Genetic authentication by RFLP versus ARMS? The case of Moldavian dragonhead (Dracocephalum moldavica L.)


Moldavian dragonhead (Dracocephalum moldavica L.), due to its pleasant lemon scent and medical effects, has acquired increasing impact as functional food. The high diversity within the genus, limited supply not keeping pace with the growing demand, the morphological similarity with other Labiatae, and trading under the common name Turkish Melissa invite adulteration by surrogate species. We have developed several verified reference accessions of D. moldavica L. along with potential surrogate species to compare different approaches of authentication, also in commercial samples. We report on three strategies of authentication—a microscopic method, based on the relative size of epidermal pavement cells versus palisade cells, and two strategies of genetic authentication based on the barcoding marker large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL). We can detect single-nucleotide exchanges between D. moldavica L. and the potential surrogate species Melissa officinalis L. and Nepeta cataria L. by restriction fragment length polymorphism (RFLP), and we show that we can use this to verify the presence of D. moldavica even in dried and highly fragmented mixtures from commercial samples. We further develop a third strategy derived from the so-called amplification refractory mutation system (ARMS), based on multiplex PCR of the rbcL marker upon addition of specifically designed intermediate primers that will generate a diagnostic second band in case of D. moldavica L., but not for the surrogate species. We demonstrate that this ARMS approach is superior to the RFLP strategy, because it safeguards against experimental fluctuations, can unequivocally verify the presence of D. moldavica in commercial samples declaring its presence, and yields a clear outcome in a one-step protocol.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    European Parliament and European Council (1997) Regulation on novel foods and novel food ingredients no. 258/97 of 27 January 1997. J Eur Comm L 43:1–5

    Google Scholar 

  2. 2.

    Ernst E (1998) Harmless herbs? A review of the recent literature. Am J Med 104:170–178

    CAS  Article  Google Scholar 

  3. 3.

    Harley RM, Atkins S, Budantsev A, Cantino PD, Conn BJ, Grayer R, Harley MM, De Kok R, Krestovskaja T, Morales R, Paton AJ, Ryding O, Upson T (2004) Labiatae. In: Kubitzki K (ed) The families and genera of vascular plants, vol 7. Springer, Berlin, pp 167–275

    Google Scholar 

  4. 4.

    Hayne FG (1822) Getreue Darstellung und Beschreibung der in der Arzneykunde gebräuchlichen Gewächse wie auch solchen, welche mit ihnen verwechselt werden können. Lucae, Berlin, VIII-32

  5. 5.

    Sultan AB, Asia HA, Eshbakova KA (2008) Flavonoids of Dracocephalum moldavica. Chem Natural Comp 44:366–367

    CAS  Article  Google Scholar 

  6. 6.

    Saeidnia S, Gohari AR, Ito M, Kiuchi F, Honda G (2005) Bioactive Constituents from Dracocephalum subcapitatum (O. Kuntze) Lipsky. Z Naturf 60c:22–24

  7. 7.

    Aziz EE, Abbass MH (2010) Chemical composition and efficiency of five essential oils against the pulse beetle Callosobruchus maculatus (F.) on Vigna radiata seeds. Am-Eurasian J Agric Environ Sci 8:411–419

    CAS  Google Scholar 

  8. 8.

    Chu SS, Liu SL, Liu QZ, Liu ZL, Du SS (2011) Composition and toxicity of Chinese Dracocephalum moldavica (Labiatae) essential oil against two grain storage insects. J Med Plant Res 5:5262–5267

    CAS  Google Scholar 

  9. 9.

    Abd El-Baky HH, El-Baroty GS (2008) Chemical and biological evaluation of the essential oil of Egyption Moldavian balm. Adv Food Sci 30:170–175

    Google Scholar 

  10. 10.

    Holm Y, Hiltunen R, Nykänen I (1988) Capillary gas chromatographic-mass spectrometric determination of the flavour composition of dragonhead (Dracocephalum moldavica L.). Flavour Fragr J 3:109–112

    CAS  Article  Google Scholar 

  11. 11.

    Sonboli A, Mojarrad M, Gholipour A, Ebrahimi SN, Arman M (2008) Biological activity and composition of the essential oil of Dracocephalum moldavica L. grown in Iran. Nat Prod Comm 3:1547–1550

    CAS  Google Scholar 

  12. 12.

    Li SM, Yang XW, Li YL, Shen YH, Feng L, Wang YH, Zeng HW, Liu XH, Zhang CS, Long CL, Zhang WD (2009) Chemical constituents of Dracocephalum forrestii. Planta Med 75:1591–1596

    CAS  Article  Google Scholar 

  13. 13.

    Lan XQ (1994) Dracocephalum moldavica linnaeus. Flora China 17:124–133

    Google Scholar 

  14. 14.

    Lyapunova PN, Salo ND, Sergienko TA (1975) An anatomical study of the herb Dracocephalum moldavica L. Farmatsija 24:15–20 (in Russian)

    Google Scholar 

  15. 15.

    Dmitruk M, Weryszko-Chmielewska E (2010) Morphological differentiation and distribution of non-glandular and glandular trichomes on Dracocephalum moldavicum L. Acta Agrobot 63:11–22

    Article  Google Scholar 

  16. 16.

    Saeidnia S, Gohari AR, Ito M, Honda G (2004) Comparison of some Dracocephalum species by phylogenetic and chemotaxonomic analysis. Intern J Biol Biotechnol 1:267–270

    CAS  Google Scholar 

  17. 17.

    Wolf HT, van den Berg H, Czygan FC, Mosandl A, Winckler T, Zündorf I, Dingerman T (1999) Identification of Melissa officinalis subspecies by DNA fingerprinting. Planta Med 65:83–85

    CAS  Article  Google Scholar 

  18. 18.

    Horn T, Barth A, Rühle M, Häser A, Jürges G, Nick P (2012) Molecular diagnostics of lemon myrtle (Backhousia citriodora versus Leptospermum citratum). Eur Food Res Technol 234:853–861

    CAS  Article  Google Scholar 

  19. 19.

    Old JM (1992) Detection of mutations by the amplification refractory mutation system (ARMS). Methods Mol Biol 9:77–84

    Google Scholar 

  20. 20.

    Schmeil O, Fitschen J, Seybold S (2006) Flora von Deutschland und angrenzender Länder : Ein Buch zum Bestimmen der wild wachsenden und häufig kultivierten Gefäßpflanzen. Quelle & Meyer Verlag, Wiebelsheim

    Google Scholar 

  21. 21.

    IDC 247.11 Melissa officinális L., IDC 249.1Dracocephalum moldavica L., Linnean herbarium (S-LINN), Department of Phanerogamic Botany of the Swedish Museum of Natural History

  22. 22.

    Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochem Bull 19:11–15

    Google Scholar 

  23. 23.

    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    CAS  Article  Google Scholar 

  24. 24.

    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  Google Scholar 

  25. 25.

    Felsenstein J (1985) ConWdence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  26. 26.

    Hohmann B (2007) Mikroskopische Untersuchung pflanzlicher Lebensmittel und Futtermittel. Behrs Verlag, Hamburg

    Google Scholar 

  27. 27.

    Nadeau JA (2009) Stomatal development: new signals and fate determinants. Curr Opin Plant Biol 12:29–35

    CAS  Article  Google Scholar 

  28. 28.

    Kaufmann M, Wink M (1994) Molecular systematics of the Nepetoideae (family Labiatae): phylogenetic implications from rbcL gene sequences. Biosci Rep 49:635–645

    CAS  Google Scholar 

  29. 29.

    Li X, Ding X, Chu B, Ding G, Gu S, Qian L, Wang Y, Zhou Q (2007) Molecular authentication of Alisma orientale by PCR-RFLP and ARMS. Planta Med 73:67–70

    CAS  Article  Google Scholar 

  30. 30.

    Diao Y, Lin XM, Liao CL, Tang CZ, Chen ZJ, Hu ZL (2009) Authentication of Panax ginseng from its Adulterants by PCR-RFLP and ARMS. Planta Med 75:557–560

    CAS  Article  Google Scholar 

Download references


We acknowledge Angelika Piernitzki and Joachim Daumann, Botanical Garden of the University, for excellent horticultural support.

Conflict of interest


Compliance with Ethics Requirements

This article does not contain any studies with human or animal subjects.

Author information



Corresponding author

Correspondence to Peter Nick.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Horn, T., Völker, J., Rühle, M. et al. Genetic authentication by RFLP versus ARMS? The case of Moldavian dragonhead (Dracocephalum moldavica L.). Eur Food Res Technol 238, 93–104 (2014).

Download citation


  • Amplification refractory mutation system (ARMS)
  • Dracocephalum moldavica L.
  • Molecular authentication
  • Restriction fragment length polymorphism (RFLP)
  • Ribulose-bisphosphate carboxylase oxygenase large subunit (rbcL)