Editor's note: American Botanical Council (ABC) Chief Science Officer
Stefan Gafner, PhD, co-authored the Planta
Medica article with Iffat Parveen, PhD; Natascha Techen, PhD;
and Ikhlas Khan, PhD; researchers at the internationally-respected National
Center for Natural Products Research (NCNPR) at the University of Mississippi
(a US Food and Drug Administration [FDA]-funded Center of Excellence in the
area of medicinal plant analysis), and Susan Murch, PhD, an expert in the
analysis of botanical ingredients at the University of British Columbia in
Canada. Gafner has produced the following brief summary of the paper.
Interested parties are encouraged to obtain the original
article from the journal.
Background
In July 2016, the respected, peer-reviewed medicinal plant journal Planta Medica published an online paper1
written by various experts in medicinal plant analysis. The paper reviews the strengths
and limitations of DNA barcoding analytical methods — a subject that has
received significant global attention since the New York attorney general’s (NY
AG’s) now-infamous and highly flawed DNA analysis of various herbal dietary
supplements, which was covered by The New
York Times and other major news outlets in 2015.
Based on the DNA
analysis, the NY AG took  regulatory action against four major retailers for
allegedly selling mislabeled herbal supplements. (The products produced and
sold by retailer GNC were later deemed by the NY AG to be compliant with state
and federal regulations and allowed back onto retail shelves in New York;
actions against Target, Walgreens, and Walmart are still pending.) The NY AG
has been criticized for relying solely on DNA barcoding analysis as the basis
for his regulatory actions. These criticisms have come from a variety of sources:
from plant analytical experts in academia, government, and industry, to
industry sources and ABC publications.
As noted in the
paper, which is summarized below, due to various well-known limitations, DNA
barcoding methods should not be used as the sole analytical method to determine
the identity of botanical ingredients in finished herbal formulations (e.g., dietary
supplements). Rather, DNA barcoding should be used as part of a larger array of
methodologies, including microscopic and chemical methods.
'Strengths and Limitations of DNA Barcoding': A
Summary
The
authors of the Planta Medica article
begin with an overview of DNA barcoding and discuss the steps (extraction,
amplification, and sequencing) involved in the technology, as well as its
strengths and limitations for plant identification.
DNA barcoding, which involves the use of short genomic
regions to distinguish species of animals, plants, fungi, bacteria, and other
organisms, has become an increasingly popular technology to determine the
authenticity of botanical ingredients in herbal medicines and dietary
supplements. Plant DNA barcoding, initially used predominantly in academia to
determine the relationship among species (phylogeny), is now used by many
groups, including dietary supplement manufacturers, contract analytical laboratories,
government agencies (e.g., the FDA), and, as noted previously, the office of the
NY AG. The outcome of DNA barcoding varies depending on the method of DNA
extraction, primer choice, amplification, and sequencing. In addition, the
success of the technology depends on the presence of high-quality DNA and the
absence of interfering compounds in the material analyzed.
A number of extraction methods are used in practice. The
choice of the method depends on the material to be analyzed. Different plant
parts (DNA is more difficult to obtain from bark or root compared to leaves or
flowers) and metabolite compositions (e.g., presence of polysaccharides or
polyphenols) will affect the extraction efficiency. The choice of the genomic
region is crucial for the successful outcome of the approach. Some regions are
difficult to amplifyin certain plants (e.g., matK) or have a low resolving power (i.e., they do not adequately distinguish
among certain species). Others, like the widely used ITS region, may
differentiate plants readily, but there may be several copies with varying DNA
sequences within the same plant (e.g., in valerian [Valeriana officinalis, Caprifoliaceae] and related
species). For example, ITS regions from fungal species that may be present as an
endophyte (living within the plant) or ectophyte (living at the surface of the
plant) may be preferentially amplified.
The quality of DNA present in botanical material is highly
dependent on the various steps used to process the material (e.g., drying,
grinding, sterilization, storage, extraction, etc.). In many cases, DNA
is degraded or absent altogether. The universal primers used for DNA barcoding
are designed to amplify the target genomic region of as many plant species as
possible, but variations in the sequence in which the primers anneal (bind) may
introduce an amplification bias (i.e., in a mixture of botanically derived
ingredients, one material is amplified preferentially over the other). The
presence of plant polyphenols, such as tannins, in the amplification step also may
be problematic, since these metabolites inhibit the polymerases that amplify
the DNA.
The
final step in DNA barcoding is the sequencing of the DNA. The Sanger sequencing
method has been the method of choice in the past, and works well in cases in
which a single botanical ingredient is present. Drawbacks of the Sanger sequencing
method include the difficulty of obtaining accurate results for mixtures of
ingredients, the low throughput, and the need for greater amounts of DNA
compared to next-generation sequencing (NGS). NGS allows simultaneous analysis
of multiple DNA fragments (e.g., when multiple plant species are present, such
as in herbal preparations used in traditional Chinese medicine or Ayurvedic
medicine).
There
are, however, some notable advantages to using DNA barcoding compared to other
methods of plant species identification. While botanical taxonomy remains the
basis of plant classification when a whole plant can be examined in its natural
habitat (or as a pressed herbarium species), botanical ingredients are
predominantly sold in cut, powdered, or extracted form. For cut or powdered
crude raw material, DNA barcoding offers a reliable way to authenticate samples
in addition to methods such as organoleptic evaluation, botanical microscopy,
and chemical analysis, especially in cases in which species distinction may be
challenging.
For materials with fragmented DNA, the amplification of
shorter genomic regions, so-called mini-barcodes, may enable identification of
the plant material. In order for mini-barcoding to be successful, the analyst
has to design species-specific primers resulting in a much shorter amplicon (the piece of DNA that is amplified) compared to the
universal barcoding. To apply mini-barcoding, the analyst has to have an idea
of the composition of the material, and by using species-specific primers may fail
to detect adulterant material. Some plant materials may not be suitable for the
design of mini-barcodes (i.e., a small amplicon may not contain sufficient sequence
variation to distinguish among species).
The main limitations of current DNA
barcoding techniques are the possibility of erroneous results when DNA is
degraded (or when large amounts of DNA from other species is present), the
reliance on databases in which the material from which the DNA sequence was
obtained may not have been authenticated properly, and its inability to
distinguish plant parts, which is a legal requirement specified in the FDA’s current
good manufacturing practices (cGMPs) for dietary supplements.
As
with every analytical method used for quality control of botanical ingredients,
DNA barcoding methods need to be properly validated in order to ensure that the
results are accurate and reproducible. Validation requirements specific to DNA
barcoding of plant ingredients are a matter of debate, and the establishment of
guidelines to validate such methods is much-needed.
Overall,
DNA barcoding is a helpful tool to determine the authenticity of botanical
material in whole, cut or powdered form. However, based on its inherent
limitations, it should be used in combination with other identification
methods, such as microscopic, macroscopic, organoleptic, and chemical methods
of analysis.
—Stefan Gafner, PhD
Reference -
Parveen I, Gafner S, Techen N, Murch SJ, Khan IA. DNA barcoding for the
identification of botanicals in herbal medicine and dietary supplements:
strengths and limitations [published online July 8, 2016]. Planta Med. 2016. doi:10.1055/s-0042-111208.
Available at: www.thieme-connect.com/products/ejournals/pdf/10.1055/s-0042-111208.pdf. Accessed August 10, 2016.
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