Planta Med 2009; 75(8): 864-869
DOI: 10.1055/s-0029-1185397
Biochemistry, Molecular Biology and Biotechnology
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

Molecular Identification of Hypericum perforatum by PCR Amplification of the ITS and 5.8S rDNA Region

Caroline Howard1 , Paul D. Bremner2 , Mark R. Fowler2 , Belinda Isodo2 , Nigel W. Scott2 , Adrian Slater1
  • 1School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University, Leicester, U. K.
  • 2Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, Leicester, U. K.
Weitere Informationen

Publikationsverlauf

received Sept. 5, 2008 revised Dec. 2, 2008

accepted January 19, 2009

Publikationsdatum:
04. März 2009 (online)

Abstract

The nuclear ribosomal internal transcribed spacer (ITS) sequences of eight Hypericum species were used to design H. perforatum-specific PCR primers by identification of short “microcode” sequences characteristic of the target species. These were tested with three vouchered H. perforatum DNA samples and eight samples from other species within the Hypericum genus. The most efficient primer combination, FO2 and HRI‐S, amplified the genomic DNA from all three H. perforatum samples but not from any of the others apart from H. delphicum. The primer pairing was then tested against seven commercially available ornamental varieties of Hypericum; a positive result was obtained only with the H. perforatum sample. Three consumer products retailed as “St. John's wort” herbal remedies were sampled, two of which gave a positive result for H. perforatum. The assay was sensitive enough to detect 0.75 ng H. perforatum present as just 0.1 % of the total DNA. This method has the potential to be replicated in other plant species and presents a novel use for DNA barcoding data.

References

  • 1 Southwel I A, Bourke C A. Seasonal variation in hypericin content of Hypericum perforatum L. (St. John's wort).  Phytochemistry. 2001;  56 437-441
  • 2 Hunt E J, Lester C E, Lester E A, Tackett R L. Effect of St. John's wort on free radical production.  Life Sci. 2001;  69 181-190
  • 3 McGarry H, Pirotta M, Hegarty K, Gunn J. General practitioners and St. John's wort: a question of regulation or knowledge?.  Complement Ther Med. 2007;  15 142-148
  • 4 Pilkington K, Boshnakova A, Richardson J. St John's wort for depression: time for a different perspective?.  Complement Ther Med. 2006;  14 268-281
  • 5 Calapai G, Crupi A, Firenzuoli F, Inferrera G, Squadrito F, Parisi A, De Sarro G, Caputi A. Serotonin, norepinephrine and dopamine involvement in the antidepressant action of Hypericum perforatum.  Pharmacopsychiatry. 2001;  34 45-49
  • 6 Zanoli P. Role of hyperforin in the pharmacological activities of St. John's wort.  CNS Drug Rev. 2004;  10 203-218
  • 7 DeSmet P. Herbal medicine in Europe – relaxing regulatory standards.  N Engl J Med. 2005;  352 12
  • 8 Gaster B, Holroyd J. St John's wort for depression: a systematic review.  Arch Intern Med. 2000;  160 152-156
  • 9 Hebert P DN, Ratnasingham S, deWaard J R. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species.  Proc Royal Soc London B. 2003;  270 S96-S99
  • 10 Chase M W, Cowan R S, Hollingsworth P M, van dan Berg C, Madrinan S, Petersen G, Seberg O, Jorgsensen T, Cameron K M, Carine M, Pedersen N, Hedderson T AJ, Conrad F, Salazar G A, Richardson J E, Hollingsworth M L, Barraclough T G, Kelly L, Wilkinson M. A proposal for a standardised protocol to barcode all land plants.  Taxon. 2007;  56 295-299
  • 11 Pennisi E. Taxonomy – wanted: a barcode for plants.  Science. 2007;  318 190-191
  • 12 Hollingsworth P M. DNA barcoding plants in biodiversity hotspots: Progress and outstanding questions.  Heredity. 2008;  101 1-2
  • 13 Lahaye R, van der Bank M, Bogarin D, Warner J, Pupulin F, Gigot G, Maurin O, Duthoit S, Barraclough T G, Savolainen V. DNA barcoding the floras of biodiversity hotspots.  Proc Natl Acad Sci USA. 2008;  105 2923-2928
  • 14 Kress W, Wurdack K J, Zimmer E A, Weigt L A, Janzen D H. Use of DNA barcodes to identify flowering plants.  Proc Natl Acad Sci USA. 2005;  102 8369-8374
  • 15 Crockett S L, Douglas A W, Scheffler B E, Khan I A. Genetic profiling of Hypericum (St. John's wort) species by nuclear ribosomal ITS sequence analysis.  Planta Med. 2004;  70 929-935
  • 16 Hajibabaei M, Smith A M, Janzen D H, Rodriquez J J, Whitfield J B, Hebert P DN. A minimalist barcode can identify a specimen whose DNA is degraded.  Mol Ecol Notes. 2006;  6 959-964
  • 17 Summerbell R C, Levesque C A, Seifert K A, Bovers M, Fell J W, Diaz M R, Boekhout T, de Hoog G S, Stalpers J, Crous P W. Microcoding: the second step in DNA barcoding.  Philos Trans R Soc Lond B Biol Sci. 2005;  360 1897-1903
  • 18 Chenna R, Sugawara H, Koike T, Lopez R, Gibson T J, Higgins D G, Thompson J D. Multiple sequence alignment with the clustal series of programs.  Nucleic Acids Res. 2003;  31 3497-500

Adrian Slater

School of Allied Health Sciences
Faculty of Health and Life Sciences
De Montfort University
The Gateway

Leicester LE1 9BH

United Kingdom

Telefon: + 44 11 62 57 78 43

eMail: ads@dmu.ac.uk