Menu
×
News
Get Involved
About Us
Our Members
Enhancing Quality Control of Botanical Medicine in the 21st Century from the Perspective of Industry: The use of chemical profiling and DNA barcoding to ensure accurate identity

Introduction

Herbal products are growing increasingly popular in North America, including those derived from North American and European herbal traditions, Traditional Chinese Medicine, and Ayurveda. However, there are problems with many products on the market today. Misidentification of plant species, adulteration with counterfeit ingredients, insufficient quantities of the known primary active ingredients, and spiking with marker compounds commonly occur.

Manufacturers of many consumer products that include medicinal plant ingredients have an obligation to ensure that the products they sell are genuine and safe; marketers of food products containing so-called “medicinal botanicals,” including dietary supplements in the United States, usually have no regulatory requirements to ensure that their products are effective (unless certain limited health-related claims are made).

Adulteration via species substitution may occur accidentally or intentionally using closely related or completely unrelated species. Thus, the first step in quality control must be proper identification of each ingredient. Botanical medicinal materials are identified by their organoleptic (color, taste, fragrance, etc.), morphological (shape), microscopic, and/or chemical chromatographic characteristics, e.g., by the use of thin-layer chromatography (TLC) and/or other chromatographic methods. Someone who is not sufficiently knowledgeable of the plants in question will not be able to accurately identify botanical ingredients. Many closely related species share morphological features and/or common names, which can lead to potential confusion and accidental adulteration. Furthermore, most herbs are sold partially processed — dried, cut into pieces, shredded, or even powdered — such that macroscopic morphological identification of the plant part (flowers, leaves, roots, etc.) is no longer possible, although microscopic and chromatographic identification can still be performed.*

Reliable analytical methods are needed to supplement these typical protocols for identification of botanical medicinal materials. Chemical profiling using TLC, high-performance TLC (HPTLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC) is common, and such profiles are documented in herbal monographs found in resources such as the American Herbal Pharmacopoeia, the United States Pharmacopeia, the Pharmacopoeia of the People’s Republic of China, and the Journal of the Association of Official Analytical Chemists. In addition, techniques such as near-infrared (NIR) and nuclear magnetic resonance (NMR) spectroscopy are becoming more common in the scientific community. However, it must be considered that the chemical profile of an herb may vary due to factors such as growth stage, plant part, geography, and post-harvest processing and storage, which is why multiple reference materials must be used to statistically overcome such variations.

DNA barcoding is growing in popularity as a means of species identification.1 In October 2011, the US Food and Drug Administration (FDA) formally approved the use of DNA barcoding for the identification of seafood in order to counteract the widespread practice of substituting and mislabeling cheaper or undesirable species of fish and seafood as more expensive species.2 Simultaneous with this announcement, FDA released a validated laboratory method for the DNA barcoding of fish species for the purposes of regulatory compliance.3 We propose that DNA barcoding be added in the future to the quality control toolbox for medicinal botanical identification, alongside organoleptic, microscopic, and chemical profiling.

What Is DNA Barcoding?

DNA barcoding is the use of a short region of DNA to identify species.4 The first step to obtaining a DNA barcode is the extraction of DNA from a small sample of the specimen. Second, the selected barcode region undergoes polymerase chain reaction (PCR) amplification, or copying. Third, the PCR-amplified product is purified and sequenced (the order of nucleotides read). Finally, the DNA sequence is compared to the sequences in a library to identify the species in question. Figure 1 illustrates the DNA barcoding process.

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.

 

 

References

1.     Cimino M. Ensuring the specific identity and quality of herbal products by the power of DNA. HerbalGram. 2010;86:50-57.

2.     DNA-based seafood identification. US Food and Drug Administration website. Available at: www.fda.gov/Food/FoodSafety/Product-SpecificInformation/Seafood/DNAspeciation/default.htm. Accessed July 6, 2012.

3.     Single laboratory validated method for DNA-barcoding for the species identification of fish for FDA regulatory compliance. Available at: www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ucm237391.htm. Accessed July 6, 2012.

4.     Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc R SocLond B. 2003;270:313-321.

5.     Hollingsworth PM, Graham SW, Little DP. Choosing and using a plant DNA barcode. PLoS One. 2011;6(5):e19254.

6.     CBOL Plant Working Group. A DNA barcode for land plants. Proc Natl Acad Sci USA. 2009;106(31):12794-12797.

7.     China Plant BOL Group. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc Natl Acad Sci USA. 2011;108(49):19641-19646.

8.     Li DZ, Liu JQ, Chen ZD, et al. Plant DNA barcoding in China. J Syst Evol. 2011;49(3):165-168.

9.     Hollingsworth PM. Refining the DNA barcode for land plants. Proc Natl Acad Sci USA. 2011;108(49):19451-19452.

10.  Chen SL, Yao H, Han JP, et al. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One. 2010;5(1):e8613.

11.  Ratnasingham S, Hebert PDN. BOLD: The Barcode of Life Data System (www.barcodinglife.org). Mol Ecol Notes. 2007;7:355-364.

12.  Yao H, Song JY, Liu C, et al. Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS One. 2010;5(10):e13102.

13.  Liu C, Liang D, Gao T, et al. PTIGS-IdIt, a system for species identification by DNA sequences of the psbA-trnHintergenic spacer region. BMC Bioinformatics. 2011;12(Suppl 13):S4.

14.  Lou SK, Wong KL, Li M, But PPH, Tsui SKW, Shaw PC. An integrated web medicinal materials DNA database: MMDBD (Medicinal Materials DNA Barcode Database). BMC Genomics. 2010;11:402.

15.  Liu C, Shi LC, Xu XL, et al. DNA barcode goes two dimensions: DNA QR code web server. PLoS One. 2012;7(5):e35146.

16.  Chen SL, Pang XH, Yao H, Han JP, Luo K. Identification system and perspective for DNA barcoding Traditional Chinese Materia Medica. World Science and Technology – Modernization of Traditional Chinese Medicine. 2011;(5):747-754 and references therein.

17.  Special Issue: Plant DNA barcoding in China. J Syst Evol. 2011;49(3):165-283.

18.  Li M, Cao H, But PPH, Shaw PC. Identification of herbal medicinal materials using DNA barcodes. J Syst Evol. 2011;49(3):271-283.

19.  Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China. Beijing: Chemical Industry Press; 2010.

20.  Brown RP, Gerbarg PL, Ramazanov Z. Rhodiola rosea. A phytomedicinal overview. HerbalGram. 2002;56:40-52.

21.  Ma YC, Zhu J, Benkrima L, et al. A comparative evaluation of ginsenosides in commercial ginseng products and tissue culture samples using HPLC. J Herbs Spices Med Plants. 1996;3(4):41-50.

22.  Ma YC, Luo M, Malley L, Doucet M. Distribution and proportion of major ginsenosides and quality control of ginseng products. Chinese Journal of Medicinal Chemistry. 1996;6(1):11-21.

23.  Ma J, Ma YC, Wang D, et al. Simultaneous quantification of Panax and Epimedium species using Rapid Resolution Liquid Chromatography (RRLC). Nat Prod Commun. 2011;6(5):581-586.

24.  Ma YC, Wang XQ, Hou FF, et al. Rapid resolution liquid chromatography (RRLC) analysis for quality control of Rhodiola rosea roots and commercial standardized products. Nat Prod Commun. 2011:6(5):645-650.

25.  Ma YC, Wang XQ, Hou FF, et al. Simultaneous quantification of polyherbal formulations containing Rhodiola rosea L. and Eleutherococcus senticosus Maxim. using rapid resolution liquid chromatography (RRLC). J Pharm Biomed Anal. 2011;55:908-915.

26.  Ma YC, Wang XQ, Hou FF, et al. Rapid resolution liquid chromatography (RRLC) analysis and studies on the stability of Shuang-Huang-Lian preparations. J Pharm Biomed Anal. 2011;54:265-272.

27.   Ma J, Ma YC, Cai C, et al. Simultaneous quantification of Echinacea species, Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae combinations by rapid resolution liquid chromatography. Nat Prod Commun. 2011;6(5):639-643.