levels were analyzed to evaluate hepatic collagen production after β-Cyf administration. The current data showed a significant increase in hepatic HYP levels in β-Cyf -treated rats in comparison to the control group. However, high levels of HYP levels dropped due to BEO administration before β-Cyf treatment. The liver of rats supplemented with BEO alone showed significant changes in the levels of most products of lipid peroxidation and protein oxidation as compared to the control group (Figure 2).

Antioxidant enzyme activity (SOD, CAT, GPx, GR, and GST) and GSH levels are decreased significantly in liver homogenates of β-Cyf treated animals as compared to control. Otherwise, a significant induction in the same parameters was detected in the animals that received BEO plus β-Cyf as compared to the β-Cyf treated group. The use of BEO alone resulted in significant improvement in these indices (Figure 3).

Hepatic enzymes as AST, ALT, ALP, and LDH are concerned as biomarkers of hepatotoxicity. Rats administered with β-Cyf exhibited alterations in AST, ALT, and ALP activities as well as protein content, total protein, albumin, globulin, and total bilirubin in both rat liver homogenate and serum as compared to the reference group (p < 0.05) while LDH activity is elevated significantly. Rats treated with BEO plus β-Cyf displayed significant modulation in enzyme activities and biochemical parameters as compared with the β-Cyf -treated group. Administration of BEO alone induced significant changes in some of these parameters (Table 6).

In the liver tissue of rats treated with β-Cyf, qPCR results demonstrated significant increases in mRNA levels of pro-inflammatory cytokines TNF-α and IL-6 when compared to the control group. Animals pretreated with BEO then intoxicated with β-Cyf showed significant downregulation in the studied genes (Figure 4).

The DNA damage in the liver of rats in different groups was assessed using a comet test. In comparison to control (G1) and other treated groups, administration of β-Cyf (G3) resulted in a substantial increase in DNA damage (p < 0.05), as evidenced by an increase in tail length and tail moment. Because BEO was given before β-Cyf intoxication, the induction of DNA injury was reduced (G4). The control group (G1) and the BEO group (G2), on the other hand, showed no difference in DNA damage (tail length) (Figure 5).

The cells in the G0/G1 phase were arrested after treatment with β-Cyf, as evidenced by a considerable (p < 0.05) increase in the number of cells in this phase compared to the reference group. However, the β-Cyf treatment significantly reduced the number of cells in the S phase and G2/M phase (p < 0.05). Further, the number of cells in each cell cycle was recovered to a level comparable to the control group after treatment with BEO (Figure 6).