| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
The Science of Botanical Supplements for Human Health: A View from the NIH Botanical Research Centers |
1 From The Center for Research on Botanical Dietary Supplements (DFB, MPW, CAL, LW, JB, AKSS, GAK, PAM, ESW, QL, SCH, WJM, and JPP), the Department of Food Science and Human Nutrition (DFB, CAL, AKSS, PAM), the Interdepartmental Genetics Graduate Program (CAL and LW), the Department of Genetics, Development, and Cell Biology (LW and ESW), the Department of Chemistry (JB and GAK), the Department of Horticulture (MPW), and the Department of Agronomy (MPW), Iowa State University, Ames, IA; the US Department of Agriculture-Agricultural Research Service, North Central Regional Plant Introduction Station (MPW), Ames, IA; the Department of Microbiology, University of Iowa, Iowa City, IA (WJM and JPP); and the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT (QL and SCH)
2 Presented at the workshop "The Science of Botanical Supplements for Human Health," held at Experimental Biology 2007, Washington, DC, 28 April 2007. 3 Supported by grant number P01 ES012020 from the National Institute of Environmental Health Sciences (NIEHS) and the Office of Dietary Supplements (ODS) and by grant 95P50AT004155 from the National Center of Complementary and Alternative Medicine (NCCAM) and ODS, NIH. The contents are the responsibility of the authors and do not necessarily represent the views of the funding agency. 4 Reprints not available. Address correspondence to DF Birt, Department of Food Science and Human Nutrition, Iowa State University, 215 MacKay Hall, Ames, IA 50011. E-mail: dbirt{at}iastate.edu.
ABSTRACT
Ongoing studies have developed strategies for identifying key bioactive compounds and chemical profiles in Echinacea with the goal of improving its human health benefits. Antiviral and antiinflammatory–antipain assays have targeted various classes of chemicals responsible for these activities. Analysis of polar fractions of E. purpurea extracts showed the presence of antiviral activity, with evidence suggesting that polyphenolic compounds other than the known HIV inhibitor, cichoric acid, may be involved. Antiinflammatory activity differed by species, with E. sanguinea having the greatest activity and E. angustifolia, E. pallida, and E. simulata having somewhat less. Fractionation and studies with pure compounds indicate that this activity is explained, at least in part, by the alkamide constituents. Ethanol extracts from Echinacea roots had potent activity as novel agonists of TRPV1, a mammalian pain receptor reported as an integrator of inflammatory pain and hyperalgesia and a prime therapeutic target for analgesic and antiinflammatory drugs. One fraction from E. purpurea ethanol extract was bioactive in this system. Interestingly, the antiinflammatory compounds identified to inhibit prostaglandin E2 production differed from those involved in TRPV1 receptor activation.
Key Words: Alkamide • antiinflammatory • antipain • antiviral • cichoric acid • Echinacea • Echinacea angustifolia • Echinacea pallida • Echinacea purpurea • Echinacea sanguinea • Echinacea simulate • HIV • TRPV1 receptor
CENTER ORGANIZATION
The primary goal of the Iowa Center for Research on Botanical Dietary Supplements is to improve our understanding of the characteristics of Echinacea, Hypericum, and Prunella that contribute to human health and thereby pave the way for optimizing these supplements for study in future clinical trials. Our center focuses on infection with an emphasis on antiviral, antiinflammatory, and antipain activities. This article summarizes some of our work on Echinacea.
A central strategy of the Iowa center has been to use biological diversity to help to identify active constituents and determine mechanisms of action. It is tempting to consider the diversity of these plant genera and the complexity of their constituents as barriers to understanding their potential health benefits. However, the range of variation in these plants, when systematically analyzed, provides a strong foundation on which to develop the strategies and tools needed to produce the most efficacious products for a growing body of consumers.
The center is organized into 3 cores: Germplasm and Phytochemical Profiling; Separations, Structure, Bioavailability; and Administration, Data Management, Statistics, and Bioinformatics. Three projects are supported: defining antiviral activities in Echinacea, Hypericum, and Prunella species; antiinflammatory activity of Echinacea, Hypericum, and Prunella species; and pain-receptor-mediated antiinflammatory activity of Echinacea and Hypericum species.
PRODUCTION OF WELL-CHARACTERIZED PLANT MATERIAL
A unique resource of the center is our collection of a genetically diverse set of well-documented plant populations of Echinacea and the conservation of these genetic stocks at the US Department of Agriculture-Agricultural Research Service North Central Regional Plant Introduction Station (NCRPIS). This resource gives us strength in controlling the production of plant materials and links with our expertise in genomic analysis and broad-based plant metabolic profiling and our ability to integrate complex datasets by using bioinformatics and other statistical tools. The NCRPIS, which is located at Iowa State University, is one of the main active gene banks in the US National Plant Germplasm System, and it conserves extensive collections of known-source medicinal plants, with an emphasis on Echinacea and Hypericum.
Since the late 1990s, the NCRPIS has acquired >150 distinct wild populations (or accessions) of Echinacea, representing all recognized species and varieties from throughout their ranges, including 2 federally endangered taxa, E. laevigata and E. tennesseensis. The genetic integrity of these populations is preserved by regenerating seed samples under controlled conditions in screened field cages with insect pollinators, typically honeybees (1). To ensure unbiased sampling of Echinacea populations for regeneration, research has been directed toward understanding seed dormancy and methods to overcome it (2, 3).
During seed regeneration, taxonomic identities are verified and populations are characterized for phenotypic traits with a standardized descriptor list. Phenotypic descriptors along with images and detailed passport data describing each accession are available from the Germplasm Resources Information Network database (4). Seeds of all available accessions are distributed for research and educational purposes at no cost to the user.
Echinacea samples used by center researchers have typically been produced from NCRPIS accessions. By using these accessions produced under known conditions, we minimize both the genetic and the environmental components of biochemical variation in the resulting products, which increases the overall repeatability of bioassays. Long-term, replicated field plantings of the 3 primary medicinal species, E. angustifolia, E. pallida, and E. purpurea, were established in 2003 to optimize root production and determine the effects of disease and shading on plant survival and productivity. Dried roots from these plantings have been a major source of plant material for our research projects. Additional accessions have also been supplied to researchers as root samples from plants used for seed regeneration, after successful completion of the seed-production process, and as leaf and pollen samples. After production and processing, all plant samples for center use are inventoried with a standardized coding system, and unextracted dried plant materials are packaged in nitrogen and stored frozen at –20°C. In addition to the supply of plant materials for bioassays, a carefully selected, diverse array of 
40 Echinacea accessions is being characterized biochemically for alkamides and caffeic-acid derivatives and genetically for both nuclear and chloroplast DNA variation. These characterization data should be valuable for describing the extent of chemical variation, elucidating taxonomic relations, and providing a framework for phylogenetic analysis and future bioassay.
ANTI-HIV ACTIVITIES OF ECHINACEA
The antiviral activities of Echinacea extracts and its metabolic constituents are surprisingly poorly studied. A recent, prominent study identifying a lack of efficacy of E. angustifolia extracts against rhinovirus infection has discouraged additional clinical antiviral studies on botanical extracts (5). However, recent in vitro studies within our center identifying antiviral activities in Echinacea against HIV are promising. Constituents responsible for the anti-HIV activity in our plant extracts were identified through bioactivity-driven fractionation studies. A example of our success with this approach is shown here for E. purpurea.
Initially, extracts from 6 Echinacea species were tested for inhibition of replication of the HIV molecular clone pNL4-3. All HIV infectivity assays were performed in the HeLa37 cell line that expresses the viral receptor and co-receptors (6). Known titers of HIV were added to cells in the presence of the botanical extract. Cells were fixed and immunostained for viral antigens at 40 h after infection. Cells immunopositive for HIV were represented as percentage of control values. Extracts from E. purpurea consistently provided the most robust inhibition of HIV with little or no cellular cytotoxicity. Increasing concentrations of E. purpurea extract had antiviral activity with a 50% inhibitory concentration (IC50) of 2.4 µg/mL (Figure 1
). This species contains the anti-HIV compound cichoric acid and thus was anticipated to inhibit HIV replication (7). To determine whether constituents other than cichoric acid had antiviral activity, E. purpurea extracts were fractionated. Seven fractions were generated; no fraction had detectable cytotoxicity or endotoxin contamination (Figure 2A). Fraction 1, the most polar, had significant anti-HIV activity; the other fractions had no antiviral activity. HPLC analysis of fraction 1 documented that a series of caffeic acid derivatives and other polyphenolics including cichoric acid were present (Figure 2B). Subfractionation into 9 subfractions yielded 6 with anti-HIV activity (Figure 3); HPLC analysis of these subfractions showed that each fraction was composed of different constituents. Subfractions 1–6 all contained numerous constituents including cichoric acid; subfractions 1–3, 1–4, 1–7, and 1–8 exhibited some antiviral activity and contained multiple constituents that absorbed light at 330 nm and likely are polyphenolics. Interestingly, subfraction 5 absorbed light at 254 nm but not at 330 nm and may contain glycosylated flavanoids. We anticipate that we will successfully continue to use this bioactivity-driven fractionation approach to identify the botanical compounds responsible for the antiviral activity.
|
|
|
The antiinflammatory activity of Echinacea extracts, fractions, and constituents was assessed with RAW264.7 cells treated with and without lipopolysaccharide, and prostaglandin E2 (PGE2) accumulation was measured. This widely used screen for antiinflammatory activity is based on PGE2 production arising from cyclooxygenase-1 and -2 activation, a key event in inflammation. Initial studies showed that soxhlet ethanol extracts of Echinacea provided the greatest antiinflammatory activity. Activity of soxhlet extracts of E. purpurea, E. angustifolia, E. pallida, E. tennesseensis, E. simulata, and E. sanguinea at 15 µg/mL, which were harvested during the fall of 2003, 2004, and 2005, did not differ significantly by repeat extraction or harvest. E. sanguinea inhibited PGE2 production to the greatest extent; E. angustifolia, E. pallida, and E. simulata inhibited less; and E. tennesseensis and E. purpurea extracts were not inhibitory at this concentration (Figure 4) but were at higher concentrations (8). Concentrations of alkamides and ketones in these extracts were determined, but these compounds did not simplistically explain bioactivity. For example, E. angustifolia and E. purpurea were rich in Bauer Amide 8, which was not abundant in E. pallida. There was relatively little cytotoxicity of Echinacea even at doses 10-fold higher than those assayed for antiinflammatory activity. All extracts reported here were negative for endotoxin contamination.
|
). Our observations are consistent with observations of others showing antiinflammatory activity with alkamide extracts containing Bauer alkamides 8 or 9 (9). However, because E. pallida does not contain amides 8 or 9; other compounds, possibly ketones, must contribute the activity.
|
). Subfractionation of E. angustifolia fraction 3 showed that the less polar subfractions have antiinflammatory activity, with subfractions 3D and 3E exhibiting the greatest reduction in PGE2 accumulation (data not shown). Alkamides are present in subfractions 3B-E: 3D contains alkamides 5, 8, 9, and 14; 3E contains 10 and 11; 3B contains alkamide 1; and 3C contains alkamides 1, 2, 3, 5, 12, 13, and 14.
This series of studies suggests that Echinacea alkamides may contribute to observed antiinflammatory activity because they are readily identified in the active extracts and exhibit appreciable activity. However, results with purified alkamides indicate that single compounds failed to completely account for antiinflammatory activity and, thus, may interact with each other or with other compounds to explain the observed antiinflammatory activity.
MEDIATION OF PAIN-RECEPTOR ACTIVITY BY ECHINACEA
TRPV1 in pain and inflammation
TRPV1 (transient receptor potential channel, vanilloid subfamily member 1, VR1) (10) is a ligand-gated cation channel pain receptor. It was initially cloned by using capsaicin, the potent compound of hot peppers, which is a strong ligand (11). TRP channels are extremely nonselective; TRPV1 is activated by factors including capsaicin, protons, noxious heat (>42°C), the endocannabinoid anandamide, lipoxygenase product, and ethanol (12). It is considered to be a key integrator of external stimuli, both chemical and physical, into the common signal of inward ion currents (11). Receptors from the TRP channel family, as well as other classes of receptors such as CB, ASIC, and Trek-1, are predominantly expressed in sensory tissues, such as nociceptors and skin. These receptors are thought to provide feedback to sense, transmit, and integrate pain and associated inflammatory responses (11). Growing evidence indicates that TRPV1 acts as an integrator of inflammatory pain and hyperalgesia, making it an excellent potential target for analgesic or antiinflammatory agents. Because of its importance in pain and inflammatory responses, TRPV1 protein structure-activity relations are being analyzed extensively to determine which TRPV1 sites are important for interaction with each of its ligands. TRPV1 is also desensitized by its ligands. Desensitization of TRPV1 after its activation in these neurons is crucial in blocking pain transmission (11). Therefore, compounds that serve as agonists have potential use as analgesics or antiinflammatory agents. In addition, capsaicin can produce a hypotensive effect in spontaneously hypertensive rats, indicating that activation of TRPV1 may contribute to the treatment of hypertension (13).
Echinacea species comparison
Because of reported effects on inflammatory pain, we investigated the effects of Echinacea extracts on TRPV1-dependent inward ion currents by using transient expression of TRPV1 in frog oocytes (11). In this system, TRPV1 cRNA is injected into healthy oocytes and is expressed transiently; then the TRPV1-expressing oocytes are bathed in Echinacea extracts or capsaicin for 10 s. Whole-cell currents are recorded by a 2-electrode voltage clamp recording system. Extracts of roots of E. angustifolia evoked a current 10-fold greater than a saturating dose of capsaicin (Figure 6). Echinacea extracts rapidly desensitize the TRPV1 channel even in the absence of added calcium, which suggests that TRPV1 activation may occur through a mechanism different from that of capsaicin. Leaf and flower extracts also activated TRPV1.
|
FUTURE OBJECTIVES
Our center's overarching objectives include identifying compounds contributing to the antiviral, antiinflammatory, and pain-control effects of Echinacea and to Echinacea toxicity. We further seek to assess the influence of plant species and population on bioactive constituents and to understand their mechanisms of action, in particular, the cellular signaling pathways and critical receptors and the effects of their interactions. We will assess the bioavailability of key constituents of Echinacea supplements to fill this important gap, because bioavailability likely plays a key role in translating the results of bioassays to potential human health effects.
ACKNOWLEDGMENTS
The contributions of the authors were as follows—DFB: directs the Center and supervised the research of CAL; MPW: co-leads the Germplasm and Phytochemistry Core; CAL: conducted the studies reported in Figures 4 and 5; LW: conducted the studies reported in Figure 6; JB: synthesized alkamides; AKSS: conducted fractionation of Echinacea; GAK: supervised the research of JB; PAM: supervised the research of AKSS; ESW: supervised the research of LW; QL: instructed the Wurtele laboratory in the oocyte TRVP1 studies; SCH: consulted with the Wurtele laboratory in the interpretation of the oocyte TRVP1 studies; WJM: supervised the research of JPP; JPP: conducted the studies reported in Figures 1, 2, and 3.
DFB served on the National Toxicology Program (NIEHS) Board of Scientific Counselors during most of the time that this research was being conducted. The remaining authors had no financial or personal interests in any company or organization sponsoring the research, including advisory board affiliations.
REFERENCES
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
4 independent oocytes for each value). Current responses were normalized in each cell to responses obtained with capsaicin (10 µmol/L). Spinach extract (0.25 mg/mL) was used as the control. *P < 0.05, **P < 0.01, significantly different when compared with spinach control (Student's t test). Extracts evoked no responses in water-injected cells.