Table of Contents
The Importance of Natural Product Characterization
Reproducible research on natural products (NPs) requires the use of replicable, well-characterized NP preparations (BC Sorkin, et al., 2020) [1] as well as rigorous and transparent reporting (JJ Gagnier et al., 2011) [2]; appropriate implementation of these processes is a prerequisite for NP integrity. The characterization and standardization of NPs (such as dried source materials, extracts, or foods) is challenging because of their chemical complexity and inherent variability, as well as because of the very limited evidence for the contributions of specific chemical constituents to biological effects of interest, whether beneficial or deleterious. Some important considerations and recommendations for NP and dietary supplement characterization in biomedical research are described and linked below. Careful attention to these issues will be critical for researchers applying for funding from the National Institutes of Health's Office of Dietary Supplements (NIH ODS).
NIH Policies and Guidelines to Promote NP Characterization and Improve Research Rigor
To enhance rigor and reproducibility in NIH-supported research, authentication and/or validation of the key biological and chemical resources used in awards has been required since January, 2016 (NOT-OD-16-011). NIH has since elaborated upon these grant criteria (https://nexus.od.nih.gov/all/2016/01/29/authentication-of-key-biological-andor-chemical-resources-in-nih-grant-applications/) and provided clarification on authentication of key resources (https://grants.nih.gov/grants/guide/notice-files/NOT-OD-17-068.html).
NCCIH Policy: Natural Product Integrity
The NIH National Center for Complementary and Integrative Health (NCCIH) has developed guidelines specific for the design and conduct of experiments that use NPs or their bioactive constituents. These guidelines are applicable to all investigators seeking funding for such research from NCCIH. An extensive array of information is provided at https://www.nccih.nih.gov/research/nccih-policy-natural-product-integrity.
ODS Product Integrity Requirements for Dietary Supplement Research Funding
Dietary supplement ingredients and products used in research must be rigorously identified and characterized to ensure reproducibility and to support scientific progress. ODS requires co-funding awardees to demonstrate the integrity of the nutrient, botanical, or other NP interventions used in research. ODS guidelines for demonstrating product integrity are similar to those of NCCIH and focus on authentication of identity and assessments of the composition, purity, and stability of the dietary intervention(s). More information can be found at https://ods.od.nih.gov/Funding/FundingFAQ.aspx#information.
Methodological Resources for NP Investigators
Botanical Authentication and Selection for Research
Proper assessment and documentation of ingredient identity and/or source is a critical part of developing a standardized dietary supplement or herbal to be used in research or marketed commercially. Guidance on the documentation needed to demonstrate authenticity differs between materials grown or collected by the applicant and purchased materials.
Analytical methods used for botanical authentication must be carefully designed, controlled, and validated to ensure they are sufficiently sensitive and specific to reliably distinguish the species of interest from related species. Furthermore, the method(s) used for authentication should be sufficiently broad to enable the detection of unanticipated contamination or adulteration. Use of material with an associated herbarium voucher is ideal for documenting authenticity (TM Culley, 2013) [3]. Botanical voucher specimens are preserved aboveground and/or belowground structures of an expertly identified representative plant that is associated with specific batches of plant matter used to prepare dietary ingredients and/or NP research materials. Several references describing good practices in authenticating botanical materials and archiving voucher specimens are provided in BC Sorkin et al., 2020 [1].
Importantly, targeted quantification of a biomarker compound or qualitative methods of authenticating plant material, such as organoleptic, anatomical, or molecular characteristics (e.g., chemical fingerprints), are not always sufficient for the rigorous characterization of NPs used in research. Familiarity with the botanical and pharmacognosy literature is helpful for anticipating potential contaminants/adulterants and determining how they may be distinguished, but this literature may not provide useful guidance for the detection of novel adulterants. A review by JJ Kellogg et al. (2019) [4] outlines approaches for rigorous selection and characterization of botanical NPs for research.
Botanical and Dietary Supplement Reference Materials
Reference materials (RMs) are important resources for optimizing and verifying analytical measurements. An RM is homogenous and stable with respect to one or more specified properties (e.g., purity, identity, content). For food and dietary supplement RMs, the specified properties are often quantified values for select macro- and micro-nutrients, marker compounds, toxins, contaminants, and/or adulterants. Matrix-based and calibration RMs are powerful tools for developing new analytical methods, verifying measurements of identified constituents in novel matrices, supporting quality assurance, and performing instrument calibrations.
A limited number of RMs for dietary supplement ingredients are commercially available, and fewer still are available for products in an oral dosage formulation. The ODS Analytical Methods and Reference Materials Program supports the development of RMs and certified RMs (CRMs) that are specifically designed to support NP and dietary supplement research. CRMs are RMs whose quantified values for key analytes meet additional metrological criteria for traceability and assessment of measurement uncertainty. ODS-supported RMs and CRMs can be found here: https://ods.od.nih.gov/Research/AMRMReferenceMaterials.aspx.
Depending on the analytical application, researchers may also utilize compendial or analytical standards to assess the identity and/or composition of their test reagents. Standards for botanical ingredients may lack quantitative information on content and stability, but they are useful as representative examples of the complex chemistry of NP preparations such as ground roots or powdered extracts.
Analytical Method Validation
Fit-for-purpose analytical methods are needed to ensure that the dietary ingredients used in commercial products are correctly identified and that NP research materials are sufficiently characterized to enable research to be interpreted and replicated. Ensuring the accuracy and precision of quantitative measurements for phytochemicals, nutrients, and possible contaminants is vital, so analytical method selection and validation requires careful consideration (EM Mudge et al., 2016) [5]. Analysts employ multiple approaches to ensure method performance, ranging from evaluation of in-house or published methods against certified RMs to performing formal validation studies on laboratory methods. AOAC International has published guidelines for validation of methods for dietary supplement characterization and botanical identification (https://academic.oup.com/aoac-publications/book/45491/chapter/392389499
). Additional relevant information can be found in the U.S. Food and Drug Administration's (FDA) 2019 Guidelines for the Validation of Chemical Methods in Food, Feed, Cosmetics, and Veterinary Products, 3d Edition (https://www.fda.gov/media/81810/download).
Similar to quantitative analytical methods for specific chemical constituents, verifying that qualitative methods for botanical identification and characterization (such as chemical fingerprinting or genomic analyses) reliably yield accurate answers is necessary before the method and its results can be useful. LaBudde and Harnly (2012) [6] describe validation criteria and procedures for qualitative botanical identification.
Consensus Methods for Select Botanical and Nutritional Products
Prescribed and validated methods of analysis for constituents of certain NPs and dietary supplements are made available from a number of standard setting organizations:
- Quantitative analytical methods for select botanicals, nutrients, dietary ingredients, and finished dietary supplement products can be found in the AOAC International Official Methods of Analysis (AOAC International, Gaithersburg, MD USA). ODS supported AOAC International to develop method performance requirement documents and Official Methods of Analysis for prominent dietary supplement ingredients. These guidelines and analytical methods are available through the ODS Analytical Methods and Reference Materials Program: https://ods.od.nih.gov/Research/AMRMAnalyticalMethods.aspx
- Official raw-material and finished-product monographs for many botanicals and nutrients, along with tests for determining whether or not specifications are met, are available in the United States Pharmacopeia’s Dietary Supplements Compendium (United States Pharmacopeial Convention, Rockville, MD USA).
- Specifications and corresponding analytical tests are available in the French and English editions of the European Pharmacopoeia (European Pharmacopoeia Commission, EDQM, Strasbourg, France).
- An English version of the 10th edition of the Pharmacopoeia of the People’s Republic of China (9th Chinese Pharmacopoeia Commission, Beijing, PRC) contains monographs for numerous botanicals used in Traditional Chinese Medicine and is available through the United States Pharmacopoeia (https://www.usp.org/products/chinese-pharmacopoeia).
Considerations of Dosage Form Disintegration and Dissolution
Research on dietary supplements and NPs often involves oral administration of a commercially available or custom formulated dosage form of the ingredient(s) or constituent(s) of interest. However, although the vitamin, mineral, or NP constituent(s) of interest may be known to be soluble in aqueous solutions over a wide pH range, the excipients and dosage-formulation matrix used in dosage form preparation may significantly influence bioavailability. For example, capsule shell materials have been found to interact with green tea catechins and reduce their disintegration and dissolution (N Glube et al., 2013 [7]; PA Gusev et al., 2020 [8]). The United States Pharmacopeia (USP) has developed general guidance on conducting disintegration and dissolution studies of oral dosage formulations of dietary supplements (USP General Chapter <1711> [9]; USP General Chapter <2040> [10]), and certain dietary supplement and NP preparations have unique USP specifications for determining dissolution. However, dietary supplements are not required by law to meet USP standards. Investigators are therefore strongly encouraged to consider, prior to initiation of tests of pharmacokinetics or efficacy, whether disintegration and dissolution (requisite first steps towards bioavailability) have been evaluated for the putative bioactive(s) in their oral dosage preparations.
Additional Resources
NIH-Supported Resources for Natural Product Researchers
Select References for Designing, Conducting, and Reporting NP Studies
Researchers should note that implementation of the recommendations described above will greatly facilitate compliance with the following:
- The Consolidated Standards of Reporting Trials (CONSORT) statement (http://www.consort-statement.org/consort-2010) has been extended to trials using both herbal (JJ Gagnier et al., 2006) [12] and traditional Chinese medicinal products (C-W Cheng et al., 2017)[13]. These consensus statements provide important recommendations for planning and reporting clinical trials, as well as helpful information for interpreting the biomedical literature on NP health effects.
- The FDA’s Botanical Drug Development Guidance for Industry, while not legally applicable to botanical dietary supplements, contains useful guidance for all researchers planning clinical research on botanical products.
- Reporting guidelines for clinical pharmacokinetic studies such as those described by Kanji et al. (2015) [14], while not specific to NP research, may be extremely valuable to those assessing the pharmacokinetics of NP constituents in preparation for the conduct of clinical trials.
- Demasi and Jefferson (2021) [15] describe the critical importance of rigorously and transparently reporting the chemistry and formulation of placebos for both research replicability and the appropriate interpretation of clinical trial results.
- Barrett, et al. (2011) [16] describe the critical importance of assessing both clinical trial participants’ expectancy (for intervention efficacy) and the effectiveness of masking verum (hypothesized effective or test intervention) and placebo (control) when interpreting the outcomes of NP clinical trials.
- Betz and Hardy (2014) [17] discuss challenges associated with evaluating herbal clinical trials and provide recommendations to authors on underappreciated aspects of trial design such as botanical nomenclature, justification of clinical endpoint(s), posology, and description of the product and placebo.
Considerations to Improve the Rigor/Integrity of Research Through Prospective Research Registration
The World Health Organization has stated that registration of research “…will improve research transparency and will ultimately strengthen the validity and value of the scientific evidence base” (WHO, 2021) [18]. Registration of the planned sample size and primary and secondary outcomes for a definitive pre-clinical experiment or for a clinical trial enables subsequent determination of whether the reported results correspond to the experimental design. Conversely, failure to report the planned primary outcomes raises the possibility that the reported outcomes derive from secondary analyses of data that failed to reject the null hypothesis. Additionally, when assessing the published literature, it is useful to check published reports against the information registered for a given experiment or clinical trial. Researchers planning clinical trials, or definitive preclinical experiments that might be considered in designing a later clinical trial, should plan to register their studies.
Preclinical in vivo studies can be registered at:
https://preclinicaltrials.eu/ OR
https://www.animalstudyregistry.org/asr_web/index.action OR
https://osf.io/registries
Many peer-reviewed scientific journals will not consider publishing the results of a clinical trial that was not previously registered. Registration of NIH-supported clinical trials and some other human subjects research is required prior to trial initiation at Clinicaltrials.gov.
References
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