Advanced
Protective Effect of Korean Medicinal Plants on Ethanol-Induced Cytotoxicity in HepG2 Cells
Protective Effect of Korean Medicinal Plants on Ethanol-Induced Cytotoxicity in HepG2 Cells
Natural Product Sciences. 2013. Dec, 19(4): 329-336
Copyright © 2013, The Korean Society of Pharmacognosy
  • Received : August 22, 2013
  • Accepted : November 02, 2013
  • Published : December 31, 2013
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Eun Jeong Song
Nam Yee Kim
Moon Young Heo
myheo@kangwon.ac.kr

Abstract
The purpose of this study is to evaluate cytoprotective effect of Korean medicinal plants on alcoholinduced cytotoxicity in liver cells. Out of the 120 plant extracts tested in this study, 53 plant extracts enhanced alcohol-induced cytotoxicity in liver cells by 50~80%, while other 11 plant extracts including Crataegus pinnatifida reduced cytotoxicity by 1~68%. The results of DPPH free radical test and LDL lipid peroxidation test on the plant extracts that sharply reduced cytotoxicity in liver cells shows that Crataegus pinnatifida and Cinnamomum cassia had antioxidative effect. This study reports that the plant extracts that enhance or reduce ethanol-induced cytotoxicity in liver cells can be research objects as cytotoxic plants or cytotoxicity-protective plants.
Keywords
Introduction
Liver diseases including alcoholic steasis, viral hepatitis, cirrhosis and liver cancer are prevalent in about 10% of the world population (Bondy, 1992 ; Cederbaum, 2001 ; Mishra ., 2011) . This has triggered a lot of studies for the application of plant-originated natural substances, which has relatively low side effects, to liver diseases. At present, about 50% of medicines for liver diseases used for clinical purposes are natural substances or derivatives of natural substances (Paterson and Anderson, 2005 ; Teuten ., 2005 ; Rollinger ., 2006) .
So far, silymarin contained in milk thistle ( Silybum marianum ) (Raškovic ., 2011) , glycyrrhizin contained in licorice ( Glycyrriza glabra ) (Ashfaq ., 2011) and wogonin contained in Scutellaria baicalensis (Guo ., 2007) has been reported as medicines for chronic viral hepatitis as well as curcumin contained in Curcuma longa (Rivera-Espinoza and Muriel, 2009) for carbon tetrachlorideinduced cytotoxicity, reveratrol contained grapes (Rajasekaran ., 2011) for prevention of liver cancer and rhein for hepatic fibrosis (Guo ., 2002) . Out of these substances, silymarin and naringenin are especially well-known for their inhibitory effect on alcohol-induced cytotoxicity in liver cells (Zhang ., 2013) . Since these cytotoxicity-protective compounds have antioxidative, antiviral and anticarcinogenic effects, they are expected to contribute to developing clinically significant medicines for liver diseases in the future (Ikeda ., 2006 ; Ghosh ., 2011) .
There have been a lot of studies on plant extracts or compounds that have protective effect of substances such as CCl 4 , galactosamine and paracetamol on cytotoxicity in liver cells (Girish ., 2012) . Nevertheless, there have been only a handful of reports on natural substances that have preventive effect on ethanol-induced hepatotoxicity (Kuma and Ali, 2000 ; Arteel ., 2002 ; McKim ., 2002) . Therefore, this study has found plant extracts that have protective effect on ethanol-induced cytotoxicity in liver cells and provided the basic information on extracting alcoholic liver disease-preventive substances in the future based on the result of screening of bioactivity of 120 plant extracts. This study also reports the plant extracts that enhance ethanol-induced cytotoxicity in liver cells.
Experimental
General – 120 plant materials were obtained from the Korean herbal medicine market in Chuncheon, South Korea and each specimen was deposited in the herbarium of College of Pharmacy, Kangwon National University. Each dried plant material (100 g) was soaked in 300 mL of 80% methanol aquous solution at room temperature for 7 days. After filtration, the methanolic filtrate was evaporated to dryness under vacuum. These extracts without further purification were used to study their effect on cytoxicity, lipid peroxidation and scavenging activity of free radical. Materials such as methyl alcohol, ethyl alcohol, acetaminophen, vitamin C (vit-C), low-density lipoprotein (LDL) and dimetyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (MO, USA). Each specimen for free radical scavenging and antioxidation tests was dissolved in DMSO and final concentration of DMSO was adjusted to 1% (v/v) and the same amount of DMSO was used in the control experiment.
Cell culture method – The cell line used for this experiment was HepG2 cell (HB-8065™) purchased from ATCC and stored in a liquid nitrogen tank of College of Pharmacy, Kangwon National University. This cell was cultured according to the experimental purpose through frequent subcultures. For cell culture, MEM medium that contains 10% FBS, 1% L-glutamine, 1% penicillinstreptomycin. Most reagents used for cell culture were purchased from GIBCO (Life Technologies, CA, USA).
Ethanol-induced cytotoxicity and cytoprotective effect test – To evaluate cytotoxicity and cytoprotective effect, the cytotoxicity of ethanol in HepG2 cells was measured by using a microplate reader (Cole, 1986) . 25,000 cells were put respectively in each well. After the culture in the medium 80~90 μl in a CO 2 incubator for 24 hours, ethanol 10 μl or the mixture of ethanol 10 μl and plant extract 10 μl was added to make 100 μl in total. Final concentration of plant extract was 0.4% in the culture with 1.3% ethanol. After the culture for 20 hours, MTT reagent 15 μl was added. After the culture for 4 hours, the medium was removed. After dissolution in DMSO 200 μl, the absorbance was measured at 570 nm. Vitamin C and acetaminophen were used under the same condition as the substances for comparison of activities. The cytoprotective effect (%) of plant extract was calculated by substracting the viability (%) of control from the viability (%) of treatment.
Free radical scavenging assay – DPPH method was used to test free radical scavenging ability of a plant extract, and vitamin C was used as the substance for comparison of activities. DPPH (1,1-Diphenyl-2-picryl hydrazil) contains radicals in molecules, which make a stable complex in combination with other free radicals. After adding the test solution 2 ml of the prescribed concentration, which was made by dissolving the extract in DMSO, in DPPH 2 ml of final concentration 60 μmol, mixing it for 5 minutes and leaving it for 20 minutes, the absorbance was measured at 520 nm (Fugita ., 1988) . Final concentration of plant extract was 150 μg/ml, and vitamin C was 7.5 μg/ml in the reaction mixture.
Antioxidative effect test by oxidized LDL – Antioxidative effect of oxidized LDL generated by lipid peroxidation by using Thiobarbituric acid (TBA) method (Ohtka ., 1979) . In the test group, LDL (2 mg/ml) 10 μl, 150 mM sodium chloride 470 μl and 100 μM copper sulfate 10 μl were added in the test solution 10 μl, which was made of the plant extract of the prescribed concentration. vitamin C was used as the substance for comparison of activities. Final concentration of plant extract was 150 μg/ml, and vitamin C was 7.5 μg/ml in the reacting solution. In the negative control group, DMSO 10 μl was added instead of the test solution. In the blank test group, distilled water 10 μl was added instead of the test solution. All the above solutions were made to be 500 μl. The reacting solutions were mixed immediately with a vortex mixer and cultured in a 37 ℃ incubator for 3 hours. After putting each reacting solution 400 μl respectively in a 15 ml tube, stopping oxidation by dividing 4% BHT ethanol solution 25 μl, adding 10% phosphotungstic acid 500 μl and 0.7% thiobarbituric acid 250 μl dissolved in 0.5 M sulfuric acid, they were heated in a 95 ℃ water bath for 50 minutes. After cooling in running water, vortex for 5 minutes by putting n-butanol 500 μl and centrifugation at 4,000 rpm for 15 minutes, the supernatant was measured at 535 nm.
Statistics process – Most experiments were conducted 3 times, and a significance test was conducted for acquired data by using Student’s t-test.
Results
Effect of herbal medicine extracts on ethanolinduced cytotoxicity – As shown in Table 1 below, the activities of 120 herbal medicines in HepG2 cells were investigated to find natural substances that reduce or enhance alcohol-induced cytotoxicity in liver cells. The cell viability in 0.4% test solution added in 1.3% ethanol was different from the cell viability in 1.3% ethanol, which is the final concentration of the plant extracts. It has been confirmed that if (+) value gets higher, alcoholinduced cytotoxicity in liver cells is reduced more, while if (–) value gets higher, alcohol-induced cytotoxicity in liver cells is enhanced more. As shown in Table 1 below, out of the 120 investigated herbal medicines, 109 extracts, which compose of 91% of the total specimens, enhanced alcohol-induced cytotoxicity in liver cells by up to 79.7%. Among these, the plant extracts that enhanced cytotoxicity by over 75% are Acanthopanax sessiliflorus , Polygala tenuifolia , Panax notoginseng , Chenastragalus membra-naceus , Alisma orilutale . Anemarrhena asphodeloides , Gentiana triflora l and Achyranthes japonica i. Especially, Dimocarpus longan , Schisandra chinensis , Achyranthes japonica and Prunus mume enhanced cytotoxicity by over 80%.
Effects of Korean indigenous plant extracts on ethanol-induced cytotoxicity in HepG2 cells
PPT Slide
Lager Image
# 0.4% extract in 1.3% ethanol at final concentration, ## Cell viability of 1.3% ethanol as positive control was 84.3 ± 2.3%, ### Cytoprotective effect (B) = A . 84.3, *P < 0.05 **p < 0.01 : 1.3% ethanol vs sample treated (Student's t-test)
On the contrary, Crataegus pinnatifida , Cinnamomum cassia , Pinus koraiensis , Fagopyrum esculentum , Asparagus cochinchinensis , Alpinia officinarum , Areca cathechu , Elsholtzia ciliata , Pueraria lobata , Plantago asiatica and Quisqualis indica reduced ethanol-induced cytotoxicity. Among these, Crataegus pinnatifida and Cinnamomum cassia reduced cytotoxicity by 68.2% and 53.3% respectively. vitamin C and Trolox used for substances for comparison of activities reduced cytotoxicity in liver cells, while acetaminophen, which is a cytotoxic substance, enhanced cytotoxicity in liver cells. Among the investigated specimens, most plant extracts enhanced ethanol-induced cytotoxicity in liver cells, while a handful of plant extracts reduced ethanol-induced cytotoxicity in liver cells.
Antioxidative effect of Crataegus pinnatifida and Cinnamomum cassia Table 2 and 3 show DPPH free radical scavenging effect and LDL antioxidative effect of Crataegus pinnatifida and Cinnamomum cassia extracts that have high protective effect on ethanol-induced cytotoxicity. The final concentration of Crataegus pinnatifida and Cinnamomum cassia extracts was 150 μg/ml. Table 2 shows that all of the two extracts have significant free radical scavenging effect (p < 0.01). vitamin C used as the substance for comparison of activities showed significant scavenging effect under similar level to the extracts in 7.5 μg/ml concentration. Table 3 shows that like DPPH free radical test, the final concentration of Crataegus pinnatifida and Cinnamomum cassia extracts was 150 μg/ml. Crataegus pinnatifida and Cinnamomum cassia had significant inhibitory effect (p < 0.01), showing as strong antioxidative effect as vitamin C (7.5 μg/ml) used as the substance for comparison of activities.
Free radical scavenging effect ofCrataegus pinnatifidaandCinnamomum cassia
PPT Slide
Lager Image
Free radical scavenging effect of Crataegus pinnatifida and Cinnamomum cassia
LDL oxidation inhibition ofCrataegus pinnatifidaandCinnamomum cassia
PPT Slide
Lager Image
LDL oxidation inhibition of Crataegus pinnatifida and Cinnamomum cassia
PPT Slide
Lager Image
Inhibition of DPPH radical scavenging and LDL oxidation of Crataegus pinnatifida (CP) and Cinnamomum cassia (CC). **P < 0.01, % inh = (positive – treated) / positive × 100
Fig. 1 shows the comparison between the antioxidative effect of Crataegus pinnatifida (150 μg/ml) and Cinnamomum cassia extracts (150 μg/ml) and that of Vitamin C (7.5 μg/ml). DPPH free radical scavenging effect was Cinnamomum cassia 50.2% > vitamin C 35.9% > Crataegus pinnatifida 29.7%. LDL antioxidative effect was Crataegus pinnatifida 94.8% > vitamin C 80.3% > Cinnamomum cassia 65.0%.
Discussion
Ethanol is a chemical substance that induces fatty liver, liver cirrhosis or liver cancer in human bodies. Therefore, it is required to find a proper pharmaceutically active material that reduces ethanol-induced cytotoxicity in liver cells. This study aimed at finding Korean plant extracts that have protective effect on ethanol-induced cytotoxicity in liver cells.
Table 1 shows that as a result of adding 0.4% plant extract in 1.3% ethanol, 109 out of the 120 plant extracts enhanced ethanol-induced cytotoxicity in liver cells, while only 11 reduced cytotoxicity. Therefore, it has been confirmed that most plant extracts enhance ethanolinduced cytotoxicity in liver cells. Acetaminophen, which is a cytotoxic substance used for the experiments of this study also enhanced ethanol-induced cytotoxicity in liver cells. Therefore, it is considered that when intaking ethanol-containing substances (including plant extracts, nutritive substances and medicines), alcohol-induced cytotoxicity is likely to be enhanced. Especially, it is required to study the synergism of ethanol-induced cytotoxicity incurred by all kinds of plant components intaken as liqueur. It is also worth to pay attention to some plant extracts that reduce ethanol-induced cytotoxicity as protective agents against cytotoxicity in live cells. Considering that antioxidants such as vitamin C reduce ethanol-induced cytotoxicity, it is expected that antioxidative components contained in these extracts have inhibitory effect on cytotoxicity. It is reported that polyphenol extract of cocoa or green tea has inhibitory effect on ethanol-induced liver damage (Arteel ., 2002 ; McKim ., 2002) .
Cytotoxicity of ethanol is related to acetaldehyde or ROS generated by catalystic activities of alcohol dehydrogenase and microsomal cytochrome P450 involved in the metabolism of ethanol [Arteel, 2003] . Alcohol, as a strong CYP2E1 inducing agent, generates superoxide radical and hydrogen peroxide in metabolic process, and is oxidized into acetaldehyde, generating free radicals (Arteel, 2008) . These radicals causes lipid peroxidation and single strand breaks of DNAs along with various types of genetic toxicity including cytotoxicity and adduct formation (Navasumrit ., 2001) .
HepG2 cells used in this study are metabolizing cells subcultured in human liver cancer cells (Todaro, 1963 ; Westerink ., 2007) . Not quite sure but it is considered that the enhancement of ethanol-induced cytotoxicity in these cells was caused by the activities of plant extracts in liver cells, which caused the induction of CYP2E1 or the generation of active oxygen in metabolic process or otherwise by the inhibition of activation of antioxidant enzyme such as catalase and SOD of HepG2 cells.
Since long before, oriental societies where natural plants have been recoginized as relatively nontoxic components have used them as not only foods but also folk medicines and health functional foods. Indeed, there have been a lot of reports on the side effects of plants themselves or metabolites such as cytotoxicity in live cells (Bunchorntavakul ., 2013) . In the aspect of clinical medicine, it is reported that the intake of plant extracts such as Polygonum multiflorum induce hepatitis (Furukawa ., 2010) . In this study, Polygonum multiflorum was also a plant extract that strongly enhances ethanol-induced cytotoxicity by 74% ( Table 1 ).
Meanwhile, only 11 plant extracts including Crataegus pinnatifida have been confirmed to reduce ethanolinduced cytotoxicity. Among these, Crataegus pinnatifida and Cinnamomum cassia had the best inhibitory effect. Vitamin C used as the substance for comparison of activities also reduced cytotoxicity. As a result of DPPH free radical test ( Table 2 ) and LDL oxidation test ( Table 3 ) on Crataegus pinnatifida and Cinnamomum cassia that reduced cytotoxicity most, their inhibitory effect has been confirmed. Vitamin C used as the substance for comparison of activities also had antioxidative effect in a low concentration level (7.5 μg/ml) ( Fig. 1 ). Such a result shows that the inhibitory effect of Crataegus pinnatifida and Cinnamomum cassia extracts on the ethanol-induced cytotoxicity is highly associated with the antioxidative effect of these extracts.
Oxidative stress generated by the metabolism of ethanol and the reduction of antioxidant defence mechanism may cause liver damage (Nordmann, 1992 ; French, 2001) . Among antioxidants such as vitamin C, Trolox or BHT, vitamin C had more protective effect on H 2 O 2 -induced cytotoxicity in NIH3T3 and HepG2 cells as well as inhibitory effect on oxidative DNA damage (Kim ., unpublished) . Intraperitoneal administration of vitamin C and vitamin E in rats before administering ethanol had more inhibitory effect on free radicals and DNA strand breaks than the ethanol-only group (Navasumrit ., 2000) . Therefore, it is considered that high antioxidative plant extracts reduce alcohol-induced oxidative cytotoxicity by scavenging ROS generated in alcohol oxidation process or otherwise by enhancing the activation of antioxidant enzyme such as superoxide dismutase, catalase and glutathione peroxidase.
Crataegus pinnatifida that contains polyphenols, procyanidines, chlorogenic acid and flavonoids (Chu ., 2003 ; Cui ., 2006) and Cinnamomum cassia that contains cinnamaldehyde and eugenol (Lin ., 2003) have antioxidative effect. Therefore it is required to study CYP2E1 expression, ROS generation and activity change of antioxidant enzyme of the above two plant extracts in animal models as studies on the mechanism of ethanolinduced cytotoxicity (Isayama ., 2003 ; Mikstacka ., 2002 ; Ohashi ., 2005) .
It is reported that medicinal plants or herbal and dietary supplements induce a spectral diversity of cytotoxicity in liver cells. Therefore, it is required to understand their toxicity risk and pathological physiology and evaluate safety of natural products that enhance ethanol-induced cytotoxicity. Therefore, we need to be careful to use plantoriginated extracts for cytological and medical purposes. This study has not resulted in detailed findings on plant extract's specific enhancing or reducing effect on ethanolinduced cytotoxicity.
We need to be careful with the use of plant extracts including those specified in this study such as Dimocarpus longan , Schisandra chinensis , Achyranthes japonica and Prunus mume for human bodies, especially, in case of oriental societies, with the intake of liqueur, which is made by maturing medicinal plants in ethanol, because of the toxicity of its congeners. In addition, it is also required to find natural substances such as Crataegus pinnatifida and Cinnamomum cassia that reduce ethanol-induced cytotoxicity in liver cells, investigate bioactive substances and derivatize them to develop cytoprotective substances.
Acknowledgements
This research was partially supported by Kangwon National University (2012) and The Institute of New Drug Development, KNU.
References
Arteel G.E. (2003) Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology 124 778 - 790    DOI : 10.1053/gast.2003.50087
Arteel G.E. (2008) Alcohol-induced oxidative stress in the liver: in vivo measurements. Methods Mol.. Biol. 447 185 - 97
Arteel G.E , Uesugi T. , Bevan L.N. , Gäbele E. , Wheeler M.D. , McKim S.E. , Thurman R.G. (2002) Green tea extract protects against early alcohol-induced liver injury in rats. Biol. Chem. 383 663 - 70    DOI : 10.1515/BC.2002.068
Ashfaq U.A. , Masoud M.S. , Nawaz Z. , Riazuddin S. (2011) Glycyrrhizin as antiviral agent against hepatitis C virus. J. Transl. Med. 9 112 -    DOI : 10.1186/1479-5876-9-112
Bondy S.C. (1992) Ethanol toxicity and oxidative stress. Toxicology Letter 63 231 - 241    DOI : 10.1016/0378-4274(92)90086-Y
Bunchorntavakul C. , Reddy K.R. (2013) Herbal and dietary supplement hepatotoxicity. Aliment Pharmacol Ther. 37 3 - 17    DOI : 10.1111/apt.12109
Cederbaum A.I. (2001) Introduction. Serial review: Alcohol, oxidative stress, and cell injury. Free Radical Biology & Medicine 31 1524 - 1526    DOI : 10.1016/S0891-5849(01)00741-9
Cole S.P.C. (1986) Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother. Pharmacol. 17 259 - 263    DOI : 10.1007/BF00256695
Cui T. , Nakamura K. , Tian S. , Kayahara H. , Tian Y.L. (2006) Polyphenolic content and physiological activities of Chinese hawthorn extracts. Biosci. Biotechnol. Biochem. 70 2948 - 2956    DOI : 10.1271/bbb.60361
Chu C.C. , Lee M.J. , Liao C.L. , Lin W.L , Yin Y.F. , Tseng T.H. (2003) Inhibitory effect of hot-water extract from dried fruit of Crataegus pinnatifida on low-density lipoprotein (LDL) oxidation in cell and cell-free systems. J. Agric. Food Chem. 51 7583 - 7588    DOI : 10.1021/jf030407y
French S.W. (2001) Intragastric ethanol infusion model for cellular and molecular studies of alcoholic liver disease. J. Biomed. Sci. 8 20 - 27    DOI : 10.1007/BF02255967
Fugita Y. , Uera I. , Morimoto Y. , Nakajima M. , Hatano C. , Okuda T. (1988) Studies on inhibition mechanism of auto-oxidation by tannins and flavonoids. Yakugaku Zasshi 108 129 - 135
Furukawa M. , Kasajima S. , Nakamura Y. , Shouzushima M. , Nagatani N. , Takinishi A. , Taguchi A. , Fujita M. , Niimi A. , Misaka R. , Nagahara H. (2010) Toxic hepatitis induced by show-wu-pian, a Chinese herbal preparation. Intern Med. 49 1537 - 1540    DOI : 10.2169/internalmedicine.49.3509
Ghosh N. , Ghosh R. , Mandal V. , Mandal S.C. (2011) Recent advances in herbal medicine for treatment of liver diseases. Pharm. Biol. 49 970 - 988    DOI : 10.3109/13880209.2011.558515
Girish C. , Pradhan S.C. (2012) Indian herbal medicines in the treatment of liver diseases: problems and promises. Clin Pharmacol. 26 180 - 189
Guo M.Z. , Li X.Z. , Xu H.R. , Mei Z.C. , Shen W. , Ye X.F. (2002) Rhein inhibits liver fibrosis induced by carbon tetrachloride in rats. Acta. Pharmacol. Sin. 23 739 - 744
Guo Q. , Zhao L. , You Q. , Yang Y. , Gu H. , Song G. , Lu N. , Xin J. (2007) Anti-hepatitis B virus activity of wogonin in vitro and in vivo. Antiviral Res. 74 16 - 24    DOI : 10.1016/j.antiviral.2007.01.002
Ikeda K. , Arase Y. , Kobayashi M. , Saitoh S. , Someya T. , Hosaka T. , Sezaki H. , Akuta N. , Suzuki Y. , Suzuki F. , Kumada H. (2006) A long-term glycyrrhizin injection therapy reduces hepatocellular carcinogenesis rate in patients with interferon-resistant active chronic hepatitis C: a cohort study of 1249 patients. Dig. Dis. Sci. 51 603 - 609    DOI : 10.1007/s10620-006-3177-0
Isayama F. , Froh M. , Bradford B.U. , McKim S.E. , Kadiiska M.B. , Connor H.D. , Mason R.P. , Koop D.R. , Wheeler M.D. , Arteel G.E. (2003) The CYP inhibitor 1-aminobenzotriazole does not prevent oxidative stress associated with alcohol-induced liver injury in rats and mice. Free Radic. Biol. Med. 35 1568 - 81    DOI : 10.1016/j.freeradbiomed.2003.09.007
Kim N.Y. , Song E.J. , Kim H.P. , Heo M.Y. Protective effect of green tea extract and EGCG on cytotoxicity and DNA damage of ethanol in NIH/3T3 and HepG2 cells. unpublished.
Kumar A. , Ali M. (2000) A new steroidal alkaloid from the seeds of Holarrhena antidysenterica. Fitoterapia 71 101 - 104    DOI : 10.1016/S0367-326X(99)00111-2
Lin C.C. , Wu S.J. , Chang C.H. , Ng L.T. (2003) Antioxidant activity of Cinnamomum cassia. Phytother. Res. 17 726 - 730    DOI : 10.1002/ptr.1190
McKim S.E. , Konno A. , Gäbele E. , Uesugi T. , Froh M. , Sies H. , Thurman R.G. , Arteel G.E. (2002) Cocoa extract protects against early alcohol-induced liver injury in the rat. Arch. Biochem. Biophys. 406 40 - 46    DOI : 10.1016/S0003-9861(02)00425-3
Mikstacka R. , Gnojkowski J. , Baer-Dubowska W. (2002) Effect of natural phenols on the catalytic activity of cytochrome P450 2E1. Acta. Biochim. Pol. 49 917 - 25
Mishra B.B. , Tiwari V.K. (2011) Natural products: an evolving role in future drug discovery. Eur. J. Med. Chem. 46 4769 - 4807    DOI : 10.1016/j.ejmech.2011.07.057
Navasumrit P. , Ward T.H. , Dodd N.J. , O'Connor P.J. (2000) Ethanolinduced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure. Carcinogenesis 21 93 - 99    DOI : 10.1093/carcin/21.1.93
Navasumrit P. , Ward T.H. , O'Connor P.J. , Nair J. , Frank N. , Bartsch H. (2001) Ethanol enhances the formation of endogenously and exogenously derived adducts in rat hepatic DNA. Mutat. Res. 479 81 - 94    DOI : 10.1016/S0027-5107(01)00156-7
Nordmann R. , Ribiere C. , Rouach H. (1992) Implication of free radical mechanisms in ethanol-induced cellular injury. Free Radic. Biol. Med. 12 219 -    DOI : 10.1016/0891-5849(92)90030-K
Ohashi Y. , Yamada K. , Takemoto I. , Mizutani T. , Saeki K. (2005) Inhibition of human cytochrome P450 2E1 by halogenated anilines, phenols, and thiophenols. Biol. Pharm. Bull. 28 1221 - 1223    DOI : 10.1248/bpb.28.1221
Ohtka H. , Ohishi N. , Yaki K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95 351 - 358    DOI : 10.1016/0003-2697(79)90738-3
Paterson I. , Anderson E.A. (2005) Chemistry. The renaissance of natural products as drug candidates. Science 310 451 - 453    DOI : 10.1126/science.1116364
Rajasekaran D. , Elavarasan J. , Sivalingam M. , Ganapathy E. , Kumar A. , Kalpana K. , Sakthisekaran D. (2011) Resveratrol interferes with Nnitrosodiethylamine-induced hepatocellular carcinoma at early and advanced stages in male Wistar rats. Mol. Med. Rep. 4 1211 - 1217
Rašković A. , Stilinović N. , Kolarović J. , Vasović V. , Vukmirović S. , Mikov M. (2011) The protective effects of silymarin against doxorubicin-induced cardiotoxicity and hepatotoxicity in rats. Molecules 16 8601 - 8613    DOI : 10.3390/molecules16108601
Rivera-Espinoza Y. , Muriel P. (2009) Pharmacological actions of curcumin in liver diseases or damage. Liver Int. 29 1457 - 1466    DOI : 10.1111/j.1478-3231.2009.02086.x
Rollinger J.M. , Langer T. , Stuppner H. (2006) Strategies for efficient lead structure discovery from natural products. Curr. Med. Chem. 13 1491 - 1507    DOI : 10.2174/092986706777442075
Todaro G.J. , Green H. (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17 299 - 313    DOI : 10.1083/jcb.17.2.299
Teuten E.L. , Xu L. , Red C.M. (2005) Two abundant bioaccumulated halogenated compounds are natural products. Science 307 917 - 920    DOI : 10.1126/science.1106882
Westerink W.M. , Schoonen W.G. (2007) Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol In Vitro 21 1581 - 1591    DOI : 10.1016/j.tiv.2007.05.014
Zhang A. , Sun H. , Wang X. (2013) Recent advances in natural products from plants for treatment of liver diseases. Eur. J. Med. Chem. 63 570 - 577    DOI : 10.1016/j.ejmech.2012.12.062