Rapid Identification and Verification of Indirubin-Containing Medicinal Plants

Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ) phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC) was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture), but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.


Introduction
Chronic myelocytic leukemia (CML) is a malignant cancer that destroys the blood and marrow [1].In 1967, a group of scientists discovered that the traditional Chinese medicine prescription, Danggui Luhui Wan, which contains 11 Chinese herbal medicines, had a significant curative effect on CML [2,3].More recently, indirubin from the Chinese herbal medicine Indigo Naturalis (Qingdai), prepared from the leaves of Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, was found to be the active ingredient [4][5][6][7][8][9][10][11].Indirubin has since been found in additional Chinese herbal medicines derived from each of these species, respectively, including Isatidis Folium (the leaf of Is. tinctoria), Polygoni Tinctorii Folium (the leaf of P. tinctorium), and Baphicacanthis Cusiae Rhizoma et Radix (the root and rhizome of S. cusia) [12][13][14].However, few studies have determined the presence and containing of indirubin in the above medicinal plants and their adulterants.These adulterants include P. hydropiper, P. chinense, Clerodendrum cyrtophyllum, Indigofera tinctoria, and S. dimorphotricha (Figure 1) and are extremely difficult to discriminate from their true medicinal counterparts morphologically [15][16][17][18].The confusion between true medicinal plants and their adulterants has adverse effects on the clinical efficacy and safety of traditional medicines.It is thus paramount that traditional medicinal herbs and their active components can be reliably and costeffectively discriminated from their false counterparts.
In order to achieve the rapid identification and verification of the indirubin-containing medicinal plants, two important problems must be resolved.First, the original plant species known to contain indirubin must be effectively  discriminated from their adulterants.DNA barcoding technology offers the best technique for this to date, involving specific amplification of a short, standardized DNA fragment with universal primers across multiple samples [19,20].In recent years, several candidate DNA regions, such as matK, rbcL, psbA-trnH, and ITS/ITS2, have been assessed for their potential as DNA barcodes in plants [21][22][23][24][25]. Analysis of 50,790 plant ITS2 sequences revealed this region to be highly effective in discriminating medicinal plants and their closely related species [23,26], more so than matK, rbcL, and psbA-trnH.As such, ITS sequences have been recommended as the core barcode region for seed plants [27].Here, we reveal the first rapid and effective method to reliably discriminate indirubin-containing species in traditional medicinal formulations from their adulterants, using ITS2 barcoding.Secondly, it is necessary that the different plant organs comprising traditional medicinal materials (Figure 1) can be analysed for indirubin levels effectively.High performance liquid chromatography (HPLC) offers an effective screening technique in this regard.Samples comprising 30∼40 mg of dried leaves or 60∼70 mg of roots were crushed into powder in 2 mL microfuge tubes at 30 Hz using stainless steel ball milling for 1 min.Total genomic DNA was extracted using a Plant Genomic DNA Kit (Tiangen Biotech Co., China) with modifications as follows.Initial incubation was at 65 ∘ C in 750 L GP1 (Tiangen) buffer for 1 h for dried leaves or 5 h for roots and rhizomes.The remaining steps followed the manufacturer's protocol.

Materials and Methods
For matK, rbcL, psbA-trnH, and ITS2 DNA barcodes, universal primers and general PCR reaction conditions were used as presented in Table S1 (see Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/484670) [23,28].PCRs were in a 25 L reaction mixture, containing 30-100 ng of genomic DNA template, 12.5 L 2 × Tag PCR Master Mix (Aidlab Biotechnologies Co., China), and 1 L of forward and reverse primers (2.5 mol/L).After PCR, a 4 L aliquot was examined by 0.5% TBE agarose gel electrophoresis, and purified PCR products were sequenced in both directions using the primers used for PCR on an ABI3730XL sequencer (Applied Biosystems Co., USA).

Cloning and Sequencing of the ITS2
Region.The ITS2 PCR products of S. cusia and S. dimorphotricha were unsuccessfully directly sequenced.Therefore, purified products (TIANquick Midi Purification Kit; Tiangen Biotech Co., China) were ligated into the pMD18-T vector (Takara Biotech Co., China) and transformed into E. coli DH5 cells using standard recombinant DNA techniques.Positive transformants were selected on LB containing 0.1 mg/mL ampicillin and confirmed with colony PCR using the above PCR conditions.Four positive clones from each sample were sequenced on an ABI3730XL sequencer.In total, 40 clones from 13 samples of S. cusia and 7 clones from 3 samples of S. dimorphotricha (excluding fungal sequences) were obtained.

Sequence Analyses.
Sequence editing and contig assembly were performed using CodonCode Aligner v4.25 (CodonCode Co., USA).The ITS2 region was obtained based on the HMMER annotation method to remove the 5.8S and 28S sections at both ends of the sequences [29].
Obtained DNA sequences were aligned and the intraspecific variation and interspecific divergence calculated by Kimura two-parameter method.Phylogenetic trees were constructed using the Neighbor-Joining method with molecular evolutionary genetics analysis (MEGA) software version 5.0 [30].

HPLC Analyses.
The reference standard of indirubin was purchased from Shanghai Yuanye Bio-Technology Company (HPLC-tested purity >98%).1.25 mg of indirubin was dissolved in N,N-dimethyl formamide in a 25 mL volumetric flask.Five milliliters of solution was transferred to another 50 mL volumetric flask containing N,N-dimethyl formamide to make a standard stock solution of 5 g/mL indirubin.Two-three replicate samples from each tested plant organ and species were randomly sampled for indirubin content.Specifically, 75 mg of powdered crude materials was sonicated in 10 mL of N,N-dimethyl formamide for 30 min at room temperature and filtered.A 20 L aliquot of the filtrate was applied to a HPLC column (Angilent TC-C 18 , 5.0 m, 4.6 mm × 250 mm).The optimum separation of HPLC was carried out with a mobile phase composed of methanol-water (75 : 25, v/v) at a flow-rate of 1 mL/min.Peaks were detected at a wavelength and column temperature of 290 nm and 25 ∘ C, respectively.

Efficiency of Amplification and Identification for Four
Candidate Barcodes.For all eight species tested, 24 samples were selected randomly for amplification efficiency.The efficiency of amplification of matK, rbcL, psbA-trnH, and ITS2 was 62.5%, 79.2%, 100%, and 100%, respectively.Highquality bidirectional sequences were obtained for all PCR products.All the GenBank No. were listed in Table S2 and Table 1.Alignment revealed no interspecific divergence between S. cusia and S. dimorphotricha using both psbA-trnH and rbcL.In addition, matK had low amplification efficiency (62.5%), making this less applicable for barcoding of these species.In comparison with the other barcodes, all 24 samples were successfully classified into eight species using the ITS2 sequence.Therefore, only the ITS2 barcode was used for further analysis.

Measurement of DNA Divergence for ITS2. Song et al.
(2012) used sequence-tagged pyrosequencing and genomewide analyses to describe intragenomic variations of ITS2 regions from 178 plant species.This study defined "major variants" as any variant whose relative variant abundance (RVA) was greater than 5% [31].In this study we obtained 40 clones of S. cusia and only 2 of them (KJ939104, KJ939105) showed significant differences when compared with the other sequences.The remaining 38 sequences were considered major variants of ITS2 in this paper.
In this study, 86 sequences of ITS2 were obtained from all samples.Two ITS2 sequences (EU196919, JN235085) of P. tinctorium were downloaded from GenBank.The sequence length, GC average content, haplotype number and number of variable sites in each species (MEGA 5.0 software) are presented in Table 2. ITS2 sequence length ranged from 191 bp to 263 bp and GC average content ranged from 45.9% to 73.6%.The GC average contents of clones of S. cusia (73.6%) and S. dimorphotricha (73.0%) were at least 4% greater than those of the other species.Based on the variable sites, Is. tinctoria, P. tinctorium, and S. cusia were divided into 5, 1, and 15 haplotypes, respectively.
Interspecific and intraspecific distances using Kimura two-parameter method are shown in Table 3.The maximum intraspecific distances of Is. tinctoria, P. tinctorium, and S. cusia were 0.027, 0.000, and 0.036, respectively, while the minimum interspecific distance was 0.401.Furthermore, the minimum interspecific distances between any one of these Interspecific distance between Is. tinctoria and its adulterants 0.514∼0.684 Interspecific distance between P. tinctorium and its adulterants 0.025∼0.755 Interspecific distance between S. cusia and its adulterants 0.065∼0.931three species and its adulterants were 0.514, 0.025, and 0.065, respectively.

Identification of ITS2
Using NJ Tree.Phylogenetic analysis demonstrated that every species clustered into their own clade, supported with at least 81% bootstrapping (Figure 2).In addition, three closely related species of the genus Polygonum were strongly supported (99%, 81% and 100% bootstrap, resp.), and clustered into a larger branch with 97% bootstrap.All cloned sequences of S. cusia (96% bootstrap) and S. dimorphotricha (92% bootstrap) in Acanthaceae family formed a larger group (93% bootstrap).

Detection of Indirubin in Crude
Drugs and Their Adulterants.HPLC detected indirubin in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (Figure 3).Indirubin was not detected in the roots and rhizomes of these three taxa or in the leaves of their adulterants (P.hydropiper, P. chinense, C. cyrtophyllum, In. tinctoria, and S. dimorphotricha).

Discussion
In previous studies, the identification methods of medicinal plants including Is. tinctoria, P. tinctorium, and S. cusia have primarily focused on characterization of morphology, chromatographic fingerprints, and microstructures [32][33][34].However, these methods all have their disadvantages.The recent, rapid development of DNA molecular marker techniques provides a powerful tool for the accurate identification of medicinal materials.In recent years, DNA barcoding has been successfully employed in species identification of medicinal herbs, with the ITS2 barcode exhibiting remarkable stability and accuracy in this field.ITS/ITS2 regions were demonstrated to successfully distinguish Corni Fructus (the flesh of Cornus officinalis) from its adulterants [35].Xin et al. (2013) presented the ITS2 barcode as a powerful tool for tracing Goji (the fruit of Lycium barbarum) [36], while it was also used to accurately identify Ephedrae Herba (the stem taken from three species of Ephedra) and their closely related species [37].Consequently, the rapidly developing DNA barcoding can effectively supplement the traditional identification methods.In this study, the ITS2 region was selected from four candidate barcodes to identify three species and their adulterants because of 100% amplification efficiency herein, high interspecific divergence, and low intraspecific variation.Based on the ITS2 barcode, the maximum intraspecific distance of the three species (Is.tinctoria, P. tinctorium and S. cusia) was less than the minimum interspecific distance, not only among the three species, but also among each species and its adulterants.Furthermore, the NJ tree indicated that Is. tinctoria, P. tinctorium, and S. cusia were clustered into their own monophyletic group, separated from the other species.Moreover, NJ tree analysis using ITS2 reliably distinguished individuals of the genus Polygonum and the family of Acanthaceae, supporting the powerful identification ability of ITS2 barcode in plants.S .c u s ia K J 9 3 9 1 1 7 S .c u s i a K J 9 3 9 1 1 6 S .c u s i a K J 9 3 9 1 2 4 S .c u s i a K J 9 3 9 1 2 0 S .c u s i a K J 9 3 9 1 2 1 S .c u s i a K J 9 3 9 1 2 3 S .c u s i a K J 9 3 9 1 3 8 S .c u s i a K J 9 3 9 1 3 9 S .c u s i a K J 9 3 9 1 4 3 S .c u s i a K J 9 3 9 1 3 2 S .c u s ia K J 9 3 9 1 3 5 S .c u s ia K J 9 3 9 1 2 2 S .cu si a K J9 S .c u si a K J9 3 9 1 1 4 S .c u si a K J9 3 9 1 0 8 S .c u s ia K J 9 3 9 1 3 3 S .c u s i a K J 9 3 9 1 0 9 S .c u s i a K J 9 3 9 1 1 1 S .c u s i a K J 9 3 9 1 2 9 S .c u s i a K J 9 3 9 1 2 6 S .c u s i a K J 9 3 9 1 4 2 Therefore, both the results of nearest distance method and NJ tree strongly support that ITS2 as DNA barcode can successfully distinguish Is. tinctoria, P. tinctorium, and S. cusia from each other and from their respective adulterants.

S . c u s i a
The demonstrated anticancer function of indirubin in the treatment of Chronic myelocytic leukemia (CML), warrants its further investigation and ability to be identified accurately in natural medicines.In this study, HPLC detection found that indirubin could only be detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia and Indigo Naturalis.Meanwhile it could not be found in the root tissues of these species, or in the adulterant species tested herein.These results confirm that Is. tinctoria, P. tinctorium, and S. cusia cannot be replaced by their adulterants as indirubin-containing tinctures.And not only that, the species used for traditional medicinal herbs can be extremely disordered because of a general divergence in regional customs and species identification abilities [38].All of these strongly supported the need for accurate discrimination of these ineffective false "pseudo"medicines.

Conclusion
Together with HPLC detection of indirubin in various organs, ITS2 DNA barcoding enables the rapid, efficient, and costeffective discrimination of the truly effective preparations of medicinal plants from their noneffective organs and adulterants that do not contain indirubin.This provides an efficient  and new method to verify indirubin-containing medicines for the natural treatment of CML.

4 Evidence-
Based Complementary and Alternative Medicine

Figure 2 :
Figure2: Phylogenetic tree of all the medicinal plants containing indirubin and their adulterants constructed with the ITS2 sequences using NJ method (Bootstrap scores ≥50%).The samples marked with blue represent the medicinal plants containing indirubin, and the others represent their adulterants.
Leaves of P. hydropiper

Figure 3 :
Figure 3: HPLC profiles of N,N-dimethyl formamide extract prepared from different organs of medicinal plants and five of their usual adulterants.Each profile was made up of two-three replicate samples from each tested plant organ and species.

2
Evidence-Based Complementary and Alternative Medicine

Table 2 :
Sequence characteristics of the related species.

Table 3 :
Data of interspecific and intraspecific distances of the related species.