Ronald Prior, PhD03.01.11
Antioxidants are growing up. They have emerged from their adolescence into the prime of maturity. In doing so, they have proven themselves to be versatile performers. In fact, these natural product compounds traditionally referred to as “antioxidants” may deserve a new name—”antiAGEnts.”
In the past decade, antioxidants have captured the consumer imagination—for the most part, for the better. They have become a part of the consumer vocabulary and have helped shape the landscape of nutrition products, from conventional supplements to specialty foods to cosmetics. During this time, antioxidant research has been robust. Central to the findings are: (1) that phyto-compounds traditionally called “antioxidants” demonstrate diverse characteristics; and (2) that “antioxidants” are implicated in a complex array of interconnected mechanisms in vivo.
The diversification of antioxidants’ role has important implications for natural products and the varied forms they take in the anti-aging marketplace.
The New Science of Antioxidants
There is continued interest in and questions regarding the antioxidant capacity of the diet and in vivo antioxidant status and effects on health outcomes. There is increasing evidence that the postprandial (after meal) state is an important contributing factor to chronic disease. A decrease in plasma antioxidant capacity has been observed following a meal containing macronutrients but no sources of antioxidants. The role of fruit and vegetable phenolic compounds to protect health and lower disease risk through their actions in mitigating fed-state metabolic and oxidative stressors is of interest.
Berries such as blueberries, grapes and strawberries have been shown to reduce postprandial oxidative stress. More research is needed in this area, but data from several studies summarized by Dr. Burton-Freeman suggest that consuming phenolic-rich fruits increases the antioxidant capacity of the blood. And when they are consumed with high fat and carbohydrate “pro-oxidant and pro-inflammatory” meals, they may counterbalance their negative effects. Given the content and availability of fat and carbohydrates in the Western diet, regular consumption of phenolic-rich foods, particularly in conjunction with meals, appears to be a prudent strategy to maintain oxidative balance and health.
It is becoming increasingly apparent that phenolic-rich foods may impact health outcomes through other mechanisms in addition to strictly antioxidant effects. Studies suggest that oxidative stress and systemic inflammation are involved in the pathogenesis of ischemic stroke and consuming a diet with a high total antioxidant capacity has been related to reduced inflammation, along with increased circulating antioxidants. In a cross-sectional and randomized intervention study of 41,620 men and women, a diet rich in total antioxidant capacity was associated with a reduction in incidence of ischemic stroke and to a lesser extent in all types of stroke. In another study, positive associations were observed between dietary antioxidant capacity and adiponectin concentration and a negative relationship with inflammatory markers. As a result, an adiponectin-mediated route through which antioxidant-rich foods exert beneficial effects against inflammation and cardiovascular diseases can be hypothesized.
Consumption of a Mediterranean diet, especially rich in virgin olive oil, has also been associated with higher levels of plasma antioxidant capacity. In addition, plasma total antioxidant capacity may lead to a reduction in body weight after three years of intervention in a high cardiovascular risk population.
In another study, a group of patients with Type 2 diabetes was given a polyphenol-rich antioxidant supplement, and a decrease in LDL and an increase in HDL was observed. In addition, a byproduct of lipid peroxidation (plasma MDA) was decreased in the study group compared to the placebo group, and an increase in antioxidant defense was observed based upon increases in total plasma GSH and antioxidant capacity. These observations indicated that the polyphenol-rich antioxidant supplement may have been important in antagonizing effects on oxidative stress and lipid peroxidation in patients with Type 2 diabetes and might be beneficial in preventing cardiovascular complications.
Based on positive associations observed for total fruit and vegetable intakes and what was termed total antioxidant performance, Talegawkar et. al. suggested that it might be prudent to focus on increasing consumption of fruit, vegetables, nuts and seeds to increase total antioxidant capacity.
In this vein, accurately testing the antioxidant level in natural products and ex vivo samples remains an important task, which is why the Oxygen Radical Absorbance Capacity (ORAC) method remains relevant. And here’s why: (1) ORAC is an established standard that already plays a valuable role in the dietary supplement and functional food industries; (2) significant improvements have been made to the method; and (3) there appears to be a vital link between ORAC level and bioassay efficacy markers.
The ORAC Method Today
Briefly, ORAC was developed as an analytical tool for estimating the antioxidant capacity of substances, with an obvious application to natural products. It was an important advancement in commercially available analysis and has become a de facto standard in the natural products industry. Let’s consider some important facts:
• From the beginning, the ORAC method was considered a starting spot—not a definitive endpoint—for comprehensive antioxidant analysis.
• The original ORAC method is not a universal standard—it favors certain antioxidant substances over others (e.g., anthocyanins over carotenoids) due to the use of only one free radical source (peroxyl radical), which was chosen initially because it is the most common radical source found in the human body.
• There now exists a complementary suite of assays based on a unified ORAC chemistry that broadens the analytical scope of antioxidant testing.
This evolution process has resulted in the Total ORAC suite. It expands the ORAC platform to measure antioxidant capacity against five primary reactive oxygen species (commonly referred to as radicals)—peroxyl, hydroxyl, peroxynitrite, superoxide anion and singlet oxygen.
While Total ORAC is subject to some of the same limitations as the original ORAC method, it substantially improves broad-spectrum antioxidant analysis. It also gives evidence of the diverse antioxidant potential of natural products against radicals other than peroxyl. In some instances this potential may be specialized (e.g., carotenoids vs. singlet oxygen); in other cases, there is balanced antioxidant performance against multiple radicals. Compounds such as resveratrol and standardized ingredients such as green tea extract exhibit significant broad-spectrum antioxidant capacity.
The significance of Total ORAC is that while these primary radicals may all broadly contribute to the same outcomes—oxidative damage and corresponding disease states—they have unique characteristics that need to be addressed. Like position players on a sports team, antioxidant substances have different skill sets. And it is meaningful and valuable to know how antioxidants will perform against different opponents.
A New Measure of Performance: ‘Cardinal Wellness Factors’
There has been a steady and justified call for analytical tools that help establish the efficacy of natural products. Industry participants note the structural challenges of conducting full-scale clinical studies to validate product efficacy, which puts even greater impetus on finding reliable and cost effective solutions. There are at least three valuable applications of commercial-grade efficacy methods.
Quality control (QC). There has never been a greater proliferation of natural product ingredients, or a greater range of QC challenges. For example, grape seed extract comes from multiple geographic sources, varies widely in standardized specifications, and defies easy validation. Robust, efficient efficacy tests that address the performance characteristics of grape seed extract samples can provide a new tool to add to other foundational QC benchmarks for suppliers and manufacturers.
Product formulation. Companies have built premium ingredients and products around ORAC for the better part of a decade, and have begun to do so with Total ORAC. With a reliable suite of outcome-based efficacy tests, they can begin to do the same thing with anti-aging formulations. Such tests would deliver valuable guidance in the development of condition-specific formulations—with performance results unique to the actual formulation.
Consumer education. Consumers continue to seek better information about the natural products they purchase. Cell-based efficacy tests deliver valid pre-clinical test results for actual products that target primary wellness concerns. At present, most products rely upon scientific information that is generic to constituent ingredients. Product- and condition-specific validation benefits consumers and the companies that provide it.
Cell-Based Assays
Cell-based assays have been developed to determine product potency, functionality and mode of action of nutraceutical and biopharmaceutical products
Live human cell lines are grown in a laboratory environment. These cells are then exposed to certain harmful substances, such as free radicals, ultraviolet light irradiation and inflammatory agents. Other substances (such as antioxidant-containing natural products) are then introduced to see if they protect the cells from these harmful sources of damage.
Cell-based assays are well suited to the current industry imperative because: (1) they are well established in research; (2) they are versatile; and (3) they represent a cost-effective pre-clinical alternative to in vivo animal or human testing. As with any assay short of in vivo testing, there are limitations. But if these limitations are recognized and kept in focus, cell-based assays bring companies one step closer to in vivo clinical testing.
References furnished upon request.
About the authors: Ronald Prior, PhD, is an adjunct professor, Dept. Food Science, University of Arkansas, Fayettevile, AR, and science consultant for Brunswick Laboratories. Boxin Ou, PhD, is the chief science officer at Brunswick Laboratories, Inc. David Bell, MBA, is the president of Bell Advisory Services. Qiuyan Zhao, MD, MBA, is the director of Biology & Immunology at Brunswick Laboratories, Inc. HuiLin Wei, MD, PhD, is a chief scientist at Brunswick Laboratories, Inc. If you have any questions regarding the information or references in this article, contact Mr. Bell at 617-575-9822 or dnb@belladvisory.com.
Brunswick Laboratories puts natural products to the test using its cell-based assays.
At Brunswick Labs, cell-based assays have been modified to make them applicable to a range of product formats and high-throughput. This represents a fundamental step forward in commercially viable cell-based methods in the natural products industry. A critical feature of the tests is that they address primary wellness factors. Each test uses a specific marker—a measureable biological parameter—to quantify performance.
For this study, Brunswick researchers selected three cell-based assays: Cellular Antioxidant Assay (CAA), Cellular Anti-inflammation Assay (NF-kB) and Cellular UVA/UVB protection (UVA/UVB). A central objective of the cell-based testing program is to provide analytical tools for industry that are relevant for a range of products, from pure compounds to consumer products.
Brunswick tested several standardized benchmarks with each of the Cardinal Wellness Factors. Samples were selected from each of these categories: reference compounds, extracts and concentrated foods. These included:
• Pure compounds: naringenin
• Food extracts: cocoa, grape seed
• Complete foods: acai, camu camu
Here is what Brunswick researchers found. Quercetin was used as positive control in cell-based assays. The quercetin equivalent (QE) was then calculated by reference to the curve established with pure quercetin in those assays. All results are expressed using the QE per gram sample (QE/g). This allows for useful comparison across different product types.
The NF-kB assay demonstrates the sample’s ability to inhibit NF-kB, an important transcription factor that adjusts the gene expression for many inflammatory factors. As the results show, NF-kB activity is inhibited by all tested samples at different levels. The cocoa extract has the highest potentiality to inhibit inflammatory factors among these five samples.
The CAA assay demonstrates the sample’s ability to reduce cellular free radical levels generated by AAPH (2,2’-azobis-2-methyl-propanimidamide dihydrochloride), a water-soluble azo compound that is used extensively as a free radical generator. As shown in the table, all samples demonstrate significance, with the cocoa extract exhibiting the most powerful free radical scavenging activity, followed by grape seed extract and naringenin.
The UVA/UVB assay demonstrates the sample’s ability to reduce UVA/UVB-induced free radicals in skin cells. In this assay, a human skin cell line was cultured to confluency, testing compounds, positive controls (antioxidants) and negative controls (vehicles alone) were added to the cells, and cells were then exposed to UVA/UVB light treatment. Free radicals generated upon UVA/UVB irradiation were measured. The decreased amount of free radical generation was calculated and expressed as free radical scavenging activity. Four of the five samples demonstrated significant free radical reduction.
Going a step further, Brunswick researchers also saw interesting results when they converted to standard dosages. For example, a standard dose of grape seed extract might be 200 mg, while a standard dose of acai powder might be 5 grams. On a dose-equivalent basis, NF-kB performance then becomes 84 QE/g for grape seed extract and 115 QE/g for camu camu powder. The same “recommended dose” conversion can also be extended to the other assays as well.
Conclusions
The central message from this study is that natural products in a variety of standardized forms demonstrate significant efficacy against primary wellness factors. This is indicative of the multi-faceted anti-aging effects of natural products—in particular, those with a complex matrix of active constituents.
Another meaningful result of this study is the broad correlation between Total ORAC and cell-based outcomes. Although preliminary, these results support the view that Total ORAC is a useful tool for suggesting not only antioxidant, but also other important anti-aging outcomes.
References furnished upon request.
In the past decade, antioxidants have captured the consumer imagination—for the most part, for the better. They have become a part of the consumer vocabulary and have helped shape the landscape of nutrition products, from conventional supplements to specialty foods to cosmetics. During this time, antioxidant research has been robust. Central to the findings are: (1) that phyto-compounds traditionally called “antioxidants” demonstrate diverse characteristics; and (2) that “antioxidants” are implicated in a complex array of interconnected mechanisms in vivo.
The diversification of antioxidants’ role has important implications for natural products and the varied forms they take in the anti-aging marketplace.
The New Science of Antioxidants
There is continued interest in and questions regarding the antioxidant capacity of the diet and in vivo antioxidant status and effects on health outcomes. There is increasing evidence that the postprandial (after meal) state is an important contributing factor to chronic disease. A decrease in plasma antioxidant capacity has been observed following a meal containing macronutrients but no sources of antioxidants. The role of fruit and vegetable phenolic compounds to protect health and lower disease risk through their actions in mitigating fed-state metabolic and oxidative stressors is of interest.
Berries such as blueberries, grapes and strawberries have been shown to reduce postprandial oxidative stress. More research is needed in this area, but data from several studies summarized by Dr. Burton-Freeman suggest that consuming phenolic-rich fruits increases the antioxidant capacity of the blood. And when they are consumed with high fat and carbohydrate “pro-oxidant and pro-inflammatory” meals, they may counterbalance their negative effects. Given the content and availability of fat and carbohydrates in the Western diet, regular consumption of phenolic-rich foods, particularly in conjunction with meals, appears to be a prudent strategy to maintain oxidative balance and health.
It is becoming increasingly apparent that phenolic-rich foods may impact health outcomes through other mechanisms in addition to strictly antioxidant effects. Studies suggest that oxidative stress and systemic inflammation are involved in the pathogenesis of ischemic stroke and consuming a diet with a high total antioxidant capacity has been related to reduced inflammation, along with increased circulating antioxidants. In a cross-sectional and randomized intervention study of 41,620 men and women, a diet rich in total antioxidant capacity was associated with a reduction in incidence of ischemic stroke and to a lesser extent in all types of stroke. In another study, positive associations were observed between dietary antioxidant capacity and adiponectin concentration and a negative relationship with inflammatory markers. As a result, an adiponectin-mediated route through which antioxidant-rich foods exert beneficial effects against inflammation and cardiovascular diseases can be hypothesized.
Consumption of a Mediterranean diet, especially rich in virgin olive oil, has also been associated with higher levels of plasma antioxidant capacity. In addition, plasma total antioxidant capacity may lead to a reduction in body weight after three years of intervention in a high cardiovascular risk population.
In another study, a group of patients with Type 2 diabetes was given a polyphenol-rich antioxidant supplement, and a decrease in LDL and an increase in HDL was observed. In addition, a byproduct of lipid peroxidation (plasma MDA) was decreased in the study group compared to the placebo group, and an increase in antioxidant defense was observed based upon increases in total plasma GSH and antioxidant capacity. These observations indicated that the polyphenol-rich antioxidant supplement may have been important in antagonizing effects on oxidative stress and lipid peroxidation in patients with Type 2 diabetes and might be beneficial in preventing cardiovascular complications.
Based on positive associations observed for total fruit and vegetable intakes and what was termed total antioxidant performance, Talegawkar et. al. suggested that it might be prudent to focus on increasing consumption of fruit, vegetables, nuts and seeds to increase total antioxidant capacity.
In this vein, accurately testing the antioxidant level in natural products and ex vivo samples remains an important task, which is why the Oxygen Radical Absorbance Capacity (ORAC) method remains relevant. And here’s why: (1) ORAC is an established standard that already plays a valuable role in the dietary supplement and functional food industries; (2) significant improvements have been made to the method; and (3) there appears to be a vital link between ORAC level and bioassay efficacy markers.
The ORAC Method Today
Briefly, ORAC was developed as an analytical tool for estimating the antioxidant capacity of substances, with an obvious application to natural products. It was an important advancement in commercially available analysis and has become a de facto standard in the natural products industry. Let’s consider some important facts:
• From the beginning, the ORAC method was considered a starting spot—not a definitive endpoint—for comprehensive antioxidant analysis.
• The original ORAC method is not a universal standard—it favors certain antioxidant substances over others (e.g., anthocyanins over carotenoids) due to the use of only one free radical source (peroxyl radical), which was chosen initially because it is the most common radical source found in the human body.
• There now exists a complementary suite of assays based on a unified ORAC chemistry that broadens the analytical scope of antioxidant testing.
This evolution process has resulted in the Total ORAC suite. It expands the ORAC platform to measure antioxidant capacity against five primary reactive oxygen species (commonly referred to as radicals)—peroxyl, hydroxyl, peroxynitrite, superoxide anion and singlet oxygen.
While Total ORAC is subject to some of the same limitations as the original ORAC method, it substantially improves broad-spectrum antioxidant analysis. It also gives evidence of the diverse antioxidant potential of natural products against radicals other than peroxyl. In some instances this potential may be specialized (e.g., carotenoids vs. singlet oxygen); in other cases, there is balanced antioxidant performance against multiple radicals. Compounds such as resveratrol and standardized ingredients such as green tea extract exhibit significant broad-spectrum antioxidant capacity.
The significance of Total ORAC is that while these primary radicals may all broadly contribute to the same outcomes—oxidative damage and corresponding disease states—they have unique characteristics that need to be addressed. Like position players on a sports team, antioxidant substances have different skill sets. And it is meaningful and valuable to know how antioxidants will perform against different opponents.
A New Measure of Performance: ‘Cardinal Wellness Factors’
There has been a steady and justified call for analytical tools that help establish the efficacy of natural products. Industry participants note the structural challenges of conducting full-scale clinical studies to validate product efficacy, which puts even greater impetus on finding reliable and cost effective solutions. There are at least three valuable applications of commercial-grade efficacy methods.
Quality control (QC). There has never been a greater proliferation of natural product ingredients, or a greater range of QC challenges. For example, grape seed extract comes from multiple geographic sources, varies widely in standardized specifications, and defies easy validation. Robust, efficient efficacy tests that address the performance characteristics of grape seed extract samples can provide a new tool to add to other foundational QC benchmarks for suppliers and manufacturers.
Product formulation. Companies have built premium ingredients and products around ORAC for the better part of a decade, and have begun to do so with Total ORAC. With a reliable suite of outcome-based efficacy tests, they can begin to do the same thing with anti-aging formulations. Such tests would deliver valuable guidance in the development of condition-specific formulations—with performance results unique to the actual formulation.
Consumer education. Consumers continue to seek better information about the natural products they purchase. Cell-based efficacy tests deliver valid pre-clinical test results for actual products that target primary wellness concerns. At present, most products rely upon scientific information that is generic to constituent ingredients. Product- and condition-specific validation benefits consumers and the companies that provide it.
Cell-Based Assays
Cell-based assays have been developed to determine product potency, functionality and mode of action of nutraceutical and biopharmaceutical products
Live human cell lines are grown in a laboratory environment. These cells are then exposed to certain harmful substances, such as free radicals, ultraviolet light irradiation and inflammatory agents. Other substances (such as antioxidant-containing natural products) are then introduced to see if they protect the cells from these harmful sources of damage.
Cell-based assays are well suited to the current industry imperative because: (1) they are well established in research; (2) they are versatile; and (3) they represent a cost-effective pre-clinical alternative to in vivo animal or human testing. As with any assay short of in vivo testing, there are limitations. But if these limitations are recognized and kept in focus, cell-based assays bring companies one step closer to in vivo clinical testing.
References furnished upon request.
About the authors: Ronald Prior, PhD, is an adjunct professor, Dept. Food Science, University of Arkansas, Fayettevile, AR, and science consultant for Brunswick Laboratories. Boxin Ou, PhD, is the chief science officer at Brunswick Laboratories, Inc. David Bell, MBA, is the president of Bell Advisory Services. Qiuyan Zhao, MD, MBA, is the director of Biology & Immunology at Brunswick Laboratories, Inc. HuiLin Wei, MD, PhD, is a chief scientist at Brunswick Laboratories, Inc. If you have any questions regarding the information or references in this article, contact Mr. Bell at 617-575-9822 or dnb@belladvisory.com.
Brunswick Laboratories puts natural products to the test using its cell-based assays.
At Brunswick Labs, cell-based assays have been modified to make them applicable to a range of product formats and high-throughput. This represents a fundamental step forward in commercially viable cell-based methods in the natural products industry. A critical feature of the tests is that they address primary wellness factors. Each test uses a specific marker—a measureable biological parameter—to quantify performance.
For this study, Brunswick researchers selected three cell-based assays: Cellular Antioxidant Assay (CAA), Cellular Anti-inflammation Assay (NF-kB) and Cellular UVA/UVB protection (UVA/UVB). A central objective of the cell-based testing program is to provide analytical tools for industry that are relevant for a range of products, from pure compounds to consumer products.
Brunswick tested several standardized benchmarks with each of the Cardinal Wellness Factors. Samples were selected from each of these categories: reference compounds, extracts and concentrated foods. These included:
• Pure compounds: naringenin
• Food extracts: cocoa, grape seed
• Complete foods: acai, camu camu
Here is what Brunswick researchers found. Quercetin was used as positive control in cell-based assays. The quercetin equivalent (QE) was then calculated by reference to the curve established with pure quercetin in those assays. All results are expressed using the QE per gram sample (QE/g). This allows for useful comparison across different product types.
The NF-kB assay demonstrates the sample’s ability to inhibit NF-kB, an important transcription factor that adjusts the gene expression for many inflammatory factors. As the results show, NF-kB activity is inhibited by all tested samples at different levels. The cocoa extract has the highest potentiality to inhibit inflammatory factors among these five samples.
The CAA assay demonstrates the sample’s ability to reduce cellular free radical levels generated by AAPH (2,2’-azobis-2-methyl-propanimidamide dihydrochloride), a water-soluble azo compound that is used extensively as a free radical generator. As shown in the table, all samples demonstrate significance, with the cocoa extract exhibiting the most powerful free radical scavenging activity, followed by grape seed extract and naringenin.
The UVA/UVB assay demonstrates the sample’s ability to reduce UVA/UVB-induced free radicals in skin cells. In this assay, a human skin cell line was cultured to confluency, testing compounds, positive controls (antioxidants) and negative controls (vehicles alone) were added to the cells, and cells were then exposed to UVA/UVB light treatment. Free radicals generated upon UVA/UVB irradiation were measured. The decreased amount of free radical generation was calculated and expressed as free radical scavenging activity. Four of the five samples demonstrated significant free radical reduction.
Going a step further, Brunswick researchers also saw interesting results when they converted to standard dosages. For example, a standard dose of grape seed extract might be 200 mg, while a standard dose of acai powder might be 5 grams. On a dose-equivalent basis, NF-kB performance then becomes 84 QE/g for grape seed extract and 115 QE/g for camu camu powder. The same “recommended dose” conversion can also be extended to the other assays as well.
Conclusions
The central message from this study is that natural products in a variety of standardized forms demonstrate significant efficacy against primary wellness factors. This is indicative of the multi-faceted anti-aging effects of natural products—in particular, those with a complex matrix of active constituents.
Another meaningful result of this study is the broad correlation between Total ORAC and cell-based outcomes. Although preliminary, these results support the view that Total ORAC is a useful tool for suggesting not only antioxidant, but also other important anti-aging outcomes.
References furnished upon request.
