J. Pharm. Technol. Res. Manag.

Evaluation of oleoresin in carbon tetrachloride induced hepatotoxicity in rats

Ravinder Khatri, Kashish Bhardwaj, Akshay Sharma, Srijana Tamang, Kiran , Umesh Chettri Arvind Sharma

KEYWORDS

Ginger oleoresin, hepatotoxicity

PUBLISHED DATE Nov. 2, 2018
PUBLISHER The Author(s) 2018. This article is published with open access at www.chitkara.edu.in/publications.
ABSTRACT

The present study evaluated the hepatoprotective activity of ginger oleoresin against Carbontetrachloride induced liver toxic damage in rats. Rats were divided into six groups. Hepatotoxicity was induced by the administration of a single intraperitoneal dose (2ml/kg) of Carbontetrachloride in experimental rats. Post-treatment with Ginger oleoresin at 300 and 600mg/kg dose given by oral routewas carried out to find their protective effectsagainst carbontetrachloride induced hepatic injury. Biochemical parameterfor oxidative stress, inflammation and lipid profile along with genotoxicity and histological changes in rat serum and liver were studied. Silymarin was used as standard hepatoprotective agent. Extracted oleoresin dose dependently provided hepatoprotective effects.The hepatoprotective action of ginger oleoresin may be related to its free radical scavenging,anti-inflammatory and hypolipidemic activity and concluded to be partly mediated by its active constituent’s 6-gingerol, shogaol and zingerone. -phosphate; CCl3*, Trichloromethyl free radical; CCl3OO*, Trichloromethyl peroxy radical; ROS, Reactive oxygen species; iNOS, inducible nitric oxide synthase; NO, Nitric oxide, VLDL, Very low density lipoprotein.

INTRODUCTION

Liver is the largest gland of body that plays a pivotal role in regulating various physiological processes in the body such as metabolism, secretion and storage. Hepatotoxicity is a term that indicates damage to the cells, tissues, and structure or liver functions. Currently, millions of people are suffering from hepatic damage known to be induced by alcohol, chemicals, and drugs, infections, and immune response [1]. Genetic condition modifies susceptibility to various types of causative factors.[2] Chemicals, in the form of some drugs such as paracetamol, and antitubercular drugs (isoniazid, rifampicin), toxic compounds like carbontetrachloride (CCl4), thioacetamide, dimethylnitrosamine, D-galactosamine/lipopolysaccharide, gamma radiations, cadmium and arsenic heavy metals, mycotoxin (aflatoxin) are unquestionably various risk factors for hepatic injury[3-5].

Carbontetrachloride (CCl4) is a clear, colorless, volatile, heavy and noninflammable liquid. [3,6]CCl4is a lipophillic molecule metabolized in liver and spread easily in the lipid compartments of the body. CCl4is a well-known hepatotoxin or classic model used extensively to investigate the hepatotoxicity in animals by initiating lipid peroxidation [1,7-8]. Lipid peroxidation induce membrane disintegration of liver hepatocytes, which in turn increases the release of cytosolic enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)[9-10]. CCl4 also causes the activation of immune system through the infiltration of inflammatory cells to the site of injury, responsible for the release of pro-inflammatory cytokines such as tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6) and c-reactive protein (CRP), which further enhance hepatotoxicity through repeated cycle of inflammation[10] Administration of CCl4 increased triglycerides(TG), total cholesterol (TC), low density lipoprotein (LDL), whereas lowered the high density lipoprotein (HDL)[11,12]. CCl4 induced hepatotoxicity lead toaccumulation of Ca2+ in mitochondria, activate many membrane damaging enzymes, causes disruption of mitochondrial metabolism, decreased ATP synthesis and damage micro-filaments that support cell structure[3,13]. Free radicals of CCl4 induced an increase in the number of Agyrophillic nucleolar organizer region (AgNORs) and enhance activity of telomerase enzyme hepatic architecture [14]. CCl4 caused various histological changes to the liver, including cell necrosis, change in hepatic architecture, Kupffer cell hyperplasia, central vein congestion, inflammation, fatty changes, infilteration of the liver by lymphocytes and hydropic degeneration of hepatic cells is common [15-16].

A flavonoligan mixture of milk thistle, Silymarin (Silybum marianum), is an vital herbal hepatoprotective drug[17]. Altered cytoplasmic membrane architecture of Silymarin prevented the penetration of hepatotoxic substances, such as carbon tetrachloride, thioacetamide and D-galactosamine, into cells[17-18]. Hepatoprotective effects of silymarin are due to several mechanisms including antioxidation, membrane stabilization, immunomodulation, inhibition of lipid peroxidation, anticarcinogen, stimulated protein synthesis and enhanced liver detoxification [19-23].

Ginger oleoresin also known as gingerin, is widely used herbal remedies obtained by percolating the powdered rhizomes of Zingiber Officinale,which is belonging to the family of Zingiberaceae[24]. Oleoresin ginger is dark golden brown viscous oil. It consists of essential oils, organic soluble resins and other non-volatile pungent components, which comprised homologous series of phenolic ketones such as gingerols, shogaols and zingerone[25-26]. These components have reported antioxidant, anti-inflammatory effects, antitumorogenic, antipyretic, antimicrobial cardiotonic, analgesic and antitussive effects[27-32]. 6-gingerol was effective in preventing hepatic and renal damage in type-II diabetic db/db mice by reducing oxidative stress[30]. 6-Gingerol-rich fraction lowers total bilirubin and further restores ALT, AST, ALP, and gamma glutaryl transpeptidase (GGT) enzymes the sensitive indicators of hepatocellular damage and dysfunction [33-34]. 6-gingerol decreasedsignificantly liver weight index and restore the levels of triglycerides, total cholesterol and free fatty acid in the plasma and liver, which may be the primary protective mechanism exerted by the compound in high fat diet fed hamsters[35]. 6-Shogoal was comparatively more significant in decreasing the hepatic malondialdehyde (MDA) than 6 gingerol possibly due to its dominant antioxidant property. 6-Shogaol is a, 5- lipoxygenase inhibitor [36], contributed to the increased intrahepatic vascular resistance of the cirrhotic rat liver which resulted in prevention from hepatotoxin induced necroinflammatory injury[37-38]. 6-Shogoal exhibited a significant hepatoprotective activity by reducing serum activities of AST, ALT and ALP, in to diclofenac sodium intoxicated rats [39]. Zingerone was found as a hepatoprotective agent due to its free radicals scavenging and anti-inflammatory ability [27, 32]. Zingerone prevents lipid peroxidation in rat liver microsomes[40]. Therefore ginger oleoresin can be used for developing new drugs to treat drug/chemical-induced liver toxicity.

Page(s) 93-113
URL http://dspace.chitkara.edu.in/jspui/bitstream/123456789/788/1/JPTRM%206-2-1.pdf
ISSN Print : 2321-2217, Online : 2321-2225
DOI 10.15415/jptrm.2018.62007
CONCLUSION

Present study demonstrated that hepatoprotective effects of ginger oleoresin dueto its ability to reduce the rate of lipid peroxidation, increased antioxidant defense status, anti-inflammatory, hypolipidemic activity and to guard against the pathological changes of the liver induced by CCl4 intoxication. The hepatoprotective activity of Ginger oleoresin is concluded to be partly mediated by its active constituent’s 6-gingerol, shogaol and zingerone which showed superior effect on boosting the antioxidant capacity, anti-inflammatory and hypolipidemic activity. Thus the study demonstrated experimental evidences and clearly justifies the traditional claims and use of Ginger oleoresin in the treatment of liver diseases.

REFERENCES
  • Al-Harbi, N.O., Imama F., Nadeema, A. M.M. Al-Harbi, Iqbal M., Ahmad S.F, International Immunopharmacology 21 (2014) 383–388. https://doi.org/10.1016/j.intimp.2014.05.014
  • P.D. Bose, M.P. Sarma, S. Medhi, B.C. Das, H. Sain, P. Kar, J Gastroenterol Hepatol 26 (2011) 312–318. https://doi.org/10.1111/j.1440-1746.2010.06355.x https://doi.org/10.1111/j.1440-1746.2010.06305.x
  • Deepa, K.I., Mandlikb S.K., Naika S.R., Environmental toxicology and pharmacology 37 (2013) 118–133.
  • Domenicali M., Caraceni P., Giannone F., Baldassarre M., Lucchetti G., C. Quarta et al, J Hepatol 51 (2009) 991–999. https://doi.org/10.1016/j.jhep.2009.09.008
  • Larson A.M., Polson J., Fontana R.J., Davern T.J., Lalani E., L.S. Hynan et al, Hepatology 42 (2005) 1364–1372. https://doi.org/10.1002/hep.20948
  • Alkreathy H.M., Khan R.A., Khan M.R., Sahreen S. S, BMC Complementary Altern Medi 14 (2014) 1–7. https://doi.org/10.1186/1472-6882-14-1
  • Weber L.W., Boll M., Stampfl A., Crit Rev Toxicol 33 (2003) 105–136. https://doi.org/10.1080/713611034
  • Recknagel R.O., Glende E.A, Dolak J.A. JA, R.L. Waller RL, Pharmacol Ther 43(1989) 139–154. https://doi.org/10.1016/0163-7258(89)90050-8
  • Suzek H., Celik I., Dogan A., Yildirim S., Informa Healthcare 22(2015) 1–7.
  • Mohamed N.Z., Abd-Alla H.I., Hanan F.A., Mantawy M., Ibrahim N., Hassan S.A., Journal of Applied Pharmaceutical Science 2(2014) 87–100.
  • Boll M., Weber L.W.D., E. Becker, Stampfl A., Z Naturforsch 56 (2001a) 283–290. https://doi.org/10.1515/znc-2001-3-419
  • Andritoiu C.V., Andritoiu V., Cuciureanu M., NicaBadea D., N. Bibire, M. Popa, Rom J Morphol Embryol 55 (2014) 835–847.
  • Nicotera P., Bellomo G., Orrenius S., Chem Res Toxicol 3 (1990) 484–494. https://doi.org/10.1021/tx00018a001
  • Khanna A.K., Ansari M.A., M. Kumar, Khanna A., Clin Mol Pathol 49(2003) 209–213.
  • Moreira P.R. PR, Maioli M.A., H.C. Medeiros, M. Guelfi, F.T. Pereira, F.E. Mingatto, Biological Research 13(2014) 47–49.
  • Al-Sayed E., O. Martiskainen, H.S. Seif el-Din, A. Abdel-Nasser, O.A. Hammam, N.M. El-Lakkany, BioMed Research International Volume 24 (2014) 1–9. https://doi.org/10.1155/2014/245171 https://doi.org/10.1155/2014/146769
  • Abenavoli L., R. Capasso, N. Milic, F. Capasso, Phytother Res 24 (2010) 1423–1432. https://doi.org/10.1002/ptr.3207
  • Basiglio C.L., S. Pozzi, E.J. Mottino, M.G. Roma, Chem Biol Interact 179 (2009) 297–303. https://doi.org/10.1016/j.cbi.2008.12.008
  • Kim S.H., H.J. Cheon, N. Yun, S.T. Oh, E. Shin, K.S. Shim, S.M. Lee, J Pharm Sci 109 (2009) 119–127. https://doi.org/10.1254/jphs.08299FP https://doi.org/10.1254/jphs.08189FP https://doi.org/10.1254/jphs.08067SC
  • Pradhan S.C., C. Girish, Indian J Med Res 124(2006) 491–504.
  • Lieber C.S., M.A. Leo, Q. Cao, C. Ren, L.M. DeCarli, J Clin Gastroenterol 37 (2003) 336–339. https://doi.org/10.1097/00004836-20031000000013
  • Skottova N., V. Krecman, V. Simanek, Phytother Res 13 (1999) 535–537. https://doi.org/10.1002/(SICI)1099-1573(199909)13:63.0.CO;2-W
  • S. Luper, Altern Med Rev 3 (1998) 410–421.
  • Harimurtia N., N. Nhestriciab, S.Y. Subardjo, S. Yuliania, Indonesian journal of agriculture 4 (2011) 33–39.
  • Singh G.,S. Maurya, C. Catalan, M.P. de Lampasona, Flavour Fragr J 20 (2005) 1–6. https://doi.org/10.1002/ffj.1373
  • Deline G.D., Cereal Foods World 30 (1985) 697–699.
  • Kumar L., S. Chhibber, K. Harjai, PLoS One 9(2014) 1–8. https://doi.org/10.1371/journal.pone.0115807 https://doi.org/10.1371/journal.pone.0114540 https://doi.org/10.1371/journal.pone.0114754 https://doi.org/10.1371/journal.pone.0109872 https://doi.org/10.1371/journal.pone.0110482 https://doi.org/10.1371/journal.pone.0111492 https://doi.org/10.1371/journal.pone.0112108 https://doi.org/10.1371/journal.pone.0112991 https://doi.org/10.1371/journal.pone.0111137 https://doi.org/10.1371/journal.pone.0110539 https://doi.org/10.1371/journal.pone.0111098 https://doi.org/10.1371/journal.pone.0110405 https://doi.org/10.1371/journal.pone.0111759 https://doi.org/10.1371/journal.pone.0106744 https://doi.org/10.1371/journal.pone.0113566
  • Huetal R., PLoS One 7 (2012) 1–10. https://doi.org/10.1371/journal.pone.0046550
  • Dugasani S., M.R. Pichika, V.D. Nadarajah, M.K. Balijepalli, S. Tandra, J.N. Korlakunta, J Ethnopharmacol 127(2010) 515–520. https://doi.org/10.1016/j.jep.2009.10.004
  • Singh A.B., N. Singh, R. Maurya, A.K. Srivastava, Int J Med Sci 1(2009) 536–544.
  • Young H.Y., Y.L. Luo, H.Y. Cheng, W.C. Hsieh, J.C. Liao, W.H. Peng WH, J Ethnopharmacol 96 (2005) 207–210. https://doi.org/10.1016/j.jep.2004.09.009
  • Shin S.G., J.Y. Kim, H.Y. Chung, J.C. Jeong, J Agric Food Chem 53 (2005) 7617–7622. https://doi.org/10.1021/jf051014x
  • Salihu M., B.O. Ajayi, I.A. Adedara, E.O. Farombi, Journal of Dietary Supplements 1 (2016) 1–16. 34. Sabina E.P., S.J. Pragasam, S. Kumar, M. Rasool, J Chin Integr Med 9 (2011) 1264–1269. https://doi.org/10.3736/jcim20111116
  • Tzeng T.F., S.S. Liou, C.J. Chang, I.M. Liu, Nutrients 7(2015) 999–1020. https://doi.org/10.3390/nu7020999 https://doi.org/10.3390/nu7105423 https://doi.org/10.3390/nu7095369
  • Flynn D.L., M.F. Rafferty, A.M. Boctor, Prostaglandin Leukot Med 24(1986) 195–198. https://doi.org/10.1016/0262-1746(86)90126-5
  • Titos E., J. Claria, A. Planguma, M. Lopez-Parra, A. Gonzalez-Periz, J Leukocyte Biol 78 (2005) 871–878. https://doi.org/10.1189/jlb.1204747
  • Graupeara M., J.C. Garcia-Pegan, E. Titos, J. Claria, A. Massaguer, J. Bosch, Gastroenterol 122 (2002) 387–393. https://doi.org/10.1053/gast.2002.31040
  • Alqasouami S., H. Yusufolgu, A. Farraj, A. Alam, International Journal of Pharmacology 7 (2011) 868–873. https://doi.org/10.3923/ijp.2011.868.873
  • Reddy A.C., B.R. Lokesh, Mol Cell Biochem 111 (1992) 117–124.
  • Ohkawa H., N. Ohishi, K. Yagi, Anal Biochem 95 (1979) 351–358.
  • Jollow D.J., J.R. Mitchell, N. Zampaglione, J.R. Gillete, Pharmacology 11 (1974) 151–169. https://doi.org/10.1159/000136485
  • Misra H.P., I. Fridovich, J Biol Chem 247(1972) 3170–3175.
  • Aebi H., Methods Enzymol 2 (1974) 673–678.
  • Ramanathan M., C.S. Babu, A. Justin, S. Shanthakumari, J Exp Biol 50 (2012) 391–397.
  • Green L.C., D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannenbaum Anal Biochem 126 (1982) 131–138. https://doi.org/10.1016/0003-2697(82)90118-X
  • D. Trere, A. Zilbering, D. Dittus, P. Kim, P.C. Ginsberg, I. Daskal, Clin Mol Pathol 49 (1996) M209–213. https://doi.org/10.1136/mp.49.4.M209
  • Gonzalez F.J., Pharmacol Rev 40 (1988) 243–288.
  • Hikino H., Y. Kiso, N. Kato, Y. Hamada, T. Shioirietal, J Ethanopharmacol 14 (1985) 31–39. https://doi.org/10.1016/0378-8741(85)90092-3 https://doi.org/10.1016/0378-8741(85)90025-X
  • Feng Y., K.Y. Siu, X. Ye, N. Wang, M.F. Yuen, C.H. Leung, Y. Tong, S. Kobayashi, Chin Med 5 (2010) 33. https://doi.org/10.1186/1749-8546-5-33
  • Gumieniczek A., Diab Res Clin Pract 68 (2005) 89–95. https://doi.org/10.1016/j.diabres.2004.09.018
  • Nevin K.G., P.L. Vijayammal, Fitoterapia 74 (2005) 578–580. https://doi.org/10.1016/S0367-326X(03)00148-5
  • Taniyama Y., K.K. Griendling, Hypertension 42 (2003) 1075–1081. https://doi.org/10.1161/01.HYP.0000100443.09293.4F
  • Rip J.W., Rupar, C.A., K. Ravi, K.K. Carroll, Prog Lipid Res 24 (1985) 269–309. https://doi.org/10.1016/0163-7827(85)90008-6
  • Tadeusz J., J. Teresa, N. Krzysztof, Curr Top Biophys 25 (2001) 33–38.
  • Janero D.R., Free Radic Biol Med 9 (1990) 515–540. https://doi.org/10.1016/0891-5849(90)90131-2
  • Manibusan M.K., M. Odin, D.A. Eastmond, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 25 (2007) 185–209. https://doi.org/10.1080/10590500701569398
  • Halliwell B. , S. Chirico, Am J Clin Nutr 57 (1993) 715–724. https://doi.org/10.1093/ajcn/57.5.715S
  • Vaca C.E., J.M. Wilhelm, M. Harms-Rihsdahl, Mutat Res Rev Gen Toxicol 195 (1988) 137–149. https://doi.org/10.1016/0165-1110(88)90022-X
  • Bellik Y. Y, Asian Pac J Trop Dis 4(2014) 40–44. https://doi.org/10.1016/S2222-1808(14)60311-X https://doi.org/10.1016/S2221-1691(14)60206-9
  • D. Bandoniene, D. Gruzdiene, P.R. Venskutonis, M. Murkovic, Eur J Lipid Sci Technol 104 (2002) 286–292. https://doi.org/10.1002/1438-9312(200205)104:53.0.CO;2-O
  • S.K. Verma, J. Singh, R. Khamesra, A. Bordia, Indian J Med Res 98 (1993) 240–242.
  • Siddaraju M.N., S.M. Dharmesh, Mol Nutr Food Res 51 (2007) 324–332. https://doi.org/10.1002/mnfr.200600202
  • JosephJ. A., U.P. Ayyappan, S.R. Sasidharan, S. Mutyala, K.S. Goudar, A. Agarwal, Pharmacognosy Res 6 (2014) 320–325. https://doi.org/10.4103/0974-8490.138284
  • Suresh S.V., S.H. Mishra, Indian J Exp Biol 46 (2008) 447–452.
  • Maritim A.C., R.A. Sanders, J.B. Watkins, J Nutr Biochem 14 (2003) 288–294. https://doi.org/10.1016/S0955-2863(03)00036-6
  • Proctor P.H., E. McGinness, Arch Dermatol 122 (1986) 507–508. https://doi.org/10.1001/archderm.1986.01660170031013 https://doi.org/10.1001/archderm.122.5.507
  • Skrzydlewska E., R. Farbiszewski, Alcohol 14 (1997) 431–437. https://doi.org/10.1016/S0741-8329(96)00149-8
  • Huang J. He, B., X. Ban, J. Tian, L. Zhu, Y. Wang, J Ethnopharmacol 141 (2012) 104–110. https://doi.org/10.1016/j.jep.2012.02.006
  • Williams A.T., R.F. Burk, Semin Liver Dis 10 (1990) 279–284. https://doi.org/10.1055/s-2008-1040483
  • Kobayashi M., M. Yamamoto, Adv Enzyme Regul 42(2006) 113–140. https://doi.org/10.1016/j.advenzreg.2006.01.007
  • C. Webb, D. Twedt D, Vet Clin North Am Small Anim Pract 38 (2008) 125–135. https://doi.org/10.1016/j.cvsm.2007.10.001
  • Zhu R., Y. Wang, L. Zhang, Q. Guo, Hepatol Res 42 (2012) 741–749. https://doi.org/10.1111/j.1872-034X.2012.00996.x
  • Chakraborty D., M. Mukherjee, S. Sikdar, A. Paul, S. Ghosh, A.R. Khuda-Bukhsh, Toxicol Lett 210 (2012) 34–43. https://doi.org/10.1016/j.toxlet.2012.01.002
  • Hemmings S.J., V.B. Pulga, S.T. Tran, R.E. Uwiera, Cell Biochem Funct 20(2002) 47–59. https://doi.org/10.1002/cbf.934
  • Mohamed N.Z., H.I. Abd-Alla, F.A. Hanan, M. Mantawy, N. Ibrahim, S.A. Hassan, Journal of Applied Pharmaceutical Science 2 (2014) 87–100.
  • Badger D.A., J.M. Sauer, N.C. Hoglen, C.S. Jolley, I.G. Sipes, Toxicol Appl Pharmacol 141 (1996) 507–519 https://doi.org/10.1006/taap.1996.0316 78. Morio L.A., H. Chiu, K.A. Sprowles, P. Zhou, D.E. Heck, M.K. Gordon, D.L. Laskin Toxicol Appl Pharmacol 172 (2001) 44–51. https://doi.org/10.1006/taap.2000.9133
  • Leist M., F. Gantner, S. Jilg, A. Wendel, J Immunol 154(1995) 1307–1316.
  • J. Li, T.R. Billiar, Am J Physiol 276 (1999) G1069–1073. https://doi.org/10.1152/ajpcell.1999.276.5.C1069
  • Ippoushi K., H. Ito, H. Horie, K. Azuma, Planta Med 71(2005) 563–566. https://doi.org/10.1055/s-2005-864160
  • Pan M.H., M.C. Hsieh, J.M. Kuo, C.S. Lai, H. Wu, S. Sang, Mol Nutr Food Res 52 (2008) 527–537. https://doi.org/10.1002/mnfr.200700157 https://doi.org/10.1002/mnfr.200700380 https://doi.org/10.1002/mnfr.200700515
  • Mascolo N., R. Jain, S.C. Jain, F. Capasso, J Ethnopharmacol 27 (1989) 129–140. https://doi.org/10.1016/0378-8741(89)90085-8
  • Boll M., L.W. Weber, E. Becker, A. Stampfl, Z Naturforsch 56 (2001) 111–121. https://doi.org/10.1515/znc-2001-1-218 https://doi.org/10.1515/znb-2001-0201
  • Jadon A., M. Bhadauria, S. Shukla, J Ethnopharmacol 109 (2007) 214–218. https://doi.org/10.1016/j.jep.2006.07.033
  • Zhou D.,J. Ruan, Y. Cai, Z. Xiong, W. Fu, A. Wei, J Ethnopharmacol 129 (2010) 232–237. https://doi.org/10.1016/j.jep.2010.03.016
  • Rajesh MG and Latha MS. Preliminary evaluation of the anti hepatotoxic activity of Kamilari, apoly herbal formulation. J Ethnopharmacol. 2004; 91:99–104. https://doi.org/10.1016/j.jep.2003.12.011
  • Girish C., S.C. Pradhan, J Pharmacol Pharmacother 3 (2012) 149–155.
  • Trere D., Micron 31 (2000) 127–131. https://doi.org/10.1016/S0968-4328(99)00069-4
  • . Fahmy S.R., S.A. Hamdi, H.A. Abdel-Salam, Aust J Basic Appl Sci 3 (2009) 2118–2129.
  • Sai K., C.A. Tyson, D.W. Thomas, J.E. Dabbs, R. Hasegawa, Y Kurokawa, Cancer Lett 87 (1994) 1–7. https://doi.org/10.1016/0304-3835(94)90402-2
  • . Khan R. A., D. Ahmed, Food Chem Toxicol 47 (2009) 1393–1399. https://doi.org/10.1016/j.fct.2009.03.020