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Studies of Proleva
ROS Study: Report - Inhibition of ROS formation by Proleva Objective: To evaluate whether the reportedly high ORAC value of this product translates into a physiological effect in a cell-based assay. Background for this study: Oxidative stress is a condition in cells which is characterized by an excess of reactive oxygen species (ROS). An excess of these molecules leads to oxidative damage which plays a role in many disease processes. Many products are marketed to help combat oxidative damage, often relying on a standardized ORAC value for marketing claims. The ORAC test is a chemical assay that measures the capacity for neutralization of oxygen radicals. Assay principle: Based on standard immunological assays, we have modified an existing method for measurement of ROS production to study natural products with respect to their ability to inhibit ROS formation in a cell-based assay. This method may supplement a standard ORAC test by documenting an anti-inflammatory effect in a cellular system. The method is based on challenging human cells with an inflammatory stimuli to produce damaging reactive oxygen radicals. Changes in the oxidative stress level in each cell is monitored by a ROS-sensitive dye, DCF-DA. The colorless precursor to this dye is able to penetrate cells, and only after exposure to free oxygen radicals is it transformed to a fluorescent molecule that is retained inside the cells. The amount of fluorescence inside a given cell is proportionate to the amount of oxygen radicals produced inside the cell after the cell has encountered an inflammatory stimulus. A reduction in fluorescence in cells pre-loaded with a natural product is an indication of a protective effect of the natural product against reactive oxygen species. Method: Freshly purified human neutrophils were preincubated with a botanical extract over a wide range of dilutions, loaded with DCF-DA, and then challenged with a high dose of hydrogen peroxide to induce severe oxidative stress. Intracellular levels of DFC-DA fluorescence intensity in untreated versus challenged cells, in the presence versus absence of the test product, were analyzed by flow cytometry. A standard curve of DCF-DA fluorescence intensity as a result of treatment with known amounts of hydrogen peroxide was used to produce an estimation of the effectiveness of a given natural product in terms of quenched hydrogen peroxide molecules. An extract of the test product was prepared by adding 0.5g of the test product to a 5 mL of phosphate buffered saline. This mixture was vortexed repeatedly and allowed to sit at room temperature for 1 hour. Prior to use, undissolved particles were removed by centrifugation and subsequent filtration. This liquid was used to prepare a series of 100 fold dilutions of the test product. Aliquots of these dilutions were applied to the neutrophils, and then incubated at 37oC for 90 minutes. Following a wash to remove botanicals and other compounds that interfere with the oxidation marker, cells were loaded with 10 mM DC-FDA (Molecular Probes, Eugene OR) for 1 hour at 37oC. All samples, except for the untreated control samples, were then exposed to 167 mM H2O2 for a period of 45 minutes to induce severe oxidative stress. Samples were washed to remove the peroxide, and transferred to cold RPMI, and stored on ice in preparation for immediate analysis by flow cytometry. Data was collected in triplicate for controls, and duplicate for each sample concentration. Results are expressed as a percent of the average, background corrected, response for that particular assay. Dose levels are reported in volumetric parts per billion. Statistical significance was determined using Student’s test.
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