Paula Brown & Dr. Wendy Applequist09.01.10
After alluding to the need for reference materials back in January and spending the next two columns on materials of a chemical nature, it is time to address botanical reference materials. To recap, “reference material” is a term that refers to a substance whose property values are sufficiently homogenous and stable with respect to specified properties and whose fitness is well established for its intended use in a measurement process. Within the context of identity testing, a botanical reference material (BRM) is a substance whose botanical identity and genuineness has been established, beyond doubt, to the genus, species and, if necessary, varietal level.
Dr. Wendy Applequist of the Missouri Botanical Garden has graciously agreed to entertain the naïve questions of this chemist concerning BRMs and their use.
How do you confirm the identity of a plant?
The most direct means of confirming botanical identity is by morphology. Botanists describe plant species and distinguish them from related species using combinations of morphological features, not chemical constituents or (normally) DNA sequences. The validity or fitness for purpose of botanical identification conducted in this manner is undoubted, whereas very few chemical assays are validated for that purpose. And, of course, botanical ID is cheap.
It sounds like the perfect method, but aren’t there complications?
Plant species are described using morphological characters of the whole plant, especially reproductive characters. For example, in flowering plants the parts studied include at least the stems or twigs, leaves and flowers, and for some plants, such as umbels that have few useful floral characters, the fruits. Authors of botanical literature, such as regional floras or monographs of specific groups, study pressed herbarium specimens that have all of these parts. The dichotomous keys they publish to enable non-specialists to identify plants are written with the assumption that the user will have similar material.
In the botanicals industry, you seldom have such luck. Purchased crude botanicals may include only leaves, roots or bark, with no flowers or fruits, so the keys in botanical literature are of little use. Some plants are still easily identified from isolated parts, if one is familiar with them. We have no difficulty recognizing garlic or ginger. Even similar species can sometimes be distinguished with experience; for example, root and rhizome anatomy can distinguish black cohosh from several potentially substituted American and Asian species. However, it is often impossible to identify bark or roots precisely, either because there are no species-specific morphological features or because no adequate comparative research has been done to find them. Furthermore, crude botanicals are often broken, chopped or sliced. If material is too finely broken, potentially useful morphological features are obliterated. To rely on morphology, you must make the effort to buy material in identifiable condition.
When a BRM is less than intact how do you know it is correctly identified?
For a botanical material to be used as a BRM for confirming identity it must itself be definitively identified first and vouchers must be preserved. A botanist, or someone who is adequately familiar with the plant to identify it, must confirm its identity either from live plants at the time of collection or from preserved voucher material adequate for the purpose, and it must come with a Certificate of Analysis (CofA) that provides information on its identification.
Voucher material, for this purpose, is any material collected at the same time and place as a collection of bulk material and preserved as evidence of that single collection’s identity. Usually, a pressed herbarium specimen is the most suitable voucher; sometimes, as for garlic bulbs or chamomile flowers, the commercially harvested parts alone are easy to identify. Voucher specimens or samples should be available for viewing, at least through the supply of high-quality images, for independent confirmation of identity.
What are the characteristics of a “good” BRM?
Some commercially available BRMs commingle material of a single species from several different sources. This approach is based on the idea that the BRM will display something closer to the full range of characteristics of the plant species than would material from any single source. This can be confusing, as the BRM will then possess a combination of characters not seen in any individual batch. In any case, if such a BRM were used to support identification, each collection locality or source of included material would need to be individually vouchered.
Sometimes, especially for certain imported plant materials, no voucher specimen or information on prior botanical identification is available. If an unvouchered BRM is not botanically identifiable from the characteristics present in the material itself, it is not fit for the purpose of supporting botanical identity. Not all BRMs have the same purpose and as such the accompanying documentation will vary; always be sure you use a BRM as it is intended.
Botanical research on many genera remains incomplete. Some bulk plant materials not currently identifiable by morphological or anatomical characters might prove to be identifiable following careful, genus-wide anatomical studies or the use of modern techniques such as scanning election microscopy (SEM). In the future, DNA barcoding may allow quick and easy authentication of both BRMs and commercial batches for many plants, although for each genus, the distribution of genetic variation within and among species will have to be explored to validate the method.
How can you use a BRM within the context of GMP compliance?
Just as a chemical standard can be used as a comparator to confirm the identity of a particular compound, a BRM can be compared to a raw botanical to assist with botanical identification. While identification of a pressed herbarium specimen is facilitated by comparison to other herbarium specimens, if you are identifying a cut herb or isolated fruits, a reference material that is in the same form will be much more helpful.
Naturally, there are several catches. A chemical standard is a substance that always has the same nature: berberine is berberine is berberine. If two compounds labeled berberine exhibit different characteristics, such as different chromatograms, you know one must be mislabeled. Living species however are normally variable in size, color, form and chemical content. If the BRM and your purchased material look a little different, that’s not necessarily proof the material is misidentified. On the other hand, if the BRM and your purchased material look identical, that may not be adequate proof they are the same species.
Some willow species, for example, have bark anatomy that’s identical even at the microscopic level. Morphological examination can then tell you only that the bulk sample is “consistent with” the stated identity. Section 402(g)(2) of the Federal Food, Drug and Cosmetic Act allows the continued use of such species as there is no validated means of proving identity. Confirmation of expected chemical composition obviously gains importance when botanical identity is uncertain.
The upshot is that you can’t rely solely on comparison to a BRM to identify a species. For each species to be identified you have to dig into the botanical literature and find out what species, if any, are potential substitutes and what characters, if any, can be used to distinguish among them. You then have to be sure those identifying characters will actually be visible in the sort of material you plan to authenticate.
If BRMs are not definitive, are they worth using?
Absolutely; morphological characters can be hard to visualize from text descriptions. Botanical literature might simply describe a plant as having short straight hairs, but when you put two leaves from different samples together under a dissecting microscope, you might be able to see instantly that their hairs look very different. The most valuable function of a BRM is to clarify your understanding of a botanical’s characteristics. Always remember it is important to examine a botanical material, not only what a botanical material is intended to be, but also for the absence of characters found only in related species or a known adulterant. Studying a BRM of a common adulterant might help you to confirm its absence.
I would like to thank Dr. Applequist for agreeing to answer my questions about BRMs. And to the reader’s of this column: Hiring a botanist as a consultant might make life easier for you, as well as for the botanist!
