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There are many similarities between research on combinatorial chemistry and natural products and research on dietary supplements and botanicals in the NIH Botanical Research Centers. The technologies in the centers are similar to those used by other NIH-sponsored investigators. All centers rigorously examine the authenticity of botanical dietary supplements and determine the composition and concentrations of the phytochemicals therein, most often by liquid chromatography–mass spectrometry. Several of the centers specialize in fractionation and high-throughput evaluation to identify the individual bioactive agent or a combination of agents. Some centers are using DNA microarray analyses to determine the effects of botanicals on gene transcription with the goal of uncovering the important biochemical pathways they regulate. Other centers focus on bioavailability and uptake, distribution, metabolism, and excretion of the phytochemicals as for all xenobiotics. Because phytochemicals are often complex molecules, synthesis of isotopically labeled forms is carried out by plant cells in culture, followed by careful fractionation. These labeled phytochemicals allow the use of accelerator mass spectrometry to trace the tissue distribution of 14C-labeled proanthocyanidins in animal models of disease. State-of-the-art proteomics and mass spectrometry are also used to identify proteins in selected tissues whose expression and posttranslational modification are influenced by botanicals and dietary supplements. In summary, the skills needed to carry out botanical centers’ research are extensive and may exceed those practiced by most NIH investigators.
Under the provisions of the Dietary Supplement Health and Education Act, dietary supplements in use on or before October 25, 1994, may be sold in the United States (1). One provision of DSHEA was the creation of the Office of Dietary Supplements (ODS) at NIH to promote scientific study on dietary supplements. As part of this mandate, ODS supports several NIH Botanical Research Centers whose purpose is to conduct state-of-the-science research on the efficacy, safety, and mechanisms of action of botanicals and other ingredients used in dietary supplements. Research on botanical supplements in centers represents therefore a particularly complex set of endeavors. The purpose of this presentation is to describe some of the complexities involved in studying botanical ingredients and to show how research in the botanicals centers is being carried out and is setting new standards for all of NIH research.
The similarities and differences among foods, dietary supplements, botanicals, and drugs must be distinguished. They are all xenobiotics. Foods are plants, animals, and other previously living forms that are safe although they are not necessary nutritious. Dietary supplements are food-derived and are also generally regarded as safe under conditions of use although they are not necessarily nutritious. Botanicals and their extracts used as ingredients in dietary supplements are generally associated with alleged improvements in health and risk of disease; they are assumed to be safe although they may also have unanticipated toxic effects when consumed as dietary supplements rather than under conditions of use associated with traditional medicine applications. Unlike vitamins and minerals, botanical supplements are rarely significant sources of nutrients. Drugs are designed to be efficacious for specific health uses and safety is evaluated in the context of risk-benefit relationships. They are rarely significant sources of nutrients.
Much of NIH-funded research is characterized by a need to understand the molecular or mechanistic basis of disease and of the agents that alleviate or treat the disease. To achieve these goals there is an emphasis on reducing complex problems to manageable sizes—a reductionist approach. A criticism often cast at dietary supplement research is that a dietary supplement is not a single compound nor is its composition tightly controlled. However, its complex nature may be its strength (2). It may contain multiple compounds with primary bioactivity as well as adjunct bioactivity (ie, properties that enhance the delivery or activity of the bioactive components). An example of this is (−)-epigallocatechin-3-gallate (EGCG), the most bioactive component of green tea. On its own, EGCG is unstable and readily degraded before it is consumed, well before reaching its biological target. It has greater biologic activity in green tea than alone because of the presence of other catechins (3). Investigators outside of botanicals research are familiar with the need to provide carriers for bioactive agents.
In research being carried out at botanical centers, investigators first perform experiments to identify the principal bioactive components by fractionating extracts of the dietary supplement or botanical and then testing the activity of each fraction in a suitable cell culture model or high-throughput assay system. This approach is the same as carried out in drug companies and university research laboratories that examine compounds generated by combinatorial chemistry or from natural nonedible products. Once compounds with possible bioactivity have been identified (whether from dietary supplements and botanicals or high-throughput assays), they are subjected to standard absorption, distribution, metabolism, and excretion experiments in small and then larger animals. Investigators also carry out dose-range experiments in the whole animal model and determine whether toxicity occurs with acute or long-term exposure. What distinguishes botanical centers research from other NIH research is that botanicals investigators then examine the activity of the whole dietary supplement at doses that deliver the same amount of the bioactive material as isolated from the supplement. This enables the investigator to assess whether other components of the dietary supplement or botanical matrix have synergistic effects.
Fractionation of botanicals and dietary supplements is carried out at the University of Illinois-Chicago (UIC; to isolate compounds with estrogenic activity), Iowa State University (to isolate bioactive compounds from Echinacea and Hypericum species), and Pennington Biomedical Research Center-Rutgers University (to isolate compounds that suppress the metabolic syndrome). Identification of the bioactive compounds requires the use of reverse- and normal-phase HPLC, high-resolution mass spectrometry, 1H- and 13C-nuclear magnetic resonance spectrometry, and other modern analytical methods.
The high-throughput methods used to find the bioactivities of phytochemicals in botanicals are identical to those used to examine synthetic drugs. For instance, at the UIC botanicals center, phytoestrogen activity is examined using an in vitro receptor binding assay and cultured cell approach using a human breast tumor MCF-7 cell line with an alkaline phosphatase or luciferase reporter genes (4). The same center has used ultrafiltration mass spectrometry to assess the binding of phytoestrogens to estrogen receptors (Fig 1) (5).
Determining the composition of the dietary supplement preparations is vital to botanical centers research. To ensure that research studies use dietary supplements that reflect those available to the public, NCCAM set up a working group to review the applications it receives (6). Applications that have inadequate analytical methods, do not have a product with known composition, or do not have plans to assess degradation are not approved for funding until these limitations are dealt with.
Several of the botanical centers are engaged in clinical trials of botanical agents from the United States and other countries with different medical cultures. The Memorial-Sloan-Kettering Cancer Center specializes in materials used in Chinese traditional medicine. In such cases, much use is made of liquid chromatography–mass spectrometry (LC-MS) to validate the composition of the dietary supplements as well as the concentrations of the bioactive compounds in blood, urine, and other biological fluids and tissues.
Most methods used to study bioavailability, pharmacokinetics, and metabolism of specific compounds in dietary supplements and botanicals are based on the qualitative and quantitative use of LC-MS. These studies have been carried out in humans and small and large animals as well as in vitro models. Richard van Breemen’s group at UIC used Caco-2 cells combined with LC-MS to estimate the rate of intestinal uptake and metabolism of specific plant phytochemicals (Fig 2) (7,8). Chao-Chen Wang at the University of Alabama at Birmingham (UAB) applied a microsampling technique with LC-MS to determine the concentrations of genistein and its 7-O-β-D-glucuronide in the aqueous humor of rats fed genistein in the diet (Fig 3) (9). Elsa Janle at Purdue University applied an automated collection system to obtain blood samples from nonanesthetized, free-moving rats (10).
The eclectic nature of the botanical centers in some cases allows for unusual combinations of methods. Investigators at the Purdue-UAB botanicals center are applying accelerator mass spectrometry (AMS) to follow 2 very rare radioactive isotopes (14C and 41Ca) in biological experiments. The extreme sensitivity of AMS allows for the detection of one 14C or 41Ca atom in 1015 atoms of nonradioactive carbon or calcium. Connie Weaver at Purdue University administered a single dose of 41Ca to postmenopausal women to examine the effects of phytoestrogen dietary supplements on the turnover of calcium in bone; this study has been going on for 5 y in the same subjects (11). Mary Ann Lila, at University of Illinois-Urbana-Champaign, manufactures 14C-labeled complex phytochemicals such as proanthocyanidins with plant cell culture systems (12). Although these can be used in conventional radiotracer experiments to assess uptake and metabolism, the extreme sensitivity of AMS allows for careful tissue-by-tissue analysis of proanthocyanidin metabolism using only very small doses (~50 nCi).
The combination of dietary supplements and botanicals research with modern proteomics and protein MS for several years seemed to many an oxymoron. However, if dietary supplements are to have biological action, they will affect protein abundance and activity. Helen Kim and colleagues at UAB fed rats a diet containing grape seed extract (GSE) for 6 wk and identified 14 brain proteins that were changed by GSE (13). They applied a combination of 2D-isoelectric focusing and SDS-PAGE resolution of the brain proteins, image analysis of stained gels, peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and peptide sequencing by LC-tandem MS (Fig 4). These studies identified creatine kinase as a candidate brain protein undergoing changes in posttranslational modifications that were induced by GSE.
Tissue profiling and imaging are other MS applications being carried out in botanical centers. Stephen Barnes at UAB is examining the spatial changes in protein expression and modification in the lens as part of a project on the role of phytochemicals in the formation of cataracts. The lens proteins are unique in that they are synthesized once and remain in the lens from womb to tomb. Compounds that alter the rate of light-induced singlet oxygen production in the lens may protect or enhance oxidation and oligomerization of the lens proteins and hence cause the loss of their chaperone function that maintains protein solubility. These experiments are being carried out using MALDI-TOF MS (Fig 5) and high-resolution Fourier transform-ion cyclotron resonance mass spectrometry (Fig 6).
Botanicals and dietary supplements may affect gene expression. DNA microarray analyses are being carried out by Ski Chilton’s group at Wake Forest University to examine the effects of n-3 fatty acids in fish oil supplements to determine the biochemical pathways that undergo changes in regulation. As for many other areas of NIH clinical research, the effects of single nucleotide polymorphisms may segregate subjects consuming dietary supplements and botanicals into responders and nonresponders. Similarly, supplements may have their effects epigenetically by altering gene methylation (14) and by modulating acetylation-deacetylation (15,16) and potentially lysine methylation-demethylation of histones.
Research in the botanical centers demands the application of the best technologies in current use. The demands brought by the complex nature of the dietary supplements and botanicals are setting new standards that will have impact throughout NIH-funded research.
Research support for the Purdue University-University of Alabama at Birmingham Botanical Center for Age-related Disease is provided by a grant (P50 AT00477, Connie M. Weaver, PI) from the National Center for Complementary and Alternative Medicine (NCCAM) and the NIH Office of Dietary Supplements (ODS). Research Support for the Pennington Biomedical Research Center LSU System-Rutgers University Center for the Study of Botanicals and Metabolic Syndrome is provided by a grant (P50AT002776, William T. Cefalu, PI) from the NCCAM and ODS. Funds for purchase of the mass spectrometers came from several NCRR Shared Instrumentation grants (S10 RR06487, RR11329, RR13795, RR17261, RR19231). Other funds were provided by the UAB Health Services Foundation General Endowment Fund. Support for the University of Illinois at Chicago Botanical Center was provided by NIH grant P50 AT00155 jointly funded by ODS, NCCAM, and the Office for Research on Women’s Health. This publication was made possible by grant number P01 ES012020 from the National Institute of Environmental Health Sciences and ODS and by grant 95P50AT004155 from NCCAM and ODS. The contents are the responsibility of the authors and do not necessarily represent the views of the funding agency.
We thank David Stella for providing the unpublished data in Figure 5 and Kerri Barrett, Shannon Eliuk, and Matthew Renfrow for the unpublished data in Figure 6. Stephen Barnes is an appointed member of the National Advisory Council for NCCAM. Diane Birt served on the National Toxicology Program (NIEHS) Board of Scientific Counselors during most of the time that this research was being conducted. Floyd H Chilton serves on the Board of Directors and is a shareholder of Pilot Therapeutics Inc. Norman R Farnsworth is a consultant to Pharmavite LLC. Ilya Raskin is a consultant for Phytomedics, Inc. Connie M Weaver is on the advisory boards of Pharmavite and Wyeth. She receives grant support from Wyeth, and reviews research proposals for the United Soybean Board. All authors contributed text and provided editorial advice for the manuscript.