Kanter M, Coskun O, Budancamanak M. Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats. World J Gastroenterol 2005; 11(42): 6684-6688 [PMID: 16425366 DOI: 10.3748/wjg.v11.i42.6684]
Corresponding Author of This Article
Dr. Mehmet Kanter, Department of Histology and Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey. firstname.lastname@example.org
Article-Type of This Article
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats
Mehmet Kanter, Omer Coskun, Mustafa Budancamanak
Mehmet Kanter, Omer Coskun, Department of Histology and Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey
Mustafa Budancamanak, Department of Rheumatology, Faculty of Medicine, Yuzuncu Yil University, Van, Turkey
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Mehmet Kanter, Department of Histology and Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey. email@example.com
Telephone: +90-284-2357641 Fax: +90-284-2352730
Received: July 23, 2005 Revised: September 25, 2005 Accepted: September 27, 2005 Published online: November 14, 2005
AIM: To investigate the effects of Nigella sativa L (NS) and Urtica dioica L (UD) on lipid peroxidation, antioxidant enzyme systems and liver enzymes in CCl4-treated rats.
METHODS: Fifty-six healthy male Wistar albino rats were used in this study. The rats were randomly allotted into one of the four experimental groups: A (CCl4-only treated), B (CCl4+UD treated), C (CCl4+NS treated) and D (CCl4+UD+NS treated), each containing 14 animals. All groups received CCl4 (0.8 mL/kg of body weight, sc, twice a week for 60 d). In addition, B, C and D groups also received daily i.p. injections of 0.2 mL/kg NS or/and 2 mL/kg UD oils for 60 d. Group A, on the other hand, received only 2 mL/kg normal saline solution for 60 d. Blood samples for the biochemical analysis were taken by cardiac puncture from randomly chosen-seven rats in each treatment group at beginning and on the 60th d of the experiment.
RESULTS: The CCl4 treatment for 60 d increased the lipid peroxidation and liver enzymes, and also decreased the antioxidant enzyme levels. NS or UD treatment (alone or combination) for 60 d decreased the elevated lipid peroxidation and liver enzyme levels and also increased the reduced antioxidant enzyme levels. The weight of rats decreased in group A, and increased in groups B, C and D.
CONCLUSION: NS and UD decrease the lipid per-oxidation and liver enzymes, and increase the anti-oxidant defense system activity in the CCl4-treated rats.
Citation: Kanter M, Coskun O, Budancamanak M. Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats. World J Gastroenterol 2005; 11(42): 6684-6688
Carbon tetrachloride (CCl4) is one of the oldest and most widely used toxins for experimental induction of liver fibrosis in laboratory animals. This model has been used in various studies on examined the deposition of extracellular matrix in the fibrotic and cirrhotic liver[2,3].
CCl4 is a selective hepatotoxic chemical agent. CCl4-induced reactive free radicals initiate cell damage through two different mechanisms of covalent binding to the membrane proteins and cause lipid peroxidation. A number of investigators have utilized this chemical to produce liver cirrhosis in experimental animals. Production of reactive oxygen species and lipid peroxidation induced by iron overload, cholestatic injury and intoxication by ethanol and CCl4 is associated with liver fibrosis and cirrhosis. These effects are partially prevented by antioxidant compounds including α-tocopherol[4,8], silymarin and salvianolic acid.
The seed of Nigella sativa L (NS), an annual Ranun-culaceae herbaceous plant, has been used traditionally for centuries in the Middle East, Northern Africa, Far East and Asia for the treatment of asthma. NS contains more than 30 of a fixed oil and 0.40-0.45 w/w of a volatile oil. The volatile oil has been shown to contain 18.4-24% thymoquinone and 46% many monoterpenes such as p-cymene, and α-pinene. Recently conducted clinical and experimental researches have shown many therapeutic effects of NS extracts such as immunomodulator, anti-inflammatory and anti-tumour agents.
Urtica dioica L (UD) is a plant belonging to the plant family Urticaceae. Its seeds are widely used in folk medicine in many parts of Turkey, especially in the therapy of advanced cancer patients. Polar extract of the UD contains lignans (+)-neoolivil, (-)-secoisolariciresinol, dehy-drodiconiferyl alcohol, isolariciresinol, pinoresinol, and 3,4-divanillyltetrahydrofuran, and has antiinflammatory effects and stimulates the proliferation of human lymphocytes.
The present study aimed to investigate the preventive effects of NS and UD on lipid peroxidation, antioxidant enzyme systems and some liver enzymes in CCl4-treated rats.
MATERIALS AND METHODS
Plant materials and extraction procedure
The NS and UD seeds were purchased from a local herb store, Zonguldak, Turkey. Voucher specimens were kept at the Department of Biochemistry, Zonguldak Karaelmas University, Zonguldak, Turkey for the future reference. The seeds of NS were powdered in a mixer, placed in a distillation flask and the volatile oil with 0.2 % yield was collected by a steam distillation. The fixed oil of UD was extracted with the help of a rotary evaporator using diethyl ether as solvent.
Treatment of rats
Fifty-six male Wistar albino rats, weighing 150-200 g, averaging 16 wk old, were used in this study. The rats were randomly allotted into one of the four experimental groups: A (CCl4-only treated), B (CCl4+UD treated), C (CCl4+NS treated) and D (CCl4+UD+NS treated), each containing 14 animals. All groups received CCl4 (Merck; 153.82 g/moL, 1.59 kg, Germany, 0.8 mL/kg of body weight, sc, twice a week for 60 d). In addition, B, C and D groups also received the daily ip injection of 0.2 mL/kg NS or/and 2 mL/kg UD oils for 60 d. Group A, on the other hand, received only 2 mL/kg normal saline solution for 60 d. The animals were housed in macrolon cages under standard laboratory conditions (light period 7.00 a.m. to 7.00 p.m., 21±1 oC, rat chow and tap water freely available). All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. The experiment lasted for 60 d.
Blood samples for the biochemical analysis were taken from each treatment group at beginning and on the 60th d of the experiment. Rats from which blood samples were taken were excluded from the experiment to eliminate the haemorrhage- and stress-induced complications. At the end of the experiment, rats in all groups were starved overnight, and sacrificed under chloralhydrate (6 mL of 7% chloralhydrate/kg, Sigma, St. Louis, MO, USA) anaesthesia. Blood samples were collected by cardiac puncture using heparinised syringes. Leukocytes and plasma components were seperated by centrifugation of the blood. Erythrocytes were washed three times with 0.9% NaCl solution and packed, and then stored at -70 °C until study.
Blood MDA (mmol/L) was determined by the double heating method of Draper and Hadley. The principle of the method is spectrophotometric measurement of the colour produced during the reaction to thiobarbituric acid (TBA) with MDA. For this purpose, 2.5 mL of 100 g/L trichloroacetic acid solution was added to 0.5 mL erythrocytes in each centrifuge tube and placed in a boiling water bath for 15 min. After cooled in tap water, the mixture was centrifuged at 1 000 r/min for 10 min, and 2 mL of the supernatant was added to 1 mL of 6.7 g/L TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured using a Shimadzu UV-1601 (Japan) spectrophotometer at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA-TBA complex 1.56×105 /cm, and expressed in µmol/g Hb erythrocytes and µmol/g tissue protein.
Blood GSH concentration was measured by the method described by Beutler et al. Briefly, 200 μL of whole blood was added to 1.8 mL of distilled water. Three ml of the precipitating solution was mixed with the hemolysate. The mixture was allowed to stand for approximately 5 min and then filtered. Two milliliters of filtrate were taken and added into another tube, and then, 8 mL of the phosphate solution and 1 mL of the DTNB [5,5’dithiobis-(2-nitrobenzoic acid)] were added. A blank was prepared with 8 mL of the phosphate solution, 2 mL of the diluted precipitating solution (three parts to two parts of the distilled water), and 1 mL of the DTNB reagent. A standard solution of glutathione was prepared (40 mg/100 mL). The optical density was measured at 412 nm with a spectrophotometer.
Serum ceruloplasmin p-phenylenediamine (PPD) oxidase activity was measured according to Sunderman and Nomoto. At pH 5.4, ceruloplasmin catalyze the oxidation of PPD to yield a colored product. The rate of formation of the colored oxidation product was proportional to the concentration of serum ceruloplasmin if a correction was made for nonenzymatic oxidation of PPD.
Vitamin E was analyzed colorimetrically with 2,4,6-tripridyl-s-triazin and FeCl3 after the extraction with absolute ethanol and xylene. Serum vitamin C level was determined after derivatisation with 2,4-dinitrophenylhydrazine. The levels of β-carotene at 425 nm and retinol at 325 nm were detected after the reaction of serum : ethanol : hexane at the ratio of 1 : 1 : 3 respectively.
Alanine aminotransferase (ALT), aspartate amino-transferase (AST) and alkaline phosphatase (ALP) levels were determined by an autoanalyzer (Roche-Hittachi, Japan) using commercial kits (Roche, Basel, Switzerland).
The data were expressed as mean±SD and analysed by repeated measures of variance. Tukey test was used to test for differences among means when ANOVA indicated a significant (P≤0.05) F ratio.
The levels of blood MDA, antioxidants of all groups and the levels of serum liver enzymes are shown in Table 1, Table 2, Table 3, respectively. The CCl4 treatment for 60 d significantly (P<0.05) increased the MDA and liver enzymes, and also decreased (P<0.05) the antioxidant levels. NS or UD treatment (alone or in combination) for 60 d significantly (P<0.05) decreased the elevated MDA and liver enzyme levels and also increased (P<0.05) the reduced antioxidant levels. The weight of rats decreased (P<0.05) in group A, and increased (P<0.05) in groups B, C, and D (Table 4).
Table 4 Weights (g) of rats in CCl4+NaCl-treated (A), CCl4+UD treated (B), CCl4+NS treated (C) and CCl4+UD+NS treated (D) rats (mean±SD).
Treatment of animals with CCl4 is known to cause severe hepatic injury. In our study, we showed that repeated CCl4 treatment for 60 d increased the lipid peroxidation and liver enzymes, and also decreased the antioxidant enzyme levels. It has been suggested that the lipid peroxidation may be a link between tissue injury and liver fibrosis by modulating collagen gene expression. It was reported that CCl4 is suitable to induce lipid peroxidation in experimental animals within a few minutes after administration and its long-term use results in liver fibrosis and cirrhosis by lipid peroxidation pathway. It is generally thought that CCl4 toxicity is due to reactive free radical (CCl3.), which is generated by its reductive metabolism by hepatic cytochrome P450. The reactive intermediate is believed to cause lipid peroxidation and breakdown of cellular membranes.
Recent experimental studies have investigated the role of antioxidative vitamins, minerals, drugs and plant-derived compounds in the prevention and therapy of liver fibrosis. Parola et al showed that an increased liver content of vitamin E leads to a significant degree of protection against carbon tetrachloride-induced chronic liver damage and cirrhosis in rats. Ianas et al have described the all-round beneficial action of a selenium preparation upon the organism in rats exposed to CCl4 as well as a strong antioxidative effect, confirming the essential role of selenium in maintaining cellular integrity.
Several plant derived compounds such as colchicine (Colchicum dispert), silymarin (Slybum marianum), polyenylphosphatidyl choline (soy bean), ellagic acid (cruciferous vegetables), Gingko biloba composita and recently Sho-saiko-to (extract of seven herbs in Chinese folk medicine) have been proposed as antioxidants and antifibrotics in the treatment of chronic liver disease[28-30]. The antioxidative and hepatoprotective effects of chitosan against CCl4-induced liver toxicity in rats have been under investigation by measuring thiobarbituric acid reactive substances (TBARS) and antioxidant enzyme activities. The antioxidant systems such as antioxidant vitamins (A, C, and E), superoxide dismutase (SOD), catalase, glutathione (GSH), ceruloplasmin and glutathione peroxidase (GSH-Px) protect the cells against lipid peroxidation, which is the base of many pathologic processes [32,33].
In our study, we found that NS or UD treatment (alone or combination) for 60 d decreased the elevated MDA and liver enzyme levels and also increased the reduced antioxidant enzyme levels in CCl4-treated rats. Previously performed clinical and experimental investigations have shown that NS has a protective effect against oxidative damage in isolated rat hepatocytes. It was found that the fixed oil of NS has both antioxidant and anti-eicosanoid effects greater than thymoquinone which is its active constituent. Furthermore, NS has antioxidant activity by suppressing the chemiluminescence in phagocytes.
Recently, Turkdogan et al observed that NS has a significant hepatoprotective effect in CCl4-administrated rabbits, ans that hepatocellular degenerative and necrotic changes are slight without advanced fibrosis and cirrhotic process in NS-treated group. However, Turkdogan et al found that NS can prevent liver fibrosis and cirrhosis, suggesting that NS protects liver against fibrosis possibly through immunomodulator and antioxidant activities.
There are no comprehensive studies on the therapeutic effects of UD. Only one study reported that UD extract can inhibit in vitro prostate cancer cell proliferation. It has been suggested that the extract of UD is effective in inducing glutathione S-transferase, SOD and catalase activity in the forestomach and SOD and CAT activity in the lung at both dose levels. However, Turkdogan et al showed that NS and UD can significantly prevent CCl4-induced hepatotoxicity in rats. Our biochemical results demonstrated that NS and UD treatment prevented CCl4-induced hepatotoxicity in rats by decreasing the lipid peroxidation and increasing the antioxidant defense system activity. However, Kanter et al also showed the NS and UD increase the antioxidant defense system activity in experimentally CCl4-treated rats.
In conclusion, NS and UD decrease lipid peroxidation and liver enzymes, and increase antioxidant defense system activity in the CCl4-treated rats. They also prevent weight lose induced by the CCl4 treatment. Further studies are required to evaluate the possible hepatoprotective effect of NS and UD which are traditionally used as a medicine for many complaints including liver diseases.
Science Editor Wang XL and Guo SY Language Editor Elsevier HK
Tsukamoto H, Matsuoka M, French SW. Experimental models of hepatic fibrosis: a review.Semin Liver Dis. 1990;10:56-65.
Hernandez-Munoz R, Diaz-Munoz M, Chagoya de Sanchez V. Possible role of cell redox state on collagen metabolism in carbon tetrachloride-induced cirrhosis as evidenced by adenosine administration to rats.Biochim Biophys Acta. 1994;1200:93-99.
Muriel P. Nitric oxide protection of rat liver from lipid peroxidation, collagen accumulation, and liver damage induced by carbon tetrachloride.Biochemical Pharmacology. 1998;56:773-779.
Parola M, Leonarduzzi G, Biasi F, Albano E, Biocca ME, Poli G, Dianzani MU. Vitamin E dietary supplementation protects against carbon tetrachloride-induced chronic liver damage and cirrhosis.Hepatology. 1992;16:1014-1021.
Bacon BR, Britton RS. The pathology of hepatic iron overload: a free radical--mediated process?Hepatology. 1990;11:127-137.
Parola M, Leonarduzzi G, Robino G, Albano E, Poli G, Dianzani MU. On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis.Free Radic Biol Med. 1996;20:351-359.
Kamimura S, Gaal K, Britton RS, Bacon BR, Triadafilopoulos G, Tsukamoto H. Increased 4-hydroxynonenal levels in experimental alcoholic liver disease: association of lipid peroxidation with liver fibrogenesis.Hepatology. 1992;16:448-453.
Halim AB, el-Ahmady O, Hassab-Allah S, Abdel-Galil F, Hafez Y, Darwish A. Biochemical effect of antioxidants on lipids and liver function in experimentally-induced liver damage.Ann Clin Biochem. 1997;34:656-663.
Mourelle M, Muriel P, Favari L, Franco T. Prevention of CCL4-induced liver cirrhosis by silymarin.Fundam Clin Pharmacol. 1989;3:183-191.
Hu YY, Liu P, Liu C, Xu LM, Liu CH, Zhu DY, Huang MF. Actions of salvianolic acid A on CCl4-poisoned liver injury and fibrosis in rats.Zhongguo YaoLi XueBao. 1997;18:478-480.
el Tahir KE, Ashour MM, al-Harbi MM. The respiratory effects of the volatile oil of the black seed (Nigella sativa) in guinea-pigs: elucidation of the mechanism(s) of action.Gen Pharmacol. 1993;24:1115-1122.
El-Kadi A, Kandil O. The black seed (Nigella sativa) and immunity: its effect on human T cell subset.Fed Proc. 1987;46:1222.
Houghton PJ, Zarka R, de las Heras B, Hoult JR. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation.Planta Med. 1995;61:33-36.
el Daly ES. Protective effect of cysteine and vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in rats.J Pharm Belg. 1998;53:87-93; discussion 93-5.
Riehemann K, Behnke B, Schulze-Osthoff K. Plant extracts from stinging nettle (Urtica dioica), an antirheumatic remedy, inhibit the proinflammatory transcription factor NF-kappaB.FEBS Lett. 1999;442:89-94.
Wagner H, Willer F, Kreher B. [Biologically active compounds from the aqueous extract of Urtica dioica].Planta Med. 1989;55:452-454.
Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation.Methods Enzymol. 1990;186:421-431.
Buetler E, Dubon O, Kelly BM. Improved method for the determination of blood glutathione.J Lab Clin Med. 1963;61:882-888.
Sunderman FW, Nomoto S. Measurement of human serum ceruloplasmin by its p-phenylenediamine oxidase activity.Clin Chem. 1970;16:903-910.
Martinek RG. Method for the determination of vjitamin E (total tocopherols) in serum.Clin Chem. 1964;10:1078-1086.
Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids.Methods Enzymol. 1979;62:3-11.
Suzuki J, Katoh N. A simple and cheap methods for measuring serum vitamin A in cattle using only a spectrophotometer.Nihon Juigaku Zasshi. 1990;52:1281-1283.
Terblanche J, Hickman R. Animal models of fulminant hepatic failure.Dig Dis Sci. 1991;36:770-774.
Parola M, Pinzani M, Casini A, Albano E, Poli G, Gentilini A, Gentilini P, Dianzani MU. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells.Biochem Biophys Res Commun. 1993;194:1044-1050.
Sherlock S. [Drugs and the liver].G Clin Med. 1970;51:753-759.
De Groot H, Sies H. Cytochrome P-450, reductive metabolism, and cell injury.Drug Metab Rev. 1989;20:275-284.
Ianăş O, Olinescu R, Bădescu I, Simionescu L, Popovici D. The influence of "selenium organicum" upon the hepatic function of carbon tetrachloride poisoned rats.Rom J Intern Med. 1995;33:113-120.
Seitz HK, Pöschl G. Antioxidant drugs and colchicine in the treatment of alcoholic liver disease. In: Arroyo V, Bosch J, Rodes J. Treatments in Hepatology. First ed.Barcelone Masson. 1995;271-276.
Schuppan D, Hahn EG. Clinical studies with silymarin: fibrosis progression is the end point.Hepatology. 2001;33:483-484.
Shimizu I. Sho-saiko-to: Japanese herbal medicine for protection against hepatic fibrosis and carcinoma.J Gastroenterol Hepatol. 2000;15 Suppl:D84-D90.
Jeon TI, Hwang SG, Park NG, Jung YR, Shin SI, Choi SD, Park DK. Antioxidative effect of chitosan on chronic carbon tetrachloride induced hepatic injury in rats.Toxicology. 2003;187:67-73.
Williams RJ. Zinc: what is its role in biology?Endeavour. 1984;8:65-70.
Bray TM, Bettger WJ. The physiological role of zinc as an antioxidant.Free Radic Biol Med. 1990;8:281-291.
Daba MH, Abdel-Rahman MS. Hepatoprotective activity of thymoquinone in isolated rat hepatocytes.Toxicol Lett. 1998;95:23-29.
Haq A, Abdullatif M, Lobo PI, Khabar KS, Sheth KV, al-Sedairy ST. Nigella sativa: effect on human lymphocytes and polymorphonuclear leukocyte phagocytic activity.Immunopharmacology. 1995;30:147-155.
Türkdoğan MK, Ozbek H, Yener Z, Tuncer I, Uygan I, Ceylan E. The role of Urtica dioica and Nigella sativa in the prevention of carbon tetrachloride-induced hepatotoxicity in rats.Phytother Res. 2003;17:942-946.
Türkdoğan MK, Ağaoğlu Z, Yener Z, Sekeroğlu R, Akkan HA, Avci ME. The role of antioxidant vitamins (C and E), selenium and Nigella sativa in the prevention of liver fibrosis and cirrhosis in rabbits: new hopes.Dtsch Tierarztl Wochenschr. 2001;108:71-73.
Konrad L, Müller HH, Lenz C, Laubinger H, Aumüller G, Lichius JJ. Antiproliferative effect on human prostate cancer cells by a stinging nettle root (Urtica dioica) extract.Planta Med. 2000;66:44-47.
Ozen T, Korkmaz H. Modulatory effect of Urtica dioica L. (Urticaceae) leaf extract on biotransformation enzyme systems, antioxidant enzymes, lactate dehydrogenase and lipid peroxidation in mice.Phytomedicine. 2003;10:405-415.
Kanter M, Meral I, Dede S, Gunduz H, Cemek M, Ozbek H, Uygan I. Effects of Nigella sativa L. and Urtica dioica L. on lipid peroxidation, antioxidant enzyme systems and some liver enzymes in CCl4-treated rats.J Vet Med A Physiol Pathol Clin Med. 2003;50:264-268.