Overview of the Nutraceutical Industry
As we understand more about human health, we continue to develop new treatments and lifestyles that are aimed at improving athletic performance and increasing not only life span but also health span. Preventative measures against the common ailments and concerns of aging and strategies aimed at maximizing physical and mental performance have driven the rapid growth of the nutraceutical industry that includes functional foods, dietary supplements, and other products that claim to provide benefits beyond basic nutrition.
With the changing landscape of the industry and increasing competition between various nutraceuticals and their providers, there is also an increase in awareness and skepticism of their efficacy. These products are far less regulated by the FDA compared to pharmaceuticals, and there is a desire and need for scientific validation of their claimed benefits.
A time- and cost-efficient way to obtain these scientific data is the implementation of in vitro assays. This blog post gives an overview of current roles of in vitro assays for product development and ways they can evolve to support and revolutionize the increasing demands of this growing industry.
The Current Role of In Vitro Assays in the Nutraceutical Industry
In vitro assays play a pivotal role in the development of nutraceutical products. These laboratory-based techniques allow researchers to examine the biological effects of products in a controlled environment, away from the full complexity of a living organism. The primary advantage of in vitro assays lies in their ability to provide rapid and detailed insights into the mechanisms of action, potential efficacy, and safety of nutraceutical compounds. By employing these assays early in the development process, nutraceutical companies can identify promising candidates and eliminate those with less favorable profiles, thereby optimizing the product pipeline. The data generated also helps in understanding how these compounds interact at a cellular level, providing a foundation for subsequent, in vivo studies and clinical trials.
Antioxidant Assays
Antioxidant activity is one popular claim of many products, and assays that assess this claim focus on measuring the ability of products to neutralize free radicals or reactive oxygen species (ROS). The DPPH and ABTS assays are examples of this, and they are widely used due to their simplicity, rapidity, and low cost. In this assay, DPPH or ABTS (both ROS compounds) are combined with the test product to measure depletion of the DPPH or ABTS in solution. These are colorimetric tests; changes in the absorption/emission profiles of the solutions on a spectrophotometer are proportional to amount of ROS elimination. The major drawback of these experiments is that they are conducted outside of a biological context – they involve an isolated system of compounds in a tube. Testing the antioxidant efficacy of a product within the context of a cell would provide more useful and physiologically relevant information.
Cytotoxicity Assays
Cytotoxicity assays determine the safety profiles of products, which are necessary to bring products to the market. By identifying toxic compounds early in the development process, cytotoxicity assays help streamline the selection of safer and more effective ingredients. This ensures that only the best candidates move forward in the development pipeline, ultimately leading to higher-quality nutraceutical products.
Briefly, cells are treated with the products, followed by measurements like cell viability, cell proliferation, and cell-death rates. Several techniques are employed in cytotoxicity assays, including MTT, XTT, and LDH assays. MTT and XTT assays measure cell viability based on mitochondrial activity, while LDH assays detect cell membrane integrity by measuring lactate-dehydrogenase release. Quantitative approaches to ascertaining cytotoxicity data yield information about not only whether a compound is toxic, but also the dose range in which it is safe. Accurate interpretation of cytotoxicity data is crucial for ensuring that nutraceutical products are safe for consumption. These data help inform regulatory submissions and supports claims of product safety and efficacy.
Anti-inflammatory Assays
Products that claim to have anti-inflammatory effects are introduced to cultured cells that are treated with an agent that is known to induce inflammation. These cells are either treated with the product before or after induction of inflammation, and the levels of inflammatory markers, such as cytokines TNF-alpha and IL-6, are quantified. These assays can also be colorimetric; supernatants from the cultures of product-treated cells are tested with enzyme-linked, immunosorbent assays (ELISAs) for cytokine quantification.
Although these cytotoxicity, anti-oxidant, and anti-inflammatory assessments are relatively simple assays, they provide a starting point to characterize the effects of nutraceutical products on human health. However, translation of these data to actual results is unclear. This murkiness in addition to the FDA’s relatively loose requirements for marketing a nutraceutical product contributes to the public’s skepticism of and uncertainty in the industry.
Future Trends in In Vitro Assay Technologies for Nutraceuticals
As the nutraceutical industry continues to grow and competition between companies and their products intensifies, predictive scientific data will be crucial in differentiating leading brands and products from the milieu. As with pharmaceutical companies, the key to achieving this is companies prioritizing and investing in the scientific tools and developments necessary to create physiologically relevant models that can be used to predict efficacy. Contract research organizations (CROs) often collaborate with companies in the health and wellness industry to provide specialized expertise and resources. These partnerships enable the systematic testing and refinement of product ideas, ensuring that they are both effective and safe for consumer use.
The choice of cell types and formats is critical in designing in vitro models. Commonly used cell lines include human epithelial cells (e.g., Caco-2, which are used to model intestinal absorption), and hepatic cell lines (e.g., HepG2) for studying metabolism and detoxification. Immune cell lines, such as macrophages and lymphocytes, are often employed to investigate immunomodulatory effects. However, these immortalized cell lines have biological characteristics that are different from their counterparts in a human. Instead, the use of iPSCs or primary cells derived directly from patients would be more translatable and predictive.
In addition to traditional, 2D cell cultures, advanced formats like 3D cell cultures (like using 3D bioprinting) and co-culture systems in which multiple cell types are cultured together (like organ-on-a-chip platforms) are increasingly being used. These formats add complexity to better mimic in vivo environments, providing more relevant data on how nutraceuticals act within intricate, biological systems.
New assay types can also be used to capture more comprehensive datasets to understand how biological systems respond to the products. The integration of omics technologies, such as genomics, proteomics, and metabolomics, is providing deeper insights into the molecular mechanisms of action of nutraceuticals. High-content imaging and high-throughput screening further enhance the ability to analyze multiple parameters simultaneously, leading to more robust and reliable results. One such strategy is cell painting, which can be used for many purposes, from safety profiling to determining mechanisms of action.
Conclusion
With the advent of new nutraceutical products, new approaches are needed to better understand, characterize, and predict their effects on health. By leveraging scientific research, companies can make informed decisions that minimize the risks of running into regulatory hurdles and market-acceptance challenges, yielding enhanced product quality and performance. Currently, in vitro assays, though accepted as part of the product-development process, are typically conducted in simple, minimalist systems that do not predict clinical outcomes. New approaches to studying products can improve the chances of success. Scientific innovations can enhance the predictive power of in vitro assays, leading to more effective and safer nutraceutical products.
