Molecular Authentication of Radix Behen Albi (“Bahman Sefid”) Commercial Products Reveals Widespread Adulteration

Document Type : Original paper


1 Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen, Sweden.

2 Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Department of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

3 Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4 Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen, Sweden. The Natural History Museum, University of Oslo, Oslo, Norway.


Background and objectives: The roots of Centaurea behen L., (Asteraceae) known as Radix Behen Albi are used as an aphrodisiac, anti-lithiasis and general tonic. It is available as dried or powdered roots in the herbal markets of Iran. Confirming the identity of this medicinal root using conventional methods is challenging because of lack of the diagnostic characters and market samples are easy to misidentify or adulterate. Methods: This study aimed to authenticate 13 Radix Behen Albisamples purchased from different herbal markets in Iran and to identify the potential adulterants through DNA barcoding. Nuclear (nrITS) and plastid (trnL-F spacer, matK and rbcL) DNA regions were used as barcoding markers. A reference database was compiled using sequences from herbarium voucher specimens and publicly available sequences. Results: Among used barcode regions nrITS was the best marker for species identification followed by trnL-F spacer. MatK and rbcL were able to identify samples to the family level. This study showed that none of the market samples belonged to the authentic Centaurea behen L. Sixty-nine percent of samples were Cousinia spp. (Asteraceae), 23% Korshinskya spp. (Apiaceae) and 8% Crambe spp. (Brassicaceae). This substitution does not only hinder consumers obtain the desired medicinal effects of Radix Behen Albi but also raises concerns about the pharmacovigilance of this medicinal root sold in the markets. Conclusion: The present study shows the need for monitoring and authentication of crude herbal drugs in the markets of Iran, and that DNA barcoding is a suitable tool for this purpose.


Main Subjects

[1] Mishra P, Kumar A, Nagireddy A, Mani DN, Shukla AK, Tiwari R, Sundaresan V. DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant Biotechnol J. 2016; 14(1): 8-21.
[2] Global Industry Analyst, Inc. Herbal Supplements and Remedies Market Trends. [Accessed 2015]. Available from:
[3] Newmaster SG, Grguric M, Shanmughanandhan D, Ramalingam S, Ragupathy S. DNA barcoding detects contamination and substitution in North American herbal products. BMC Med. 2013; 11(1): 1-13.
[4] De Boer HJ, Ichim MC, Newmaster SG. DNA barcoding and pharmacovigilance of herbal medicines. Drug Saf. 2015; 38(7): 611-620.
[5] Heinrich M. Quality and safety of herbal medical products: regulation and the need for quality assurance along the value chains. Br J Clin Pharmacol. 2015; 80(1): 62-66.
[6] World Health Organization. Regulatory situation of herbal medicine. A worldwide review. Geneva: World Health Organization, 1998.
[7] World Health Organization. Quality control methods for herbal materials. [Accessed 2011]. Available from:
[8] Chen S, Pang X, Song J, Shi L, Yao H, Han J, Leon C. A renaissance in herbal medicine identification: From morphology to DNA. Biotechnol Adv. 2014; 32(7): 1237-1244.
[9] Schlick-Steiner BC, Steiner FM, Seifert B, Stauffer C, Christian E, Crozier RH. Integrative taxonomy: a multisource approach to exploring biodiversity. Annu Rev Entomol. 2010; 55: 421-438.
[10] Li M, Cao H, But PPH, Shaw PC.  Identification of herbal medicinal materials using DNA barcodes. J Syst Evol. 2011; 49(3): 271-283.
[11] Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci. 2003; 270 (1512): 313-321.
[12] Hajibabaei M, Singer GAC, Hebert PDN, Hickey DA. DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet TIG. 2007; 23(4): 167-172.
[13] Kool A, De Boer HJ, Krüger A, Rydberg A, Abbad A, Björk L, Martin G. Molecular identification of commercialized medicinal plants in southern Morocco. PloS One. 2012; Article ID 39459.
[14] Ghorbani A, Saeedi Y, De Boer HJ. Unidentifiable by morphology: DNA barcoding of plant material in local markets in Iran. PLoS One. 2017; Article ID 0175722.
[15] De Boer HJ, Ghorbani A, Manzanilla V, Raclariu AC, Kreziou A, Ounjai S, Osathanunkul M, Gravendeel B. Metabarcoding of orchid products reveals widespread illegal orchid trade and adulteration. Proc R Soc B. 2017; Article ID 20171182.
[16] Khare CP. Centaurea behen   L.  In:  Indian herbal remedies, rational western therapy Ayurvedic and other traditional usage, botany. New Delhi: Springer, 2004.
[17] Lev E, Amar Z. Practical materia medica of the medieval eastern Mediterranean according to the Cairo Genizah. Leiden: Brill Academic Publishers, 2008.
[18] Dannenfeldt KH. Leonhard Rauwolf: Sixteenth-century physician, botanist and traveler. Cambridge: Harvard University Press, 1968.
[19] Amiri MS, Joharchi MR. Ethnobotanical investigation of traditional medicinal plants commercialized in the markets of Mashhad, Iran. Avicenna J Phytomed. 2013; 3(3): 254-271.
[20] Imtiyaz S, Tariq M, Chaudhary S. Aphrodisiacs used in Unani system of medicine. J Biol Sci Opin. 2013; 1(3): 239-242.
[21] Mozaffarian V. Identification of medicinal and aromatic plants of Iran. Tehran: Farhang-e Moaser, 2013.
[22] Joharchi MR, Amiri MS. Taxonomic evaluation of misidentification of crude herbal drugs marketed in Iran. Avicenna J Phytomed. 2012; 2(2): 105-112.
[23] Ghasemi Dehkordi NA, Sajadi SE, Ghanadi A, Amanzadeh Y, Azadbakht M, Asghari GR, Amin G, Haji Akhoundi A, Taleb A. Iranian herbal pharmacopoeia. Tehran: Ministry of Health and Medical Education, 2003.
[24] Ghahreman A, Okhovvat AR. Matching the old descriptions of medicinal plants with the scientific ones. Tehran: Tehran University Press, 2004.
[25] Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987; 19(1): 11-15.
[26] Sun Y, Skinner DZ, Liang GH, Hulbert SH. Phylogenetic analysis of sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theor Appl Genet. 1994; 89(1): 26-32.
[27] Ford CS, Ayres KL, Toomey N, Haider N, Stahl JA, Kelly LJ, Wikstrom N, Hollingsworth PM, Duff RJ, Hoot SB, Cowan RS, Chase MW, Wilkinson MJ. Selection of candidate coding DNA barcoding regions for use on land plants. Bot J Linn Soc. 2009; 159(1): 1-11.
[28] Bellstedt DU, Linder HP, Harley EH. Phylogenetic relationships in Disa based on non-coding trnL-trnF chloroplast sequences: evidence of numerous repeat regions. Am J Bot. 2001; 88(11): 2088-2100.
[29] Levin RA, Wagner WL, Hoch PC, Nepokroeff M, Pires JC, Zimmer EA, Sytsma KJ. Family-Level Relationships of Onagraceae Based on Chloroplast rbcL and ndhF Data.  Am J Bot. 2003; 90(1): 107-115.
[30] Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, Bermingham E. Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proc Natl Acad Sci U S A. 2009; 106(44): 18621-18626.
[31] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7): 1870-1874.
[32] Selvaraj D, Shanmughanandhan D, Sarma RK, Joseph JC, Srinivasan RV, Ramalingam S. DNA Barcode its effectively distinguishes the medicinal plant Boerhavia diffusa from Its adulterants. Genom Proteom Bioinf.  2012; 10(6): 364–367.
[33] Zhao S, Chen X, Song J, Pang X, Chen S. Internal transcribed spacer 2 barcode: a good tool for identifying Acanthopanacis cortex. Front Plant Sci. 2015; Article ID 840.
[34] Zuo Y, Chen Z, Kondo K, Funamoto T, Wen J, Zhou S. DNA barcoding of Panax species. Planta Med. 2011; 77(2): 182-187.
[35] Sui X, Huang Y, Tan Y, Guo Y, Long C. Molecular authentication of the ethnomedicinal plant Sabia parviflora and its adulterants by DNA barcoding technique. Planta Med. 2011; 77(5): 492-496.
[36] Sass C, Little DP, Stevenson DW, Specht CD. DNA Barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of Cycads. PloS One. 2007; Article ID e1154.
[37] Parmentier I, Duminil J, Kuzmina M, Philippe M, Thomas DW, Kenfack D, Chuyong GB, Cruaud C, Hardy OJ. How effective are DNA barcodes in the identification of African rainforest trees? PloS One. 2013; Article ID e54921.
[38] Vivas CV, Moraes RCS, Alves-Araújo A, Alves M, Mariano-Neto E, Van den Berg C, Gaiott FA. DNA barcoding in Atlantic forest plants: what is the best marker for Sapotaceae species identification? Genet Mol Biol. 2014; 37(4): 662-670.
[39] Bergsten J, Bilton DT, Fujisawa T, Elliott M, Monaghan MT, Balke M, Hendrich L, Geijer J, Herrmann J, Foster GN, Ribera I, Nilsson AN, Barraclough TG, Vogler AP. The Effect of geographical scale of sampling on DNA barcoding. Syst Biol. 2012; 61(5): 851-869.
[40] Luo A, Lan H, Ling C, Zhang A, Shi L, Ho SYW, Zhu C. A simulation study of sample size for DNA barcoding. Ecol Evol. 2015; 5(24): 5869-5879.
[41] López-Vinyallonga S, Mehregan I, Garcia-Jacas N, Tscherneva O, Susanna A, Kadereit JW. Phylogeny and evolution of the Arctium-Cousinia complex (Compositae, Cardueae-Carduinae). Taxon. 2009; 58(1): 153-171.
[42] Negaresh K, Rahiminejad MR. A taxonomic revision of Centaurea sect. microlophus (Asteraceae, Cardueae-Centaureinae) and three new records for the flora of Iran. Nord J Bot. 2015; 33: 335-353.
[43] Zare M, Khosravi AR, Joharchi MR. Distribution patterns of the genus Cousinia (Asteraceae) in Iran. Iran J Bot. 2013; 19(1): 127-141.
[44] De Boer HJ, Ouarghidi A, Martin G, Abbad A, Kool A. DNA barcoding reveals limited accuracy of identifications based on folk taxonomy. PloS One. 2014; Artcle ID e84291.
[45] Zhu X, Zhang Y, Liu X, Hou D, Gao T. Authentication of commercial processed Glehniae Radix (Beishashen) by DNA barcodes. Chin Med. 2015; 10(35): 1-9.
[46] Ouarghidi A, Powell B, Martin GJ, De Boer H, Abbad A. Species substitution in medicinal roots and possible implications for toxicity of herbal remedies in Morocco. Econ Bot. 2012; 66(4): 370-382.
[47] Posadzki P, Watson L, Ernst E. Contamination and adulteration of herbal medicinal products (HMPs): an overview of systematic reviews. Eur J Clin Pharmacol. 2013; 69(3): 295-307.
[48] Sun Z, Chen S. Identification of cortex herbs using the DNA barcode nrITS2. J Nat Med. 2013; 67(2): 296-302.
[49] Guo H, Wang W, Yang N, Guo B, Zhang S, Yang R, Yuan Y, Yu J, Hu S, Sun Q, Yu J. DNA barcoding provides distinction between Radix Astragali and its adulterants. Sci China Life Sci. 2010; 53(8): 992-999.