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The Science of Botanical Supplements for Human Health: A View from the NIH Botanical Research Centers |
1 From the Biotechnology Center for Agriculture and the Environment, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ(IR, DMR, AP, and BS), and the Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA (WTC)
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 Phytomedics Inc (Jamesburg, NJ); the NIH Center for Dietary Supplements Research on Botanicals and Metabolic Syndrome, grant 1-P50 AT002776-01; the Fogarty International Center of the NIH under U01 TW006674 for the International Cooperative Biodiversity Groups; and Rutgers University. The contents are the responsibility of the authors and do not necessarily represent the views of the funding agency.
4 Address reprint requests and correspondence to I Raskin, Biotechnology Center for Agriculture and the Environment, Foran Hall, Cook College, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901-8520. E-mail: raskin{at}aesop.rutgers.edu.
ABSTRACT
Botanical preparations have been used medicinally for thousands of years. Many commercially available botanical products are being marketed in the United States with little or no publicly available scientific validation of efficacy or consistency. For botanicals to be reliable for research purposes and consumer products, they must be standardized with sufficient quality controls to ensure consistent composition, safety, and potency. This includes uniform cultivation of source plants with controls to monitor for contamination from other species, pesticides, and environmental toxins. The active components of botanicals must be identified by activity-guided fractionation with the use of in vitro assays that require little test material followed by validation in vivo. Concentrations of active compounds within the botanicals can then be accurately measured to ensure the delivery of a dependable dose in the final product. The use of bioenhancing agents may be considered for compounds with poor bioavailability. Standardization of botanical therapeutics can only be achieved when the active compounds are identified and biological activity is confirmed, thus ensuring a consistent product.
Key Words: Botanicals • standardization • bioactivity • bioassay-guided fractionation • LC-MS analysis • bioenhancers
INTRODUCTION
Botanical medicine has been used throughout history similar to the way modern pharmaceuticals are used today (ie, to improve human health). Fossil evidence suggests that plants were used medicinally in prehistoric times (1). Although the first use of plant-derived pharmaceuticals may be difficult to pinpoint, the use of botanicals for human health is the basis for the modern pharmaceutical industry (2, 3).
Botanicals and the purification of their active ingredients have played a significant role in the history of mankind. The purification of an early botanical from opium poppy (Papaver somniferum) was described in great detail in 1806 in a report summarizing >57 studies (4). The botanical was very effective for pain and insomnia, leading to a world social crisis when its addictive nature was realized and a war ignited between China and England in the mid 19th century (4).
STANDARDIZATION OF BOTANICALS
Standardization of botanicals refers to the production of a plant preparation that is consistent in terms of composition and efficacy. The process begins with the source of the raw material. Botanicals should be well characterized and produced with the same quality as drugs but contain multiple compounds, many of which may not be identified.
Botanical products that are encapsulated dried plant material are generally regarded as being low quality because they are typically standardized on the basis of the dry weight of the plant material. Most botanical extracts are produced from a defined part of a plant and represent a large collection of compounds. Ideally, compounds within the extract that are responsible for the overall activity have been identified. The extract can then be standardized on the basis of the content of these compounds. Hypericin, for example, is thought to be the active compound in extracts from St John's Wort (Hypericum perforatum), which is used to prevent and treat some forms of depression (5). St John's Wort extracts can be standardized for hypercin content, and consistent doses can be obtained from different batches of plant material. Despite the apparent simplicity of this botanical, a study comparing 10 products showed variations from the label claim for hypericin ranging from 22% to 140% (6; GH Constantine, Y Karchesy, unpublished observations, 1999), which could account for variations in study results. In contrast, Ginseng products contain 2 families of active compounds—ginsenosides and eleutherosides—and are generally standardized for the total content of each class of compound. Although this form of standardization provides a means of comparing products, it does not ensure that large variations do not exist within each class of compound that could affect the effectiveness of a preparation. In another example, the widely advertised diet supplements from the South African plant Hoodia gordonii were found to contain no putative active ingredients and were likely derived from different plants (7).
If little or no information exists about the active constituents or mechanism of action of an herbal preparation, a less specific approach is taken, such as standardization by chromatographic fingerprinting. A consistent general profile can be used without regard for peak identity, or a marker compound can be used that may not be related to activity. The American Herbal Pharmacopoeia (8) advocates the use of marker compounds for characterizing products used in marketed dietary supplements but requires that a botanical monograph contain a high-performance thin layer chromatography fingerprint, which although useful for establishing a baseline chromatographic profile has significant specificity and sensitivity limitations. Other chromatographic techniques can be used for this purpose, each with its own advantages and limitations, such as HPLC with an ultraviolet or photodiode array detector, LC-MS, LC-MS/MS, and GC-MS.
Identifying active compounds in botanicals is often a challenge even though the process is based on simple principles. Usually, active compounds are isolated from a complex matrix by activity-guided fractionation, which uses chromatographic techniques such as HPLC for separating compounds, followed by activity measurements made with an in vitro assay related to in vivo activity [eg, measuring the inhibitory activity of aldose reductase, associated with diabetes (9)]. The process is complicated when botanicals have activity related to complex metabolic disorders that involve multiple metabolic pathways. Sensitive in vitro assays are essential to the process because fractionation can produce hundreds of fractions requiring testing in submilligram quantities. The active components may be major components of the botanical or minor components with high activity.
A major hypothetical advantage of botanicals over conventional single-component drugs is the presence of multiple active compounds that together can provide a potentiating effect that may not be achievable by any single compound. This advantage presents a unique challenge for the process of activity-guided fractionation, however, because the relative activity of fractions may decrease with greater purity and may even be lost entirely. The potentiating activity of the individually active components of botanicals can be assessed by recombining the fractions after separation followed by confirmation of biological activity. The complexity of the process increases when multiple in vitro assays are used for activity-guided fractionation, each yielding a different set of active compounds. Alternatively, the interactions of the botanical components could be negative, as in the case of the diminished bioavailability of caffeine resulting from the flavonoids in tea (10).
Compounds that are identified by activity-guided fractionation must be tested in appropriate animal models to confirm in vivo activity. If the active compounds are present in low concentrations, obtaining sufficient material for animal testing can be a serious problem. Some compounds are available commercially and may be purchased and tested directly. When the compounds are not available, they must be isolated from the plant extract on a larger scale than is typically used for initial activity-guided fractionation. The isolation may involve chromatographic procedures amenable to larger-scale separations, including various forms of solvent partitioning, medium-pressure liquid chromatography, and variations of countercurrent chromatography. Depending on the compound and information about its chemistry, it may be possible to synthesize the compound. Synthesis of the compounds may be difficult and expensive yet more economical and efficient than isolating a comparable amount from a plant. Pure active compounds are generally obtained for testing and quality control.
QUALITY CONTROL OF BOTANICALS
The composition of the total botanical extract must be standardized and free of any potential hazards. Raw plant materials for botanicals are obtained from plants that are cultivated specifically for producing the botanical, are obtained as a byproduct from the production of another product, or are collected from the wild. For example, ginseng is grown specifically for the production of related botanical products, whereas grapeseed extract is a byproduct of the commercial winemaking industry (11). Botanicals have been collected as they grow in their native environments, but this practice raises concerns about the consistency of end products and is considered an irresponsible collection technique that can lead to environmental destruction and the endangerment of species when not restricted to research purposes (12).
Plants grown specifically for the production of botanical extracts for basic research should ideally originate from a characterized and uniform genetic source with a taxonomic record of the genus, species, and cultivar or other additional identifiers. Records should be maintained for the source of the seed, locations and conditions of cultivation, and exposure to possible chemical treatments such as pesticides. Ideally, botanicals should be cultivated under controlled conditions such as hydroponics within climate-controlled greenhouses yielding consistent plant material. Only with tight control over the entire process of botanical production can seed-to-pill standardization be achieved (3). When source plants are procured in various regions of the world, records of plant identification, maintenance of voucher specimens, and biochemical profiling may suffice. Materials from international sources must also be carefully monitored for contamination from a variety of sources including coharvested weed plants, toxic phytochemicals, and heavy metals. Plants that were not directly treated with pesticides can become contaminated by chemical drift. Depending on the formulation, humidity, and temperature, pesticides can drift from the site of intended application by as much as 4.8 km (3 miles) (13).
Because the environment can significantly affect phytochemical profiles and the efficacy of the botanical end product (14), botanical extracts can vary from year to year and may be significantly affected by temperature, drought, or flood as well as by geographical location. Therefore, biochemical profiling must be used to ensure that a consistent material is being used to produce a botanical. It can also be used to reject a particular crop (Figure 1
). A variety of molecular techniques including restriction fragment length polymorphism, random amplification of polymorphic DNA, and DNA sequencing can be used to authenticate plant material and detect adulterant plant species (11, 15). Each technique has advantages and disadvantages in terms of cost, accuracy, reproducibility, time, and taxonomic level of identification. New technologies and advances in molecular plant systematics suggest that these techniques could become standard regulatory criteria.
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The processing often necessary to concentrate active compounds to a sufficient level can negatively affect their solubility and bioavailability. The active compounds within botanical preparations are very often hydrophobic and tend to precipitate at high concentrations, so attempting to improve efficacy by increasing concentration can be counterproductive. The use of solubilizers and bioenhancers is a logical consideration for many botanicals just as for drugs. A wide range of enhancers is available, each with specific solubility properties.
One widely used enhancer is Capmul MCM C10, a glyceryl monocaprate produced from edible fats and oils and commonly used in lip products. In a rat study examining the enteric bioavailability of the antibiotic ceftriaxone, Capmul increased bioavailability by as much as 80% (16). In a similar study, coenzyme Q10 was formulated with self-emulsifying drug delivery systems, which are mixtures of an oil, a surfactant, a cosurfactant, and the active substance used for improving the bioavailability of lipophilic compounds. The optimized formulation doubled the bioavailability of coenzyme Q10 in dogs (17). A similar formulation of a lipid-based self-microemulsifying drug delivery system was used to enhance the bioavailability of a silymarin preparation from Silybum marianum (milk thistle) for liver disease in rabbits. Bioenhancers are effectively used for pharmaceuticals, dietary supplements, and botanicals.
EXAMPLE OF THE DEVELOPMENT OF A BOTANICAL AND ITS STANDARDIZATION
An ethanolic extract of Artemisia dracunculus was evaluated as a potential botanical to be used for preventing and treating type 2 diabetes. The extract was active in both chemically induced diabetic mice and a genetic model that develops severe type 2 diabetes (18). The plants used for the production of the extract were grown hydroponically. The extract was initially characterized by LC-MS with a photodiode array detector and an electron impact mass detector using spectral database matching. The chromatograms of the extract, measured at 254 nm, and the total ion current electron impact mass spectrometry (EI-MS) are given in Figure 2. The crude extract was fractionated into 10 simple fractions based on HPLC retention. The active window was defined by using activity-guided fractionation and in vitro assays such as glucose uptake into muscle cells (Z Wang, X Zhang, DM Ribnicky, WT Cefalu, unpublished observations, 2004). Because the identity of the compounds from spectral matching of chromatographic peaks was only tentative, initial standardization of the extract for scientific quality control was based on the area of the most abundant peaks in the retention time period thought to contain the active compounds (active window, Figure 2A). It was decided that for the standardized product, peak areas should not vary >10% from the reference extract. Standardization by chromatographic fingerprinting may be useful and sufficient and is currently being practiced for some commercial botanical products.
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). Compound identification was confirmed with the use of commercial standards, mass fragmentation pattern libraries, or a combination of LC-MS, LC-MS/MS, and NMR (9). The abundance of these compounds is used for direct standardization of the extract in addition to ultraviolet spectra, thereby decreasing the likelihood that unknown compounds can interfere with the analysis. Electrospray ionization, a sensitive detection method, was used to enhance the level of standardization by confirming the molecular weights of the active compounds and providing additional chemical information about active compounds (eg, the caffeoylquinate peak of 515 m/z shown in Figure 2C is not detectable by ultraviolet detection or EI-MS). Assays were then used to validate the activity component of the standardization process. Hence, this is an example of a standardization technique that has evolved together with the development of the botanical drug. Multicomponent botanical preparations are a rich resource with a potential as future ingredients in botanical dietary supplements. Scientific validation of botanical therapeutics is necessary to ensure consistent results between research studies and botanical products. Although many challenges exist at every level of development, the identity of the active compounds and the validation of their efficacy will be a requisite for the quality control and standardization of botanicals.
ACKNOWLEDGMENTS
All authors contributed to the writing and reviewing of the manuscript. DMR and IR are consultants for Phytomedics Inc.
REFERENCES
This article has been cited by other articles:
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  W. T Cefalu, J. Ye, A. Zuberi, D. M Ribnicky, I. Raskin, Z. Liu, Z. Q Wang, P. J Brantley, L. Howard, and M. Lefevre Botanicals and the metabolic syndrome Am. J. Clinical Nutrition, February 1, 2008; 87(2): 481S - 487S. [Abstract] [Full Text] [PDF]
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