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Planta Med 2021; 87(04): 314-324
DOI: 10.1055/a-1336-1685
Natural Product Chemistry and Analytical Studies
Original Papers

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea

Sara M. Handy 
1   Center for Food Safety and Applied Nutrition, Office of Regulatory Science, U. S. Food and Drug Administration, College Park, Maryland, United States
,
Rahul S. Pawar 
1   Center for Food Safety and Applied Nutrition, Office of Regulatory Science, U. S. Food and Drug Administration, College Park, Maryland, United States
,
Andrea R. Ottesen   *
1   Center for Food Safety and Applied Nutrition, Office of Regulatory Science, U. S. Food and Drug Administration, College Park, Maryland, United States
,
Padmini Ramachandran
1   Center for Food Safety and Applied Nutrition, Office of Regulatory Science, U. S. Food and Drug Administration, College Park, Maryland, United States
,
Satyanarayanaraju Sagi
1   Center for Food Safety and Applied Nutrition, Office of Regulatory Science, U. S. Food and Drug Administration, College Park, Maryland, United States
,
Ning Zhang
1   Center for Food Safety and Applied Nutrition, Office of Regulatory Science, U. S. Food and Drug Administration, College Park, Maryland, United States
,
Erica Hsu
2   Joint Institute of Food Safety and Applied Nutrition, University of Maryland, College Park, Maryland, United States
,
David L. Erickson
2   Joint Institute of Food Safety and Applied Nutrition, University of Maryland, College Park, Maryland, United States
3   DNA4 Technologies LLC, Halethorpe, Maryland, United States
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Abstract

The use of DNA-based methods to authenticate botanical dietary supplements has been vigorously debated for a variety of reasons. More comparisons of DNA-based and chemical methods are needed, and concordant evaluation of orthogonal approaches on the same products will provide data to better understand the strengths and weaknesses of both approaches. The overall application of DNA-based methods is already firmly integrated into a wide array of continually modernizing stand alone and complementary authentication protocols. Recently, the use of full-length chloroplast genome sequences provided enhanced discriminatory capacity for closely related species of Echinacea compared to traditional DNA barcoding approaches (matK and rbcL). Here, two next-generation sequencing approaches were used: (1) genome skimming and (2) PCR amplicon (metabarcoding). The two genetic approaches were then combined with HPLC-UV to evaluate 20 commercially available dietary supplements of Echinacea representing “finished” products. The trade-offs involved in different DNA approaches were discussed, with a focus on how DNA methods support existing, accepted chemical methods. In most of the products (19/20), HPLC-UV suggested the presence of Echinacea spp. While metabarcoding was not useful with this genus and instead only resolved 7 products to the family level, genome skimming was able to resolve to species (9) or genus (1) with the 10/20 products where it was successful. Additional ingredients that HPLC-UV was unable to identify were also found in four products along with the relative sequence proportion of the constituents. Additionally, genome skimming was able to identify one product that was a different Echinacea species entirely.

Key words

next-generation sequencing (NGS) - genome skimming - botanical dietary supplements - Asteraceae

* Current address: Center for Veterinary Medicine, U. S. Food and Drug Administration, College Park, Maryland, United States


Supporting Information

    Metabarcoding results, calculations of relative ratios of plant components, links to certification for Echinacea standards, the equation that shows how the p value was calculated based on a Z-statistic, and kmer score output from Genome2-ID are available as Supporting Information.

  • Supporting Information


Publication History

Received: 10 July 2020

Accepted after revision: 11 December 2020

Article published online:
14 January 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
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  • References

  • 1 Upton R, David B, Gafner S, Glasl S. Botanical ingredient identification and quality assessment: strengths and limitations of analytical techniques. Phytochem Rev 2020; 19: 1157-1177
    CrossrefPubMedGoogle Scholar
  • 2 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. Planta Med 2016; 82: 1225-1235
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Raclariu AC, Heinrich M, Ichim MC, de Boer H. Benefits and limitations of DNA barcoding and metabarcoding in herbal product authentication. Phytochem Anal 2018; 29: 123-128
    CrossrefPubMedGoogle Scholar
  • 4 Khan IA, Smillie T. Implementing a “quality by design” approach to assure the safety and integrity of botanical dietary supplements. J Nat Prod 2012; 75: 1665-1673
    CrossrefPubMedGoogle Scholar
  • 5 Pawar RS, Handy SM, Cheng R, Shyong N, Grundel E. Assessment of the authenticity of herbal dietary supplements: Comparison of chemical and DNA barcoding methods. Planta Med 2017; 83: 921-936
    Article in Thieme ConnectPubMedGoogle Scholar
  • 6 CBOL Plant Working Group. A DNA barcode for land plants. Proc Natl Acad Sci U S A 2009; 106: 12794-12797
    CrossrefPubMedGoogle Scholar
  • 7 Fazekas AJ, Kesanakurti PR, Burgess KS, Percy DM, Graham SW, Barrett SC, Newmaster SG, Hajibabaei M, Husband BC. Are plant species inherently harder to discriminate than animal species using DNA barcoding markers?. Mol Ecol Resour 2009; 9 (Suppl. 01) 130-139
    CrossrefPubMedGoogle Scholar
  • 8 Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci 2003; 270: 313-321
    CrossrefPubMedGoogle Scholar
  • 9 Deagle BE, Jarman SN, Coissac E, Pompanon F, Taberlet P. DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match. Biol Lett 2014; 10: 20140562
    CrossrefPubMedGoogle Scholar
  • 10 Percy DM, Argus GW, Cronk QC, Fazekas AJ, Kesanakurti PR, Burgess KS, Husband BC, Newmaster SG, Barrett SC, Graham SW. Understanding the spectacular failure of DNA barcoding in willows (Salix): does this result from a trans-specific selective sweep?. Mol Ecol 2014; 23: 4737-4756
    CrossrefPubMedGoogle Scholar
  • 11 Sgamma T, Lockie-Williams C, Kreuzer M, Williams S, Scheyhing U, Koch E, Slater A, Howard C. DNA barcoding for industrial quality assurance. Planta Med 2017; 83: 1117-1129
    Article in Thieme ConnectPubMedGoogle Scholar
  • 12 Seethapathy GS, Raclariu-Manolica AC, Anmarkrud JA, Wangensteen H, de Boer HJ. DNA metabarcoding authentication of ayurvedic herbal products on the European market raises concerns of quality and fidelity. Front Plant Sci 2019; 10: 68
    CrossrefPubMedGoogle Scholar
  • 13 Raclariu AC, Ţebrencu CE, Ichim MC, Ciuperca OT, Brysting AK, de Boer H. Whatʼs in the box? Authentication of Echinacea herbal products using DNA metabarcoding and HPTLC. Phytomedicine 2018; 44: 32-38
    CrossrefPubMedGoogle Scholar
  • 14 Bruno A, Sandionigi A, Agostinetto G, Bernabovi L, Frigerio J, Casiraghi M, Labra M. Food tracking perspective: DNA metabarcoding to identify plant composition in complex and processed food products. Genes (Basel) 2019; 10: 248
    CrossrefPubMedGoogle Scholar
  • 15 Shanmughanandhan D, Ragupathy S, Newmaster SG, Mohanasundaram S, Sathishkumar R. Estimating herbal product authentication and adulteration in India using a vouchered, DNA-based biological reference material library. Drug Saf 2016; 39: 1211-1227
    CrossrefPubMedGoogle Scholar
  • 16 Cheng CM, Van Khanh T, Lin W, Ruby RM. Interlaboratory validation of a real-time PCR 24-hour rapid method for detection of Salmonella in foods. J Food Prot 2009; 72: 945-951
    CrossrefPubMedGoogle Scholar
  • 17 Blumenthal M, Gafner S, Foster S, Cardellina JH, Khan IA, Upton R. Preventing adulteration and fraud in botanical ingredients in the international marketplace: the ABC-AHP-NCNPR botanical adulterants prevention program. Planta Med 2019; 85: 1400
    PubMedGoogle Scholar
  • 18 Little DP, Jeanson ML. DNA barcode authentication of saw palmetto herbal dietary supplements. Sci Rep 2013; 3: 3518
    CrossrefPubMedGoogle Scholar
  • 19 Little DP. Authentication of Ginkgo biloba herbal dietary supplements using DNA barcoding. Genome 2014; 57: 513-516
    CrossrefPubMedGoogle Scholar
  • 20 Straub SC, Parks M, Weitemier K, Fishbein M, Cronn RC, Liston A. Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics. Am J Bot 2012; 99: 349-364
    CrossrefPubMedGoogle Scholar
  • 21 Zeng CX, Hollingsworth PM, Yang J, He ZS, Zhang ZR, Li DZ, Yang JB. Genome skimming herbarium specimens for DNA barcoding and phylogenomics. Plant Methods 2018; 14: 43
    CrossrefPubMedGoogle Scholar
  • 22 Quince C, Walker AW, Simpson JT, Loman NJ, Segata N. Shotgun metagenomics, from sampling to analysis. Nat Biotechnol 2017; 35: 833-844
    CrossrefPubMedGoogle Scholar
  • 23 Xin T, Su C, Lin Y, Wang S, Xu Z, Song J. Precise species detection of traditional Chinese patent medicine by shotgun metagenomic sequencing. Phytomedicine 2018; 47: 40-47
    CrossrefPubMedGoogle Scholar
  • 24 Bista I, Carvalho GR, Tang M, Walsh K, Zhou X, Hajibabaei M, Shokralla S, Seymour M, Bradley D, Liu S, Christmas M, Creer S. Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples. Mol Ecol Resour 2018; DOI: 10.1111/1755-0998.12888.
    CrossrefPubMedGoogle Scholar
  • 25 McDermott PF, Tyson GH, Kabera C, Chen Y, Li C, Folster JP, Ayers SL, Lam C, Tate HP, Zhao S. Whole-genome sequencing for detecting antimicrobial resistance in nontyphoidal Salmonella . Antimicrob Agents Chemother 2016; 60: 5515-5520
    CrossrefPubMedGoogle Scholar
  • 26 Raime K, Remm M. Method for the identification of taxon-specific k-mers from chloroplast genome: A case study on tomato plant (Solanum lycopersicum). Front Plant Sci 2018; 9: 6
    CrossrefPubMedGoogle Scholar
  • 27 Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol 2019; 20: 257
    CrossrefPubMedGoogle Scholar
  • 28 Breitwieser FP, Lu J, Salzberg SL. A review of methods and databases for metagenomic classification and assembly. Brief Bioinform 2019; 20: 1125-1136
    CrossrefPubMedGoogle Scholar
  • 29 Simner PJ, Miller HB, Breitwieser FP, Pinilla Monsalve G, Pardo CA, Salzberg SL, Sears CL, Thomas DL, Eberhart CG, Carroll KC. Development and optimization of metagenomic next-generation sequencing methods for cerebrospinal fluid diagnostics. J Clin Microbiol 2018; 56: e00472-18
    CrossrefPubMedGoogle Scholar
  • 30 Flagel LE, Rapp RA, Grover CE, Widrlechner MP, Hawkins J, Grafenberg JL, Alvarez I, Chung GY, Wendel JF. Phylogenetic, morphological, and chemotaxonomic incongruence in the North American endemic genus Echinacea . Am J Bot 2008; 95: 756-765
    CrossrefPubMedGoogle Scholar
  • 31 Parsons JL, Cameron SI, Harris CS, Smith ML. Echinacea biotechnology: advances, commercialization and future considerations. Pharm Biol 2018; 56: 485-494
    CrossrefPubMedGoogle Scholar
  • 32 Haria E, Perera M, Senchina D. Immunomodulatory effects of Echinacea laevigata ethanol tinctures produced from different organs. Bioscience Horizons 2016; 9: hzw001
    CrossrefPubMedGoogle Scholar
  • 33 Brown PN, Mudge EM, Paley L. Determination of phenolic constituents in Echinacea raw materials and dietary supplements by HPLC-UV: collaborative study. J AOAC Int 2016; 99: 1197-1203
    CrossrefPubMedGoogle Scholar
  • 34 Handy SM, Timme RE, Jacob SM, Deeds JR. Development of a locked nucleic acid real-time polymerase chain reaction assay for the detection of Pinus armandii in mixed species pine nut samples associated with dysgeusia. J Agric Food Chem 2013; 61: 1060-1066
    CrossrefPubMedGoogle Scholar
  • 35 Newmaster SG, Shanmughanandhan D, Kesanakurti P, Shehata H, Faller A, Noce ID, Lee JY, Rudzinski P, Lu Z, Zhang Y, Swanson G, Hanner R, Ragupathy S. Recommendations for validation of real-time PCR methods for molecular diagnostic identification of botanicals. J AOAC Int 2019; 102: 1767-1773
    CrossrefPubMedGoogle Scholar
  • 36 González-Escalona N, Hammack TS, Russell M, Jacobson AP, De Jesús AJ, Brown EW, Lampel KA. Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA. Appl Environ Microbiol 2009; 75: 3714-3720
    CrossrefPubMedGoogle Scholar
  • 37 Puente-Lelievre C, Eischeid AC. Development and evaluation of a real-time PCR multiplex assay for the detection of allergenic peanut using chloroplast DNA markers. J Agric Food Chem 2018; 66: 8623-8629
    CrossrefPubMedGoogle Scholar
  • 38 Ruvindy R, Bolch CJ, MacKenzie L, Smith KF, Murray SA. qPCR Assays for the detection and quantification of multiple paralytic shellfish toxin-producing species of Alexandrium . Front Microbiol 2018; 9: 3153
    CrossrefPubMedGoogle Scholar
  • 39 Morigengaowa. Luo JJ, Knapp R, Wei HJ, Liu BD, Yan YH, Shang H. The identity of Hypolepis robusta, as a new synonym of Hypolepis alpina (Dennstaedtiaceae), based on morphology and DNA barcoding and the new distribution. PhytoKeys 2018; 96: 35-45
    CrossrefPubMedGoogle Scholar
  • 40 Ivanova NV, Kuzmina ML, Braukmann TW, Borisenko AV, Zakharov EV. Authentication of herbal supplements using next-generation sequencing. PLoS One 2016; 11: e0156426
    CrossrefPubMedGoogle Scholar
  • 41 Wohlmuth H, Savage K, Dowell A, Mouatt P. Adulteration of Ginkgo biloba products and a simple method to improve its detection. Phytomedicine 2014; 21: 912-918
    CrossrefPubMedGoogle Scholar
  • 42 Howard C, Hill E, Kreuzer M, Mali P, Masiero E, Slater A, Sgamma T. DNA authentication of St Johnʼs wort (Hypericum perforatum L.) commercial products targeting the ITS region. Genes (Basel) 2019; 10: 286
    CrossrefPubMedGoogle Scholar
  • 43 Zhang N, Erickson DL, Ramachandran P, Ottesen AR, Timme RE, Funk VA, Luo Y, Handy SM. An analysis of Echinacea chloroplast genomes: Implications for future botanical identification. Sci Rep 2017; 7: 216
    CrossrefPubMedGoogle Scholar
  • 44 Lang D, Tang M, Hu J, Zhou X. Genome-skimming provides accurate quantification for pollen mixtures. Mol Ecol Resour 2019; 19: 1433-1446
    CrossrefPubMedGoogle Scholar
  • 45 Dodsworth S. Genome skimming for next-generation biodiversity analysis. Trends Plant Sci 2015; 20: 525-527
    CrossrefPubMedGoogle Scholar
  • 46 Ivanova NV, Dewaard JR, Hebert PDN. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol Ecol Notes 2006; 6: 998-1002
    CrossrefPubMedGoogle Scholar
  • 47 Erickson DL, Reed E, Ramachandran P, Bourg NA, McShea WJ, Ottesen A. Reconstructing a herbivoreʼs diet using a novel rbcL DNA mini-barcode for plants. AoB Plants 2017; 9: plx015
    CrossrefPubMedGoogle Scholar
  • 48 Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Res 2007; 35: e14
    CrossrefPubMedGoogle Scholar
  • 49 Bell KL, Loeffler VM, Brosi BJ. An rbcL reference library to aid in the identification of plant species mixtures by DNA metabarcoding. Appl Plant Sci 2017; 5: apps.1600110
    CrossrefPubMedGoogle Scholar