PCR amplification entails multiple cycles of a three-phase process. The double-stranded DNA is denatured (separated into its individual strands) at a high temperature. Next, the temperature is lowered and sequence-specific primers (short sequences of 20 or so nucleotides) attach to sites neighboring the target sequence. Primers are required as the DNA polymerase can only add new nucleotides to an existing piece of double-stranded DNA. Finally, the DNA polymerase uses the single strand of DNA as a template to extend the sequence from the primers. This new product then becomes the template for the next cycle. The cycles are repeated 20 to 30 times, generating thousands to millions of copies of the target DNA sequence.

Standardization, minimalism, and scalability are key factors in the application of DNA barcoding. Practically speaking, this means that one or a few standard regions of a limited number of DNA base pairs (usually 200 to 1,000) must be chosen so that they can be sequenced readily in a large and varied sample set, enabling comparison of the data and allowing for species identification.⁵ As a corollary, the inter-species variation in the DNA sequence should be much larger than the intra-species variation. In animals, a fragment of the cytochrome c oxidase 1 (CO1) gene has been accepted as the standard DNA barcode. In plants, however, no single region has been found that meets all of the criteria of universality (ease of sequencing in all land plants), sequence quality, and species discrimination. The Consortium for the Barcode of Life (CBOL) has proposed the combination of the matK and rbcL genes as the core plant barcode, though it recognizes that matK + rbcL may at times need to be supplemented with other markers.⁶ Specifically, matK cannot always be amplified and sequenced, though its species discrimination is high, while rbcL is easy to amplify and sequence, but its species discrimination is low. The internal transcribed spacers of nuclear ribosomal DNA (nr ITS/ITS2) and the chloroplast intergenic spacer psbA-trnH have been proposed as alternates to matK + rbcL,⁵ and, in fact, the China Plant Barcode of Life group has suggested the addition of ITS, or ITS2 when ITS cannot be successfully sequenced, to the core plant barcode of matK + rbcL.⁷

The biggest challenge thus far in DNA barcoding of plants has been that good, universal primers for plant marker barcodes can be difficult to design. Amplification and/or sequencing of a given marker may be possible only in certain families of plants. For a particular marker, genetic gaps between species may be large in some groups of plants, but not in others.⁸ For these reasons, it appears that several markers, alone or in combination, will be required for the DNA barcoding of plants, rather than the single CO1 marker prevalent in the DNA barcode analysis of animals.

A further problem with DNA barcoding of plants is that many plants lack barcodes altogether, and there is not yet a universal database of plant barcodes.⁹ However, as a major use of DNA barcoding is the identification of unknown specimens, non-chemistry specialists such as customs officers, producers of traditional medicines, pharmaceutical manufacturers, and forensics investigators may welcome a relatively rapid and simple — albeit still imperfect — method for the identification of botanical products.¹⁰ Nevertheless, a great deal of work is still needed before DNA barcoding of plants can be considered sufficiently reliable for widespread practical application.

GenBank is a database of all publicly available DNA sequences and is part of the International Nucleotide Sequence Database Collaboration, which also includes the DNA DataBank of Japan and the European Molecular Biology Laboratory. These three organizations exchange data on a daily basis. Databases specific to DNA barcodes include the Barcode of Life Database,¹¹ which is based on the matK + rbcL combination, as well as the IdIt-ITS2 and PTIGS (IdIt-psbA-trnH-IGS) databases, which are based on ITS2 and psbA-trnH, respectively.^12,13

Of particular interest and use to those in industries or markets that utilize medicinal plants as ingredients is the Medicinal Materials DNA Barcode Database (MMDBD).¹⁴ At the time of its 2010 publication, the database contained more than 18,000 sequences from 1,259 species, representing 66.5% and 84.5% of the medicinal materials listed in the 2005 Pharmacopoeia of the People’s Republic of China and the American Herbal Pharmacopoeia, respectively. As of May 2012, the MMDBD featured more than 31,000 barcode sequences from more than 1,650 indexed species. Core and supplementary DNA barcodes for medicinal materials listed in the above pharmacopeias and other sources are included, as well as information on adulterants and substitutes, photographs of the medicinal materials, PCR conditions, and literature references. The database can be searched by keyword or sequence similarity, and researchers can upload their DNA barcode sequences to help expand the database.^‡

Finally, Liu et al. have established a web application that will convert a DNA barcode into a two-dimensional Quick Response (QR) Code for use in practical applications — in essence, barcoding the barcode.¹⁵ The user can retrieve the DNA sequence and QR code for a species of interest, convert a sequence to a QR code and vice-versa, or search the database using a QR code to identify a sample. This leads one to envision a system in which an herbal material is labelled with its QR DNA barcode as a means of inventory tracking.

Undoubtedly, DNA barcoding of plants will improve with advances in PCR amplification and DNA sequencing technology. Identification of plants will be enhanced with better access to authenticated botanical DNA libraries that contain more species and more samples of each species.

DNA Barcoding of Botanical Medicines

According to surveys in China, medicinal plants comprise more than 11,000 species in 2,300 genera and nearly 400 families. Quick and accurate authentication of these plants and their adulterants can be difficult on an international trade scale. Shi-Lin Chen, PhD, an author of this article,  and colleagues at the Institute of Medicinal Plant Development in Beijing have been dominant in the field of DNA barcode analysis of botanical medicines. Chen et al. investigated different DNA regions for the purpose of barcoding plants found in the traditional Chinese Materia Medica, both in terms of PCR efficiency and species identification.¹⁰ The PCR efficiency for both ITS2 and psbA-trnH was greater than 90 percent. Furthermore, psbA-trnH was more successful for some plants such as ferns. The identification rate of ITS2 was 92.7% and 99.8% at the species and genus levels, respectively, for 6,685 samples from 4,800 species in 753 genera of 193 families. In contrast, psbA-trnH correctly identified only about 70 percent of the species, though it was more than 95 percent accurate at the genus level for 2,108 samples from 1,433 species in 551 genera of 135 families. They proposed the use of ITS2, supplemented by psbA-trnH, as the standard barcode for international trade and safe use of medicinal plants.

In an additional study, Yao et al. evaluated the ITS2 sequences of 50,790 plant samples available in GenBank. Species identification rates ranged from 67 percent to 88 percent.¹² A recent review article by Chen et al. summarized their work on the families Rosaceae, Fabaceae, Asteraceae, Rutaceae, Euphorbiaceae, Polygonaceae, and the genera Paris (Melanthiaceae), Lonicera (Caprifoliaceae), Dendrobium (Orchidaceae), Cistanche (Orobanchaceae), Panax (Araliaceae), and Datura (Solanaceae), as well as medicinal pteridophytes and cortex herbs (medicinal materials from the bark of stems or roots).¹⁶

The Journal of Systematics and Evolution recently published a special issue on plant DNA barcoding in China.¹⁷ In particular, Li et al. reviewed more than 125 studies on the application of DNA barcodes to the identification of more than 75 different Chinese herbal medicinal materials.¹⁸ They concluded that DNA barcoding of medicinal plants is still a work in progress, but that it holds great promise for future applications in taxonomy, biodiversity, conservation, the pharmaceutical industry, and forensics; the authors proposed that future work should focus on reliable species identification and barcoding multiple samples of each species to help build the reference database for Chinese medicinal plants. The Chinese Pharmacopoeia Commission, recognizing the value of DNA barcoding for the authentication of medicinal materials, has included protocols and DNA barcodes for some animal-derived traditional Chinese medicines in the 2010 edition of the Pharmacopoeia of the People’s Republic of China, such as Wushaoshe (Chinese rat snake; Zaocys dhumnades, Colubridae) and Qishe (Chinese moccasin; Agkistrodon acutus, Viperidae).^18,19 Work is underway on drafting guidelines for the identification of Chinese herbal medicines using DNA barcodes, potentially to be included in the 2015 edition (Hui Yao email to M. Thibault, September 5, 2012).

An Explanation of Chemical Profiling

DNA barcoding is an excellent solution for identifying raw or dried plant products. However, many botanical products are sold as liquid or powder extracts. The alcohol and heat used during the extraction process filters out or eliminates most cellular data and denatures proteins and DNA, rendering DNA barcoding unfeasible. Consequently, chemical identification of marker compounds must be utilized.

Raw herbs and extracts possess a characteristic botanical profile of phytochemicals. Initially, one or two of these phytochemicals were used as marker compounds for the purpose of qualitative and quantitative quality control, which led to spiking with low-quality or fraudulent botanical extracts containing the marker compounds by unscrupulous producers. With the technological advances of the last 20 years, simultaneous analysis for multiple chemical constituents is possible. Thus, many herbs and botanical extracts are now analyzed for several marker compounds as a means of circumventing potential spiking issues. For example, Rhodiola rosea (Crassulaceae) root extracts were formerly standardized only for salidroside. After the discovery of widespread substitution of other Rhodiola species for R. rosea, the latter extracts are now standardized for salidroside and rosavins. Rosavins are unique to R. rosea, whereas salidroside is found across the Rhodiola genus and in some plants outside the genus.²⁰

HPTLC is a simple, rapid, economical, and qualitative method of identification. It allows for the natural variability within a plant and can be used even when many chemical components of the sample are unknown. Reference compounds, plant samples, and adulterants can be compared in a parallel, high-throughput fashion. In addition, the multiple chemical components of an herb are often present in a consistent ratio to one another. HPLC commonly is used to separate and quantify these constituents, which results in a characteristic profile, or fingerprint, of the herb or extract. Manufacturers can use these profiles to help optimize their extraction procedures, such that the resultant extract has the same profile as the initial raw herb. This is beneficial to herbalists, naturopaths, integrative physicians, and other traditional medicine practitioners who have a holistic view of herbs and healing.

Over the last 20 years, there have been thousands of publications discussing the HPLC profile of popular herbs. As mentioned earlier, many pharmacopoeias include HPLC methods and profiles for quality control in the botanical industry. The Canadian Phytopharmaceuticals Corporation has established a proprietary database of HPLC profiles for more than 100 North American, South American, European, Ayurvedic, and traditional Chinese botanicals and extracts. Shown in Figure 2 are the HPLC profiles developed by this HerbalGram article’s co-author, Ma, and colleagues in the 1990s for American ginseng (Panax quinquefolius, Araliaceae), Asian ginseng (P. ginseng), and notoginseng (P. notoginseng).^21,22 Each of these species has a characteristic ratio of ginsenosides that distinguish one from the other.

With advances in technology — such as the development of Ultra High-Performance Liquid Chromatography (UHPLC, also commonly referred to as UPLC), gradient elution, multi-wavelength detectors, and other types of detectors — analytical methods have become much more powerful and simple. UHPLC offers significant time and cost savings over conventional HPLC, due to its shorter run times and concomitant reduced solvent usage. Thus, returning to the example of the three Panax species, the UHPLC profiles developed in the 2010s are completed in half the time but maintain the same appearance as the earlier HPLC profiles (Figure 3). In a further development, a method recently was established in which the three Panax species, alone or in combination with Epimedium leaves (Berberidaceae), could be quantified in just four minutes as compared to the 45 minutes required by the HPLC method.²³

Remedies developed by traditional Chinese and other herbal medicine practitioners often involve combinations of herbs. Method development for the HPLC fingerprinting of formulated or combination products represents a breakthrough in the quality control of botanical products. Individual herbs may have been analyzed by different methods, using different HPLC columns, solvent gradients, or detection wavelengths. Their profiles may overlap; hence, new methods must be developed that will distinguish the profile for each herb, yet still allow for analysis within a reasonable timeframe. The complexity of this task necessarily increases with the number of herbs present in the combination product.

As an example, consider a formulated product consisting of American ginseng roots, Epimedium koreanum (Berberidaceae) leaves, eleuthero (Eleutherococcus senticosus, Araliaceae) rhizomes, and R. rosea roots. Such a combination may be used as a Western-style “Energy Formula.” UHPLC profiles for the latter three herbs are shown in Figure 4, with relevant marker compounds labeled. Run times range from four minutes for Epimedium to eight minutes for R. rosea²⁴ and eleuthero.²⁵ The UHPLC profile for the combination product (Figure 5), while complicated, clearly shows the unique fingerprint of each herb, and the quantification of more than 20 compounds is complete in only 22 minutes in a single run.

Shuang-Huang-Lian (SHL) is a traditional Chinese formula comprised of Flos Lonicerae (Japanese honeysuckle; Lonicera japonica, Caprifoliaceae), Radix Scutellariae (Chinese skullcap; Scutellaria baicalensis, Lamiaceae), and Fructus Forsythiae (forsythia; Forsythia suspensa, Oleaceae). It is used commonly to treat upper respiratory illnesses. Ma et al. developed a UHPLC profile for SHL that is complete in seven minutes (Figure 6),²⁶ and extended the study to an “East-meets-West” SHL-Echinacea combination (E. angustifolia and E. purpurea, Asteraceae).²⁷

Technological and analytical methodology development makes possible the qualitative and quantitative analysis of multiple marker compounds in formulated products, guaranteeing the quality of these products. Very few manufacturers currently analyze combination products. Those that do are in a position to be leaders in the marketplace.

Conclusion

The current industry standards can and will change and improve according to market demands. Industry must take the lead and set the benchmark for the quality control of botanical extracts and Traditional Chinese Medicine, to counteract the erroneous belief that herbal medicines are unregulated, untested, and ineffective. Combining the applicable, reliable, and practical complementary techniques of DNA barcoding and chemical profiling for the quality control of herbal products — from raw herb to extract to finished product — will assure the delivery of high-quality, safe, and efficacious products to market. Since DNA barcoding is not yet ready for widespread implementation, an interim solution would be for botanical product manufacturers to establish specifications that require testing and conformance of the raw herbal material or extract with a pharmacopoeial monograph. This includes organoleptic, microscopic, and chemical (TLC or HPLC) profiling. Furthermore, by better validating the quality of botanical ingredients used in products that may undergo robust pharmacological or clinical studies, there should be a higher level of confidence and scientific credibility in the clinical results.

Yuan-Chun Ma, PhD, founder, president, and CEO of Canadian Phytopharmaceuticals Corporation (CPC) in Vancouver, BC, Canada, received his doctorate in Pharmaceutical Sciences from the School of Pharmacy and Biomedical Sciences at the University of Portsmouth in England. Author of over 60 research and scientific publications, Dr. Ma is a guest professor with both the Chinese Academy of Medical Sciences in Beijing and the Tongji Medical School, Huazhong University of Science and Technology in Wuhan, China. He can be contacted at info@canphyto.com.

Professor Shi-Lin Chen, PhD, is director of the Institute of Medicinal Plant Development, affiliated with the Chinese Academy of Medical Sciences and Peking Union Medical College in Beijing. He obtained his PhD from Chengdu University of Traditional Chinese Medicine and has been a Visiting Professor at Hong Kong Polytechnic University. He holds a concurrent position as the editor of such reputable Chinese medicinal research journals as China Journal of Chinese Materia Medica, Chinese Medicine, Journal of Chinese Pharmaceutical Science, Chinese Traditional and Herbal Drugs, and World Science and Technology. He has published more than 160 scientific papers.

Michelle E. Thibault, PhD, is a quality administrator at CPC. She received her doctorate in Chemistry from the University of Guelph in Canada, and is co-author of 15 papers and one patent.

Jie Ma is a PhD candidate in the School of Pharmacy and Biomedical Sciences at the University of Portsmouth and quality control manager at CPC.

* Responsible companies should require their qualified suppliers to carry out suitable identification tests, including appropriate macroscopic examination, prior to particle size reduction and also require their supplier’s quality control unit to retain samples of the whole, uncut starting material so that the companies can trace back and re-test such material at a future date in the event of a problem requiring rapid investigation, for example, a quality problem (adulteration, contamination, etc.) or, in a worst-case scenario, a product recall.

†This may seem self-evident. In the context of pharmacopoeias mentioned in the previous sentence, these factors are accounted for in the establishment of a monograph. If a material does not test in conformance with all of the qualitative and quantitative limits in the monograph, then a company producing botanical products with the intention that they should provide a therapeutic or other health benefit likely should reject the material as it would be indicative of one or more problems at source, such as having been harvested at the wrong growing stage (e.g., immature) or wrong plant part (e.g., should be all leaf but contains a high percentage of stems), or having been grown in the wrong climate such that secondary metabolites never developed due to lack of stress conditions.

‡The PPRC 2010 has 2,165 botanical monographs including Chinese Materia Medica crude drugs, crude drug preparations, prepared slices, patent Chinese traditional medicines, oils and extracts. So far the AHP has published 33 monographs.

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