Antioxidant activity and hepatoprotective effects of whey protein and Spirulina in rats


Journal Title (Medline/Pubmed accepted abbreviation): Nutrition
Year: 2010 Epub ahead of print
Volume:
Number:
Page numbers:
doi: 10.1016/J.Nut.2010.04.002

Summary of Background and Research Design

Background:Recent interest in the use of antioxidant nutritional supplements has been sparked by epidemiologic evidence suggesting that dietary antioxidants in food constituents may protect against or prevent heart disease, cancer, and the aging process. Whey protein concentrates (WPCs) derived from milk may have antioxidant, antihypertensive, antitumor, hypolipidemic, antiviral, antibacterial, and chelating properties. Similar activities have been attributed to Spirulina platensis, the only blue-green alga commercially cultivated for food.

Hypothesis/purpose of study:WPC and Spirulina alone or in combination provide hepatoprotective effects against CCl4-induced liver damage.

Subjects:Three-month-old Sprague-Dawley male rats.

Experimental design:Preclinical in vitro and animal study

Treatments and protocol:The free-radical-scavenging and metal-chelating abilities of WPC and Spirulina extract were estimated in vitro. In addition, the ability of WPC and/or Spirulina extracts to prevent liver damage and lipid peroxidation was assessed in vivo. Liver damage was induced in rats by CCl4 administration. At the end of the experimentation period (Day 31) blood samples for the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin, cholesterol, total bilirubin (TB), and direct bilirubin (DB) were drawn from test animals. The following groups were compared: Group 1, untreated control; Group 2, treated orally with CCl4 (100 mg/kg of body weight) in corn oil; Group 3, treated orally with the aqueous solution of WPC (0.5 mL/day per rat); Group 4, treated orally with the aqueous solution of Spirulina (0.5 mL/day per rat); Group 5, treated orally with WPC plus Spirulina (0.5 mL/day per rat); Group 6, treated orally with CCl4 plus WPC; Group 7, treated orally with CCl4 plus Spirulina; and Group 8, treated orally with WPC plus Spirulina plus CCl4 at the same doses.

Summary of research findings:
In vitro study
  • The 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging capacity of the Spirulina solution was higher than that of the WPC solution.
    • Further, combining WPC with Spirulina significantly increased DPPH-scavenging activity vs WPC alone or Spirulina alone at all concentrations.
  • However, Spirulina alone had a higher metal-chelating rate than WPC alone, and the combination of WPC and Spirulina was not as effective as Spirulina alone.

In vivo study

  • CCl4 induced severe toxicologic effects as indicated by the significant increases in ALT, AST, ALP, TB, DB, cholesterol, and malondialdehyde (MDA) accompanied by significant decreases in TP, albumin, and total antioxidative capacity (TAC) (P < .05 for all).
  • Animals given Spirulina alone, WPC alone, or the 2 in combination had biochemical responses that were comparable, for the most part, with untreated control animals. However, there were significant increases in TAC and decreases in cholesterol in all 3 groups (generally positive health effects). In addition, there was a significant decrease in MDA for WPC animals, which is also a positive health effect. The only biochemical change that was not in the positive health direction was a small but significant increase in ALT in the Spirulina animals. It should be noted, however, that the slight ALT increase in the Spirulina group is not likely a toxicologic concern.
  • Addition of WPC, Spirulina, or the combination of WPC plus Spirulina significantly improved virtually all of the biochemical changes induced by CCl4, except for ALT in the Spirulina-only group.
    • Moreover, WPC plus Spirulina restored serum TP, albumin, and TB to control levels.
  • In general, improvement in biochemical parameters was most pronounced in the animals that received CCl4 and were treated with WPC plus Spirulina.
  • Microscopic examination of the liver tissue was concordant with biochemical analyses.
    • Liver sections of animals treated with CCl4 showed fatty degeneration, necrosis, and apoptosis with inflammatory cells scattered around the congested blood.
    • Liver sections of animals treated with WPC alone showed marked improvement in hepatocyte architecture.
    • Liver sections of animals treated with Spirulina alone showed that most hepatocytes had normal structure in different zones.
    • Liver sections of animals treated with WPC plus Spirulina showed nearly normal hepatocytic structure with moderate obliteration in blood sinusoids.

Interpretation of findings/Key practice applications:

Treatment with WPC or Spirulina improved the studied biochemical parameters, oxidative stress markers, and histologic picture of the liver in CCl4-treated rats. Moreover, this improvement was most pronounced in the group that received the combined treatment with WPC and Spirulina. Interestingly, combined treatment with WPC and Spirulina was not as effective as Spirulina alone in the in vitro metal-chelating assay although this combination was the most protective in the in vivo study. This may be because of in vivo effects on glutathione (GSH) that cannot be mimicked in vitro. The protective effects of Spirulina against CCl4-induced liver toxicity may be attributed to its antioxidant- and free-radical-scavenging activities. Conversely, the protective effects of WPC may be attributed to its antioxidant activity due to its ability to elevate cellular GSH levels. Spirulina and WPC have the ability to increase TAC and decrease lipid peroxidation. Moreover, WPC plus Spirulina may be used as new functional foods for people with liver diseases.
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