Nutr Res Pract.  2010 Jun;4(3):183-190.

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

• Senevirathne M
• Kim SH
• Jeon YJ

Abstract

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro.

Keyword

Blueberry; enzymatic hydrolysates; water soluble compounds; H2O2 induced damage; ROS

MeSH Terms

Animals
Asian Continental Ancestry Group
Blueberry Plant
Cell Line
Cell Survival
Cricetinae
Cricetulus
DNA Damage
Endopeptidases
Fibroblasts
Humans
Hydrogen
Hydrolysis
Lipid Peroxidation
Lung
Metalloendopeptidases
Peptide Hydrolases
Phenol
Subtilisins
Endopeptidases
Hydrogen
Metalloendopeptidases
Peptide Hydrolases
Phenol
Subtilisins

Figure

• 

  Fig. 1 Total phenolic content and yield of hydrolysates obtained by treatment of different enzymes. Experiments were conducted in triplicate and data are expressed as means ± SD. Total phenolic content was evaluated via Folin-Ciocalteu method and expressed as the gallic acid equivalents.

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  Fig. 2 Cytoprotective effect of hydrolysates from blueberry on V79-4 cells. V79-4 cells were incubated (1×105 cells/mL) for 16 h and induced with 1 mM H2O2 for 24 h with or without various hydrolysates. Cell viability was evaluated by MTT assay. Hydrolysates were tested at the concentrations of ▒50 µg/mL and □100 µg/mL in triplicate. Data were expressed as means ± SD. NC: negative control, PC: positive control.

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  Fig. 3 ROS scavenging activity of enzymatic hydrolysates from blueberry in V79-4 cells. V79-4 cells were incubated (1×105 cells/mL) for 16 h and induced with 1 mM H2O2 for 30 min with or without various hydrolysates. The oxidation-sensitive dye DCFH-DA was used to determine the formation of intracellular ROS. Data were presented as % of positive control. Hydrolysates were tested at the concentrations of ▒50 µg/mL and □100 µg/mL in triplicate. Data were expressed as means ± SD. NC: negative control.

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  Fig. 4 Lipid peroxidation inhibitory effect of Alcalase and AMG hydrolysates from blueberry. V79-4 cells were incubated (1×105 cells/mL) for 16 h and induced with 1 mM H2O2 for 1 h with or without various hydrolysates. Lipid peroxidation products were evaluated by TBA. Bars filled in gray color: Alcalase hydrolysate and bars without filling: AMG hydrolysate; lipid peroxidation inhibitory effect of Alcalase: -▴- and AMG: -x-. *P < 0.05, significantly different from H2O2 alone. NC: negative control. 0 indicates and vehicle control (data not shown). The reduction of cell positive control

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  Fig. 5 Effect of Alcalase and AMG hydrolysates on DNA damage induced by H2O2 in V79-4 cells. V79-4 cells were incubated (4×104 cells/mL) for 16 h and pre-treated with or without various hydrolysates for 30 min. Then, induced with 50 µM H2O2 for 5 min and DNA damage was determined by comet assay. Bars filled in gray color: Alcalase and bars without filling: AMG; Inhibitory effect of cell damage of Alcalase hydrolysate: -▴- and AMG hydrolysate: -x-. *P < 0.05, significantly different from H2O2 alone. NC: negative control, 0 indicates positive control

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  Fig. 6 Comet images of V79-4 cells obtained for AMG hydrolysate. Measurements were made by image analysis with fluorescence microscope determining the percentage of fluorescence in the tail. (A) negative control (B) V79-4 cells treated with 50 µM H2O2 (Positive control) (C) V79-4 cells treated with 50 µg/mL AMG hydrolysate+50 µM H2O2 (D) V79-4 cells treated with 100 µg/mL AMG hydrolysate+50 µM H2O2

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  Fig. 7 Effect of hydrolysates from blueberry on apoptosis. The nuclear morphology of the cells was evaluated using a cell-permeable DNA dye, Hoechst 33342. A: Negative control, B: H2O2 treated sample (positive control), C: 1 mM H2O2+100 µg/mL of Alcalase hydrolysate, D: 1 mM H2O2+100 µg/mL of AMG hydrolysate

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