Evaluating Effectiveness of Nigella sativa L Extract on Lipopolysaccharide-D-Galactosamine Induced Hepatic Toxicity in Rats

Rakesh Kumar Sahu¹, Aayush Vaishnaw², Saket Singh Chandel²*, Neelima Yadav², Manali Rai²

¹Research Scholer, Department of Pharmacology, Dr. C.V. Raman Institute of Pharmacy,

Dr. C.V. Raman University, Kota, Bilaspur, Chhattisgarh - 495113, India.

²Faculty of Pharmacy, Dr. C.V. Raman Institute of Pharmacy,

Dr. C.V. Raman University, Kota, Bilaspur, Chhattisgarh - 495113, India.

*Corresponding Author E-mail:

ABSTRACT:

Nigella sativa is a promising natural remedy for liver problems because of its cytoprotective, hypolipidemic, and antioxidant properties. Here, we looked at the possible defense of Nigella sativa seed (NSS) against hepatotoxicity in rats caused by lipopolysaccharide and D-galactosamine. For 30 days, 36 adult Wistar albino rats were evenly and randomly split into six groups in order to achieve this goal. Lipopolysaccharide (30μg/kg b.wt., i.p.) and D-galactosamine (300mg/kg b.wt., i.p.) were given as supplements to the second group, whereas the control group received no therapy. Nigella sativa alcoholic extract (500 mg/kg b. wt orally) was added as a supplement to the third group. The fourth group was given DGalN/LPS along with 500 mg/kg.B.wt. of Nigella sativa alcoholic extract. Group VI was given Thymoquinone (30 mg/kg b.wt. orally) in combination with D-GalN/LPS, whereas Group V was given Thymoquinone (40 mg/kg b.wt. orally) as a regular medication. NSS succeeded in boosting serum reduced glutathione level along with hemoglobin level. It reduced lipid peroxides in the serum along with serum bilirubin and ESR. NSS was successful in raising both the hemoglobin and serum reduced glutathione levels. Serum bilirubin, ESR, and lipid peroxides were all decreased. NSS's anti-apoptotic and antioxidant properties effectively guarded against the hepatotoxicity of DGalN/LPS. These results are extremely important since they highlight the use of NSS in our food sector and as a traditional medicine treatment to combat liver problems

KEYWORDS: D-Galactosamine, Lipopolysaccharide, Hepatic toxicities, Nigella sativa.

INTRODUCTION:

As the primary organ for metabolism and excretion, the liver is both incredibly complicated and exquisitely simple. It is straightforward because the hepatocyte, the only type of epithelial cell that makes up the parenchyma, is distributed throughout the organ in sheets that are closely connected to a venous portal system in a straightforward and uniform manner¹. It is complicated because these hepatocytes carry out more than five hundred metabolic processes, some of which are vital for survival, some of which are crucial for preserving homeostasis, and some of which support overall health. Owing to its wide range of tasks, this organ is also linked to several illnesses². In addition to being a significant event in primary liver illnesses, hepatocyte injury which can be either permanent or reversible may also play a substantial role as a secondary factor in other human diseases. When an injury is severe enough and lasts long enough, the integrity of the cells is disrupted irreparably, which causes necrosis and cell death^3,4.

Hypoxia, portal hypertension, ascites, aberrant bile secretion, aberrant clearance of gut-absorbed proteins and ammonia, liver cell necrosis and cirrhosis, tumors, hepatocellular carcinoma, and hepatic failure are among the several signs of hepatic dysfunction⁵.

According to studies, oxidative stress and ROS generation act as mediators for a number of liver damage triggers. Hepatocytes are protected from negative effects by a number of bodily mechanisms that guard against oxidative damage in addition to high glutathione levels. An amino sugar called D-galactosamine is typically only present in vivo in acetylated form in specific structural polysaccharides⁶. A tiny dose of lipopolysaccharide and D-galactosamine, an inhibitor of hepatic RNA synthesis, can work in concert to cause fulminant hepatitis in experimental animals. For this reason, the "Galactosamine Hepatitis" model of hepatotoxicity and hepatocellular apoptosis is a useful tool for pharmacological studies⁷.

Hepatocyte apoptosis, inflammatory processes, and macrophage and granulocyte activation may ensue as a subsequent event. Apoptotic or necrotic cell death is the "point of no return" that the process irrevocably reaches once it has begun. Galactosamine LPS hepatitis is an excellent model of hepatocellular apoptosis because it is easily manipulable⁸.

The broad range of safety, effectiveness, and availability of Nigella sativa seeds (NSS) makes them a dietary desirable option⁹. It is generally known as Kalonji in India and is a member of the Ranunculaceae family. The seed and its oil have diuretic properties and also have lacto-gogue, vermifuge, and carminative properties. Black seed oil and extracts have been shown to exhibit antimicrobial and immune-stimulating qualities, as well as hypotensive and anti-inflammatory measures, as well as anticancer, antioxidant, hypoglycemic, spasmolytic, and bronchodilator effects¹⁰.

About 7% of the different esters found in N. sativa seeds are made from fatty acids and terpene alcohols. Alkaloids from the pyrazol and isoquinoline groups are present in the seeds. The examined samples contain trace levels of these substances. Furthermore, it contains roughly 33.9% carbohydrates, 5.5% fiber, and 6% water. High performance liquid chromatography analysis may identify the primary constituents of N. sativa essential oil as thymohydro-quinone, thymo-quinone, dithymo-quinone, and thymol. Thymo-quinone's primary active ingredient has antioxidant properties^11,19-22.

In light of the aforementioned factors, Nigella sativa L. seeds, often known as black cumin, have been chosen for the current study in order to assess how well they protect experimental mice from hepatic diseases brought on by lipopolysaccharide. It is widely recognized for its flavor and is regarded as a spice by the general public.

MATERIAL AND METHOD:

The Nigella sativa seed sample utilized in this study was validated and purchased locally. Among other biochemicals, D-galactosamine, lipopolysaccharide, and thymoquinone were acquired from Sigma Chemical Company in Mumbai, India. Analytical grade (AR) chemicals, acids, bases, solvents, and salts were all acquired from approved vendors and utilized in the experiments.

Making a Plant Extract:

Methanol (95%) was used to do a Soxhlet extraction on the powdered material. To get a suitable yield of 22%, the solvent was vacuum-expelled until the oily residue or remnant had no methanol odor left¹⁸.

Experimental Design:

Thirty-six healthy male Wistar albino rats (6–8 weeks old), weighing 180–230g, were procured for the study. The animals were acclimatized for two weeks under standard laboratory conditions (temperature: 22±2°C; relative humidity: 55–65%; 12h light/12 h dark cycle) with free access to a standard pellet diet and water ad libitum. The rats were randomly divided into six groups, with six animals in each group. In experimental models, D-galactosamine and lipopolysaccharide were used to induce liver injury.

All experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, under approval number SOP/CEC/IAEC/2024-25/042. The study was conducted in compliance with CPCSEA norms to ensure humane care and use of laboratory animals. As the control group, Group I was given a regular food and unlimited access to water. Eighteen hours prior to the experiment, Group II received 30μg/kg b.wt., i.p. of lipopolysaccharide and 300mg/kg b.wt., i.p. of D-galactosamine. As a pre-treatment, Group III was given 500mg/kg b.wt. of Nigella sativa alcoholic extract orally for 30 days. For 30 days before DGalN/LPS was administered, Group IV was given an oral pretreatment of 500mg/kg b.wt. of Nigella sativa alcoholic extract. Before D-GalN/LPS was administered, Group V got the normal oral pretreatment of Thymoquinone 40mg/kg b.wt. for 30 days, while Group VI received oral pretreatment of Thymoquinone 30mg/kg b.wt. for 30 days.

Collection of Blood Samples:

The rats' body weight was routinely tracked and documented during the trial. On the first, eighth, and fifteenth days, blood samples were taken from the tail vein of every animal in the group. To separate the serum on the days of collection, the obtained blood samples were centrifuged at 3000rpm for 30minutes after being allowed to clot for 30minutes. Aspartate transaminase and alanine transaminase, two liver marker enzymes, were measured for activity.

Preparation of Liver Homogenize:

After being perfused with physiologic saline, the liver was promptly removed. The organs were then blotted, dried, weighed, and homogenized to create a 1% tissue homogenate solution in Tris-HCl buffer (10mM, pH 8.0), which was used to measure a number of parameters.

Biochemical Estimation in Blood And Tissue Samples Hemoglobin level Estimation:

The standard suggested methodology was used to measure hemoglobin. Five milliliters of the reagent were used to dilute 0.02 milliliters of blood. To make sure the reaction was finished, the diluted blood was thoroughly mixed and let to stand for ten minutes. Together with the cyanmethemoglobin standard solution, the solution was measured at 540nm. The unit of measurement for blood hemoglobin was g/dl.

Erythrocyte Sedimentation Rate (ESR) Determination:

Erythrocyte sedimentation rate (ESR) determination: Bottiger's (1967) technique¹² was used to determine ESR. After mixing and drawing up to the "0" mark of a Westegren tube, the blood-3% trisodium citrate mixture (4:1 v/v) was put in a rack upright. The distance between the surface meniscus and the top of the sedimenting red cell column was measured at 10minute intervals. mm/hr was used to express the ESR.

Determination of Levels of Serum Bilirubin:

A serum White and Duncan's (1952) approach was used to estimate bilirubin levels¹³. 0.2ml of serum and 1.8ml of distilled water were placed into each of two test tubes. 0.5ml of diazo reagent and 0.5ml of 1.5% hydrochloric acid were added to the blank and unknown, respectively. Lastly, 2.5ml of methanol was added to each tube. After 30 minutes of standing in ice, the absorbance at 540 nm was measured. The aforementioned standard was diluted with methanol to create a solution with 2mg of bilirubin per 100milliliters for a standard curve. A similar method can be used to find the amount of direct reacting bilirubin by replacing 2.5ml of methanol with 2.5ml of water.

Determination of Tissue Lipid Peroxides:

The thio barbituric acid reaction, as outlined by Ohkawa et al. (1979), was used to evaluate the lipid peroxide levels of the liver samples¹⁴. 1.5ml of 20% acetic acid, 0.2ml of SDS, and 1.5ml of TBA were added to 0.5ml of liver homogenate. After adding distilled water to get the mixture up to 4.0ml, it was heated to 95°C for 60 minutes while a glass ball served as a condenser. 4.0ml of the butanol-pyridine mixture was added after cooling, and it was thoroughly shaken. Following ten minutes of centrifugation at 4,000rpm, the organic layer was removed, and its absorbance at 532nm was measured. As a rule, 1, 1', 3, 3'-tetramethoxy propane was utilized. N moles of MDA/mg protein were used to represent the lipid peroxide levels.

Determination of Total Reduced Glutathione (GSH):

The method of Moron et al. (1979)¹⁵ was used to estimate the total amount of reduced glutathione in the liver tissue. 5% TCA was used to precipitate 0.5ml of liver homogenate. After thoroughly mixing the ingredients to ensure that all of the proteins precipitated, the mixture was centrifuged. To get a final volume of 4.0ml, 2.0ml of DTNB reagent and 0.2M phosphate buffer were added to an aliquot of clear supernatant. At 412nm, the absorbance was measured against a blank that contained TCA rather than the sample. To ascertain the glutathione content, a number of benchmarks that were handled similarly were also conducted. The ratio of glutathione to moist tissue is n moles/g.

RESULTS AND DISCUSSION:

Serum Hemoglobin and ESR:

The protective effects of the plant extract and its active compound (TQ) on the hematopoietic system are demonstrated by the increased hemoglobin levels (table 1, figure 1) in Group IV and VI rats administered Nigella sativa L. alcoholic extract (MENS) and thymoquinone (TQ) prior to the D-GalN/LPS toxicities. The change in red cells in plasma, which speeds up sedimentation, may be the cause of the Group II rats' markedly elevated ESR (table 1 and figure 1).

Figure 1: Levels of Hb and ESR in different study groups

Assessing Serum Bilirubin:

Total, conjugated, and unconjugated bilirubin levels in the serum of the experimental and control groups of rats are displayed in Table 2 and Figure 2. A naturally occurring organic anion, bilirubin binds to albumin reversibly before being carried to the liver, where it is converted to glucuronyl acid and eliminated as bile. Serum bilirubin levels are measured as an indicator of hepatic function, and any abnormal rise in these levels suggests hepatobiliary disorders and serious disruptions in hepatocellular function. The hallmark of D-galactosamine-induced hepatitis is elevated serum bilirubin levels. This demonstrates the degree of hepatic dysfunction brought on by the hepatotoxic agent and is in good agreement with the elevated levels of serum bilirubin (Total, Conjugated, and Unconjugated) in Group II rats given the DGalN/LPS challenge in comparison to the Group I control rats.

A combination of reduced excretion of the pigment and impaired absorption or conjugation may be the cause of the elevated levels of unconjugated and conjugated bilirubin seen in this study. Normal Wister Kyoto rats' bilirubin levels were lowered by Nigella sativa fixed oil¹⁶. The improvement in the liver's functional state is evident from the stabilization of blood bilirubin levels in Group IV and VI rats that were pre-treated with thymoquinone and Nigella sativa alcoholic extract before DGalN/LPS was administered, as compared to Group II rats.

Figure 2: Levels of total, conjugated and unconjugated bilirubin in different study groups

Assessment of Tissue Lipid Peroxidase Level:

Lipid peroxidase is a presumed indicator of the production of free radicals and the onset of oxidative stress. It may be concluded that TQ and Nigella sativa alcoholic extract offset the aberrant rise in lipid peroxides brought on by D-GalN/LPS because Group IV and VI rats had lower levels of lipid peroxides (table 3, figure 3). The plant's anti-oxidative properties may be the cause of this.

According to earlier publications, endotoxin injection in D-GalN-sensitized mice has been shown to cause membrane damage and lipid peroxide generation in experimental animals, which lowers the levels of free radical scavengers or quenchers¹⁷.

Total reduced glutathione (GSH):

GSH levels inside cells are essential for shielding the cell from oxidative damage. Glutathione Reductase (GR) is essential for controlling GSH. Group II rats exhibit reduced GR activity (figure 3). The GR is inactivated in this group of mice because of elevated oxidative stress.

Therefore, the crucial component of the GSH redox cycle is the deactivation of GR during oxidative stress. The animals' decreased oxidative stress may be the cause of the return of GR activity in those treated with TQ and Nigella sativa extract. These medications may stop the production of hydroperoxide, which raises GR activity.

Figure 3: Levels of GSH in the liver of control and experimental groups of rats

SUMMARY AND CONCLUSION:

The present study's findings were based on research on the toxicity of D-GalN/LPS to mammals and the relative hepato-protective effectiveness of an alcoholic extract of Nigella sativa L. seed in comparison to the standard of one of the active ingredients, thymoquinone. The NSE and TQ pretreatment significantly decreased the elevation of the diagnostic marker enzymes of hepatic function that indicate changes in membrane permeability and damage during DGalN/LPS toxication. This strongly suggests that NSE and TQ have protective activity towards tissues by preventing the leakage of these enzymes to the serum. In the NSE and TQ pretreatment groups, the elevated serum bilirubin, elevated ESR, and lowered Hb levels seen in the D-GalN/LPS toxicated group were kept close to normal. The toxin-induced decrease in ESR was stopped by pretreatment with NSE and TQ. This suggests that the NSE and TQ can stop the hepatotoxin's harmful effects on the hematological system. Furthermore, the treatment of D-GalN/LPS led to a decrease in enzymatic antioxidants and an increase in lipid peroxides. Lower levels of lipid peroxides and higher levels of enzymatic antioxidants (GSH) indicated that NSE and TQ pretreatment decreased the oxidative stress caused by D-GalN/LPs, confirming their antioxidant activity and their ability to inhibit the lipid peroxidation chain reaction. As a result, the NSE and TQ pretreatment brought the biological parameter changes brought on by D-GalN/LPS toxicities back to levels that were almost normal. When compared to the conventional TQ, NSE demonstrated superior results and appeared to have a greater hepatoprotective impact against D-GalN/LPS. The NSE's efficacy is thought to be attributed to a mix of fatty acids, volatile oils, and trace elements.

CONFLICT OF INTEREST STATEMENT:

The authors declare that there is no conflict of interest regarding the publication of this study.

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Received on 14.06.2025 Revised on 11.08.2025

Accepted on 22.09.2025 Published on 11.10.2025

Available online from October 25, 2025

Res.J. Pharmacology and Pharmacodynamics.2025;17(4):263-268.

DOI: 10.52711/2321-5836.2025.00042

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Variables studies	Gr I	Gr II	Gr III	Gr IV	Gr V	Gr VI
Hemoglobin (g/dl)	13.8±1.21	7.34±1.11	14.21±0.21	11.64±1.32	13.65±1.22	11.14±1.11
ESR (mm/hr)	3.76±1.10	7.9±1.1	3.89±1.01	6.34±1.02	3.98±1.24	5.98±1.31

Different Bilirubins (mg/dl)	Gr I	Gr II	Gr III	Gr IV	Gr V	Gr VI
Total	0.39±1.01	0.98±1.1	0.41±1.23	0.59±1.21	0.39±1.01	0.57±1.01
Conjugated	0.26±1.01	0.58±1.01	0.25±1.21	0.29±1.01	0.27±1.21	0.29±1.23
Unconjugated	0.21±1.01	0.39±1.02	0.12±1.11	0.27±1.02	0.13±1.02	0.28±1.12

Variables studied	Gr I	Gr II	Gr III	Gr IV	Gr V	Gr VI
Lipid Peroxides	1.87±1.13	2.69±1.32	1.88±1.34	2.16±1.23	1.86±1.2	2.15±1.01
Reduced Glutathion	8.36±0.81	4.27±0.96	9.08±0.36	8.06±0.86	7.48±0.86	7.39±0.79