Toxicity studies
The acute toxicity investigation demonstrated that Nigella sativa oil extract was relatively non-toxic, as no treatment-related symptoms of toxicity were observed in the animals during the observation period. A chemical compound has minimal toxicity if its LD50 is 1000 mg/kg body weight (Erhirhie et al., 2018). Similarly, if a substance's LD50 is between 100 and 1000 mg/kg body weight, Lorke (1983) classifies it as relatively hazardous. In this study, the LD50 of more than 5000 mg/kg body weight indicated that the oil has a wide safety margin, and is consistent with OECD report that such compounds are relatively non-toxic (OECD Guideline For The Testing Of Chemicals, 1997).
The absence of visible adverse effects on the respiratory, neurological, and locomotor systems added to the safety of Nigella sativa oil extract. The use of data based on acute toxicity for clinical reasons is limited by the cumulative effect of drugs at low dosages. Acute toxicity is said to have a low predictive value for determining the toxicity of chemicals in humans and animals (Lorke, 1983). Furthermore, drugs might be damaging without being lethal. Acute toxicity testing, on the other hand, is the first line of defense in determining the safety profile of drugs, including medicinal plants, and its usefulness in determining therapeutic doses. It can also be used to estimate the right doses for another research. Because acute toxicity studies are limited, it is necessary to conduct additional research. In analyzing the safety profile of medicinal plants, subacute or chronic toxicity studies (for example, a 21-day toxicity study) are used.
Nigella sativa oil was given orally for 21 days had no effect on mortality. Throughout the study, daily cage-side observations revealed no adverse clinical symptoms, implying that the extract may be safe at the test levels. The weight gain in the extract-treated groups was comparable to the control group. The fact that the rats gained weight on a weekly basis implies that the extract did not decrease appetite. A large drop in mean body weight has been identified as a key indicator of toxicity (Lazic et al., 2020). Furthermore, the findings demonstrated that both absolute and relative values were significant. Furthermore, the results demonstrated that oral administration of the extract had no effect on both absolute and relative organ weights (Table 3). When toxicants were supplied that might change either the secretory activities or produce inflammation of the organs, several investigators observed that a rise or decrease in either absolute or relative organ weights reflected organ toxicity (Porwal et al., 2017).
Hematological studies
The health state of an animal can be determined using hematological measures. (Tables 4 ) show that Nigella sativa oil administered orally at the test dosages for 21 days had no effect on hematological parameters in the rats, indicating that there was no interference with red blood cell production. RBCs play a crucial role in the transfer of respiratory gases. The fact that there were no treatment-related impacts on RBC and Hb suggests that the oil extract had no negative impact on the blood's oxygen carrying capacity or the amount of oxygen given to the tissues.
The MCV, MCH, and MCHC erythrocytic indices (MCV, MCH, and MCHC) are crucial in the diagnosis of anemia (Blanc & Wolfe, 2016). Some medicinal herbs are known to cause red blood cell breakdown, which can lead to anemia (Diederich et al., 2017). Nigella sativa oil does not appear to have the ability to cause anemia, hence justifying its use in treatment of drug related disorders (Udu et al., 2021). The increased total WBC count (Table 4), suggested that the leucocytes' phagocytic function has improved. Platelets play a vital function in hemostasis, and their decline may have a detrimental effect on thrombopoietin levels (Ramadori et al., 2019). The increased platelet count after 21 days of Nigella sativa oil extract administration suggests that the extract may not create any coagulation issues, but it may have the potential to improve clotting and reduce hemorrhages.
Biochemical parameters
Biochemical parameters are helpful indicators of an organ's functional integrity. Hepatocellular injury is indicated by transerases (ALT and AST) (Kumar et al., 2021). Alanine amino transerase (ALT) is present throughout the body, including the liver, kidneys, pancreas, lungs, brain, heart, and skeletal muscles, whereas aspartate amino transerase (AST) is mostly located in the liver (Lala V, Goyal A, 2022). Alanine amino transerase is present in the cytoplasm of hepatic cells, whereas aspartate amino transerase is found in both the cytoplasm and mitochondria. The dose-related increased levels of these liver enzymes seen in this study indicate that Nigella sativa oil extract has not caused hepatocellular damage, this is consistent with (Adedapo et al., 2004), which reported that Nigella sativa’s pharmacological characteristics linked to the presence of thymoquinone and its antioxidant qualities (Shahin et al., 2018). Thymoquinone protects the liver by inhibiting iron-dependent lipid peroxidation, increasing total thiol content and glutathione levels, radical scavenging, increasing the activity of quinone reductase, catalase, superoxide dismutase, and glutathione transferase, inhibiting the activity of free radicals in both cyclooxygenase and lipoxygenase. As a result, the studies emphasize the function of ROS in liver disorders as well as the mechanisms of Nigella sativa oil in liver damage prevention (Dollah et al., 2013; Shahin et al., 2018). The current study confirms previous information protective effect of Nigella sativa oil on toxicity in animal models that might be attributed to repeated dosage exposure (Figure 2).
The variations in serum enzymes levels (Table 5), might have been produced by the oil extracts extended treatment. However, an increase in enzymes may not indicate systemic toxicity. Although amino-transferases levels suggest hepatocellular injury, they do not always indicate the degree of the damage (Contreras-Zentella & Hernández-Muñoz, 2016). The enzyme alkaline phosphatase (ALP) is found in abundance in the microvilli of the bile canaliculi and is utilized to check the plasma membrane's integrity (Dey et al., 2016). An increased ALP level in the blood is a well-known sign of cholestatic liver disease (Kozat, 2017).
The amounts of total protein, albumin, and bilirubin in the blood reflect the condition and degree of hepatic damage. The normal concentration of protein, albumin, and bilirubin found in the current study (Table 5) imply that the extract did not affect the liver's synthetic and secretory activities, and that the extract is not fully hepatotoxic. Albumin is the plasma protein with the greatest concentration. It distributes a variety of chemicals, including medications, and protects the tissues from fluid leakage (Kadry et al., 2020). Haemolytic anemia, biliary obstruction, and hepatic illness are all linked to elevated levels of bilirubin, a result of the haeme component of haemoglobin. The amounts of bilirubin measured in this investigation were unchanged, indicating that no erythrocytes were destroyed as a result of the treatment with the oil extract.
The findings might potentially imply that the NS-oil extract had no effect on hepatic cells' ability to excrete bilirubin. Urea molecule produced by protein catabolism and expelled by the kidney, is a by-product of protein catabolism (Chaklader et al., 2020). High protein diets, dehydration, acute bleeding, and shock can all cause elevated urea levels in the blood (Atata et al., 2019). The elevated quantity of urea found in this study (Table 5), especially at high dose levels (800 mg/kg body weight), suggests that the waste product's excretion ability has been compromised in the kidneys. The higher creatinine level (Table 5) was also indicative of potential nephrotoxicity. Increased creatinine levels in the blood are linked to impaired renal excretory function as a result of sickness or a toxic insult (Liu et al., 2016).
The preserved renal corpuscles and cortex (Figure 3), however, did not suggest nephrotoxicity in the histological results. Flavonoid and tannin have been shown to protect the kidneys from nephrotoxicity (Alomar, 2020; Harakeh et al., 2022). Plasma cholesterol and triglyceride levels give insight into lipid metabolism and the risk of atherosclerosis and coronary heart disease Liu et al., 2016). The lower lipid profile (Table 5) indicated that the extract has no lipolytic activity or the potential to cause atherosclerosis. Several plants that include tannins and flavonoids have been shown to reduce blood lipid levels (Michel et al., 2020). As highlighted, tannins, flavonoids, alkaloids, and terpenoids were detected in the n-hexane Nigella sativa extracted oil in this study.
The considerable reduction in serum glucose concentrations (Table 5) suggests that the extract has a hypoglycemic effect. In the liver and kidneys, gross and histological examinations indicated no abnormalities. There were also minor lesions observed in the liver, (Image 1) and were backed up by an increase in serum enzymes levels (Table 5), suggested that there was some hepatic damage. The impact of anesthesia at sacrifice of the animals may be linked to that, as the same were observed in the rats in both the treatment and control groups (Figure 3).