Sage Journals: Discover world-class research

Abstract

Background: Liver damage is indicated by histopathological and metabolic dysfunction. There has been increasing interest in natural hepatoprotective agents from medicinal herbs. The medicinal plant Cistus ladanifer widely used in phytotherapy was selected for this study. Objectives: The work aims to investigate the hepatoprotective effects of Cistus ladanifer leaf extracts in vivo against the paracetamol-induced hepatotoxicity mice and in silico using the docking method. The chemical composition of the extracts was also studied. Materials and methods: The paracetamol-induced hepatotoxicity mice were treated with aqueous and methanolic extracts of Cistus ladanifer at two doses of 150 and 300 mg/kg body. The serum levels of aspartate aminotransferase, alanine aminotransferase, triglycerides, total cholesterol, urea, and creatinine in mice-induced hepatotoxicity were measured to evaluate the hepatoprotective activity of the extracts. The histopathological alterations were also investigated. The chemical profile of methanolic and aqueous C. ladanifer extracts was determined using GC-MS. The Human Cytochrome P450 2E1(CYP2E1) was presented as a protein target and the docking was studied using Maestro version 11.5 software developed by Schrodinger, LLC. Results: The administration of C. ladanifer extracts showed hepatic protection at an oral dose of 150 and 300 mg/kg for the aqueous extract and only at 150 mg/kg for the methanolic one. The aqueous extract at both doses and the methanolic extract at 150 mg/kg didn’t occur any biochemical or histological alterations. However, the methanolic extract showed hepatic toxicity at 300 mg/kg. Various compounds were identified in the methanolic and the aqueous extracts, probably responsible of the hepatoprotective activity. The in silico study has also shown that some molecules present in both extracts presented a good affinity in the active site of Cytochrome P450 2E1 (CYP2E1). Conclusion: The results deserve more attention as C. ladanifer can be considered a source of pharmacological compounds with hepatoprotective effects. Further detailed investigation is needed to deepen the study.

Keywords

hepatoprotective effects liver damage

Introduction

The Liver plays one of the most important human body organs due to its regulation of homeostasis, and neutralizing of toxins within the body,¹ it has multi-biological functions in carbohydrate, lipid, and protein metabolism.² Liver damage is caused by metabolic dysfunction, histopathological patterns, and associated diseases such as hepatitis, steatosis, neoplasms, and others.³ Generally, the treatment of hepatic diseases causes various and serious long-term side effects;^3,4 therefore, many studies showed interest in developing effective natural agents from medicinal plants in order to treat and prevent hepatic disorders. Treatment with medicinal plants has increased worldwide, as the use of herbs in an optimal way is harmless with no serious adverse reactions.²

Cistus plants (rock rose) draw attention due to their active compounds such as phenolic compounds and terpenoids, well known for their different pharmacological activities. In Mediterranean traditional medicine, C. ladanifer have been used as herbal infusions or as decoction of the aerial parts for the prevention and the treatment of a variety of diseases and infections. It is commonly used as anti-inflammatory, anti-diarrhoeal, antiacid, antidiabetic, and antispasmodic anti-diarrhoeal, agents.^4-6 C. ladanifer has been the subject of various studies as this plant presents significant pharmacological potential such as antimicrobial, antioxidant,^7-10 anti-inflammatory, analgesic actions,¹¹ hypoglycemic and hypolipidemic,^6,12 and cytotoxic effects.^7,13,14 This plant is known for the presence of various compounds mainly Kaempferol, Punicalagingallate, Punicalin, and Punicalagin, Epigallocatechin, Ellagic acid, Gallic acid, Myricitrin and Quercetin.^14-18 These compounds are known for their pharmacological effects and can be responsible for the plant therapeutic activities. Moreover, the whole plant, but mainly the leaves, secrete a very fragrant gum—labdanum—composed mainly of flavonoids and terpenes, in particular labdan-type diterpenes,^19,20 it is traditionally used in low doses in sedative tea, coffee, and other infusions to prevent and to treat dysentery, diarrhea insomnia, catarrh, and anxiety.^7,15,21,22

C. ladanifer is known for its pharmacological properties; however, it is little explored for its hepatoprotective activity. The aim of our work is to evaluate the hepatoprotective activity of C. ladanifer extracts in vivo and in silico and to identify their chemical profile.

Material and Methods

Plant Material

Leaves of C. ladanifer were harvested from the Taza region in May 2017 (006°28.382′ E; 004°49.405′ N). The authentication of the plant was effectuated by Dr Khabbach Abdelmajid. The leaves parts of C. ladanifer were dried at room temperature then powdered by a mechanical grinder. A quantity of 10 g was then macerated overnight with 100 mL of distilled water and methanol separately. The whole mixture was filtered and the filtrate was evaporated under pressure. The extracts were conserved at +4 °C until further use. The yield of both aqueous and methanolic extracts was calculated with a percentage of 6.64 ± 0.06% and 6.24 ± 0.08% respectively.

Hepatoprotective Activity of C. ladanifer Extracts

Animals

Adult male Swiss mice aged 2 months and weighing between 20 and 25 g were used to perform the current study. Animals were obtained from the animal house at the Department of Biology, Faculty of Sciences, Sidi Mohamed Ben Abdellah University. The animals were housed in controlled laboratory conditions with a set temperature (23 ± 2◦C) and light cycle (12 h light/dark cycles). The animals were allowed free access to food and water ad libitum. The Animal Ethics Review Committee at the faculty of Sciences, Fez, Morocco, approved this study under the ethical clearance number 02/2023/LBEAS. The use of laboratory animals in all experimental procedures was conducted according to ethical guidelines for the care and use of laboratory animals.

Paracetamol Dose Regimen

In order to evaluate the hepatoprotective effect of C. ladanifer extracts, the paracetamol tablets were used, each tablet contains 1000 mg of paracetamol. The tablets were obtained from a Bab Elghoul Pharmacy. The paracetamol was made into fine powder using a mortar and pestle. The powdered paracetamol was suspended in a saline solution and was administered according to the body weight of the mice. The dose administered to the mice was set as 1000 mg/kg. Grouping of mice and treatments. Thirty mice were randomly divided into three groups and each group consists of five mice. The first group received 1 mL/kg of saline (control). Group II was given paracetamol (1000 mg/kg) orally and group III received orally both 1000 mg/kg paracetamol and 100 mg/kg of the aqueous extract, respectively. Extract was administered 3 hours after the administration of paracetamol. Paracetamol 1000 mg/kg was given to mice to induce hepatotoxicity. The treatments were continued for 7 days and on the Eighth day of the experiment; all animals were anesthetized and dissected.

Biochemical Parameters

At the end of the experimental period of the test, the animals were anesthetized with sodium pentobarbital at a dose of 30 mg/Kg before being sacrificed for blood collection. Blood samples were collected from the heart, in heparinized tubes (0.2 ml with 10 U/ml heparin) and then centrifuged at 1500 rpm for 10 min. Next, the recovered plasma was analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, urea, cholesterol, and triglycerides (TG) parameters.

Histopathological Examination

Livers and kidney of mice were excised for histopathological examination. The tissues were washed and fixed in 10% formalin solution, dehydrated with alcohol, this step involves immersing the sample in increasing concentrations of alcohol to remove water and formalin from the tissues. Afterwards, an organic solvent such as toluene is used to remove the alcohol and allow infiltration of tissues with paraffin, and then enclosed in paraplast Afterward, tissues were embedded in paraffin (heated to 56 °C for 4 h in an drying oven), and sectioned using microtome. Sections of 5 µm thickness were cut and mounted on slides. The slides are then placed on a hot plate (15 min, 40 °C) for deparaffinization. Next, sections were stained with hematoxylin-eosin before being read using a light microscope. Hematoxylin binds to DNA, allowing it to stain the nucleus. Eosin binds to basic molecules, which allows the cytoplasm to be colored.

Chemical Composition

The identification of the compounds present in the extracts was carried out using the GC-MS technique according to the protocol detailed in our previous studies.²³ The mass spectra were matched to the NIST database for identification of compounds.

Computational Modeling

Our computational modeling investigations focused on the Human Cytochrome P450 2E1 protein and specific phytochemicals.^24,25 To carry out these simulations, we utilized Maestro version 11.5 developed by Schrodinger, LLC. The procedural steps involved in our study encompassed the selection and preparation of the protein, preparation of ligands, creation of a receptor grid, and ligand docking.

Protein Preparation

The crystal structure of Human Cytochrome P450 2E1 (PDB ID: 3LC4) was obtained from the RCSB Protein Data Bank and underwent a series of steps, including examination, adjustments, and minimization through the use of the protein preparation wizard tool. The protein was subjected to preprocessing, which involved addressing missing loops and side chains using the Prime tool, with the PROPKA pH set at 7.4 for accurate ionization state predictions. Important residues were retained, followed by the addition of hydrogen atoms and the elimination of water molecules located more than 5 Å away. Subsequent energy minimization was conducted using the OPLS3e force field to achieve a protein structure in a low-energy state with an RMSD of 0.3 Å, which was then utilized in subsequent simulation investigations.²⁶

Ligand Preparation

To prepare ligands (phytochemicals extracted from our plant) for our molecular docking studies with the CYP2E1 protein, we employed the LigPrep tool. The ligands were subjected to preparation using the OPLS3e force field, and the pH was set at 7.4 ± 0.5 to generate potential ionization states.²⁷

Receptor Grid Generation

The receptor grid was generated by selecting atoms located within the binding site of the co crystallized ligand within the protein after it had been minimized. This specific region was earmarked for ligand docking and subsequent processing stages. The box has been identified under the following coordinates: X = -28.364, Y = -40.442, and Z = 30.193.²⁸

Molecular Docking

Using the receptor grid generated, the molecular docking studies were conducted in standard precision mode using the Glide tool. These investigations enabled us to uncover interactions between the ligands and the protein, along with associated docking scores.²⁸

Statistical Analysis

The data were presented as means and standard deviations. The statistical analysis was carried out by use of one-way analysis of variance, using Dunnett test along with GraphPad Prism 5 (GraphPad software, La Jolla, CA). When the P-value was less than .05, the values were statistically judged to be significant.

Results

Serum Biochemical Analysis

Effect of C. ladanifer Extracts on the Transaminases Levels and the Lipidic Profile

In order to evaluate the hepatoprotective activity of both aqueous and methanolic extracts, two doses were prepared 150 and 300 mg/kg, and the level of the AST (U/L), ALT (U/L) and lipidic profile (Total cholesterol and TG) was measured. As shown in Figure 1, the values of enzymatic activities of AST and ALT measured in the mice's serum treated with paracetamol alone were found to be significantly higher (P < .001) when compared to the group of mice treated with paracetamol along with C. ladanifer extracts. The levels of AST and ALT decreased when treated with the extracts, indicating the presence of an hepatoprotective activity from paracetamol-induced injury. However, the methanolic extract at the dose of 300 mg/kg showed some hepatic toxicity. Measurement of total cholesterol and TG levels is another indicator of the presence of an hepatic disorder. In Figure 2, decreased levels of cholesterol and TG were found in the positive control group compared to the negative presenting higher levels. When treated with paracetamol along with both C. ladanifer extracts, these levels were restored to normal in the mice's serum.

Figure 1.

Effect of aqueous and methanolic extracts of C. ladanifer on Transaminsases (ALT and AST).Values significantly different compared to negative control: *P < .05; **:P < .01; ***P < .001. Aq: Aqueous extract; MeOH: Methanolic extract. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Figure 2.

Effect of aqueous and methanolic extracts of C. ladanifer on Cholesterol and triglycerides. Aq: Aqueous extract; MeOH: Methanolic extract.

Effect of C. ladanifer Extracts on the Urea and Creatinine Levels

Urea and creatinine are measured as biomarkers of renal damage. Figure 3 shows that the negative group of mice presented high levels of urea and creatinine when compared to the positive control group. Treated mice with only paracetamol decreased the levels of the two biomarkers causing a renal damage. At tested concentrations, both aqueous and methanolic extracts increased the urea and creatinine levels in mice serum and were recovered to normal. However, mice treated with methanolic extract at 300 mg/kg showed a low concentration of urea compared to the positive control, indicating a toxicity.

Figure 3.

Effect of aqueous and methanolic extracts of C. ladanifer on urea and creatinine. Aq: Aqueous extract; MeOH: Methanolic extract.

Histopathological Alterations

The Histopathological analysis of the two organs Liver and kidney are shown in Figures 4 and 5. The histological analysis of animal kidneys didn’t reveal any pathological alterations that may be attributed to treatment with C. ladanifer aqueous extract at the doses of 150, and 300 mg/kg, and methanolic extract at 150 mg/kg respectively. The liver histology of rats treated with paracetamol at 1000 mg/kg, and the methanolic extract at the dose of 300 mg/kg demonstrated numerous pathological alterations, including sinus dilation, infiltration of inflammatory cells (lymphocytes), as well as necrosis. However, rats treated with aqueous extract at the doses of 150 and 300 mg/kg respectively and methanolic extract at the dose of 150 mg/kg didn’t show any signs of toxicity.

Figure 4.

Histopathological photos of liver slices stained with hematoxylin-eosin (*40) after oral administration of paracetamol and the extracts. Liver section of groups treated with NaCl (A), paracetamol (B), aqueous extract (100 mg/kg) + paracetamol (C), aqueous extract (300 mg/kg) +paracetamol (D), methanolic extract (100 mg/kg) + paracetamol (E), methanolic extract (300 mg/kg) +paracetamol (F). H: Hepatocyte; CV: Central vein; L: Lymphocyte; N: Necrosis.

Figure 5.

Kidney section of animal groups treated with NaCl (A), paracetamol (B), aqueous extract (100 mg/kg) + paracetamol (C), aqueous extract (300 mg/kg) +paracetamol (D), methanolic extract (100 mg/kg) + paracetamol (E), methanolic extract (300 mg/kg) + paracetamol (F). G: Glomerulus; TCP: Proximal Convoluted Tubule; TCD: Distal convoluted tubule; US: Urinary space.

Chemical Composition

The chemical composition of the methanolic and aqueous extracts showed the presence of various compounds known for their pharmacological activities. Tables 1 and 2 present the chemical composition of methanolic and aqueous extracts, respectively.

Table 1.

Chemical Composition of the Methanolic Extract of C. ladanifer.

	RT (min)	Name		Area (%)
1	4.769	L-Arabinitol		2.625
2	4.832	Hydrazine. 1.1-dimethyl-		1.215
3	7.691	Cyclohexanone. 2.2.6-trimethyl-		2.445
4	7.903	Cyclohexanone. 2.2.6-trimethyl-		1.41
5	8.272	2-Cyclohexen-1-one. 3.5.5-trimethyl-		1.875
6	8.717	Cyclohexanol. 2.6-dimethyl-		1.32
7	9.029	2-Oxatricyclo [3.3.1.1(3.7)]decane. 1-methyl-		1.215
8	9.267	Camphor		1.125
9	9.33	Isoborneol		0.945
10	9.411	Borneol		1.41
13	11.056	Bicyclo[2.2.1]heptan-2-ol. 1.7.7-trimethyl-. acetate. (1S-endo)-		2.535
14	11.575	Bornylacetate		1.32
15	12.063	1-(5.6-Dimethyl-2-pyrazinyl)-2-methyl-1-propanone		2.07
16	12.451	.alpha.-Cubebene		1.32
17	14.259	1H-Cyclopropa[a]naphthalene.		1.59
18	14.384	Bicyclo[4.4.0]dec-1-ene. 2-isopropyl-5-methyl-9-methylene-		1.59
19	14.665	Naphthalene. 1.2.3.5.6.8a-hexahydro-4.7-dimethyl-1-(1-methylethyl)-. (1S-cis)-		2.445
20	14.815	Naphthalene. 1.2.3.4.4a.7-hexahydro-1.6-dimethyl-4-(1-methylethyl)-		0.66
21	14.884	Ledol		0.945
23	15.197	1H-Cycloprop[e]azulene. decahydro-1.1.7-trimethyl-4-methylene		1.875
24	15.328	Naphthalene. 1.2.3.5.6.7.8.8a-octahydro-1.8a-dimethyl-7-(1-methylethenyl)		1.32
28	16.173	.tau.-Muurolol		1.59
29	16.467	Naphthalene. 1.2.4a.5.8.8a-hexahydro-4.7-dimethyl-1-(1-methylethyl)		1.035
30	17.361	Tetradecanoicacid		2.34
31	18.237	Bicyclo[3.1.1]heptane. 2.6.6-trimethyl		1.215
32	18.775	Bicyclo[3.1.1]heptane. 2.6.6-trimethyl-		1.035
33	18.863	Pyrrolidine. 1-(1-cyclohexen-1-yl)-		1.965
34	19.807	n-Hexadecanoicacid		1.965
35	20.414	Hexadecanal		1.215
36	21.265	2.3.3-Trimethyl-2-(3-methyl-buta-1.3-dienyl)-cyclohexanone		0.75
37	21.74	13-Tetradece-11-yn-1-ol		3.57
38	22.003	Octadecanoicacid		3.285
40	23.054	Bicyclo[4.1.0]heptane-7-carboxylic acid. 4-acetylphenyl ester		1.41
41	23.323	1.5.9-Decatriene. 2.3.5.8-tetramethyl-		2.91
42	23.467	Methyl 18-methylnonadecanoate		0.84
43	23.529	2-(2-Hydroxyphenoxy)-N-(2-methoxyphenyl)acetamide		1.215
44	23.667	(-)-Isolongifolol. methylether		2.445
45	23.973	Eicosanoicacid		4.035
48	24.761	Alloaromadendreneoxide-(1)		1.59
49	25.193	1.3-Cyclopentadiene. 2.3.4.5-tetramethyl-1-(4-pentenyl)-		1.035
52	26	4H-1-Benzopyran-4-one. 5-hydroxy-7-methoxy-2-phenyl-		1.41
53	26.482	Octadecane		1.59
54	26.857	Chrysin		3.84
59	28.521	4H-1-Benzopyran-4-one. 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-		1.5
60	28.677	2-(3-Hydroxy-4-methoxyphenyl)-3.7-dimethoxy-4H-chromen-4-one		1.875
61	29.234	Triacontylacetate		1.32
62	29.403	Octadecane		1.41
63	29.922	Vitamin E		1.32
			Total	82.965

Table 2.

Chemical Composition of the Aqueous Extract of C. ladanifer.

	RT (min)	Name	Area (%)
1	7784	Cyclohexanone, 22,6-trimethyl-	6,9
2	8953	Cyclohexanol, 3,5-dimethyl-	4,4
3	9848	Borneol	5,3
4	11,243	Propanoic acid, 2-methyl-, heptyl ester	6,25
5	11 956	2-Methoxy-4-vinylphenol	10,65
6	13 038	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-	4,4
7	13,67	Aspidospermidin-17-ol, 1-acetyl-19,21-epoxy-15,16-dimethoxy-	6,9
8	14 389	3-Methyl-2-(2-oxopropyl)furan	8,15
9	15 077	2-Butanone, 4-(4-hydroxyphenyl)-	5,95
10	15 209	Benzenepropanol, 4-hydroxy-.alpha.-methyl-, (R)-	5,65
11	15 584	Ethanone, 1-(4,5-dihydro-2-thiazolyl)-	5,3
13	16 141	2-Cyclohexen-1-one, 4-(3-hydroxy-1-butenyl)-35,5-trimethyl-, [R-[R,R-(E)]]-	5,65
15	17 817	3,5-Dihydroxytoluene	9,05
25	28 676	1H-Cycloprop[c]inden-7-ol, octahydro-	4,05
			88.60

Table 3.

Docking Results in the Active Site.

	CYP2E1 (PDB :3LC4)
	glidegscore(kcal/mol)	glideemodel(kcal/mol)	glideenergy(kcal/mol)
Chrysin	−8.571	−30.523	−30.623
1H-Cycloprop[c]inden-7-ol, octahydro-	−7.005	−26.919	−20.012
Naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1S-(1.alpha.,7.alpha.,8a.alpha.)]-	−6.768	−8.219	−8.163
3-Methyl-2-(2-oxopropyl)furan	−6.507	−29.825	−22.951
Pyrrolidine, 1-(1-cyclohexen-1-yl)-	−6.239	−28.604	−22.317
Cyclohexanol, 3,5-dimethyl-	−6.236	−24.997	−19.135
Borneol	−6.194	−17.961	−7.583
Cyclohexanone, 2,2,6-trimethyl-	−6.176	−23.385	−20.178
2-Methoxy-4-vinylphenol	−5.918	−30.399	−22.802
Eicosanoicacid	−5.861	−17.528	−21.543
3,5-Dihydroxytoluene	−5.66	−28.582	−20.908
2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-	−5.632	−16.07	−9.069
2-Cyclohexen-1-one, 4-(3-hydroxy-1-butenyl)-3,5,5-trimethyl-, [R-[R,R-(E)]]-	−5.621	−16.542	−10.296
Benzenepropanol, 4-hydroxy-.alpha.-methyl-, (R)-	−5.606	−33.927	−26.662
2-Butanone, 4-(4-hydroxyphenyl)-	−5.584	−36.502	−27.53
Ethanone, 1-(4,5-dihydro-2-thiazolyl)-	−4.958	−29.582	−22.677
1,5,9-Decatriene, 2,3,5,8-tetramethyl-	−3.136	−28.604	−25.477
Octadecanoicacid	−3.058	−28.879	−30.879
n-Hexadecanoicacid	−2.393	−40.765	−35.811
Hexadecanal	−2.124	−40.094	−39.628
Tetradecanoicacid	−2.023	−36.801	−32.78
Octadecane	−1.506	−33.199	−32.834
Propanoic acid, 2-methyl-, heptyl ester	−1.196	−25.419	−24.876
13-Tetradece-11-yn-1-ol	−0.315	−32.107	−33.82

The chemical composition of the studied extracts showed the presence of interesting compounds mainly Eicosanoic acid, Chrysin, 2-Methoxy-4-vinylphenol and 3-Methyl-2-(2-oxopropyl) furan. The compounds present in each extract act in synergy and are probably responsible of the obtained hepatoprotective activity

Molecular Docking

In our in silico study, Chrysin, 1H-Cycloprop[c]inden-7-ol, octahydro- Naphthalene, 1, 2, 3, 5, 6, 7, 8, 8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-,[1S-(1.alpha.,7.alpha.,8a.alpha.)]-, 1H-Cycloprop[c]inden-7-ol, octahydro-, and 3-Methyl-2-(2-oxopropyl)furan were the most active molecules in the active site of CYP2E1 with a glide score of −8.571, −7.005, −6.768, and −6.73 kcal/mol (Table 3). Figures 6 and 7 show the 2D and 3D viewers of different types of bonds established between the ligands and the active site.

Figure 6.

2D viewer interactivity of ligands with CYP2E1 (PDB :3LC4) active site; (A) chrysin interactivity between quercetin and vanillic acid and caspase-3 (PDB :3GJQ) active site; (B) 1H-cycloprop[c]inden-7-ol, octahydro-; (C) naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1S-(1.alpha.,7.alpha.,8a.alpha.)]-; (D) 3-Methyl-2-(2-oxopropyl)furan.

Figure 7.

Chrysin docking established Pi-Pi stacking bonds with residues PHE 478 and HEM 500 inactive site of CYP2E1, when 3-Methyl-2-(2-oxopropyl) furan established one hydrogen bond with residue ASN 206 and one Pi-Pi stacking bond with residue PHE 298 in same active site (Figure 6A, 7A and Figure 6D, 7D).

Discussion

The normal function of the liver is evaluated by various serum biomarker enzymes. In our study, we evaluated the hepatoprotective activity of C. ladanifer aqueous and methanolic extracts at two doses of 150 and 300 mg/kg. According to EL Kabbaoui et al, these two doses don’t show any sign of acute toxicity.⁶

In order to evaluate the activity of our extracts on liver damage, three groups of mice were prepared, group of negative control (Saline solution), group of positive control (paracetamol at 1000 mg/kg), and the third group (1000 mg/kg paracetamol additionally 150 and 300 mg/kg of both extracts), and different biochemical parameters were analyzed in the mice serum of different groups.

The transaminases levels of AST and ALT present a principal indication of liver dysfunction. In fact, increased levels of AST and ALT cause hypertrophy and injury in liver tissues.^29,30 Our results showed that the level of AST and ALT in mice treated with the paracetamol and both C. ladanifer extracts decreased compared to positive control group; however, the methanolic extract showed at 300 mg/kg revealed some hepatic toxicity. The transaminases activities decrease and return to normal indicates the stabilization of plasma membrane, as well as repair of hepatic tissue damages caused by the paracetamol;^1,31 this result suggests that C. ladanifer extracts protected the liver tissue from paracetamol-induced injury. Our findings are in agreement with El kabbaoui et al, 2017 that showed that aqueous extract of C. ladanifer may exhibit a potential hepatoprotective effect at 500 mg/kg, producing a decrease in the level of ALT.⁶ The measurement of the total cholesterol and TG levels presented another biochemical parameter to investigate the hepatoprotective effect of C. ladanifer extracts. Low and decreased levels of TG and cholesterol usually indicate chronic liver diseases and a reduced liver biosynthesis capacity,.³² Interestingly both aqueous and methanolic extracts at 150 and 300 mg/kg induced an elevation of total cholesterol and TG levels compared to the positive control; these two parameters were restored to normal after treatment with C. ladanifer extracts.

The kidneys are a vital organ, it filters large types of toxins due its high blood flows.³³ The examination urea and creatinine concentration are considered biomarkers of renal damage.^6,34 In our case, urea and creatinine were recovered to normal levels in mice treated with C. ladanifer extracts. The different studied parameters were restored to normal levels in mice serum treated with both paracetamol and C. ladanifer extracts. All of our findings are in agreement with the study of El kabbaoui et al study

The histopathological analysis of liver and kidney recovered from the mice treated with paracetamol alone, and both extracts were identified. The histopathological analysis of liver sections excised from treated with paracetamol 1000 mg/kg alone and with paracetamol along with methanolic extract at 300 mg/kg showed the presence of various serious histopathological alterations including sinus dilation, infiltration of inflammatory cells as well as the necrosis. No significant histological modifications were noted for liver sections recovered from the mice treated with aqueous extract at both doses of 150 and 300 mg/kg and methanolic extract at 150 mg/kg. In the same study of Kabbaoui et al, aqueous extract of C. ladanifer at 500, 700 and 1000 mg/kg showed no remarkable pathological changes. Our findings revealed that aqueous extract at 150 and 300 mg/kg and methanolic extract at 150 mg/kg showed an interesting hepatoprotective activity.⁶ However, the methanolic extract at 300 mg/kg showed a toxicity.

The GC-MS was used to identify the chemical composition of C. ladanifer both extracts, and the results showed the presence of various biomolecules. The compounds found in C. ladanifer extracts such as, chrysin, Vitamin E, Camphor, and borneol are well known for their therapeutic proprieties in alternative medicine and can probably be responsible of the hapatoprotective activity of the extract. It was demonstrated that chrysin exhibited hepatoprotective activity via various molecular and biochemical mechanisms, in liver-induced hepatotoxicity by chemical agents.^35,36 Moreover, Vitamin E has shown in various studies its hepatoprotective effect, it decreased oxidative stress in the liver, tumor necrosis factor, liver fibrosis inflammation, and lipid peroxidation.³⁷ Camphor was also reported for its hepatoprotective effect.³⁸

Cytochrome P450 2E1 (CYP2E1) is an enzyme primarily found in the liver and plays a significant role in drug metabolism and the oxidation of various endogenous and exogenous compounds, including ethanol and certain drugs. Some studies have suggested that in certain circumstances, CYP2E1 might contribute to hepatoprotection. It has been proposed that CYP2E1 may play a role in the detoxification of certain chemicals and drugs that are harmful to the liver. The in silico study showed that some compounds present in C. ladanifer mainly Chrysin, 1H-Cycloprop[c]inden-7-ol, octahydro-, Naphthalene, 1, 2, 3, 5, 6, 7, 8, 8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-,[1S-(1.alpha.,7.alpha.,8a.alpha.)]-,1H Cycloprop [c] inden-7-ol, octahydro-, and 3-Methyl-2-(2-oxopropyl)furan were the most active molecules in the active site of CYP2E1. CYP2E1 can be induced by various substances, including dietary compounds like polyphenols found in fruits and vegetables, which may have antioxidant properties and help counteract the harmful effects of CYP2E1-induced oxidative stress.^24,25

The present study which aims to determine the activity of our extracts on hepatotoxicity induced by paracetamol in mice represents certain limitations. First, the duration of the test in vivo and the animal testing that raises ethical concerns. Second, the metabolism of paracetamol in animals differs from humans, which would affect the applicability of the results to humans. In other words, animal models may not accurately reflect the effect of these same extracts on humans.

Conclusion

C. ladanifer is a medicinal plant widely used in phytotherapy for its therapeutic and pharamcological activities. In our work, we evaluated the hepatoprotective activity of two C. ladanifer extract, the aqueous, and the methanolic. Our findings are encouraging, since this plant is little explored for its hepatoprotective activity. Both extracts showed an interesting hepatoprotective activity in vivo and in silico and in paracetamol liver toxicity-induced mice; however, the methanolic one showed a hepatic toxicity at higher dose. We reported that this plant is a source of various compounds that are probably responsible for the observed activity. Further investigations are needed to deepen the study.

Author's Contributions

Conceptualization, writing the original draft, reviewing and editing: Kaoutar Bouothmany, Fatima ez-zahra Amrati, Mohamed Chebaibi, Yousef A. Bin Jardan, and Hiba-Allah Nafidi. Formal analysis, investigations, funding acquisition, reviewing, and editing: Mohammed Bourhia, El mzibri Mohammed, and Fouad mellouki. Resources, data validation, data curation, and supervision: Dalila Bousta, Fakhreldeen Dabiellil, and Laila Benbacer.

Footnotes

Acknowledgement

The authors would like to extend their sincere appreciation to the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia for funding this work through the project number (RSP2024R457).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia through the project number (RSP2024R457).

Ethical Approval

The Animal Ethics Review Committee at the faculty of Sciences, Fez, Morocco, approved this study under the ethical clearance number 02/2023/LBEAS.

Data Availability

Data will be available upon reasonable request from the authors.

ORCID iDs

Mohammed Bourhia

Fakhreldeen Dabiellil

References

Dixon

Lane

MacPhee

Philips

. Xenobiotic metabolism: the effect of acute kidney injury on non-renal drug clearance and hepatic drug metabolism. IJMS. 2014;15(2):2538-2553. doi:10.3390/ijms15022538

Ali

Sharief

Mohamed

. Hepatoprotective activity of some medicinal plants in Sudan. Evidence-Based Complementary Altern Med. 2019;2019:1-16. doi:10.1155/2019/2196315

Boro

Usha

Babu

, et al. Hepatoprotective activity of the ethanolic extract of Morus indica roots from Indian Bodo tribes. SN Appl Sci. 2022;4(2):49. doi:10.1007/s42452-021-04859-z

Bellakhdar

Claisse

Fleurentin

Younos

. Repertory of standard herbal drugs in the Moroccan pharmacopoea. J Ethnopharmacol. 1991;35(2):123-143. doi:10.1016/0378-8741(91)90064-K

Attaguile

Russo

Campisi

, et al. Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L. Cell Biol Toxicol. 2000;16(2):83-90. doi:10.1023/A:1007633824948

El Kabbaoui

Chda

El-Akhal

, et al. Acute and sub-chronic toxicity studies of the aqueous extract from leaves of Cistus ladaniferus L . in mice and rats. J Ethnopharmacol. 2017;209:147-156. doi:10.1016/j.jep.2017.07.029

Barrajón-Catalán

Fernández-Arroyo

Saura

, et al. Cistaceae aqueous extracts containing ellagitannins show antioxidant and antimicrobial capacity, and cytotoxic activity against human cancer cells. Food Chem Toxicol. 2010;48(8-9):2273-2282. doi:10.1016/j.fct.2010.05.060

Amensour

Sendra

Pérez-Alvarez

Skali-Senhaji

Abrini

Fernández-López

. Antioxidant activity and chemical content of methanol and ethanol extracts from leaves of rockrose (Cistus ladaniferus). Plant Foods Hum Nutr. 2010;65(2):170-178. doi:10.1007/s11130-010-0168-2

Zidane

Elmiz

Aouinti

, et al. Chemical composition and antioxidant activity of essential oil, various organic extracts of Cistus ladanifer and Cistus libanotis growing in Eastern Morocco. Afr J Biotechnol. 2013;12(34):5314-5320. doi:10.5897/AJB2013.12868

10.

Raimundo

Frazão

Domingues

, et al. Neglected Mediterranean plant species are valuable resources: the example of Cistus ladanifer. Planta. 2018;248(6):1351-1364. doi:10.1007/s00425-018-2997-4

11.

El Hamsas El Youbi

El Mansouri

Boukhira

Daoudi

Bousta

. In vivo anti-inflammatory and analgesic effects of aqueous extract of Cistus ladanifer L. From Morocco. Am J Ther. 2016;23(6):e1554-e1559. doi:10.1097/MJT.0000000000000419

12.

El Kabbaoui

Chda

Azdad

, et al. Evaluation of hypoglycemic and hypolipidemic activities of aqueous extract of Cistus ladaniferus in streptozotocin-induced diabetic rats. Asian Pac J Trop Biomed. 2016;6(12):1044-1049. doi:10.1016/j.apjtb.2016.09.005

13.

Bouothmany

Bourhia

Aoussar

, et al. Leaf extracts of Cistus ladanifer exhibit potent antioxidant and antiproliferative activities against liver, prostate and breast cancer cells. Applied Sciences. 2022;12(17):8603. doi:10.3390/app12178603

14.

Gaweł-Bęben

Kukula-Koch

Hoian

Czop

Strzępek-Gomółka

Antosiewicz

. Characterization of Cistus × incanus L. and Cistus ladanifer L. Extracts as potential multifunctional antioxidant ingredients for skin protecting cosmetics. Antioxidants. 2020;9(3):202. doi:10.3390/antiox9030202

15.

Papaefthimiou

Papanikolaou

Falara

Givanoudi

Kostas

Kanellis

. Genus Cistus: a model for exploring labdane-type diterpenes’ biosynthesis and a natural source of high value products with biological, aromatic, and pharmacological properties. Front Chem. 2014;2:1-19. doi:10.3389/fchem.2014.00035

16.

Barros

Dueñas

Alves

, et al. Antifungal activity and detailed chemical characterization of Cistus ladanifer phenolic extracts. Ind Crops Prod. 2013;41:41-45. doi:10.1016/j.indcrop.2012.03.038

17.

Alves-Ferreira

Miranda

Duarte

, et al. Cistus ladanifer as a source of chemicals: structural and chemical characterization. Biomass Conv Bioref. 2020;10(2):325-337. doi:10.1007/s13399-019-00448-8

18.

Fernández-Arroyo

Barrajón-Catalán

Micol

Segura-Carretero

Fernández-Gutiérrez

. High-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic compounds from a Cistus ladanifer aqueous extract: identification of phenolic compounds from Cistus Ladanifer. Phytochem Anal. 2010;21(4):307-313. doi:10.1002/pca.1200

19.

Gülz

Herrmann

Hangst

. Leaf trichomes in the genus Cistus. Flora. 1996;191(1):85-104. doi:10.1016/S0367-2530(17)30692-8

20.

Stępień

Aebisher

Bartusik-Aebisher

. Biological properties of Cistus species. Eur J Clin Exp Med. 2018;16(2):127-132. doi:10.15584/ejcem.2018.2.8

21.

Vitali

Pennisi

Attaguile

Savoca

Tita

. Antiproliferative and cytotoxic activity of extracts from Cistus incanus L. and Cistus monspeliensis L. on human prostate cell lines. Nat Prod Res. 2011;25(3):188-202. doi:10.1080/14786410802583148

22.

Barrajón-Catalán

Fernández-Arroyo

Roldán

, et al. A systematic study of the polyphenolic composition of aqueous extracts deriving from several Cistus genus species: evolutionary relationship: polyphenolic characterization of Cistus aqueous extracts. Phytochem Anal. 2011;22(4):303-312. doi:10.1002/pca.1281

23.

Moussaoui

Bourhia

Jawhari

, et al. Withania frutescens. L extract: phytochemical characterization and acute and repeated dose 28-day oral toxicity studies in mice. BioMed Res Int. 2020;2020:1-7. doi:10.1155/2020/1976298

24.

Costea

Chiţescu

Boscencu

, et al. The polyphenolic profile and antioxidant activity of five vegetal extracts with hepatoprotective potential. Plants. 2022;11(13):1680. doi:10.3390/plants11131680

25.

Bairagi

Ghule

Gilhotra

. Molecular docking, in vitro antioxidant, and in vivo hepatoprotective activity of methanolic extract of Calotropis gigantea leaves in carbon tetrachloride-induced liver injury in rats. CEI. 2022;18(2):110-126. doi:10.2174/1573408018666220511170125

26.

Aboul-Soud

MAM

Ennaji

Kumar

, et al. Antioxidant, anti-proliferative activity and chemical fingerprinting of Centaurea calcitrapa against breast cancer cells and molecular docking of caspase-3. Antioxidants. 2022;11(8):1514. doi:10.3390/antiox11081514

27.

Amrati

FEZ

Elmadbouh

OHM

Chebaibi

, et al. Evaluation of the toxicity of Caralluma europaea (C.E) extracts and their effects on apoptosis and chemoresistance in pancreatic cancer cells. J Biomol Struct Dyn. 2023;41(17):8517-8534. doi:10.1080/07391102.2022.2135595

28.

Kumar

Alfhili

Bari

, et al. Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to caspase-3. Front Cell Dev Biol. 2022;10:972111. doi:10.3389/fcell.2022.972111

29.

Costa-Silva

Lima

Silva

EJR

, et al. Acute and subacute toxicity of the Carapa guianensis Aublet (Meliaceae) seed oil. J Ethnopharmacol. 2008;116(3):495-500. doi:10.1016/j.jep.2007.12.016

30.

Amrati

FEZ

Bourhia

Slighoua

, et al. Protective effect of chemically characterized polyphenol-rich fraction from apteranthes europaea (Guss.). Murb. Subsp. maroccana (Hook.f.) Plowes on carbon tetrachloride-induced liver injury in mice. Appl Sci. 2021;11(2):554. doi:10.3390/app11020554

31.

Ouassou

Bouhrim

Daoudi

, et al. Evaluation of hepatoprotective activity of Caralluma europaea stem extract against CCl4-induced hepatic damage in wistar rats. Chan KW, ed. Adv Pharmacol Pharmaceut Sci. 2021;2021:1-8. doi:10.1155/2021/8883040

32.

Ghadir

Riahin

Havaspour

Nooranipour

Habibinejad

. The relationship between lipid profile and severity of liver damage in cirrhotic patients. Hepat Mon. 2010;10(4):285-288.

33.

Akindele

Adeneye

Salau

Sofidiya

Benebo

. Dose and time-dependent sub-chronic toxicity study of hydroethanolic leaf extract of Flabellaria paniculata Cav. (Malpighiaceae) in rodents. Front Pharmacol. 2014;5:1–11. doi:10.3389/fphar.2014.00078

34.

Nerdy

Ritarwan

. Hepatoprotective activity and nephroprotective activity of peel extract from three varieties of the passion fruit (Passiflora Sp.) in the albino rat. Open Access Maced J Med Sci. 2019;7(4):536-542. doi:10.3889/oamjms.2019.153

35.

Pingili

Pawar

Challa

Kodali

Koppula

Toleti

. A comprehensive review on hepatoprotective and nephroprotective activities of chrysin against various drugs and toxic agents. Chem-Biol Interact. 2019;308:51-60. doi:10.1016/j.cbi.2019.05.010

36.

Hermenean

Mariasiu

Navarro-González

, et al. Hepatoprotective activity of chrysin is mediated through TNF-α in chemically-induced acute liver damage: an in vivo study and molecular modeling. Exp Ther Med. 2017;13(5):1671-1680. doi:10.3892/etm.2017.4181

37.

Adikwu

Brambaifa

. Hepatoprotective effect of vitamin E. Am J Pharmacol Toxicol. 2012;7(4):154-163. doi:10.3844/ajptsp.2012.154.163

38.

Malabadi

Kolkar

Meti

Chalannavar

. Camphor tree, Cinnamomum camphora (L.); ethnobotany and pharmacological updates. Biomedicine. 2021;41(2):181-184. doi:10.51248/.v41i2.779

Hepatoprotective Effects of a Chemically-Characterized Extract from Cistus ladanifer : In Vivo and in Silico Investigations

Abstract

Keywords

Introduction

Material and Methods

Plant Material

Hepatoprotective Activity of C. ladanifer Extracts

Animals

Paracetamol Dose Regimen

Biochemical Parameters

Histopathological Examination

Chemical Composition

Computational Modeling

Protein Preparation

Ligand Preparation

Receptor Grid Generation

Molecular Docking

Statistical Analysis

Results

Serum Biochemical Analysis

Effect of C. ladanifer Extracts on the Transaminases Levels and the Lipidic Profile

Effect of C. ladanifer Extracts on the Urea and Creatinine Levels

Histopathological Alterations

Chemical Composition

Molecular Docking

Discussion

Conclusion

Author's Contributions

Footnotes

Acknowledgement

Declaration of Conflicting Interests

Funding

Ethical Approval

Data Availability

ORCID iDs

References