Introduction

The purpose of this manual is to assist purchasers or collectors of common unprocessed botanical materials in using easily observed morphological characteristics to confirm the identity of those materials. The focus is on species and plant parts that are used to produce botanical medicines or dietary supplements. Fundamentally, identification of most such materials is not qualitatively different than identification of different vegetables in the grocery store. With repeated experience, we quickly learn to recognize the size, shape, color, texture, aroma and flavor of different types of salad greens or root vegetables, so that it would be obvious if whole material had been mislabeled. Many herbs and spices can be recognized in just the same way. In fact, there is no real distinction possible between herbs and fruits or vegetables; numerous plants, such as garlic, ginger, chili peppers, and cranberries, fall into both categories.

However, at least four factors make recognition of many other botanicals more difficult. First, most of us are less familiar with such plants, just as when we first encounter an exotic fruit or vegetable, we do not know what color it usually is or what it should taste like. Second, common cultivated vegetables have often been selectively bred so that they differ conspicuously in appearance and sensory characteristics from their wild relatives. It is rarely possible to confuse them with an undesired species. By contrast, many herbs have close relatives that are not used in the same way, yet look so similar that they are easily confused. Third, while many botanicals are cultivated, most medicinal species are usually gathered from the wild, sometimes from habitats where look-alike relatives are also present. This creates the possibility that incorrectly identified plants will be included in a harvest. Finally, the characteristics that distinguish closely related plant species are often small features that are not immediately obvious to people who are not used to looking for them.

Those who handle botanicals professionally, therefore, give special attention to quality control (QC). QC encompasses much more than confirmation of identity. For example, plant material that is too young or too old, moldy, or excessively insect-damaged might be correctly identified yet of unacceptable quality. Likewise, in modern commerce, minimum standards for content of selected chemical marker compounds are often set by purchasers of certain plants and/or are a required minimum quantitative standard specified in an official pharmacopeial monograph that is the basis for the buyer’s specification requirement. Traditional methods of ensuring minimum chemical content were indirect, e.g., many roots are collected in the autumn in the belief that their potency is increased at that season. Large manufacturers of finished products now routinely perform direct chemical screening, such as high performance liquid chromatography (HPLC), to confirm the presence and quantity of a marker compound or compounds. Some also standardize their products by adjusting their manufacturing processes to ensure that every batch of finished product contains a similar quantity of the selected compounds; often accomplished by batch-specific normalization, a process by which the native extract is assayed and then diluted accordingly with an excipient in order to standardize the quantity of specified markers. Such procedures may add considerable value, but they are built upon a base of correct botanical identification: whether one is selling bulk botanical raw materials, intermediate extractives or essential oils, or a highly processed, so-called “value-added” consumer product, the most fundamental aspect of product quality is, and will always be, the assurance that the plants provided are what they are stated to be.

Methods of Identification

There are several means by which one can attempt to identify plants, or more simply, to confirm the accuracy of a previous identification.

Macroscopic taxonomic identification

The simplest and most direct of these is botanical identification of unprocessed material, often using the same characteristics that botanists would use to identify the species. In botanical keys, floras, and field guides, plants are identified mostly by features of the leaves and flowers, or sometimes fruits, which can readily be observed using a hand lens or dissecting microscope. Basic taxonomic work usually relies upon characteristics of this sort, so when we say that a particular genus has 10 species, what we mean is that the known specimens from that genus can be separated into 10 groups that share unique combinations of morphological features. The identity of any future specimen is determined by its morphology.

These characteristics can be observed either in living material or in herbarium specimens, which are (usually) pressed dried plants or plant parts glued to sheets of stiff paper and preserved for research. Most floras or taxonomic treatments of individual plant groups, such as revisions and monographs, are based on study of herbarium collections rather than live material. For many herbs in commerce the whole herb is used, usually meaning the aboveground (aerial) parts including flowers. If such material is whole or coarsely broken, it can be treated like an herbarium sheet and identified, through simple morphology alone, as accurately as it is possible to identify any plant.

However, for other botanicals the parts used do not include flowers or fruits. They may include only parts such as roots or bark, which present few obvious taxonomic characteristics. Naturally, when such material was harvested, the collector could and should have observed the whole plant carefully to ensure that undesired species were not included. Nevertheless, the purchaser’s QC Unit will often want to confirm the material’s identity independently, and new good manufacturing practices (GMPs) for herbal dietary supplements that are expected to be published by the United States Food and Drug Administration (FDA) in late 2006 will probably require such additional verification of identity. Further, it is sometimes possible to differentiate closely related species using only the gross anatomy of parts, such as roots and rhizomes, that are usually assumed to have few useful taxonomic characters. (Black cohosh, Actaea racemosa L., Ranunculaceae, syn. Cimicifuga racemosa, is an example; the rhizome and root anatomy of the desired species is visibly different from that of related species whose aerial parts, especially leaves, appear very similar.) However, many groups of related species share essentially identical root anatomy and cannot be distinguished. For other groups, the economically unimportant relatives have never been studied, so that we do not know whether they could be distinguished. In these cases, morphological examination cannot guarantee that a batch of material could not possibly belong to any species other than the expected one, only that its appearance is fully consistent with that of the expected species. If substitution of other species is unlikely (if material is produced under controlled cultivation according to suitable Good Agricultural and Collection Practices (GACPs), for example), this may provide an adequate level of confidence.

Microscopy

Micromorphology or microanatomy, the observation of cellular-level anatomical features using a light microscope, is also used as an aid to confirmation of identity. This may involve preparing slides with stained thin sections of plant parts or making wet mounts of powdered material. There is certainly no rationale for doing this when material is complete enough to be botanically identified in the normal manner: taxonomists do not make slides to observe the shape of individual petal cells and so forth when they define species or identify specimens. Microanatomy sometimes provides information that gross anatomy does not. For example, two roots that appear identical may contain differently sized xylem vessels or starch grains. However, to be relied upon, such information must be based on multiple samples of the desired species (and of related potential substitutes for comparison) from across its range, because if only a few samples had been examined, environmental or genetic variation could be overlooked. For many medicinal genera, there has not been adequate study. Microscopy was once the only means of identifying powdered material; pharmacognosists were expected to be able to recognize numerous plants by the size and shape of loose tissue fragments, crystals, hairs, etc. Here, too, it is often possible to determine only that the material is “consistent with” its supposed identity. Closely related species often have similar types of cells, and when they are powdered it may be impossible to identify them to the species level.

Chemical and molecular analysis

Other means of confirming the identity of processed material include the uses of chemical or molecular markers. The former is often done automatically during the production of so-called “standardized” products, i.e., extracts. The presence of marker compounds known to occur in the desired plant, within certain quantitative ranges, is used as a means of confirming identity. More and more, assays to determine presence of markers that should not occur in the desired plant are being added as required tests in pharmacopeial monographs. For example, based on the misidentification example described in the Foreword to this book, the European Pharmacopoeia monograph for plantain leaf (Plantago lanceolata L., Plantaginaceae), described under the pharmacopeial name “Plantaginis lanceolatae folium”, includes a test for yellow foxglove (Digitalis lanata Ehrh., Scrophulariaceae) leaves. Often, the level of a marker compound is used to estimate the quality (often associated with the presumed potency) of the material, although standard marker compounds often have not been shown to be among the plant’s active ingredients at all, and occasionally have been shown not to be. Molecular identification methods, which are a technology still in development, use various PCR (polymerase chain reaction) primers to amplify bands of DNA from powdered material. Different species, even closely related species, may have different DNA banding patterns. Chemical screening is the only test that can be performed if the identity of an extract or its constituents must be confirmed, because at that point in processing, both morphological features and DNA have been destroyed.

Both chemical and molecular methods should be validated through extensive sampling before they are used in commerce to determine botanical identity. For example, sometimes certain populations of the desired species may contain little or none of a selected marker compound. If that ingredient has been proven to be an active ingredient, its absence represents a good reason not to use the material in question, but it does not show that the material was adulterated or misidentified. It is also common for a cluster of related species (e.g., species ofHypericum or Actaea) to share numerous characteristic compounds. These related species may be similarly potent and interchangeable in traditional use, yet should not be marketed under the name of some other species. A cogent example of that practice is the recent occurrence in the botanical trade of so-called “black cohosh” derived from medicinal species of Actaea native to China, whereas industry convention in the U.S. and corresponding federal regulations recognize A. racemosa as being the only taxon that can be sold in the U.S. under the common name “black cohosh.” In such cases, a chemical test cannot be claimed to provide 100% accuracy unless someone has not only extensively surveyed the desired species, but has surveyed the potential substitutes to ensure that they are always chemically different in some way. (This has been done recently in the above example, where samples of Chinese Actaea have been assayed chemically and shown to be distinct from A. racemosa.)

Similar problems will arise in molecular testing. We know from forensic-oriented shows on television that there is plenty of DNA band variation within our own species; on the other hand, there are certain gene variants that are found both in some humans and in some apes. Extensive study is required to find markers that are alwaysand only found in a single species. Moreover, unlike mammals, some plants hybridize freely with related species. If there is a significant amount of gene flow across species boundaries in a portion of a plant’s range, it may be impossible to create a completely reliable molecular method of identification. In any case, it is worth remembering that all of the validating studies of variation within and among species will be done using samples that were assigned to species using morphological features, which remain the basis of species identity.

Microscopic, chemical and molecular methods, to one extent or another, all require considerable technical skill, combined with access to laboratory equipment and appropriate analytical reference standards, or else funds to hire outside labs. Supplies may be costly, and procedures are often time-consuming. Moreover, many of these procedures do not definitely confirm identity to the species level. There are many different viewpoints about what constitutes adequate QC of botanical materials, ranging from the slovenly to the obsessive. This author’s opinion is that it is better to rely on botanical identification, which is relatively inexpensive and quick, to determine species identity whenever possible. This means that some competent person must look at material before it has been comminuted, traditionally prepared (e.g., carbonized, charred, cooked in wine, fermented, stir-fried in honey, etc.) and/or extracted. Given an adequate chain of custody, the botanical identification made when the raw material is intact remains valid throughout later sales or processing, just as when whole wheat has been ground into flour.

Sometimes, the so-called organoleptic aspects, i.e., the morphology, color, odor, taste and mouth feel of difficult material, can only be said to be consistent with that of the desired article, either because we know that material from a particular look-alike species would display all of the same characteristics, or because we do not know whether poorly known related species might also display those characteristics. In these cases, especially if substitution is known or suspected to be a significant problem, it is preferable to combine morphological examination of the anatomical characteristics with a “chemical fingerprint”. Chemical analysis and reference to such a chemical fingerprint will be conducted automatically if a “standardized” product is to be produced or if the manufacturer’s specific QC requirements include such chemical process. For example, in some companies, particularly those with fairly advanced GMPs for dietary supplements and those making products for use as registered OTC drugs, botanical raw material specifications often require a numerous identity tests: i.e., macroscopic, microscopic, organoleptic evaluation, and chemical testing, e.g., high performance thin-layer chromatography (HPTLC). In many cases, additional identity-related tests are also required depending on the referenced monograph on which a particular specification may be based. For example, in the case of the current monograph in the United States Pharmacopeia-National Formulary for Chamomile (Matricaria recutitaL., Asteraceae) flower, in addition to macroscopic, microscopic, and TLC tests, the essential oil is distilled in order to examine not only the color of the oil (must be blue), but also to quantify the oil content (min 4 ml / kg dried drug), after which gas chromatography (GC) can be performed with the oil to determine content of bisabolan derivatives (not less than 0.15%). Finally, the USP monograph requires HPLC determination of apigenin-7-glucoside (not less than 0.3%). In addition to the aforementioned, there are other species-specific identity-related tests, which the supplier’s QC department and/or the buying company’s QC department might be required to perform before raw material can be released for use in a medicinal product (i.e., a product that is being sold as a medicine, not necessarily as a dietary supplement).

Nevertheless, despite the battery of chemical testing now available to QC laboratories, it is worthwhile to look at morphology first, if only because the tested material may prove to be inconsistent with the desired article, allowing the material to be rejected before the more expensive chemical test(s) is/are performed. Moreover, the combination of a relatively inexact morphological test with an inexact or incompletely validated chemical test may provide, together, greater confidence in the stated identity than either the morphological or chemical tests alone.

Ideally, all botanicals would be handled in this way. That is, nobody would ever purchase powdered botanicals or extracts that did not come with a certification of identity or consistency with the stated identity, as determined by appropriate botanical analysis. Chemical methods would then be necessary either to standardize ingredients or finished products to specific marker compounds for QC purposes and, further, to support the identification of material that could not be unambiguously identified by morphological or organoleptic characteristics alone. In fact, according to some official pharmacopeial monographs, the additional chemical testing is required; i.e., botanical documentation alone is not sufficient to guarantee identity, although it is a necessary initial step. Unfortunately, in practice, processed materials are often sold without such certification, and the purchaser’s choices are then to use microscopic methods (for powdered material) or chemical methods to try to confirm the material’s identity. Unless pharmacognostic literature indicates that powder can be identified to the species level by microscopy, chemical testing is probably the better choice even if it is not as extensively validated as one might like. (There is currently a growing movement within the herb industry and elsewhere to validate chemical analytical methods for determining the identity of various botanical materials.) It may provide an estimate of the quality of the material simultaneously, and if the purchaser does not have the necessary expertise and equipment, it is easier in many places to contract with an outside laboratory for HPLC or TLC (thin-layer chromatography) analytical services than microscopic examination, in which few people are skilled these days. It may also be easier to observe the presence of a botanical or pharmaceutical adulterant mixed with the correct material when chemical screening is used. For powdered plant material, fully validated molecular techniques may also prove valuable for detecting botanical adulterants.

Summary of this Book

This book describes morphological characteristics that can be observed with a hand lens or dissecting microscope, such as are commonly used by taxonomists for species identification, as well as organoleptic characteristics (e.g., color, taste, mouth feel, and odor) that are useful for confirming plant identity. It does not deal with microscopic anatomy or identification of powdered botanicals, which requires very different skills. The first part provides a brief review of basic plant structure, some practical advice on identification, an introduction to botanical nomenclature, and an explanation of the format. The second part, which constitutes the main portion of the book, contains entries for individual species follow, alphabetized by scientific name. Species have been selected based on a combination of their use in Western botanical products and their potential for misidentification (so that plants having known substitutes or similar relatives are more likely to be treated). Some very common botanicals that also fall into the category of cultivated vegetables (such as garlic) have been omitted since they are very easy to recognize and there is very little possibility that other species will be substituted. Some species of limited importance are treated only as relatives or contaminants of other species, rather than receiving complete individual entries, to save space. The index should be consulted to locate all references to a particular plant. A glossary at the back of the book defines basic botanical terminology. Finally, there is a list of references that may be of broad use in the identification of plants or raw botanicals, some of which were invaluable to the compilation of this manual. The reader may wish to add a few of these to his or her own botanical library.