# Rapid Identification and Verification of Indirubin-Containing Medicinal Plants

Authors: Zhigang Hu; Yuan Tu; Ye Xia; Peipei Cheng; Wei Sun; Yuhua Shi; Licheng Guo; Haibo He; Chao Xiong; Shilin Chen; Xiuqiao Zhang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2015)

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## Abstract

Indirubin, one of the key components of medicinal plants includingIsatis 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.


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## Body

## 1. 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 ofIsatis tinctoria,Polygonum tinctorium, andStrobilanthes cusia, was found to be the active ingredient [4–11]. Indirubin has since been found in additional Chinese herbal medicines derived from each of these species, respectively, including Isatidis Folium (the leaf ofIs. tinctoria), Polygoni Tinctorii Folium (the leaf ofP. tinctorium), and Baphicacanthis Cusiae Rhizoma et Radix (the root and rhizome ofS. cusia) [12–14]. However, few studies have determined the presence and containing of indirubin in the above medicinal plants and their adulterants. These adulterants includeP. hydropiper, P. chinense, Clerodendrum cyrtophyllum, Indigofera tinctoria, andS. dimorphotricha (Figure 1) and are extremely difficult to discriminate from their true medicinal counterparts morphologically [15–18]. 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 cost-effectively discriminated from their false counterparts.Figure 1
The relationship among indirubin-containing medicinal materials (plant organs/formulations), their plants of origin, and adulterants.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 asmatK,rbcL,psbA-trnH, and ITS/ITS2, have been assessed for their potential as DNA barcodes in plants [21–25]. 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 thanmatK,rbcL, andpsbA-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.


## 2. Materials and Methods

### 2.1. Plant Materials

A total of 57 samples from 8 species were gathered from various geographical areas in China, as detailed in Table1. All the specimens were carefully visually identified using standard expert identification parameters at the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences. The specimens were deposited in the herbarium of the Hubei University of Chinese Medicine.Table 1
Detailed description of all the samples in this study.
Species
Medicinal part
Locality
Voucher number
GenBank number of ITS2
Isatis tinctoria
Leaves
Hebei, China
YC0021MT02
KJ939152
Is. tinctoria
Leaves
Anhui, China
YC0021MT09
KJ939157
Is. tinctoria
Leaves
Chongqing, China
YC0021MT10
KJ939158
Is. tinctoria
Leaves
Chongqing, China
YC0021MT12
KJ939159
Is. tinctoria
Leaves
Chongqing, China
YC0021MT13
KJ939160
Is. tinctoria
Leaves
Yunnan, China
YC0021MT14
KJ939161
Is. tinctoria
Leaves
Beijing, China
YC0021MT15
KJ939162
Is. tinctoria
Leaves
Beijing, China
YC0021MT04
KJ939154
Is. tinctoria
Leaves
Beijing, China
YC0021MT05
KJ939155
Is. tinctoria
Leaves
Beijing, China
YC0021MT06
KJ939156
Is. tinctoria
Roots
Sichuan, China
YC0021MT01
KJ939151
Is. tinctoria
Roots
Hebei, China
YC0021MT03
KJ939153
Is. tinctoria
Roots
Beijing, China
YC0021MT20
KJ939163
Is. tinctoria
Roots
Beijing, China
YC0021MT21
KJ939164
Is. tinctoria
Roots
Beijing, China
YC0021MT22
KJ939165
Is. tinctoria
Roots
Beijing, China
YC0021MT23
KJ939166
Is. tinctoria
Roots
Sichuan, China
YC0021MT29
KJ939167
Is. tinctoria
Roots
Hubei, China
YC0021MT30
KJ939168
Polygonum tinctorium
Leaves
Beijing, China
YC0390MT04
KJ939177
P. tinctorium
Leaves
Beijing, China
YC0390MT05
KJ939178
P. tinctorium
Leaves
Beijing, China
YC0390MT01
KJ939174
P. tinctorium
Leaves
Beijing, China
YC0390MT07
KJ939179
P. tinctorium
Leaves
Beijing, China
YC0390MT09
KJ939181
P. tinctorium
Leaves
Fujian, China
PS2901MT01
FJ503014
Strobilanthes cusia
Leaves
Guangdong, China
YC0389MT01
KJ939116-KJ939119
S. cusia
Leaves
Chongqing, China
YC0389MT02
KJ939109-KJ939112
S. cusia
Leaves
Chongqing, China
YC0389MT03
KJ939113-KJ939115
S. cusia
Leaves
Hainan, China
YC0389MT04
KJ939125-KJ939127, KJ939104
S. cusia
Leaves
Yunnan, China
YC0389MT07
KJ939139, KJ939140
S. cusia
Leaves
Yunnan, China
YC0389MT08
KJ939141-KJ939143
S. cusia
Leaves
Guangxi, China
YC0389MT10
KJ939105, KJ939137, KJ939138
S. cusia
Leaves
Fujian, China
YC0389MT11
KJ939133-KJ939136
S. cusia
Leaves
Guangxi, China
YC0389MT12
KJ939120-KJ939122
S. cusia
Leaves
Guangxi, China
YC0389MT13
KJ939123, KJ939124
S. cusia
Roots and rhizomes
Hainan, China
YC0389MT05
KJ939128-KJ939130
S. cusia
Roots and rhizomes
Hainan, China
YC0389MT06
KJ939131, KJ939132
S. cusia
Roots and rhizomes
Guangdong, China
YC0389MT14
KJ939106-KJ939108
Polygonum hydropiper
Leaves
Guangdong, China
YC0509MT01
KJ939169
P. hydropiper
Leaves
Guangdong, China
YC0509MT02
KJ939170
P. hydropiper
Leaves
Guangdong, China
YC0509MT03
KJ939171
P. hydropiper
Leaves
Guangdong, China
YC0509MT04
KJ939172
P. hydropiper
Leaves
Guangdong, China
YC0509MT05
KJ939173
Polygonum chinense
Leaves
Guangxi, China
YC0510MT01
KJ939182
P. chinense
Leaves
Guangxi, China
YC0510MT02
KJ939183
P. chinense
Leaves
Guangdong, China
YC0510MT03
KJ939184
P. chinense
Leaves
Guangdong, China
YC0510MT04
KJ939185
Clerodendrum cyrtophyllum
Leaves
Guangxi, China
YC0508MT01
KJ939144
C. cyrtophyllum
Leaves
Guangxi, China
YC0508MT02
KJ939145
C. cyrtophyllum
Leaves
Guangxi, China
YC0508MT03
KJ939146
C. cyrtophyllum
Leaves
Guangxi, China
YC0508MT04
KJ939147
Strobilanthes dimorphotricha
Leaves
Guangdong, China
YC0511MT01
KJ939187, KJ939188, KJ939191
S. dimorphotricha
Leaves
Guangdong, China
YC0511MT02
KJ939189, KJ939190
S. dimorphotricha
Leaves
Guizhou, China
YC0511MT03
KJ939192, KJ939193
Indigofera tinctoria
Leaves
Guangxi, China
YC0707MT01
KJ939148
In. tinctoria
Leaves
Guangxi, China
YC0707MT02
KJ939149
In. tinctoria
Leaves
Guangxi, China
YC0707MT03
KJ939150
In. tinctoria
Leaves
Guangxi, China
PS0251MT02
GU217625


### 2.2. DNA Extraction, PCR Amplification, and Sequencing

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 750μL GP1 (Tiangen) buffer for 1 h for dried leaves or 5 h for roots and rhizomes. The remaining steps followed the manufacturer’s protocol.FormatK,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 μL reaction mixture, containing 30–100 ng of genomic DNA template, 12.5 μL 2 × Tag PCR Master Mix (Aidlab Biotechnologies Co., China), and 1 μL of forward and reverse primers (2.5 μmol/L). After PCR, a 4 μL 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).


### 2.3. Cloning and Sequencing of the ITS2 Region

The ITS2 PCR products ofS. cusia andS. 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 intoE. 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 ofS. cusia and 7 clones from 3 samples ofS. dimorphotricha (excluding fungal sequences) were obtained.


### 2.4. 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].


### 2.5. 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 μg/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 μL aliquot of the filtrate was applied to a HPLC column (Angilent TC-C18, 5.0 μm, 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.


## 2.1. Plant Materials

A total of 57 samples from 8 species were gathered from various geographical areas in China, as detailed in Table1. All the specimens were carefully visually identified using standard expert identification parameters at the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences. The specimens were deposited in the herbarium of the Hubei University of Chinese Medicine.Table 1
Detailed description of all the samples in this study.
Species
Medicinal part
Locality
Voucher number
GenBank number of ITS2
Isatis tinctoria
Leaves
Hebei, China
YC0021MT02
KJ939152
Is. tinctoria
Leaves
Anhui, China
YC0021MT09
KJ939157
Is. tinctoria
Leaves
Chongqing, China
YC0021MT10
KJ939158
Is. tinctoria
Leaves
Chongqing, China
YC0021MT12
KJ939159
Is. tinctoria
Leaves
Chongqing, China
YC0021MT13
KJ939160
Is. tinctoria
Leaves
Yunnan, China
YC0021MT14
KJ939161
Is. tinctoria
Leaves
Beijing, China
YC0021MT15
KJ939162
Is. tinctoria
Leaves
Beijing, China
YC0021MT04
KJ939154
Is. tinctoria
Leaves
Beijing, China
YC0021MT05
KJ939155
Is. tinctoria
Leaves
Beijing, China
YC0021MT06
KJ939156
Is. tinctoria
Roots
Sichuan, China
YC0021MT01
KJ939151
Is. tinctoria
Roots
Hebei, China
YC0021MT03
KJ939153
Is. tinctoria
Roots
Beijing, China
YC0021MT20
KJ939163
Is. tinctoria
Roots
Beijing, China
YC0021MT21
KJ939164
Is. tinctoria
Roots
Beijing, China
YC0021MT22
KJ939165
Is. tinctoria
Roots
Beijing, China
YC0021MT23
KJ939166
Is. tinctoria
Roots
Sichuan, China
YC0021MT29
KJ939167
Is. tinctoria
Roots
Hubei, China
YC0021MT30
KJ939168
Polygonum tinctorium
Leaves
Beijing, China
YC0390MT04
KJ939177
P. tinctorium
Leaves
Beijing, China
YC0390MT05
KJ939178
P. tinctorium
Leaves
Beijing, China
YC0390MT01
KJ939174
P. tinctorium
Leaves
Beijing, China
YC0390MT07
KJ939179
P. tinctorium
Leaves
Beijing, China
YC0390MT09
KJ939181
P. tinctorium
Leaves
Fujian, China
PS2901MT01
FJ503014
Strobilanthes cusia
Leaves
Guangdong, China
YC0389MT01
KJ939116-KJ939119
S. cusia
Leaves
Chongqing, China
YC0389MT02
KJ939109-KJ939112
S. cusia
Leaves
Chongqing, China
YC0389MT03
KJ939113-KJ939115
S. cusia
Leaves
Hainan, China
YC0389MT04
KJ939125-KJ939127, KJ939104
S. cusia
Leaves
Yunnan, China
YC0389MT07
KJ939139, KJ939140
S. cusia
Leaves
Yunnan, China
YC0389MT08
KJ939141-KJ939143
S. cusia
Leaves
Guangxi, China
YC0389MT10
KJ939105, KJ939137, KJ939138
S. cusia
Leaves
Fujian, China
YC0389MT11
KJ939133-KJ939136
S. cusia
Leaves
Guangxi, China
YC0389MT12
KJ939120-KJ939122
S. cusia
Leaves
Guangxi, China
YC0389MT13
KJ939123, KJ939124
S. cusia
Roots and rhizomes
Hainan, China
YC0389MT05
KJ939128-KJ939130
S. cusia
Roots and rhizomes
Hainan, China
YC0389MT06
KJ939131, KJ939132
S. cusia
Roots and rhizomes
Guangdong, China
YC0389MT14
KJ939106-KJ939108
Polygonum hydropiper
Leaves
Guangdong, China
YC0509MT01
KJ939169
P. hydropiper
Leaves
Guangdong, China
YC0509MT02
KJ939170
P. hydropiper
Leaves
Guangdong, China
YC0509MT03
KJ939171
P. hydropiper
Leaves
Guangdong, China
YC0509MT04
KJ939172
P. hydropiper
Leaves
Guangdong, China
YC0509MT05
KJ939173
Polygonum chinense
Leaves
Guangxi, China
YC0510MT01
KJ939182
P. chinense
Leaves
Guangxi, China
YC0510MT02
KJ939183
P. chinense
Leaves
Guangdong, China
YC0510MT03
KJ939184
P. chinense
Leaves
Guangdong, China
YC0510MT04
KJ939185
Clerodendrum cyrtophyllum
Leaves
Guangxi, China
YC0508MT01
KJ939144
C. cyrtophyllum
Leaves
Guangxi, China
YC0508MT02
KJ939145
C. cyrtophyllum
Leaves
Guangxi, China
YC0508MT03
KJ939146
C. cyrtophyllum
Leaves
Guangxi, China
YC0508MT04
KJ939147
Strobilanthes dimorphotricha
Leaves
Guangdong, China
YC0511MT01
KJ939187, KJ939188, KJ939191
S. dimorphotricha
Leaves
Guangdong, China
YC0511MT02
KJ939189, KJ939190
S. dimorphotricha
Leaves
Guizhou, China
YC0511MT03
KJ939192, KJ939193
Indigofera tinctoria
Leaves
Guangxi, China
YC0707MT01
KJ939148
In. tinctoria
Leaves
Guangxi, China
YC0707MT02
KJ939149
In. tinctoria
Leaves
Guangxi, China
YC0707MT03
KJ939150
In. tinctoria
Leaves
Guangxi, China
PS0251MT02
GU217625


## 2.2. DNA Extraction, PCR Amplification, and Sequencing

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 750μL GP1 (Tiangen) buffer for 1 h for dried leaves or 5 h for roots and rhizomes. The remaining steps followed the manufacturer’s protocol.FormatK,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 μL reaction mixture, containing 30–100 ng of genomic DNA template, 12.5 μL 2 × Tag PCR Master Mix (Aidlab Biotechnologies Co., China), and 1 μL of forward and reverse primers (2.5 μmol/L). After PCR, a 4 μL 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).


## 2.3. Cloning and Sequencing of the ITS2 Region

The ITS2 PCR products ofS. cusia andS. 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 intoE. 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 ofS. cusia and 7 clones from 3 samples ofS. dimorphotricha (excluding fungal sequences) were obtained.


## 2.4. 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].


## 2.5. 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 μg/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 μL aliquot of the filtrate was applied to a HPLC column (Angilent TC-C18, 5.0 μm, 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.


## 3. Results

### 3.1. 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 ofmatK, rbcL,psbA-trnH, and ITS2 was 62.5%, 79.2%, 100%, and 100%, respectively. High-quality 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 betweenS. cusia andS. dimorphotricha using bothpsbA-trnH andrbcL. 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.


### 3.2. Measurement of DNA Divergence for ITS2

Song et al. (2012) used sequence-tagged pyrosequencing and genome-wide 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 ofS. 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) ofP. 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 ofS. cusia (73.6%) andS. dimorphotricha (73.0%) were at least 4% greater than those of the other species. Based on the variable sites,Is. tinctoria, P. tinctorium, andS. cusia were divided into 5, 1, and 15 haplotypes, respectively.Table 2
Sequence characteristics of the related species.
Species/(number of sequences)
Length of ITS2 (bp)
GC average content (%)
Number of haplotypes
Number of variable sites
Is. tinctoria(18)
191
56.7
5
5
P. tinctorium(8)
245
68.2
1
1
S. cusia(38)
230~235
73.6
15
20
P. hydropiper(5)
244
68.9
1
1
P. chinense(4)
263
65.8
1
1
C. cyrtophyllum(4)
224
56.7
1
1
S. dimorphotricha(7)
224~233
73.0
6
13
In. tinctoria(4)
219
45.9
1
1Interspecific and intraspecific distances using Kimura two-parameter method are shown in Table3. The maximum intraspecific distances ofIs. tinctoria, P. tinctorium, andS. 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 three species and its adulterants were 0.514, 0.025, and 0.065, respectively.Table 3
Data of interspecific and intraspecific distances of the related species.
Parameter
Range
Intraspecific distances ofIs. tinctoria
0.000~0.027
Intraspecific distances ofP. tinctorium
0.000
Intraspecific distances ofS. cusia
0.000~0.036
Interspecific distance among the above three species
0.401~0.684
Interspecific distance betweenIs. tinctoria and its adulterants
0.514~0.684
Interspecific distance betweenP. tinctorium and its adulterants
0.025~0.755
Interspecific distance betweenS. cusia and its adulterants
0.065~0.931


### 3.3. Identification of ITS2 Using NJ Tree

Phylogenetic analysis demonstrated that every species clustered into their own clade, supported with at least 81% bootstrapping (Figure2). In addition, three closely related species of the genusPolygonum were strongly supported (99%, 81% and 100% bootstrap, resp.), and clustered into a larger branch with 97% bootstrap. All cloned sequences ofS. cusia (96% bootstrap) andS. dimorphotricha (92% bootstrap) in Acanthaceae family formed a larger group (93% bootstrap).Figure 2
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.


### 3.4. Detection of Indirubin in Crude Drugs and Their Adulterants

HPLC detected indirubin in the leaves ofIs. 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, andS. dimorphotricha).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.
(a)
Reference substance
(b)
Indigo Naturalis
(c)
Leaves ofP.tinctorium
(d)
Leaves ofIs.tinctoria
(e)
Roots ofIs.tinctoria
(f)
Leaves ofS.cusia
(g)
Roots and Rhizomes ofS.cusia
(h)
Leaves ofC.cyrtophyllum
(i)
Leaves ofP.chinense
(j)
Leaves ofIn.tinctoria
(k)
Leaves ofS.dimorphotricha
(l)
Leaves ofP.hydropiper


## 3.1. 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 ofmatK, rbcL,psbA-trnH, and ITS2 was 62.5%, 79.2%, 100%, and 100%, respectively. High-quality 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 betweenS. cusia andS. dimorphotricha using bothpsbA-trnH andrbcL. 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.


## 3.2. Measurement of DNA Divergence for ITS2

Song et al. (2012) used sequence-tagged pyrosequencing and genome-wide 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 ofS. 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) ofP. 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 ofS. cusia (73.6%) andS. dimorphotricha (73.0%) were at least 4% greater than those of the other species. Based on the variable sites,Is. tinctoria, P. tinctorium, andS. cusia were divided into 5, 1, and 15 haplotypes, respectively.Table 2
Sequence characteristics of the related species.
Species/(number of sequences)
Length of ITS2 (bp)
GC average content (%)
Number of haplotypes
Number of variable sites
Is. tinctoria(18)
191
56.7
5
5
P. tinctorium(8)
245
68.2
1
1
S. cusia(38)
230~235
73.6
15
20
P. hydropiper(5)
244
68.9
1
1
P. chinense(4)
263
65.8
1
1
C. cyrtophyllum(4)
224
56.7
1
1
S. dimorphotricha(7)
224~233
73.0
6
13
In. tinctoria(4)
219
45.9
1
1Interspecific and intraspecific distances using Kimura two-parameter method are shown in Table3. The maximum intraspecific distances ofIs. tinctoria, P. tinctorium, andS. 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 three species and its adulterants were 0.514, 0.025, and 0.065, respectively.Table 3
Data of interspecific and intraspecific distances of the related species.
Parameter
Range
Intraspecific distances ofIs. tinctoria
0.000~0.027
Intraspecific distances ofP. tinctorium
0.000
Intraspecific distances ofS. cusia
0.000~0.036
Interspecific distance among the above three species
0.401~0.684
Interspecific distance betweenIs. tinctoria and its adulterants
0.514~0.684
Interspecific distance betweenP. tinctorium and its adulterants
0.025~0.755
Interspecific distance betweenS. cusia and its adulterants
0.065~0.931


## 3.3. Identification of ITS2 Using NJ Tree

Phylogenetic analysis demonstrated that every species clustered into their own clade, supported with at least 81% bootstrapping (Figure2). In addition, three closely related species of the genusPolygonum were strongly supported (99%, 81% and 100% bootstrap, resp.), and clustered into a larger branch with 97% bootstrap. All cloned sequences ofS. cusia (96% bootstrap) andS. dimorphotricha (92% bootstrap) in Acanthaceae family formed a larger group (93% bootstrap).Figure 2
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.


## 3.4. Detection of Indirubin in Crude Drugs and Their Adulterants

HPLC detected indirubin in the leaves ofIs. 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, andS. dimorphotricha).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.
(a)
Reference substance
(b)
Indigo Naturalis
(c)
Leaves ofP.tinctorium
(d)
Leaves ofIs.tinctoria
(e)
Roots ofIs.tinctoria
(f)
Leaves ofS.cusia
(g)
Roots and Rhizomes ofS.cusia
(h)
Leaves ofC.cyrtophyllum
(i)
Leaves ofP.chinense
(j)
Leaves ofIn.tinctoria
(k)
Leaves ofS.dimorphotricha
(l)
Leaves ofP.hydropiper


## 4. Discussion

In previous studies, the identification methods of medicinal plants includingIs. tinctoria,P. tinctorium, andS. cusia have primarily focused on characterization of morphology, chromatographic fingerprints, and microstructures [32–34]. 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 ofCornus officinalis) from its adulterants [35]. Xin et al. (2013) presented the ITS2 barcode as a powerful tool for tracing Goji (the fruit ofLycium barbarum) [36], while it was also used to accurately identify Ephedrae Herba (the stem taken from three species ofEphedra) 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 andS. 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 thatIs. tinctoria, P. tinctorium, andS. cusia were clustered into their own monophyletic group, separated from the other species. Moreover, NJ tree analysis using ITS2 reliably distinguished individuals of the genusPolygonum and the family of Acanthaceae, supporting the powerful identification ability of ITS2 barcode in plants. Therefore, both the results of nearest distance method and NJ tree strongly support that ITS2 as DNA barcode can successfully distinguishIs. tinctoria, P. tinctorium, andS. cusia from each other and from their respective adulterants.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 ofIs. 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 thatIs. tinctoria, P. tinctorium, andS. 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.


## 5. Conclusion

Together with HPLC detection of indirubin in various organs, ITS2 DNA barcoding enables the rapid, efficient, and cost-effective 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.



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*Source: 484670-2015-05-18.xml*