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Planta Med 2023; 89(08): 824-832
DOI: 10.1055/a-1855-1778
Natural Product Chemistry & Analytical Studies
Original Papers

Discrimination of Zicao Samples Based on DNA Barcoding and HPTLC Fingerprints, and Identification of (22E)-Ergosta-4,6,8(14),22-tetraen-3-one As a Marker Compound[ # ]

Nadine Kretschmer 
1   Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Austria
,
Christin Durchschein
1   Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Austria
,
Guenther Heubl
2   Systematic Botany and Mycology, Department of Biology, Ludwig-Maximilians-University Munich, Germany
,
Eva-Maria Pferschy-Wenzig 
1   Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Austria
,
Olaf Kunert 
3   Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz, Austria
,
Rudolf Bauer 
1   Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Austria
› Author AffiliationsSupported by: Austrian Science Fund P27505
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Abstract

The unambiguous identification of plant material is a prerequisite of rational phytotherapy. Misidentification can even cause serious health problems, as in the case of the Chinese medicinal herb Zicao. Commercial material labelled “Zicao” may be derived from the roots of Arnebia euchroma (ruan zicao), Lithospermum erythrorhizon (ying zicao), or Onosma paniculata (dian zicao). All of these roots contain shikonin derivatives as main bioactive constituents, but ying zicao and dian zicao contain also hepatotoxic pyrrolizidine alkaloids in high amounts. Therefore, the use of A. euchroma with a very low pyrrolizidine alkaloid content is desirable. Confusions of the species occur quite often, indicating an urgent need for an unambiguous identification method. Discrimination of 23 zicao samples has been achieved by analyses of the nuclear internal transcribed spacer ITS2 and trnL-F intergenic spacer of the chloroplast DNA. Data were analyzed using Bioedit, ClustalX, Mega 11 and BLAST. Results indicate that ITS2 barcoding can accurately distinguish Arnebia euchroma from their adulterants. Subsequently, an HPTLC method has been developed allowing a chemical discrimination of the most widely used species. (22E)-Ergosta-4,6,8(14),22-tetraen-3-one has been identified as characteristic marker compound, allowing an unambiguous discrimination of A. euchroma and L. erythrorhizon.

Key words

Zicao - Arnebia euchroma - Lithospermum erythrorhizon - Onosma paniculata - Boraginaceae - ITS2 - trnL-F - HPTLC - (22E)-ergosta-4,6,8(14),22-tetraen-3-one

# Dedicated to Professor Dr. Gerhard Franz on the occasion of his 85th birthday.


Supporting Information

    Supplementary information is available for this publication. It includes: Alignments, BLAST search results, genetic distances, HPTLC fingerprints and data of LC-DAD-ESI-MS, NMR, and GC-MS measurements.

    Figures of HPTLC fingerprints of samples 1 – 23, developed with SS1 and SS2, LC-DAD-ESI-MS data of (22E)-ergosta-4,6,8(14),22-tetraen-3-one reference compound, LC-DAD-ESI-MS and GC-MS data of the isolated fraction containing M1, as well as tables with 1H NMR and 13C NMR data of (22E)-ergosta-4,6,8 (14),22-tetraen-3-one, and 13C NMR data of β-sitosterol are provided.

  • Supporting Information


Publication History

Received: 18 December 2021

Accepted after revision: 03 May 2022

Article published online:
22 July 2022

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
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  • References

  • 1 World Health Organization. WHO Global Report on Traditional and Complementary Medicine 2019. Geneva: World Health Organization; 2019
    Search in Google Scholar
  • 2 Stegelmeier BL, Brown AW, Welch KD. Safety concerns of herbal products and traditional Chinese herbal medicines: Dehydropyrrolizidine alkaloids and aristolochic acid. J Appl Toxicol 2015; 35: 1433-1437
    CrossrefPubMedSearch in Google Scholar
  • 3 Zhang J, Wider B, Shang H, Li X, Ernst E. Quality of herbal medicines: Challenges and solutions. Complement Ther Med 2012; 20: 100-106
    CrossrefPubMedSearch in Google Scholar
  • 4 Han J, Pang X, Liao B, Yao H, Song J, Chen S. An authenticity survey of herbal medicines from markets in China using DNA barcoding. Sci Rep 2016; 6: 18723
    CrossrefPubMedSearch in Google Scholar
  • 5 Debelle FD, Vanherweghem JL, Nortier JL. Aristolochic acid nephropathy: A worldwide problem. Kidney Int 2008; 74: 158-169
    CrossrefPubMedSearch in Google Scholar
  • 6 Röder E. Medicinal plants in China containing pyrrolizidine alkaloids. Pharmazie 2000; 55: 711-726
    PubMedSearch in Google Scholar
  • 7 Yan X, Kang H, Feng J, Yang Y, Tang K, Zhu R, Yang L, Wang Z, Cao Z. Identification of toxic pyrrolizidine alkaloids and their common hepatotoxicity mechanism. Int J Mol Sci 2016; 17: 318
    CrossrefPubMedSearch in Google Scholar
  • 8 Fu PP, Chou MW, Xia Q, Yang YC, Yan J, Doerge DR, Chan PC. Genotoxic pyrrolizidine alkaloids and pyrrolizidine alkaloid n-oxides – Mechanism leading to DNA adduct formation and tumorigenicity. J Environm Sci Health 2001; 19: 353-385
    CrossrefPubMedSearch in Google Scholar
  • 9 Andújar I, Ríos JL, Giner RM, Recio MC. Pharmacological properties of shikonin – a review of literature since 2002. Planta Med 2013; 79: 1685-1697
    Thieme ConnectPubMedSearch in Google Scholar
  • 10 Chen X, Yang L, Oppenheim JJ, Howard MZ. Cellular pharmacology studies of shikonin derivatives. Phytother Res 2002; 16: 199-209
    CrossrefPubMedSearch in Google Scholar
  • 11 Röder E, Rengel-Mayer B. Pyrrolizidine alkaloids from arnebia euchroma*. Planta Med 1993; 59: 192
    Thieme ConnectPubMedSearch in Google Scholar
  • 12 Röder E, Rengel B. Pyrrolizidine alkaloids from Lithospermum erythrorhizon . Phytochemistry 1990; 29: 690-693
    CrossrefPubMedSearch in Google Scholar
  • 13 Hebert PDN, Cywinska A, Ball SL, DeWaard JR. Biological identifications through DNA barcodes. Proc R So Lond B Biol Sci 2003; 270: 313-321
    CrossrefPubMedSearch in Google Scholar
  • 14 Qian L, Liu J, Guo L, Zheng JI, Ma S. Molecular identification of Arnebiae Radix by PCR-RFLP based on the ITS2 sequence. Chin J Pharm Anal 2019; 36: 1611-1617
    CrossrefPubMedSearch in Google Scholar
  • 15 Liu J, Zhao C, Guo L, Ma S, Zheng J, Wang J. The plant original identification of Inner Mongolia Arnebia radix based on morphology and DNA barcoding. Res Sq 2020; PPR196478
    CrossrefPubMedSearch in Google Scholar
  • 16 Xu H, Li P, Ren G, Wang Y, Jiang D, Liu C. Authentication of three source spices of Arnebiae Radix using DNA barcoding and HPLC. Front Pharmacol 2021; 12: 677014
    CrossrefPubMedSearch in Google Scholar
  • 17 Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C. Validation of the ITS2 region as a Novel DNA barcode for identifying medicinal plant species. PLoS One 2010; 5: e8613
    CrossrefPubMedSearch in Google Scholar
  • 18 Rinner B, Kretschmer N, Knausz H, Mayer A, Boechzelt H, Hao XJ, Heubl G, Efferth T, Schaider H, Bauer R. A petrol ether extract of the roots of Onosma paniculatum induces cell death in a caspase dependent manner. J Ethnopharmacol 2010; 129: 182-188
    CrossrefPubMedSearch in Google Scholar
  • 19 Doyle J, Doyle JL. Isolation of plant DNA from fresh tissue. Focus 1990; 12: 13-15
    PubMedSearch in Google Scholar
  • 20 Khan S, Qureshi I, Kamaluddin M, Alam T, Abdin MZ. Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr J Biotechnol 2007; 6: 175-178
    PubMedSearch in Google Scholar
  • 21 White TJ, Bruns T, Lee S, Taylor J. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. eds. PCR Protocols – A Guide to Methods and Applications. San Diego, United Kingdom: Academic Press; 1990: 315-322
    Search in Google Scholar
  • 22 Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 2010; 5: e8613
    CrossrefPubMedSearch in Google Scholar
  • 23 Taberlet P, Gielly L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 1991; 17: 1105-1109
    CrossrefPubMedSearch in Google Scholar
  • 24 Braeuchler C, Meimberg H, Heubl G. Molecular phylogeny of the genera Digitalis L. and Isoplexis (Lindley) Loudon (Veronicaceae) based on ITS- and trnLF sequences. Plant Syst Evol 2004; 248: 111-128
    CrossrefPubMedSearch in Google Scholar
  • 25 Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 1999; 41: 95-98
    PubMedSearch in Google Scholar
  • 26 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Bio 1990; 215: 403-410
    CrossrefPubMedSearch in Google Scholar
  • 27 Tamura K, Stecher G, Kumar S. MEGA 11 Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol 2021; 38: 3022-3027
    CrossrefPubMedSearch in Google Scholar
  • 28 Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 2010; 5: e8613
    CrossrefPubMedSearch in Google Scholar
  • 29 Sharma N, Sharma UK, Gupta AP. Devla, Sinha AK, Lal B, Ahuja PS. Simultaneous densitometric determination of shikonin, acetylshikonin, and beta-acetoxyisovaleryl-shikonin in ultrasonic-assisted extracts of four Arnebia species using reversed-phase thin layer chromatography. J Sep Sci 2009; 32: 3239-3245
    CrossrefPubMedSearch in Google Scholar
  • 30 Albrecht A, Vovk I, Simonovska B, Srbinoska M. Identification of shikonin and its ester derivatives from the roots of Echium italicum L. J Chromatogr A 2009; 1216: 3156-3162
    CrossrefPubMedSearch in Google Scholar
  • 31 Bozan B, Baser KHC, Kara S. Quantitative determination of naphthoquinones of Arnebia densiflora by TLC-densitometry. Fitoterapia 1999; 70: 402-406
    CrossrefPubMedSearch in Google Scholar
  • 32 Ding ZS, Jiang FS, Chen NP, Lv GY, Zhu CG. Isolation and identification of an anti-tumor component from leaves of Impatiens balsamina . Molecules 2008; 13: 220-229
    CrossrefPubMedSearch in Google Scholar
  • 33 Reich E, Schibli A. Report: Evaluate Proposed TLC Method with Respect to Onosma as Adulterant. Muttenz, Schweiz: CAMAG Laboratories; 2009
    Search in Google Scholar
  • 34 Hu Y, Jiang Z, Leung KS, Zhao Z. Simultaneous determination of naphthoquinone derivatives in Boraginaceous herbs by high-performance liquid chromatography. Anal Chim Acta 2006; 577: 26-31
    CrossrefPubMedSearch in Google Scholar
  • 35 Nåbo LJ, List NH, Witzke S, Wüstner D, Khandelia H, Kongsted J. Design of new fluorescent cholesterol and ergosterol analogs: Insights from theory. Biochim Biophys Acta 2015; 1848: 2188-2199
    CrossrefPubMedSearch in Google Scholar
  • 36 Sun Y, Liang X, Zhao Y, Fan J. A sensitive spectrofluorometric method for determination of ergosta-4,6,8(14),22-tetraen-3-one in rat plasma, feces, and urine for application to pharmacokinetic studies using cerium(III) as a probe. Appl Spectrosc 2013; 67: 106-111
    CrossrefPubMedSearch in Google Scholar
  • 37 Damianakos H, Kretschmer N, Sykłowska-Baranek K, Pietrosiuk A, Bauer R, Chinou I. Antimicrobial and cytotoxic isohexenylnaphthazarins from Arnebia euchroma (Royle) Jonst. (Boraginaceae) callus and cell suspension culture. Molecules 2012; 17: 14310-14322
    CrossrefPubMedSearch in Google Scholar
  • 38 Cao H, Zhang W, Liu D, Hou M. Identification, in vitro evaluation and modeling studies of the constituents from the roots of Arnebia euchroma for antitumor activity and STAT3 inhibition. Bioorg Chem 2020; 96: 103655
    CrossrefPubMedSearch in Google Scholar
  • 39 Zhao YY, Xie RM, Chao X, Zhang Y, Lin RC, Sun WJ. Bioactivity-directed isolation, identification of diuretic compounds from Polyporus umbellatus . J Ethnopharmacol 2009; 126: 184-187
    CrossrefPubMedSearch in Google Scholar
  • 40 Fujimoto H, Nakamura E, Okuyama E, Ishibashi M. Six immunosuppressive features from an ascomycete, Zopfiella longicaudata, found in a screening study monitored by immunomodulatory activity. Chem Pharm Bull (Tokyo) 2004; 52: 1005-1008
    CrossrefPubMedSearch in Google Scholar
  • 41 Zhao YY, Shen X, Chao X, Ho CC, Cheng XL, Zhang Y, Lin RC, Du KJ, Luo WJ, Chen JY, Sun WJ. Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Biochim Biophys Acta 2011; 1810: 384-390
    CrossrefPubMedSearch in Google Scholar
  • 42 Fernando D, Adhikari A, Nanayakkara C, de Silva ED, Wijesundera R, Soysa P. Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus . BMC Complement Altern Med 2016; 16: 484
    CrossrefPubMedSearch in Google Scholar
  • 43 Qiao MF, Ji NY, Liu XH, Li F, Xue QZ. Asporyergosterol, a new steroid from an algicolous isolate of Aspergillus oryzae. Nat Prod Commun 2010; 5: 1575-1578
    PubMedSearch in Google Scholar
  • 44 Gao JM, Dong ZJ, Liu JK. Constituents of Basidiomycetes Russula cyanoxantha . Acta Bot Yunnanica 2000; 22: 85-89
    PubMedSearch in Google Scholar
  • 45 Bok JW, Lermer L, Chilton J, Klingeman HG, Towers GH. Antitumor sterols from the mycelia of Cordyceps sinensis . Phytochemistry 1999; 51: 891-898
    CrossrefPubMedSearch in Google Scholar
  • 46 Jia M, Chen L, Xin HL, Zheng CJ, Rahman K, Han T, Qin LP. A friendly relationship between endophytic fungi and medicinal plants: A systematic review. Front Microbiol 2016; 7: 906
    CrossrefPubMedSearch in Google Scholar
  • 47 Nicoletti R, Fiorentino A. Plant bioactive metabolites and drugs produced by endophytic fungi of spermatophyta. Agriculture 2015; 5: 918-970
    CrossrefPubMedSearch in Google Scholar