How similar is similar enough? A sufficient similarity case study with Ginkgo biloba extract
Graphical abstract
Introduction
Botanical dietary supplements are used by approximately 18% of adults (Clarke et al., 2015), or over 40 million people, in the United States. Therefore, the safety of these products is an important public health concern. For each type of botanical dietary supplement, there are numerous products available in the marketplace. These products are complex mixtures that can vary significantly depending on the ingredients (e.g., plant part, growing conditions), formulation (e.g., presence of different excipients like stabilizing agents or fillers), processing procedures, and storage conditions. Adulteration, either economically-motivated (i.e., addition of less expensive plant material) or with pharmaceutical compounds, adds to the variability in botanical dietary supplements. A companion paper (Shipkowski et al., 2018), provides a detailed discussion of the many challenges associated with assessing the safety of botanical dietary supplements.
Studies to evaluate the toxicity (or efficacy) of a botanical dietary supplement are typically limited to a single sample (e.g., unfinished extract from a certain supplier or finished product from a specific manufacturer) based on the inherent assumption that the selected sample is representative of all products with the same name on the label. However, there has been little effort to systematically evaluate this assumption. Two major challenges in determining whether toxicological data from a tested sample can be used to evaluate the safety of an untested sample are 1) the development of methods for evaluating sufficient similarity (i.e., what are the best methods to compare complex mixtures?), and 2) the establishment of categorical or quantitative similarity criteria (i.e., what level of similarity is sufficient?).
The term sufficient similarity, also referred to as phytoequivalence in the parlance of botanical dietary supplements, applies to complex mixtures and indicates that two mixtures are similar enough that the toxicity or efficacy data from one of the mixtures (reference mixture) can be used as a surrogate for the mixture(s)-of-interest. A reference mixture can be selected based on a pre-determined quality standard or a high level of characterization (i.e., the known to which the unknown is being compared). To our knowledge, there has not been a concerted effort to evaluate sufficient similarity of botanicals using both chemical and biological-response measures. However, several organizations have made significant progress in establishing the authenticity of botanicals and identifying potential adulterants based on chemical analysis of samples, including the USDA (particularly the Food Composition and Methods Development Laboratory) (Harnly et al., 2012, 2013, 2016), the Association of Official Analytical Chemists (AOAC) International Guidelines for Validation of Botanical Identification Methods (2012), The United States Pharmacopeial Convention (2015), and the American Botanical Council (Gafner et al., 2015). These efforts have been instrumental in providing manufacturers with the tools needed to evaluate the quality of their products.
The approaches developed to evaluate authenticity and detect adulteration of botanical dietary supplements have relied on both untargeted and targeted chemistry. Untargeted chemistry is defined here as any method that is used to detect the presence and relative abundance of constituents without prior knowledge of their identity. Untargeted chemistry approaches are intended to characterize as much of the sample composition as possible. These methods make no attempt to identify or quantify individual constituents within the mixture. However, they are not truly “untargeted” in that the methods used for sample preparation and the specific detection device used will influence the range of chemical structures included in the chromatographic profile. For example, the extraction process used to prepare samples for chemical analysis by chromatography will target components of a polarity range determined by the solvent. A true untargeted approach would employ multiple solvents with a range of polarities. In contrast to untargeted chemistry, targeted chemistry uses methods that have been qualified for each constituent-of-interest and requires authentic standards to quantify select marker constituents. Targeted chemistry can serve as a complement to untargeted chemistry by confirming the identity of specific analytes noted in chromatographic profiles generated using untargeted chemistry. Targeted chemistry is often used to quantify the purported active constituent(s) or marker constituents (if the active constituent is unknown) of a botanical product to confirm that the appropriate concentration is present in a standardized sample. For example, organizations such as the U.S. Pharmacopeia (http://www.usp.org/verification-services) and NSF International (http://www.nsf.org/services/by-industry/dietary-supplements) use targeted chemistry techniques to verify that the contents of a dietary supplement match label claims in their certification programs.
While there is not a history of sufficient similarity work involving botanical dietary supplements, there have been a limited number of examples evaluating similarity of complex environmental chemical mixtures such as water disinfection byproducts, petroleum substances, and pesticide mixtures (Rice et al., 2009; Marshall et al., 2013; Murray et al., 2013). These approaches have typically relied on quantification and comparison of select constituents in the mixture (Marshall et al., 2013) or structurally-defined classes of constituents (Murray et al., 2013), inferring that the toxicologically-active constituents are captured by the chemical analysis. However, it is important to note that there is usually a very large unidentified fraction in botanical products, and the active constituents are often unknown. Therefore, targeted analysis of select chemical constituents could be inadequate for determining similarity. In addition to examples that rely on chemical analysis alone, a few studies with environmental mixtures have also included in vitro biological data in the evaluation of similarity (Schenck et al., 2009; Grimm et al., 2016). In this manuscript, we explore sufficient similarity in terms of both chemical and biological-response similarity as applied to a complex botanical mixture: Ginkgo biloba extract (GBE).
Section snippets
Sufficient similarity framework
An overview of the framework for determining sufficient similarity is presented in Fig. 1. The process begins with the hypothesis that an untested mixture or mixture-of-interest is “sufficiently similar” to a reference mixture (i.e., the mixture that has undergone comprehensive toxicological evaluation). Note, there can be multiple reference mixtures and/or multiple mixtures-of-interest. Next, the reference mixture and the mixture-of-interest undergo a chemical analysis to compare constituents
Ginkgo biloba extract: a case study to explore sufficient similarity
GBE was selected for case study development by the NTP because it offers a relatively tractable starting place. GBE is derived from Ginkgo biloba leaves, and standardized GBEs have a well-characterized chemical profile compared to many other botanicals (van Beek and Montoro, 2009). Two major chemical classes in GBE, flavonol glycosides and terpene lactones, are considered to be responsible for its bioactivity. Flavonol glycosides have antioxidant potential (Ding et al., 2009), while terpene
Strength-of-evidence
In the first method used to integrate chemistry and biological-response data, referred to as the strength-of-evidence approach, numerical values were assigned to similarity determinations and averaged across data streams. The similarity determinations from the untargeted and targeted chemistry and in vitro and in vivo biological assays are summarized in Table 2. For each measured endpoint in each data stream (untargeted chemistry, targeted chemistry, SC-PHH, Attagene, in vivo), samples were
Conclusions and the path forward
A critical challenge in preclinical safety assessments and clinical efficacy studies of botanical dietary supplements is the inherent complexity and variability in these products, which complicates comparison of data generated with one sample to untested samples in the marketplace (Pferschy-Wenzig and Bauer, 2015; Shipkowski et al., 2018). Methods for comparing across these complex mixtures are required to understand how broadly data generated from a single sample can be applied to other
Acknowledgments
We were grateful for funding from the Office of Dietary Supplements to support case study development. We would also like to thank Ikhlas Khan, Kerri LeVanseler, and James MacGregor for their participation in the 2016 NTP Workshop “Addressing Challenges in the Assessment of Botanical Dietary Supplement Safety” panel on sufficient similarity. Finally, we would like to thank Kembra Howdeshell, Nigel Walker, and Jeffrey Swartout for their review of this manuscript.
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