Contamination and substitution in North American herbal products

Few studies have attempted to estimate how prevalent herbal product contamination and substitution is in the marketplace. To date there are only a few [1–3] studies of herbal product substitution, representing <1% of the herbal products and <5% of the herbal companies in North America. These studies document species-level DNA barcode identification success for 42% to 66% of the herbal species tested. Our study demonstrates increased testing and success, including (i) species-level DNA barcode identification success to 95% of the herbal species tested, and (ii) herbal market testing by approximately 20% for both products and companies. Although we provide new estimates of herbal product contamination and substitution, we caution that these values need to be refined by further studies as we have yet to sample >1% market. However, our estimates do corroborate those of other studies. For example, Stoeckle et al.[1] investigated contamination in 131 herbal tea products representing 48 herbal species and found that although they were able to authenticate 58% of the products, 33% of the herbal teas tested were contaminated. Similar estimates from a marketplace survey [2] of 40 dietary supplement products labeled as 'black cohosh’ (Actaea racemosa) found that 75% of the samples were authentic black cohosh, whereas 25% were substituted with 4 closely related Asian Actaea species (A. asiatica, A. cimicifuga, A. dahurica, and A. simplex). We also found substitution of black cohosh for Asian Actaea, as did another study by Wallace et al.[3]. Wallace et al.[3] also documented that 50% of ginseng products labeled 'Korean ginseng (Panax ginseng)’ were actually American ginseng (Panax quinquefolius). Although the DNA barcode success rate was low (48%, see below) in Wallace’s study, they were still able to detect product substitution in 14% of the herbal samples tested, representing 7 species of herbs in 14 products. This study also detected fillers in 21% of the herbal products [3]. We found alfalfa (Medicago sativa) in 16% of the products tested, which was also previously detected in teas [1]. It is unlikely that this was a contaminant as it was found in so many of the products in addition to the main ingredient. It may be possible that this species is used by some companies as a cheap filler as it is commonly grown and bailed in agricultural areas, and available in large quantities throughout the year.

Contamination and substitution in herbal products present considerable health risks for consumers. In our study, we found contamination in several products with plants that have known toxicity, side effects and/or negatively interact with other herbs, supplements, or medications. For example, we found that one product (HP8) labeled as St. John’s wort (Hypericum perforatum) was substituted with Senna alexandrina (fabaceae); it contained only senna barcodes and no St. John’s wort barcodes. This is a serious health risk as senna is a Food and Drug Administration (FDA)-approved non-prescription herbal laxative, which is not for prolonged use as it can cause adverse effects such as chronic diarrhea, cathartic colon, liver damage, abdominal pain, epidermal breakdown and blistering [23, 24]. Senna contains several unique glycosides called sennosides that interact with immune cells in the colon [25]. We also found contamination of several herbal products (Table 1) with Parthenium hysterophorus (feverfew). Feverfew is native to Eurasia and is an invasive weed in Europe, the Mediterranean, North America and Chile. Although feverfew has been used to treat fever, migraine headaches and arthritis, it does have negative side effects such as swelling and numbness of the mouth, oral ulcers, nausea, vomiting, abdominal pain, diarrhea, and flatulence; some users experienced withdrawal syndromes when discontinuing use, such as rebound headaches and muscle and joint pain [26, 27]. Feverfew reacts with a variety of medications metabolized by the liver and may also increase the risk of bleeding, especially if taken with blood-thinning medications such as warfarin or aspirin [28, 29]. Pregnant women should not consume any amount of feverfew [29]. Feverfew can also cause allergic reactions, including contact dermatitis due to a toxin found in this plant species called parthenin [30, 31]. Although it is not known how contamination with feverfew occurred in this product, it is possible that it was a common weed in the crop used to make this herbal product.

We also found contamination of a Ginkgo product with Juglans nigra (black walnut). Wallace et al.[3] also found contamination of an Echinacea product with walnut and advised that such contamination would be particularly dangerous for a consumer with nut allergies. However, we feel it is unlikely that nuts are the source of contamination, but rather leaves that often litter a very large area surrounding a tree in mid to late summer. It is conceivable that there may have been a field of commercial herbs bordered by walnuts (a common occurrence on the landscape), and that the target crop was harvested along with walnut leaves. Walnut leaves, woods, bark and fruits all contain juglone [32], which is toxic; juglone can lead to oxidative stress or electrophilicity [33–35]. The Registry of Toxic Effects of Chemical Substances (RTECS®) [36] defines juglone as an equivocal tumorigenic agent (lungs, thorax and respiration, skin and appendages), which has been shown to promote skin tumors [37–39]. Ours and other studies [1, 3] of herbal product contamination in the marketplace have not been able to identify all the contaminants to species-level resolution due to the lack of a complete SRM barcode library for herbal plants; a complete SRM library would include herbal species, related species and known toxic plants. It is possible that there were other contaminants that were missed because they could only be identified to family rather than to species.

Unlabeled plant fillers may also be found in herbal products, and these fillers are in some cases a potential health risk for consumers. DNA barcoding in our study identified several potential fillers, including rice (O. sativa), soybean (G. max) and various grasses such as wheat (Triticum spp.). This is a health concern for people allergic to these plants, as well as for people seeking gluten free products. Wallace et al.[3] found rice and soybean fillers in natural plant and animal products and suggested that these fillers may produce a mixed signal during the sequencing process, contributing to a rather high percentage of failed sequencing reactions in capsulated products. We did not share this high percentage of sequence failures and found barcodes from both the herbal species on the label and fillers within the same product, suggesting that there may be fillers substituted for authentic herbal species. It is probable that barcoding detected rice and soybean, which is a common microcrystalline cellulose and gelatin used as additives in preparing the capsules that contain the herbal product [40]. It is also common practice in the natural products industry to use fillers such as those listed above, which are mixed with the active ingredients. Nonetheless, the consumer has a right to see all of the plant species used in producing a natural product on the list of ingredients.

Challenges and biotechnical advances in DNA barcoding of herbal products

Authentication of herbal products is challenging, but new DNA barcoding methods are providing tools for routine market analysis [41]. Several major challenges include the lack of an SRM herbal barcode library, and use of only plastid barcode regions, which has resulted in low species resolution. The original definition of a reliable DNA identification of species requires (i) recovery of a barcode sequence from the sample, (ii) representation of relevant species in the reference database, and (iii) sufficient nucleotide sequence variability to distinguish among closely related species [42]. We are designing new protocols for recovering DNA barcodes from herbal samples, which satisfies the first requirement, but the second requirement of a reference database has yet to be satisfied. This problem has been discussed in previous market studies that have tested the authenticity of herbal products without a herbal SRM barcode library [1–3]. These studies have defined the uncertainties of assigning unknown haplotypes from herbal products using incomplete reference barcode databases in GenBank and BOLD. The use of GenBank is inappropriate given that many of the DNA sequences do not have vouchers to professionally identified specimens archived in a herbarium. Therefore the reference sequence in GenBank may be from an incorrectly identified plant and there is no way to verify the specific origin of that DNA. Similarly, the 'Medicinal Materials DNA Barcode Database’ [43] provides barcodes for many species of medicinal plants for which there is no record of vouchers to confirm identity; this is an essential component of any DNA Barcode [42]. Our study is the first to build a partial herbal SRM barcode library using vouchered samples of known provenance that was used to test the identity of unknown barcodes recovered from herbal products. Although we only have 100 species in the SRM library, we expect to expand this to over 200 species by the end of 2013, and 1,000 species by 2015. However, this will only add up to 55% of the 1,800 medicinal plant species that are commercially available [44]. If we want to have reliable identifications using DNA barcodes we must build an SRM herbal barcode library that has all sister species for the 1,800 known medicinal species used in commercial products. This is one of the goals of our Herb-BOL (barcode of life) research program in the next 5 years.

The use of only plastid barcode regions is problematic for testing herbal products. Previous studies have not satisfied Hebert’s third criteria for a reliable barcode: sufficient nucleotide sequence variability to distinguish among closely related species [42]. Other studies have adopted the core plant barcodes of rbcLa and matK genes [45]. We do not recommend using matK because of associated problems we encountered with PCR amplification while working on nutmeg [46]. Other published studies have also encountered similar problems [47–51]. We recommend a tiered [21] approach to analysis as one way to overcome the issue of alignment with non-coding regions, while providing the most variability from two-barcode regions for identifying closely related taxa. The tiered approach is based on the use of a common, easily amplified, and aligned region such as rbcL that can act as a scaffold on which to place data from a highly variable region such as ITS2. The use of rbcL as a primary tier barcode is appropriate because of its universality, ease of amplification, ease of alignment, and because there is a significant body of data available for evaluation [51, 52]. We prefer to use ITS2 as the second tier as proposed by Chen et al.[16] for use in identifying medicinal plants because (i) it provides high species resolution, (ii) it is from the nuclear genome, which evolves at a different rate than the plastid genome, and (iii) it is a much shorter sequence allowing higher recovery from processed plant materials found within herbal products. The use of rbcL + ITS2 in our study resulted in 95% species resolution of barcodes recovered from herbal products, which is much higher than previous studies that used rbcL + matK (42% in Wallace et al.[3]; 66% in Stoeckle et al.[1]). DNA success rate was high in our study (91%) and in that of Stoeckle et al.[1] where they recovered barcodes from 90% of commercial herbal tea products. DNA barcode success rate was relatively low (48%) in the study by Wallace et al.[3]. This may be attributed to different manufacturing protocols, the type of plant material (for example, leaf, stem, roots and so on) used in the herbal preparation, or insufficient laboratory protocols used to extract, amplify and sequence haplotypes from herbal products. Herbal products contain plant secondary metabolites that may prevent barcode success. Herbal extracts contain complicated mixtures of organic chemicals (for example, fatty acids, sterols, alkaloids, flavonoids, glycosides, saponins, tannins and terpenes) that often result in PCR inhibition [53]. In addition, degradation at primer binding sites may also contribute to differential amplification success of selected genes in samples with potentially degraded DNA. In the Baker et al.[2] study, four dietary supplements could not be identified using the laboratory’s PCR amplification protocol, presumably because the DNA was degraded, possibly when heat was applied during the manufacturing process. Amplification failure in some samples, especially in rbcL gene, can be explained by the fact that primer sets used in this analysis may not be suitable for amplification of all species; we are developing new primer sets for mini-rbcL barcodes that will be easier to recover from degraded samples such as herbal products. The ideal protocol for barcoding herbal products will be realized as we build a more complete SRM herbal barcode library and continue to test more commercial products in the marketplace. Our study and others have documented that DNA can be routinely extracted from common forms of herbal dietary supplement extracts and powders, supporting a continued effort to explore DNA-based methods for quality assurance and quality control of herbal dietary supplements [1–3, 54].

Many herbal products contain mixtures, which are particularly difficult to barcode. This difficulty is due to varied PCR success of selected genes in samples with potentially degraded DNA due to varied gene copy number and PCR bias; the chemical and physical properties of each piece of DNA can selectively amplify certain sequences more than others [55]. Several approaches could be developed for testing herbal mixtures such as real-time multiplex PCR and the use of next-generation sequencing [56]. In our study we chose to test only single ingredient herbal products. However, some of these products could contain more species than what was labeled. In order to search for other possible DNA barcodes we extracted multiple barcodes per sample in an attempt to obtain the most accurate estimate of identity within a sample. This included building barcode sample by species curves to see how many times we needed to sample to get an accurate estimate of species in a sample. Although we arrived at a figure of five to ten samples, we are currently using other molecular methods to verify the number of barcodes recovered from all of our herbal products that will overcome PCR bias and provide appropriate methods for testing herbal mixtures. Recent innovative approaches have combined morphological, molecular, and chemical techniques in order to identify the plant and chemical composition of some previous-generation smart drugs [57]. Such a multidisciplinary approach is proposed as a method for the identification of herbal blends of uncertain composition, which are widely available and represent a serious hazard to public health [57].