With health and wellness a continuing priority, and the surging popularity of vegan and plant-based products, consumers are more informed than ever about the products they consume and what's in them. To substantiate the ingredient list on product labels, each ingredient must be appropriately identified by fit-for-purpose methodologies. Among these ingredients, the identification of botanical materials, also known as botanical authentication, is particularly complex. This complexity arises from the vast diversity of plant species, overlaps in nomenclature, and variations caused by harvest seasons and locations.

Building on the intricacies, botanical authentication becomes even more critical. It’s not just about ensuring the quality, safety, and efficacy of botanical products. The authentication process also addresses several issues that drive our industry, including regulatory compliance, supply chain transparency, and sustainable business practices. All of this ultimately affects brand reputation.

Therefore, if a brand is committed to developing superior products and upholding quality standards, it’s imperative to understand the significance of botanical authentication, its challenges, and the methodologies to address them in practice.

Navigating the Complexity of Botanical Authentication

Despite the importance of botanical identity authentication, significant challenges can arise. One challenge is the use of incorrect species. This may occur unintentionally in the form of mislabeling, due to confusion over plant names, accidental inclusion of non-target species, or difficulty in identifying species at certain stages of growth or processing (Sgamma et al., 2017). However, it can also occur intentionally for economic gain, a practice known as economic adulteration.

Adulteration manifests in various ways, with one prevalent form being the partial substitution or blending of adulterants with the intended botanicals. Such adulteration can be elusive, especially since many conventional chemical methods are calibrated for individual botanical materials and may lack the sensitivity to identify mixtures.

Given the frequency of these challenges, it’s no surprise that scientists continue to develop methods to enhance botanical authentication accuracy.

This has led us to develop a new type of DNA testing method, called Tetra-primer ARMS-PCR assay, which can be tailored to detect both the intended ingredient and any adulterant, ensuring rigorous quality standards.

A Modified Tetra-primer ARMS-PCR Method with Improved Sensitivity and Specificity

The Tetra-primer Amplification Refractory Mutation System, or Tetra-primer ARMS-PCR, employs four primers. Two outer primers are designed to amplify a larger "control" fragment, ensuring the PCR's effectiveness. In contrast, the two inner primers, capitalizing on the specificity of Taq polymerase's 3' end amplification, produce smaller "diagnostic" fragments that highlight variances in genotypes where a single nucleotide base differs—known as a single-nucleotide polymorphism (SNP).

This technique has prevalent applications in disease diagnostics and pharmacogenetics, pinpointing variations linked to certain genetic disorders or drug responses.

Our innovative adaptation has tailored this system to identify differences between target species and common adulterants, yielding “diagnostic” fragments when both are present in a sample.

For instance, using Panax ginseng root as a benchmark, our study illustrated the method's capability to discern close species like American ginseng (Panax quinquefolius) and Tianqi (Panax notoginseng) when their roots are combined with Panax ginseng root. However, the method's sensitivity and specificity became questionable when applied to highly processed materials like ginseng root extracts or red ginseng in its steamed root form.

To address these challenges, we introduced a new primer design that integrates both 5´ end random nucleotides and 3’ terminus phosphorothioate linkage modifications on the inner primers. The modified Tetra-primer ARMS-PCR showcases superior sensitivity and specificity, effectively identifying P. ginseng under optimal conditions. These modifications also exhibited marked improvements in other botanical authentications, including parsley. Collectively, our findings underscore the method's promise as a reliable, efficient, and precise tool for botanical authentication, heralding new horizons for quality assurance in the botanical sector.

This groundbreaking research emerged from a collaboration between Herbalife and Professor Pang Chui Shaw of the Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, the State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, and the School of Life Sciences, at The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.

The findings were recently published in the peer-reviewed journal, Scientific Reports. Additionally, our strategy to enhance Tetra-primer ARMS-PCR's sensitivity and specificity through oligo modifications is published under The Patent Cooperation Treaty (PCT) as of March 30, 2023 (WO 2023/049708 A1).

Conclusion

As the nexus of food science and botanical authenticity strengthens, our research marks a pivotal step, ensuring transparent and safe botanical products. This collaboration combines collective expertise to address industry challenges. With the rise in discerning consumers and plant-based products, techniques like the Tetra-primer ARMS-PCR assay are vital, steering brands toward unmatched quality and innovation.