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Evaluation of the ability of aqueous black walnut extracts to induce the production of reactive oxygen species

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  • 1 Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 2 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 3 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 4 Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 5 Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 6 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 7 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
  • | 8 Department of Chemistry, College of Liberal Arts, Hamline University, Saint Paul, MN 55104.

Abstract

Objective—To assess the in vitro capability of aqueous black walnut extracts (BWEs) to generate reactive oxygen species in water-based media ranging in makeup from a simple buffer solution to a complex solution containing serum.

Sample—3 BWEs.

Procedures—Production of reactive oxygen species by BWEs prepared in water or N-hexane was tested in PBS solution, PBS solution containing 0.5% bovine serum albumin and 5mM glucose (PBG), and RPMI-1640 medium (RPMI) containing 10% fetal bovine serum or 10% donor horse serum. Reactive oxygen species production was measured as conversion of nonfluorescent dihydrorhodamine 123 by reactive oxygen species to its fluorescent product, rhodamine-123. Hydrogen peroxide was used as a standard for reactive oxygen species activity.

Results—BWEs prepared in water generated reactive oxygen species in a dose-dependent manner over a 4-hour period, with peak activity detected when the BWEs were added as 10% (vol/vol) of the RPMI. The BWE prepared in N-hexane generated maximal reactive oxygen species activity after incubation for 3 to 4 hours when added at concentrations ranging from 0.3% to 0.5% (vol/vol) of the RPMI. The BWE prepared in water generated the highest fluorescent signal in PBS solution, whereas the BWE prepared in N-hexane generated the highest fluorescent signal in PBG.

Conclusions and Clinical Relevance—The BWEs prepared in water generated a dose-dependent induction of fluorescence in all the water-based solutions tested. These findings indicated that the BWEs, which are used to induce laminitis in horses, generate reactive oxygen species.

Abstract

Objective—To assess the in vitro capability of aqueous black walnut extracts (BWEs) to generate reactive oxygen species in water-based media ranging in makeup from a simple buffer solution to a complex solution containing serum.

Sample—3 BWEs.

Procedures—Production of reactive oxygen species by BWEs prepared in water or N-hexane was tested in PBS solution, PBS solution containing 0.5% bovine serum albumin and 5mM glucose (PBG), and RPMI-1640 medium (RPMI) containing 10% fetal bovine serum or 10% donor horse serum. Reactive oxygen species production was measured as conversion of nonfluorescent dihydrorhodamine 123 by reactive oxygen species to its fluorescent product, rhodamine-123. Hydrogen peroxide was used as a standard for reactive oxygen species activity.

Results—BWEs prepared in water generated reactive oxygen species in a dose-dependent manner over a 4-hour period, with peak activity detected when the BWEs were added as 10% (vol/vol) of the RPMI. The BWE prepared in N-hexane generated maximal reactive oxygen species activity after incubation for 3 to 4 hours when added at concentrations ranging from 0.3% to 0.5% (vol/vol) of the RPMI. The BWE prepared in water generated the highest fluorescent signal in PBS solution, whereas the BWE prepared in N-hexane generated the highest fluorescent signal in PBG.

Conclusions and Clinical Relevance—The BWEs prepared in water generated a dose-dependent induction of fluorescence in all the water-based solutions tested. These findings indicated that the BWEs, which are used to induce laminitis in horses, generate reactive oxygen species.

Contributor Notes

Supported by the American Quarter Horse Foundation and the White Fox Farm Research Fund.

Address correspondence to Dr. David Hurley (djhurley@uga.edu).