Detecting the Components of Botanical Mixtures by Single-Strand

Chapter 24

Downloaded by OHIO STATE UNIV LIBRARIES on June 19, 2012 | http://pubs.acs.org Publication Date (Web): November 17, 2011 | doi: 10.1021/bk-2011-1081.ch024

Detecting the Components of Botanical Mixtures by Single-Strand Conformation Polymorphism Analysis Michelle R. Lum1,3 and Ann M. Hirsch*,1,2 1Department

of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095-1606 2Molecular Biology Institute University of California, Los Angeles, California 90095-1606 3Current address: Department of Biology, Loyola Marymount University, Los Angeles, CA 90045-2677 *E-mail: [email protected]

Botanical supplements are increasingly being used in the United States, and of major importance is the authenticity of their ingredients. Here we describe a molecular method for species identification that takes advantage of the species variation in the internal-transcribed spacer region of the 18S-23S rDNA of plants. We show that analysis of the ITS-2 region by single-strand conformation polymorphism facilitates the detection of botanical ingredients and potentially contaminating plant material, which can subsequently be identified by DNA sequencing. Our method is useful for authenticating plant species prior to their being processed for manufacture as botanical products and also for recognizing contaminating or adulterating species in dried or powdered botanicals.

© 2011 American Chemical Society In Progress in Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


Introduction Herbal supplements, also known as botanicals, represent a market that has been growing at a rapid pace over the past 15 years. In the United States alone, it is estimated that over 80 million people use some type of botanical supplement, and in 2008, the U.S. market was an estimated $4.8 billion (1). In many parts of the world, particularly in developing countries, herbal products are utilized almost exclusively for health care, as pharmaceuticals are either too expensive or not readily available. For example, in rural and urban South Africa, approximately 60-70% of the people use some type of indigenous herbal medicine (2). The medicinal properties of botanicals result from secondary plant products contained within various tissues and organs of the plants. Phytochemicals such as alkaloids, phenylpropanoids, and terpenoids are well documented for their pharmacological activities. However, even though more information is being obtained on the bioactivity of these compounds, the efficacy of the botanical supplements used to deliver them may be questionable due to lack of standardization and defined quality control standards for cultivation and processing of the plant material. Different commercial products have been found to be highly variable in pharmacologically relevant phytochemicals following analysis (3, 4). Such variability could be due to a multitude of factors, including different harvesting and processing techniques used by diverse manufacturers. Moreover, problems also arise because of misidentification of plant material or a confusion of plant names. Hence, a major concern of consumers is the authenticity of the botanical supplements they use. This concern has been exacerbated by actual incidents where the presence of an undesirable botanical resulted in illness. In one case, two women were hospitalized because of digoxin poisoning after taking a herbal product that was contaminated with Digitalis lanata (5). In another instance, a number of patients in a clinical weight loss trial developed severe nephrotoxicity, in some cases resulting in irreversible damage and the need for kidney transplants (6). This toxicity effect was determined to be due to the presence of Aristolochiza fangchi, which had been supplied instead of Stephania tetrandra (7). Identification and authentication of botanical material used to make herbal products has traditionally relied on morphological characteristics and phytochemical markers (8). More recently, molecular methods, such as sequencing, RAPD, RFLP and microarrays have been reported (see (9) for review). However, although these methods can be used to confirm the presence of specific species, they are not as readily applied to determining and identifying multiple species in a product. We therefore sought to develop a DNA-based method to identify the plant species present in a given sample of plant material, whether it be present as a single component or a mixture. Such a technique would be useful for identifying the contents of botanical supplements and for confirming the identity of the raw material used for preparing botanical mixtures. DNA sequences give the most unambiguous results because DNA does not change in response to environmental triggers, which might be a factor when using phytochemical marker compounds for analysis. In addition, a large database of DNA sequences cataloging various gene sequences is available, particularly 352 In Progress in Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


for the Internal Transcribed Spacer (ITS) region in plants (10). We assessed the utility of the two ITS regions, ITS-1 and ITS-2, as well as the chloroplast matK gene (11) in an analysis of plant material by Single-Strand Conformation Polymorphism (SSCP). We found that the use of the ITS-2 region distinguishes between individual plant species. We further show that SSCP can be used to verify the presence of individual species in a botanical product and to reveal whether contaminating species are present, the identity of which can be subsequently determined by DNA sequencing.

Materials and Methods DNA Extraction A modified CTAB procedure (12) was used to extract genomic DNA from fresh leaves of alfalfa (Medicago sativa), red clover (Trifolium pratense), woad (Isatis indigotica), European licorice (Glycyrrhiza glabra), Chinese licorice (Glycyrrhiza uralensis), or plant material contained in a commercial red clover product and two different commercial alfalfa or European licorice dietary supplements. Aliquots of DNA were run on a 1% agarose gel to check the quality of the DNA. A repair reaction as described by LeRoy et al. (13) was used on those samples that appeared degraded as evidenced by a smear on a gel and failure to produce a PCR product.

PCR Amplification The polymerase chain reaction was carried out in a final volume of 20 µl with 1U Eppendorf Hotmaster Taq, 1x PCR buffer, 1.5 mM MgCl2, 100 ng of each primer, 1 mM of each deoxynucleotide (dATP, dCTP, dTTP, dGTP) and 1 µl of genomic DNA from fresh tissue or 4 µl of repaired DNA. The primers used, as described in Blattner et al. (14), were ITS-A and ITS-C, which amplifies the ITS-1 region between the 18S and 5.8S rRNA genes or the ITS-D and ITS-B, which amplifies the ITS-2 regions between the 5.8S and 28S rRNA genes. The matK primers were designed by aligning representative sequences from the database and designing degenerate primers to conserved regions. The forward primer F2 5′TATGSACTTGCTYATRRTCAT3′ and reverse primer R3 5′GAACYAAKATTTCCARAT3′ produce a 400 bp fragment. The reverse primer for ITS-2 or matK was phosphorylated at the 5′ end to promote single-strand digestion by lambda exonuclease (15). PCR was conducted at 94°C for 5 min, followed by 25 cycles of 30 s 94°C, 30 s 57°C, 90 s at 68°C, followed by a final extension step at 68°C for 10 min.

353 In Progress in Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


Genetic Profiling by Single Strand Conformational Polymorphism (SSCP) The PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA). Ten µl of the purified product was either heat-denatured or digested by lambda exonuclease (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at 37°C for 2 hours. The digested product was purified with the Qiagen Minielute kit and eluted in 10 µl 1 M Tris-HCl. Eight µl of denaturing loading buffer (95% formamide, 10 mM NaOH, 0.25% bromophenol blue, 0.25% xylene cyanol) were added to each eluted product and incubated at 95°C for 3 min and snap cooled on ice. The entire sample was loaded on a 0.6X MDE (Mutational Detection Enhancement) gel (Cambrex, Rockland, ME) using 0.4 mm spacers on a Vertical Gel Electrophoresis System (BRL, LifeTech, Inc., MD) and a 1X TBE (16) running buffer. Gels were run at room temperature at 7 mA and 90 V for 14 hours. DNA was visualized by soaking the gels in 1 µg/mL ethidium bromide for 30 min. Extraction, Reamplification, and Sequencing Bands were cut out of ethidium bromide-stained MDE gels and eluted at 37°C for 2 hours in an elution buffer (0.5 M NH4OAc, 10 mM MgOAc, 1 mM EDTA, 0.1% SDS). One µl eluent was used in polymerase chain reactions using the ITS-2 primers. The resulting PCR products were run on an agarose gel, purified using the Qiagen gel extraction kit, and directly sequenced using ABI Big Dye Terminator mix and automated sequencing with an ABI 3700 Capillary DNA Analyzer at the UCLA Sequencing Core Facility. Sequence Analysis Sequences were compared to the non-redundant database using NCBI BLAST and default settings.

Results We first confirmed that we could obtain amplifiable DNA from each of our samples. The ITS-2 region could readily be amplified by PCR using DNA extracted from fresh leaves of alfalfa, red clover, European licorice, Chinese licorice, and woad (Figure 1B). Bands showed similar migration, indicating that differentiation based on standard agarose electrophoresis would not distinguish amng the species (Figure 1B). Genomic DNA of the commercial products was found to be degraded when observed on an agarose gel (Figure 1A). Repair reactions on the DNA isolated from the red clover product and first alfalfa product had to be done before a PCR product could be obtained (Figure 1C). SSCP had previously been successfully applied to the analysis of bacterial and fungal populations, as well as to plants. We desired to extend its application by determining the sensitivity of the method for distinguishing different plant species as well as for detecting the presence of contaminating plant material. We initially looked at five species commonly used in botanicals: alfalfa, red clover, 354 In Progress in Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


European licorice, Chinese licorice, and woad. SSCP analysis of the ITS-2 region for the five different species tested showed that the species could be differentiated based on the migration of single-stranded DNA (Figure 2). In contrast, when we used the ITS-1 region, a distinction between European and Chinese licorice could not be detected (data not shown), similar to the results obtained with the matK gene (Figure 3). A comparison of DNA products generated by heat denaturation (Figure 2A) versus single-strand digestion by lambda exonuclease (Figure 2B) showed that exonuclease digestion was more efficient at generating ssDNA products and produced fewer bands in Chinese licorice. Therefore, either method of ssDNA generation was effective at distinguishing between species, but exonuclease digestion was generally better.

Figure 1. Agarose gel electrophoresis of genomic DNA and the ITS-2 region. (A) Genomic DNA isolated from the contents of commercial alfalfa or red clover capsules was severely degraded as indicted by a smear on an agarose gel. (B) The ITS-2 region was successfully amplified from DNA isolated from fresh leaves and from capsules of the second alfalfa product, but not from the DNA from the first alfalfa product or the red clover product. (C) After a repair reaction, the ITS-2 region could be amplified from DNA from the alfalfa and red clover commercial products.



Figure 2. SSCP analysis of the ITS-2 region can be used to distinguish between different plant species. (A) SSCP analysis of PCR products after heat denaturation showed both single-stranded (ssDNA) and double-stranded DNA (dsDNA) products, which illustrated a unique migration pattern for each of the species tested. (B) Lambda exonuclease digestion was more efficient at generating ssDNA.

To verify that SSCP could distinguish species in a mixture, we prepared a simulated plant contamination event by mixing alfalfa and woad genomic DNA. We found that the amplified alfalfa and woad PCR products could be differentiated based on the ssDNA or dsDNA bands generated by PCR amplification of the ITS-2 region and SSCP analysis (Figure 4). To test the sensitivity of this technique for detecting contaminating plant material, we mixed alfalfa DNA with decreasing amounts of woad DNA. The woad contaminant could still be detected at a 1:5000 woad:alfalfa dilution (Figure 4).



Figure 3. The use of matK in SSCP analysis distinguished alfalfa, red clover, licorice, and woad products. However, no obvious difference in band migration was seen between European licorice and Chinese licorice.

We next applied the SSCP technique to commercial products. We analyzed a red clover product and two different commercial products of alfalfa and European licorice. SSCP analysis of each supplement showed bands corresponding to those expected for each product (Fiure 5). However, the analysis of the commercial supplements also revealed faster or slower migrating bands that might be contaminants (Figure 5). The identity of the single-strand PCR products generated from each of the supplements was determined by extraction of the individual bands, PCR reamplification, and then direct sequencing.



Figure 4. SSCP can detect a woad “contaminant”. SSCP analysis of alfalfa containing a simulated woad contaminant using heat denaturation showed that the presence of the two different species could be detected using the ITS-2 region. The woad “contaminant” was detected even when present at a 1:5000 dilution (lower arrow).

The DNA sequence of the bands corresponding to the expected size for the species specified in the product were found to be identical to alfalfa, red clover or European licorice sequences in the database. Sequencing of the bands of the potential contaminants resulted in some ambiguous base calls, suggesting a mixture of species was present. Nevertheless the contaminant in the red clover product was most similar to a Trifolium species (92%), albeit one distinct from T. pratense. The additional band in the first alfalfa product was found to have greatest similarity to species of Taraxacum (dandelion) (93%). The sequence of the contaminant in the second alfalfa supplement was most similar to red clover (95%). The sequences of the contaminants in the European licorice products were most similar to Cimicifuga racemosa (black cohosh) (97%) and Stellaria media (chickweed) (94%) (data not shown). Cloning and then sequencing the PCR product from the potential contaminating bands rather than direct sequencing would likely result in more robust identifications.



Figure 5. SSCP analysis using lambda exonuclease of commercial alfalfa and red clover products. SSCP analysis of DNA from the commercial alfalfa, red clover, and European licorice products produce a PCR product that migrates to a distance similar to that of DNA from plants of alfalfa, red clover, or European licorice. However, each of the commercial products contains additional faster or slower migrating bands. Asterisks have been placed to the left of these putative contaminants.

Discussion Molecular approaches for identifying the components of dietary supplements have been described before, but few can readily detect multiple components in the product. PCR-based methods that have been commonly used for botanical authentication are PCR-RFLP, RAPD, direct sequencing, etc. (9, 17). Mihalov et al. (18) used PCR combined with sequencing and DNA fingerprinting to identify ginseng in commercial samples. Through direct sequencing of a PCR-generated DNA band, they identified a gene sequence that indicated that the sample contained soybean. However, such an analysis cannot be used when multiple species are present. Another common way to analyze botanicals is RAPD analysis, which relies on comparisons of known molecular fingerprints. However, this technology is not feasible for identifying species for which a profile does not 359 In Progress in Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


already exist. We and others (for examples, see (19–21)) have used PCR-RFLP to detect contaminating material. In this method, a PCR fragment is digested by a restriction enzyme to give multiple fragments. Its drawback is the number of steps involved and the possibility that adulterants contain the same restriction site as the species of interest. Single-strand conformation polymorphism analysis was developed for the detection of mutations, specifically in human DNA (22, 23). However, it has become a useful tool in the study of communities and has successfully been used in population biology for the analysis of bacterial, fungal, and sponge populations (24, 25). Although a few examples of SSCP applied to assess plant genotype have been published (26, 27), overall this method has not been widely utilized for plants, especially to species with medicinal properties, except for cinnamon (28). Because SSCP detects even small base differences between species, we believe it to be an ideal procedure for detecting and identifying contaminating and/or adulterating species in botanicals. Here we show that SSCP can be used for discriminating different plant species from one another and for identifying whether a botanical sample or dietary supplement contains multiple species either as contaminants or adulterants. We found that the ITS-2 region worked well for differentiating plant species and also provided better resolution than ITS-1 or matK. For example, SSCP analysis of the nuclear ITS-2 region showed distinct variation for each of the five plant species tested, including European and Chinese licorice, both members of the genus Glycyrrhiza. Our results analyzing plant mixtures showed that the individual components of the mixture could be distinguished, demonstrating that this method could be used to assess the contents of a botanical product. Our analysis of commercial products using SSCP, with confirmation by sequencing, illustrates that SSCP is an effective technique for confirming species in mixed populations. Moreover, high-throughput methods of capillary SSCP have been developed (29), and these could be adapted to analysis of botanicals, although sequence information would be less readily obtainable due to the lack of a standard way of collecting the DNA fragments from the capillary for subsequent sequencing. The main drawback with our method, or any other DNA-based protocol is the need to have amplifiable DNA. Although we have shown previously, and in this report, that even severely degraded DNA can be repaired and amplified, products that exist solely as plant extracts lack DNA and hence cannot be authenticated or identified using these techniques. However, the SSCP method can be applied to any botanical product that contains intact cellular material and for assessing plant material prior to its processing for botanical products. We have shown that SSCP combined with sequencing allows for the differentiation and identification of specific species, even those that are very closely related. Therefore, in conjunction with more traditional methods of authentication of botanicals such as microscopic analysis or the assessment of marker compounds, SSCP could be used to demonstrate unequivocally whether contaminants or adulterants are present, and moreover, facilitate their exact identification.


Acknowledgments This research was funded in part by NIH/NCCAM 5 P50 AT00120 to the Center for Dietary Supplements Research Botanicals (CDSRB) at UCLA.

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Detecting the Components of Botanical Mixtures by Single-Strand

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