Evaluation of the hepatoprotective effect of combination between fermented camel milk and Rosmarinus officinalis leaves extract against CCl4 induced liver toxicity in mice

  • Houda Hamed
  • khaled Bellassoued
  • Abdelfattah El Feki
  • Ahmed Gargouri
Original Article


The present study was conducted to evaluate the in vitro antioxidant activity of fermented camel milk with Lactococcus lactis subsp. cremoris (FCM-LLC) alone, Rosmarinus officinalis extract (R) alone and their combination and to investigate their hepatopreventive effects against CCl4 liver damage in mice. The antioxidant activity in vitro of FMC-LLC supplemented with R exhibited the highest free radical scavenging and ferric reducing power activities. The results showed that the pretreatment with a combination of FMC-LLC and R significantly alleviated the increased levels of hepatic markers and the elevated lipid levels induced by CCl4 in mice. Meanwhile, the enzymatic antioxidants activities (superoxide dismutase, glutathione peroxidase, and catalase) and GSH level in liver significantly were increased while the malondialdehyde level was significantly improved by pretreatment with FMLLC plus R. These data suggest that FCM-LLC in combination with R. officinalis extract possesses better antioxidant and hepatoprotective activity than FMC-LLC alone.


Carbon tetrachloride Fermented camel milk Hepatoprotection Lacotococus lactis subsp. cremoris Rosmarinus officinalis 



This research was supported by the Tunisian Ministry of higher Education and Scientific Research.


  1. Abd El-Ghany MA, Motawee MM, El-Kewawy HEM (2012) Biological effects of yoghurt with rosemary on injured liver rats. Aust J Basic Appl Sci 6:525–532Google Scholar
  2. Abdel-Wahhab MA, Abdel-Galil MM, Ellithey MM (2005) Melatonin counteracts oxidative stress in rats fed ochratoxin A–contaminated diet. J Pineal Res 38:130–135. CrossRefPubMedGoogle Scholar
  3. Abu Taher Sagor MD, Mahmud Reza H, Tabassum N, Sikder B, Ulla A, Subhan N, Ashraful Alam Md (2016) Supplementation of rosemary leaves (Rosmarinus officinalis) powder attenuates oxidative stress, inflammation and fibrosis in carbon tetrachloride (CCl4) treated rats. Curr Nutr Food Sci 12:288–295. CrossRefGoogle Scholar
  4. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126. CrossRefPubMedGoogle Scholar
  5. ATSDR “Agency for Toxic Substance and Diseases Registry” (1994) Toxicological profile for carbon tetrachloride, U.S. Department of Health and Human Services, Public Health Service, TP-39/02. Atlanta, CAGoogle Scholar
  6. Benzie I, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power: the FRAP assay. Anal Biochem 239:70–76. CrossRefGoogle Scholar
  7. Beyer W, Fridovich I (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal Biochem 161:559–566. CrossRefPubMedGoogle Scholar
  8. Council of European Communities (1986) Council instructions about the protection of living animals used in scientific investigations. Off J Eur Communities L358(JO 86/609/CEE):1–18Google Scholar
  9. Couto N, Wood J, Barber J (2016) The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol Med 95:27–42. CrossRefPubMedGoogle Scholar
  10. Ellman GL (1959) Tissue sulphydryl groups. Arch Biochem Biophys 82:70–77CrossRefGoogle Scholar
  11. Fahmy NM, Al-Sayed E, Abdel-Daim MM, Karonen M, Singab AN (2016) Protective effect of Terminalia muelleri against carbon tetrachloride-induced hepato and nephro-toxicity in mice and characterization of its bioactive constituents. Pharm Biol. 54:303-13. CrossRefPubMedGoogle Scholar
  12. Flohe L, Gunzler WA (1984) Assays of glutathione peroxydase. Methods Enzymol 105:114–21CrossRefGoogle Scholar
  13. Gad AS, Abd El-salam MH (2010) The antioxidant properties of skim milk supplemented with rosemary and green tea extracts in response to pasteurisation, homogenisation and the addition of salts. J Dairy Technol 63:349–355. CrossRefGoogle Scholar
  14. Gigante D, Buchweitz M, Helbig E, Almeida AS, Arau JO, Neumann NA, Victoria C (2007) Randomized clinical trial of the impact of a nutritional supplement “multimixture” on the nutritional status of children enrolled at preschools. J Pediatr 83:363–369. CrossRefGoogle Scholar
  15. Guven A, Guven A, Gulmez M (2003) The effect of kefir on the activities of GSH-Px, GST, CAT, GSH and LPO levels in carbon tetrachloride-induced mice tissues. J Vet Med B 50:412–416. CrossRefGoogle Scholar
  16. Huang H-L, Wang Y-J, Zhang Q-Y, Liu B, Wang F-Y, Li J-J, Zhu R-Z (2012) The hepatoprotective effects of baicalein against CCl4-induced acute liver injury in mice. World J Gastroenterol 18: 6605–6613. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hamed H, Chaari F, Ghannoudi Z, ElFeki A, Chaabouni Ellouz S, Gargouri A (2017a) Beneficial effects of fermented camel milk by Lactococcus lactis subsp. cremoris on cardiotoxicity induced by carbon tetrachloride in mice. Biomed Pharmacother 97:107–114. CrossRefPubMedGoogle Scholar
  18. Hamed H, Chaari F, Krichen F, Ncir M, Chaabouni S, El Feki A, Gargouri A (2017) Evaluation in vitro of the angotensin convert enzyme(ACE)-inhibitory, antioxidant, and antimicrobial activities of Fermented camel Milks. (Submitted) Google Scholar
  19. Hamed H, Bellassoued K, Brahmi N, Ghannoudi Z, Elfeki A (2018a) Modulatory effects of rosemary leaves aqueous extract against oxidative stress and related damages in experimental model of CCl4-induced cardiac toxicity in mice. Appl Physiol Nutr Metab. CrossRefPubMedGoogle Scholar
  20. Hamed H, Gargouri M, Boulila S, Chaari F, Ghrab F, Kallel R, Ghannoudi Z, Boudawara T, Chaabouni S, Feki AE, Gargouri A (2018b) Fermented camel milk prevents carbon tetrachloride induced acute injury in kidney of mice. J Dairy Res 85:251–256. CrossRefPubMedGoogle Scholar
  21. Janero R (1990) Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 6:515–540. CrossRefGoogle Scholar
  22. Liu J, Jia M, Yao Lu, Wang Q (2016) The hepatoprotective effects of baicalein against CCl-induced acute liver injury in mice. Int J Clin Exp Med 9:23206–23213. CrossRefGoogle Scholar
  23. Lowry OH, Rosebrugh NJ, Farr AL (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275 PMID:14907713 PubMedPubMedCentralGoogle Scholar
  24. Kumar HK, Razack S, Nallamuthu I, Khanum F (2014) Phytochemical analysis and biological properties of Cyperus rotundus L. Ind Crops Products 52:815– 826. CrossRefGoogle Scholar
  25. Marcason W (2007) Is supplementation of B vitamins still recommended to reduce the risk of heart disease? J Am Diet Assoc 107:525–531CrossRefGoogle Scholar
  26. Moslehishad M, Mirdamad S, Ehsani MR, Ezzatpanah H, Moosavi-Movahed AA (2013) The proteolytic activity of selected lactic acid bacteria in fermenting cow’s and camel’s milk and the resultant sensory characteristics of the products. Int J Dairy Technol 66:279–285. CrossRefGoogle Scholar
  27. Nishino T, Shibahara-Sone H, Kikuchi-Hayakawa H (2000) Transit of radical scavenging activity of milk products prepared by maillard reaction and Lactobacillus Strain Shirota fermentation through the hamster intestine. J Dairy Sci 83:915–922. CrossRefPubMedGoogle Scholar
  28. Panda SH, Ray RC (2007) Lactic acid fermentation of beta-carotene rich sweet potato (Ipomoea batatas L.) into lacto-juice. Plant Foods Hum Nutr 62:65–70. CrossRefPubMedGoogle Scholar
  29. Prieto P, Pineda M, Aguilar M (1999) Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 269:337–341. CrossRefPubMedGoogle Scholar
  30. Ribereau-Gayon P, Stonestreet E (1968) Les dosages des anthocyanes dans le vin rouge. Bull Soc Chim Fr 9:2649–2652Google Scholar
  31. Seifert WF, Bosma A, Hendriks HF, van Leeuwen RE, van Thiel-de Ruiter GC, Seifert-Bock I (1995) Beta-carotene (provitamin A) decreases the severity of CCl4-induced hepatic inflammation and fibrosis in rats. Liver 15(1):1–8. CrossRefPubMedGoogle Scholar
  32. Son S, Lewis BA (2002) Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: structure-activity relationship. J Agric Food Chem 50:468–472. CrossRefPubMedGoogle Scholar
  33. Soni B, Visavadiya NP, Madamwar D (2008) Ameliorative action of cyanobacterial phycoerythrin on CCl4-induced toxicity in rats. J Toxicol 248:59–65. CrossRefGoogle Scholar
  34. St-Onge MP, Farnworth ER, Jones PJH (2000) Consumption of fermented and nonfermented dairy products: effects on cholesterol concentrations and metabolism. Am J Clin Nutr 71:674–681CrossRefGoogle Scholar
  35. Venukumar MR, Latha MS (2002) Antioxidant activity of Curculigo orchioides carbon tetrachloride induced hepatopathy in rats. J Clin Biochem 17:80–87. CrossRefGoogle Scholar
  36. Wang R, Xia F, Kai Z, Xin Z, Huayi S (2016) Preventive activity of banana peel polyphenols on CCl4-induced experimental hepatic injury in Kunming mice. Exp Ther Med. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Wunjuntuk K, Kettawan A, Charoenkiatkul S, Rungruang T (2016) Parboiled germinated brown rice protects against CCl4-induced oxidative stress and liver injury in rats. J Med Food 19(1):15–23. CrossRefPubMedGoogle Scholar
  38. Yagi K (1976) A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 15(2):212–216. CrossRefPubMedGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2019

Authors and Affiliations

  • Houda Hamed
    • 1
  • khaled Bellassoued
    • 1
  • Abdelfattah El Feki
    • 1
  • Ahmed Gargouri
    • 2
  1. 1.Laboratory of Animal Ecophysiology, Faculty of SciencesSfax UniversitySfaxTunisia
  2. 2.Research Unit of Toxicology, Environmental Microbiology and Health, Faculty of Science of SfaxUniversity of SfaxSfaxTunisia

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