Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.


Introduction
Herbal medicines have been used for thousands of years to promote human health and cure diseases. Nowadays, herbal medicines occupy an indispensable position in the medical treatment of human diseases across the world. The market for herbal medicinal products has increased substantially and consistently in past years [1]. Because the efficacy and safety of herbal medicines is highly dependent on the proper use of authentic material, the accurate identification of herbs is a major concern [2]. However, discriminating between species used for herbal medicines and related species is very difficult, due to similarities in the morphology of processed tissues, such as roots, stems, leaves, fruits, and seeds [3,4].
In the past, the authentication of the ingredients of herbal medicines has relied on personal skills and has lacked objectivity and accuracy [4]. However, over the last two decades, technological ± 0.0012% in A. anomala, and 0.0017% ± 0.0012% in A. japonica (Table 2). Inter-species variability ranged from 0.0423% ± 0.0021% to 0.0476% ± 0.0053% (Table 2). A species-specific insertion/deletion (indel) mutation was detected at one site in A. japonica, and species-specific nucleotide substitutions were detected at 18 sites in A. dahurica, 10 sites in A. anomala, and 16 sites in A. japonica (Table 2). These species-specific nucleotide polymorphisms were used to develop SCAR markers to discriminate among the three Angelica species (Figure 1).   We additionally inferred the phylogenetic relationships among the three Angelica species related to Angelicae Dahuricae Radix, using ITS sequencing ( Figure S1). The Kimura 2-parameter (K2P) model was selected for the phylogenetic analysis. The phylogenetic tree constructed using the maximum likelihood (ML) method revealed a monophyletic group for each species with high bootstrap values (99-100%; Figure S1). Samples of A. anomala and A. japonica were more closely related with each other than with those of A. dahurica ( Figure S1). Overall, these data suggest that the three Angelica species are identifiable on the basis of sequence variability in the ITS region.

Development of Species-Specific SCAR Markers
To develop species-specific SCAR markers, comparative sequence analysis of the ITS regions of all plant samples was performed, and three species-specific primer pairs were designed ( Table 3). The forward primers and reverse primers were designed using the ITS1 and ITS2 region, respectively. Hence, the resultant PCR amplicons contain the 5.8S ribosomal-RNA region (Figure 1). The size of the PCR fragments generated using these primers was unique for each species (       Table 3.  Table 1. Lanes M and N represent the 100 bp DNA ladder and no template control, respectively. Arrows indicate the sizes of the PCR products, and arrowheads indicate the sizes of different molecular weight bands of the DNA ladder.

Development of A Multiplex SCAR Assay for the Authentication of Herbal Medicines
Differences in PCR product sizes are key for species identification. In this study, we developed a multiplex SCAR assay to distinguish Angelicae Dahuricae Radix from other related species using all three species-specific SCAR primer pairs in a single PCR reaction. Each primer pair specifically amplified the DNA of the corresponding species, yielding distinct species-specific PCR products ( Figure 3). Additionally, for the mixed template DNAs composed of two to three different species combinations (A. dahurica + A. anomala; A. dahurica + A. japonica; A. anomala + A. japonica; A. dahurica + A. anomala + A. japonica), authentic materials and adulterants were successfully detected at the species level, depending on the sizes of PCR products when PCR amplification was carried out using all three primer pairs (ADA-F/ADA-R, AAN-F/AAN-R, and AJA-F/AJA-R) in a single reaction. Thus, each PCR reaction containing mixed template DNAs produced distinct PCR products representative of the corresponding species ( Figure 4). These data demonstrate that the primers designed in this study are capable of discriminating among the three Angelica species related to Angelicae Dahuricae Radix in a multiplex PCR.  Table 3.  Table 1. Lanes M and N represent the 100 bp DNA ladder and no template control, respectively. Arrows indicate the sizes of the PCR products, and arrowheads indicate the sizes of different molecular weight bands of the DNA ladder.

Development of A Multiplex SCAR Assay for the Authentication of Herbal Medicines
Differences in PCR product sizes are key for species identification. In this study, we developed a multiplex SCAR assay to distinguish Angelicae Dahuricae Radix from other related species using all three species-specific SCAR primer pairs in a single PCR reaction. Each primer pair specifically amplified the DNA of the corresponding species, yielding distinct species-specific PCR products ( Figure 3). Additionally, for the mixed template DNAs composed of two to three different species combinations (A. dahurica + A. anomala; A. dahurica + A. japonica; A. anomala + A. japonica; A. dahurica + A. anomala + A. japonica), authentic materials and adulterants were successfully detected at the species level, depending on the sizes of PCR products when PCR amplification was carried out using all three primer pairs (ADA-F/ADA-R, AAN-F/AAN-R, and AJA-F/AJA-R) in a single reaction. Thus, each PCR reaction containing mixed template DNAs produced distinct PCR products representative of the corresponding species ( Figure 4). These data demonstrate that the primers designed in this study are capable of discriminating among the three Angelica species related to Angelicae Dahuricae Radix in a multiplex PCR.  The multiplex SCAR assay was subsequently used to investigate the current commercial distribution status of Angelicae Dahuricae Radix, and to verify the reproducibility of the developed SCAR markers. A total of 20 commercial samples of Angelicae Dahuricae Radix were purchased from herbal markets in China and Korea (Table S1). Although the amplification signal intensity of one commercial sample (sample number 8; Figure 5) was weaker than that of the other samples, all of the 20 commercial samples were identified as genuine Angelicae Dahuricae Radix (dried roots of A. dahurica) ( Figure 5 and Table S1). These results suggest that the multiplex SCAR assay developed   The multiplex SCAR assay was subsequently used to investigate the current commercial distribution status of Angelicae Dahuricae Radix, and to verify the reproducibility of the developed SCAR markers. A total of 20 commercial samples of Angelicae Dahuricae Radix were purchased from herbal markets in China and Korea (Table S1). Although the amplification signal intensity of one commercial sample (sample number 8; Figure 5) was weaker than that of the other samples, all of the 20 commercial samples were identified as genuine Angelicae Dahuricae Radix (dried roots of A. dahurica) ( Figure 5 and Table S1). These results suggest that the multiplex SCAR assay developed The multiplex SCAR assay was subsequently used to investigate the current commercial distribution status of Angelicae Dahuricae Radix, and to verify the reproducibility of the developed SCAR markers. A total of 20 commercial samples of Angelicae Dahuricae Radix were purchased from herbal markets in China and Korea (Table S1). Although the amplification signal intensity of one commercial sample (sample number 8; Figure 5) was weaker than that of the other samples, all of the 20 commercial samples were identified as genuine Angelicae Dahuricae Radix (dried roots of A. dahurica) ( Figure 5 and Table S1). These results suggest that the multiplex SCAR assay developed in this study is capable of rapidly and effectively discriminating between authentic Angelicae Dahuricae Radix and its related herbal species.
Molecules 2018, 23, x FOR PEER REVIEW 7 of 13 in this study is capable of rapidly and effectively discriminating between authentic Angelicae Dahuricae Radix and its related herbal species.  (Table S1). Lanes M and N represent the 100 bp DNA ladder and no template control, respectively. Arrows indicate the sizes of PCR products, and arrowheads indicate the sizes of different molecular weight bands of the DNA ladder.

Discussion
The dried roots of A. dahurica and its variety formosana, collectively referred to as Angelicae Dahuricae Radix, are commonly used as a traditional herbal medicine. Related species of A. dahurica, including A. anomala and A. japonica, are morphologically similar in appearance to A. dahurica. A. anomala is considered as an important traditional medicinal plant and is used to treat several conditions, including inflammation, pain, and poisoning [27]. Angelica japonica has additionally been regarded as a potential herbal medicine, due to its antitumor actions [28]. However, A. anomala and A. japonica are not authorized as authentic medicinal plants, nor are they registered in the Korean Pharmacopoeia or the Pharmacopoeia of the People's Republic of China, as their safety and usage have not yet been established [14][15][16][17].
The introduction of a SCAR marker based on DNA barcoding has been a key technique for the precise identification of medicinal plants at the species level, and for the authentication of the botanical origins of herbal medicines [29,30]. In order to develop SCAR markers for distinguishing A. dahurica and A. dahurica var. formosana from A. anomala and A. japonica, we amplified and sequenced the following candidate DNA barcodes: The ITS region of nuclear rDNA and the matK, rbcL, and psbA-trnH regions of chloroplast DNA. Using this DNA barcode sequence analyses, we developed three SCAR markers that can identify two authentic medicinal plant taxa for Angelicae Dahuricae Radix (A. dahurica and its variety A. dahurica var. formosana) and two adulterant species, A. anomala and A. japonica, from the ITS region of nuclear rDNA (Figures 1 and 2). However, we could not develop SCAR markers to differentiate between A. dahurica and its variety formosana, because the  (Table S1). Lanes M and N represent the 100 bp DNA ladder and no template control, respectively. Arrows indicate the sizes of PCR products, and arrowheads indicate the sizes of different molecular weight bands of the DNA ladder.

Discussion
The dried roots of A. dahurica and its variety formosana, collectively referred to as Angelicae Dahuricae Radix, are commonly used as a traditional herbal medicine. Related species of A. dahurica, including A. anomala and A. japonica, are morphologically similar in appearance to A. dahurica. A. anomala is considered as an important traditional medicinal plant and is used to treat several conditions, including inflammation, pain, and poisoning [27]. Angelica japonica has additionally been regarded as a potential herbal medicine, due to its antitumor actions [28]. However, A. anomala and A. japonica are not authorized as authentic medicinal plants, nor are they registered in the Korean Pharmacopoeia or the Pharmacopoeia of the People's Republic of China, as their safety and usage have not yet been established [14][15][16][17].
The introduction of a SCAR marker based on DNA barcoding has been a key technique for the precise identification of medicinal plants at the species level, and for the authentication of the botanical origins of herbal medicines [29,30]. In order to develop SCAR markers for distinguishing A. dahurica and A. dahurica var. formosana from A. anomala and A. japonica, we amplified and sequenced the following candidate DNA barcodes: The ITS region of nuclear rDNA and the matK, rbcL, and psbA-trnH regions of chloroplast DNA. Using this DNA barcode sequence analyses, we developed three SCAR markers that can identify two authentic medicinal plant taxa for Angelicae Dahuricae Radix (A. dahurica and its variety A. dahurica var. formosana) and two adulterant species, A. anomala and A. japonica, from the ITS region of nuclear rDNA (Figures 1 and 2). However, we could not develop SCAR markers to differentiate between A. dahurica and its variety formosana, because the nucleotide sequences of all four DNA barcodes were identical in A. dahurica and A. dahurica var. formosana (Figures 1 and 2). These results indicate that these DNA barcode regions are not capable of distinguishing between A. dahurica and A. dahurica var. formosana, and strongly support previously reported results which reported that DNA barcoding is sufficient as a molecular marker at inter-specific and intergeneric levels [10,[31][32][33]. However, we did not consider distinguishing these two authentic plant taxa because the roots of the two plant taxa are official herbal materials listed in the National Pharmacopoeias of Korea and China [15]. Additional genomic fingerprinting analyses, such as RFLP, RAPD, inter simple sequence repeats (ISSRs), or the next-generation sequencing of whole genomes, may be attempted to acquire genetic information for identifying A. dahurica and A. dahurica var. formosana.
Sequence analyses of A. dahurica, A. anomala, and A. japonica showed the largest number of species-specific nucleotide polymorphisms (44 of 690 bp) in the ITS region, followed by matK (5 of 1276 bp), psbA-trnH (3 of 332 bp), and rbcL (2 of 1503 bp) (data not shown), suggesting a greater applicability of the ITS region as a DNA barcode than the chloroplast regions for discriminating among the three Angelica species, which is consistent with the report of Yuan et al. [25]. The ITS region is a popular DNA barcode for species identification due to its high inter-specific distance. However, the intra-specific distance of the ITS region is also high in comparison with other DNA barcode regions [33]. To avoid intra-specific nucleotide variation from being counted as a species-specific nucleotide polymorphism, we collected plant samples from at least three different sites (Table 1). In addition, we also confirmed that the nuclear rDNA ITS sequences of A. dahurica were identical to those registered by Yuan et al. (GenBank accession nos. JX022904, JX022905, and JX022940) and did not show any nucleotide sequence variability in the species-specific primer regions (data not shown) [25]. These results suggest that the SCAR primers to identify A. dahurica are stable to amplify authentic Angelicae Dahuricae Radix.
Although using DNA barcodes for species identification is generally advantageous, it has some drawbacks [10,29,34]. The principal requirement for DNA barcoding is high quality DNA [29]. During the processing of herbal medicines, DNA is likely degraded and fragmented due to drying at high temperature and extreme pH [29]. Poor DNA quality obstructs the amplification of DNA fragments of sufficient size for species identification [10,25,33,34]. In this respect, developing species-specific SCAR markers targeting short DNA sequences, rather than reading the complete sequence of DNA barcode regions, provides a more efficient method for the identification of herbal medicines [35]. We designed primers to target short fragments of the ITS region (<400 bp), which enable markers to successfully amplify low-quality DNA [31]. The primer pairs designed in this study successfully amplified species-specific DNA fragments of different sizes in both SCAR ( Figure 2) and multiplex SCAR (Figure 3) assays. We verified the ability of the multiplex SCAR assay to amplify and discriminate among a mixture of template DNAs (Figure 4), and used the assay to analyze commercially distributed Angelicae Dahuricae Radix ( Figure 5). From the verification of the botanical origins of 20 samples of commercially processed Angelicae Dahuricae Radix purchased from Korean and Chinese herbal markets, we did not confirm adulteration or contamination (Table S1 and Figure 5). To verify the discriminability of SCAR markers, PCR products amplified from commercial samples of Angelicae Dahuricae Radix using the multiplex SCAR assay were sequenced to confirm the botanical origins and sequence identities of the samples. The sequences of all the commercial samples were identical to those of the control plants (data not shown). Although we did not confirm the adulteration or contamination of inauthentic herbal materials originating from A. anomala and A. japonica in this study, additional consistent monitoring assays are needed to prevent the adulteration and contamination of Angelicae Dahuricae Radix in herbal markets. The commercial sample number 8 obtained from the Chinese herbal medicine market showed weak amplification intensity in the multiplex SCAR assay ( Figure 5). It is possible that this sample was subjected to harsh treatment, such as hot air drying during processing, which degraded its DNA. Overall, we predict that the multiplex SCAR assay developed in this study will prove to be advantageous in reducing both the time and cost involved in DNA barcoding, thus allowing researchers to discriminate between genuine Angelicae Dahuricae Radix and adulterants.

Plant Material and Herbal Medicines
Three or four plant samples each of A. dahurica, A. dahurica var. formosana, A. anomala, and A. japonica were used in this study (Table 1). The scientific name of the plant was listed in accordance with The Plant List (http://www.theplantlist.org/). All plant samples were collected from multiple native habitats or farming fields in Korea and China, and stored at a temperature of −70 • C before further analysis. Commercially available samples of herbal medicine were purchased from various herbal markets across Korea and China. Plant samples of A. dahurica, A. anomala, and A. japonica were saved as specimens, and deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbariorum code KIOM) under unique voucher numbers (Table 1).

Genomic DNA Extraction
Genomic DNA was extracted from frozen leaves or dried specimens using a DNeasy ® Plant Mini Kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions, and stored at a temperature of −20 • C. The concentration of the DNA samples was measured using a spectrophotometer (Nanodrop ND-1000, Nanodrop, Wilmington, DE, USA), and the final concentration of all DNA samples was adjusted to approximately 15 ng/µL for PCR amplification.

PCR Amplification and Sequencing of The ITS Region
The ITS region was amplified using the universal primers ITS1 (5 -TCCGTAGGTGAACCTG CGG-3 ) and ITS4 (5 -TCCTCGCTGATTGATATGC-3 ) [36]. PCRs were performed in a total reaction volume of 40 µL, comprising 10mM Tris-HCl (pH 9.0), 2.5 mM MgCl 2 , 200 µM of each dNTP, 10 mM (NH 4 ) 2 SO 4 , 0.5 U Taq DNA polymerase (Solgent, Daejeon, Korea), 0.5 µM of each primer, and 15 ng of template DNA, on a ProFlex PCR System (Applied Biosystems, Life Technologies, Foster City, CA, USA). The following conditions were used for PCR amplification: An initial denaturation at a temperature of 95 • C for 2 min, followed by 35 cycles of denaturation at a temperature of 95 • C for 30 s, annealing at a temperature of 55 • C for 30 s, an extension at a temperature of 72 • C for 45 s, and a final extension at a temperature of 72 • C for 5 min. The PCR products were separated by gel electrophoresis on a 1.5% agarose gel alongside a 100 bp DNA ladder (Solgent), and visualized using Ecodye™ Nucleic Acid Staining Solution (Biofact, Daejeon, Korea). The identifiable PCR products were extracted from the agarose gel using a QIAquick gel extraction kit (QIAGEN, Valencia, CA, USA). The nuclear rDNA-ITS regions extracted from the agarose gel were sub-cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and transformed into Escherichia coli JM109 competent cells (RBC, Taipei, Taiwan), following the manufacturer's instructions [37]. The transformed cells were plated on Luria Broth (LB) agar media containing 100 µg/mL ampicillin, 40 µg/mL X-gal, and 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and incubated at a temperature of 37 • C for 18 h. Recombinant clones were confirmed by colony PCR using vector-based primers, T7 and SP6. At least three white colonies for each sample were sequenced using the Sanger method.

Nucleotide Sequence, Phylogenetic Analyses and SCAR Marker Development
To avoid errors during PCR amplification and sequencing, and to identify potential chimeric sequences, the sequence quality was checked by comparing sequences from three white colonies from each sample. The ITS sequences were verified using the NCBI BLAST search tool, and deposited in the GenBank database under the following accession numbers: Approximately 700 bp of all 15 ITS sequences were aligned using ClustalW, and manually edited using BioEdit ver. 7.2.5 (North Carolina State University, Raleigh, NC, USA) [38] to identify species-specific indels and nucleotide substitutions. Inter-and intra-species genetic distances were calculated using MEGA ver. 7.0, following the K2P model with 1000 bootstrap replicates [39]. A best-fit substitution model was selected using MEGA for phylogenetic analysis, and a phylogenetic tree was constructed using the ML method using MEGA, based on the selected model, using all gaps or missing data. The ITS sequence of Heracleum moellendorffii Hance (GenBank accession number: MH188445) served as an outgroup in the phylogenetic analysis.
To develop SCAR markers, species-specific primer pairs were designed using species-specific nucleotide substitutions to amplify short DNA fragments of different sizes for each species (Table 3). PCRs were conducted in a total reaction volume of 30 µL, comprising 10mM Tris-HCl (pH 9.0), 2.5 mM MgCl 2 , 200 µM of each dNTP, 10 mM (NH 4 ) 2 SO 4 , 0.5 U Taq DNA polymerase (Solgent), 0.5 µM of each species-specific forward and reverse primers (Table 3), and 15 ng of template DNA, using the following conditions: An initial denaturation at a temperature of 95 • C for 2 min, followed by 35 cycles of denaturation at a temperature of 95 • C for 30 s, annealing at a temperature of 68 • C for 30 s, an extension at a temperature of 72 • C for 40 s, and a final extension at a temperature of 72 • C for 5 min. The PCR products were visualized by gel electrophoresis on a 1.5% agarose gel, as described in Section 4.3.

Development of Multiplex SCAR Assay and Monitoring of Commercial Herbal Medicines
To develop the multiplex SCAR assay, all three species-specific primer pairs were combined in a single PCR reaction. Optimal PCR conditions were determined by altering various parameters, including the duration and temperature of annealing, the number of amplification cycles, the concentrations of primer, and the combinations and concentrations of template DNAs. The quality of PCR products was verified by gel electrophoresis on a 1.5% agarose gel, as described in Section 4.3.
To validate the multiplex PCR method and to verify the authenticity of the commercial Angelicae Dahuricae Radix samples, 20 commercially available Angelicae Dahuricae Radix samples were purchased and examined using the multiplex SCAR assay. Approximately 15 ng of genomic DNA was used in a 30 µL PCR reaction, comprising 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl 2 , 200 µM of each dNTP, 10 mM (NH 4 ) 2 SO 4 , 0.5 U Taq DNA polymerase (Solgent), 0.5 µM of each three species-specific primer pair, and 15 ng of template DNA. The protocol used for amplification in the multiplex PCR was the same as that described in Section 4.4. The quality of PCR products was verified by gel electrophoresis on a 1.5% agarose gel, as described in Section 4.3.
To examine the ability of SCAR markers to identify adulterants in processed herbal medicines, template DNAs of two or more Angelica species were mixed in a 1:1 ratio (w:w) and subjected to multiplex PCR, as described in Section 4.5. The PCR products were separated by gel electrophoresis on a 2% agarose gel and visualized using Ecodye™ Nucleic Acid Staining Solution (Biofact).

Supplementary Materials:
The following are available online, Figure S1: A phylogenetic tree showing the relationships among 15 samples of three Angelica species based on sequences of the internal transcribed spacer (ITS) regions; Table S1: A list of plant samples and commercial herbal medicines investigated using a multiplex SCAR assay developed in this study.