Part 1 Detection Of Cistanches Herba (Rou Cong Rong) Medicinal Products Using Species-Specific Nucleotide Signatures

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Xiao-Yue Wang 1, Rong Xu 1, Jun Chen 1, Jing-yuan Song 1, Steven-G Newmaster 2, Jian-ping Han 1*, Zheng Zhang 1* and Shi-lin Chen 3

1 Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medicinal Science and Peking Union Medicinal College, Beijing, China, 2 NHP Research Alliance, Biodiversity Institute of Ontario (BIO), University of Guelph, Guelph, ON, Canada,

3 Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

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Cistanches Herba is a medicinal plant that has detoxification properties and is commonly used in Asia. Owing to the imbalance between supply and demand, adulterants are frequently added for profit. However, there is no regulatory oversight because quality control tools are not sufficient for identifying heavily processed products. Thus, a novel molecular tool based on nucleotide signatures and species-specific primers was developed. The ITS2 regions from 251 Cistanches Herba and adulterant samples were sequenced. On the basis of SNP sites, four nucleotide signatures within 30∼37 bp and six species-specific primers were developed, and they were validated by artificial experimental mixtures consisting of six different species and different ratios. This method was also applied to detect 66 Cistanches Herba products on the market, including extracts and Chinese patent medicines. The results demonstrated the utility of nucleotide signatures in identifying adulterants in mixtures. The market study revealed 36.4% adulteration: 19.7% involved adulteration with Cynomorium songaricum or Cistanche Sinensis, and 16.7% involved substitution with Cy. songaricum, Ci. sinensis, or Boschniakia rossica. The results also revealed that Cy. songaricum was the most common adulterant in the market. Thus, we recommend the use of species-specific nucleotide signatures for regulating adulteration and verifying the quality assurance of medicinal product supply chains, especially for processed products whose DNA is degraded.

Keywords: Cistanches Herba, Chinese patent medicine, nucleotide signature, degraded DNA, medicine quality control

INTRODUCTION

Cistanches Herba (Rou Cong Rong) is a well-known Pharmacopoeia-recorded medicine in Asia (Chinese Pharmacopoeia Commission, 2015; Japan Pharmacopeial Convention, 2016); this medicine is derived from the dried succulent stems of Cistanche deserticola Y. C. Ma or Cistanche tubulosa tonic, as it is not toxic and can be taken for long periods of time (Li et al., 2016). Furthermore, Cistanches Herba was bestowed with the honor of being named “Desert Ginseng” because of its great medicinal value, especially in strengthening male sexual function (Zhang and Su, 2014; Gu et al., 2016). There are more than 100 Chinese patent medicines recorded in the Chinese Pharmacopoeia Commission (2015) and in other local official promulgated standards (Wang et al., 2012). As the population of elderly individuals increases, there is considerable demand for Cistanches Herba and its medicinal products. However, raw material resources are becoming increasingly scarce. In fact, the two original species of Cistanches Herba have been added to the China Plant Red Data Book as state-protected wild plants (category II) (Fu, 1991). The medicinal materials on the market are mainly cultivated in northwestern China.

Owing to the considerable imbalance between the supply and demand of Cistanches Herba, many adulterants have entered the market; these adulterants are inconsistent with standards and can threaten drug security. The known adulterants include the dried succulent stems of Cynomorium songaricum Rupr. (Cynomorii Herba, Suo Yang in Chinese), Cistanche sinensis Beck, Orobanche coerulescens Stephan, and Boschniakia rossica (Cham. et Schlecht.) Fedtsch. et Flerov (Sun et al., 2012). These adulterants have morphological characteristics similar to those of Cistanches Herba, making traditional taxonomic identification difficult, particularly after the material is processed into medicinal products. Microscopic identification is not available because Cistanches Herba has no definitive or unique microscopic characteristics. Current analytical chemistry tools are not sufficient for detecting adulteration of Cistanches Herba because similar compounds also exist within the known adulterants Ci. salsa (Lei et al., 2001; Chen et al., 2007) and Ci. sinensis (Liu et al., 2013). Therefore, the development of a rapid molecular method for the authentication of Cistanches Herba and its products is urgently needed for proper quality control systems in Chinese patent medicine and other medicinal product industries.

Chen et al. first suggested internal transcribed spacer 2 (ITS2) as a universal barcode for medicinal plants (Chen et al., 2010). Sun et al. verified the ITS2 region as a preferable DNA barcode for identifying Cistanches Herba and its adulterants (Sun et al., 2012). However, ITS2 cannot be used to distinguish Chinese patent medicine from degraded DNA. Recently, an increasing number of studies have shown that the “mini barcode” is a useful method for amplifying degraded DNA (Hajibabaei et al., 2006; Meusnier et al., 2008; Dubey et al., 2011; Lo et al., 2015). However, nucleotide signatures are more appropriate than mini barcodes, as the formers refer to one or more nucleotides that are unique to one species and can be effectively utilized by many molecular techniques, such as DNA probes, microfluidics, and loop-mediated isothermal amplification (de Boer et al., 2015). Han et al. developed nucleotide signatures for Panax ginseng, Angelica Sinensis, and Lonicera japonica and successfully identified the associated Chinese patent medicines (Liu et al., 2016; Wang et al., 2016a; Gao et al., 2017).

functional products containing Cistanches Herba. Specifically, we (1) developed four nucleotide signatures and six specific primer pairs for differentiating authentic Cistanches Herba and known adulterants, (2) validated the four nucleotide signatures in experiments including mixtures of known taxonomic vouchers used to prepare Cistanches Herba products that contain both authentic and adulterated ingredients, and (3) performed a market survey of 66 Cistanches Herba products via their nucleotide signatures.

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MATERIALS AND METHODS

Sample Collection and Preparation

In total, 251 samples were collected from Inner Mongolia, Xinjiang, and Ningxia, among other areas; these samples included 214 Cistanches Herba and 37 adulterants and are detailed in Supplementary Table S1. Corresponding voucher samples were validated by taxonomists and deposited in the Herbarium of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China. A total of 35 batches of powders, spices, and extracts of Cistanches Herba were purchased from online stores and brick-and-mortar drugstores in Beijing and Chengdu (Table 1). In total, 31 batches of Chinese patent medicine containing Cistanches Herba were purchased from different drugstores (Table 2), and the declared compositions of different Chinese patent medicines are shown in Supplementary Table S3. The different morphological characteristics of different dose forms are shown in Figure 1.

Mixed samples: Powders of Ci. deserticola, Ci. tubulosa, Cy. songaricum, Ci. sinensis, B. rossica, and O. coerulescens were artificially mixed in different combinations (at a ratio of 1:1) before extraction. The details of these mixed samples are shown in the legend of Figure 3. In addition, the powders of four adulterants were mixed with the genuine Ci. deserticola at different weight ratios: 10:1, 50:1, 100:1, 200:1, 500:1, 1000:1,

2000:1, 5000:1, 10000:1, 15000:1, 20000:1, 25000:1, 30000:1,

40000:1, 50000:1 and 60000:1 (Table 3). And the two genuine products are mixed in the same proportions.

Decoction: Slices of Ci. deserticola and Cy. songaricum was used to prepare the decoction. The slices (10 g) were boiled in 300 mL of double-distilled water for 30, 60, 90, 120, 150, 180, 210, and 240 min and then used for DNA extraction.

DNA Extraction, Polymerase Chain Reaction (PCR) Amplification, and Sequencing

Specimens, decoction, and mixed samples: The samples (40– 50 mg) were ground into fine powders via a Retsch MM400 laboratory mixer mill (Retsch Co., Germany) at a frequency of 30 Hz. The genomic DNA was subsequently extracted with a Plant Universal Genomic DNA Kit (Tiangen Biotech Beijing Co., China) according to the manufacturer’s instructions. ITS2 was amplified by the universal primers 2F/3R (Chen et al., 2010).

in parallel per batch. The Chinese patent medicine powder was washed with 700 µL of prewash buffer [100 mM Tris-HCl, pH 8.0; 20 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0;

700 mM NaCl; 2% polyvinylpyrrolidone (PVP)-40; and 0.4% β-mercaptoethanol] several times until the supernatant was clear and colorless, after which the mixture was centrifuged at 7500 × g for 5 min at room temperature. The precipitate was subsequently used to extract the genomic DNA via the Plant Universal Genomic DNA Kit (Tiangen Biotech Beijing Co.) according to the manufacturer’s instructions. In the end, DNA from each batch was concentrated into one tube. Six species-specific primer pairs—SYF1/SYR1, HMRCF/HMRCR, GHRCF/GHRCR, CCRF/CCRR,

SCRF/SCRR, and LDF/LDR—were designed via Primer Premier

6.0 software (Premier Co., Canada) to amplify Cistanches Herba and its adulterants (the details are shown in Table 4). PCR was performed in a 50 µL-volume reaction containing 1 µL of

KOD FX (Toyobo Co., Japan), 25 µL of 2 × PCR buffer, 10 µL of dNTPs (2 mM), 1.5 µL of each primer (10 µM), and 4 µL (∼50 ng) of DNA template; the remaining volume consisted of double-distilled water. The reactions were performed by

a thermal cycler (VeritiTM 96-Well Thermal Cycler, Applied Biosystems Co., USA); the thermal programs are listed in Table 4. The PCR products were examined via 3% agarose gel electrophoresis and purified for bidirectional sequencing with an ABI 3730XL sequencer (Applied Biosystems Co.) in accordance with the Sanger sequencing method. Then, the sensitivity of the six primers was tested via the quantitative real-time PCR (qRT-PCR) assay with a CFX96 Real-time System (Bio-rad Lab., USA). The cycle thresholds were automatically calculated by the system via the “PCR Baseline Subtracted Curve Fit” model. qRT-PCR was performed in a 15 µL-volume reaction containing 7.5 µL of SYBRⓍR Premix Ex TaqTM (Tli RNaseH Plus)

(Takara Bio Co., Japan), 1.0 µL of each primer (10 µM), and 1.0 µL of DNA template with the remaining volume consisting of double-distilled water. The qRT-PCR was performed with three technical replicates based on which standard error was calculated.

Sequence analysis: The sequences were edited and manually assembled via CodonCode Aligner 5.1.4 (CodonCode Co., USA).

ITS sequences from GenBank were annotated via the Hidden Markov model (HMM) to obtain the ITS2 sequences (Keller et al., 2009). The sequences were then aligned by MEGA 5.0 software via the “Muscle” alignment method (Edgar, 2004; Tamura et al., 2011).

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RESULTS

Development of Nucleotide Signatures and Species-Specific Primer Pairs for Cistanches Herba and Adulterants

The PCR amplification and sequencing success rates of the 251 samples were 100% when the primer pair 2F/3R was used. The aligned length of the Cy. songaricum ITS2 sequences were 229 bp (Supplementary Figure S1). Analysis of the sequences from the herbarium species and those of closely related species retrieved from GenBank (Supplementary Table S2) revealed two single nucleotide polymorphism (SNP) sites for Cy. songaricum. On the basis of the SNPs, one Cy. songaricum-specific 30- bp nucleotide signature (5′-caattatttg aggtgcattg taagaagcgt-3’) was developed (Figure 2A). Basic Local Alignment Search Tool (BLAST) results in NCBI demonstrated that this nucleotide signature was unique to Cy. songaricum (Table 5). With a similar analysis of the sequences from closely related species, one to two SNPs were discovered from Ci. sinensis, B. rossica, and O. coerulescens. On the basis of the SNPs, the nucleotide signatures for the other three adulterants were also developed similarly, including a 34 bp signature (5′-cgatggtctc ccgtgcgcga，ggatgcacgg ccgg-3′) for Ci. Sinensis, a 37 bp signature (5′- acactggcct cccgtgcgca acgacgtgcg gccggtc-3’) for B. rossica, and a 31 bp signature (5′-gtctgtcgtg tcggatggtg ttgcttgttg g-3′) for O. coerulescens (Figures 2BD). BLAST analysis in NCBI also revealed that these nucleotide signatures were specific and not present in any other species (Table 5).

Cistanches Herba and its adulterants could be amplified simultaneously from mixtures via the 2F/3R universal primer pair. Thus, we designed species-specific primers for nucleotide signature amplification by aligning the ITS2 sequences. Four specific primer pairs—SYF1/SYR1, CCRF/CCRR, SCRF/SCRR, and LDF/LDR—were designed to amplify the nucleotide signatures of Cy. songaricum, B. rossica, Ci. sinensis, and O. coerulescens, respectively (Table 4). The lengths of the amplicons were 123, 72, 131, and 71 bp, respectively.

In addition, a total of 214 ITS2 sequences from experimental Cistanches Herba materials were analyzed. Two short specific primers—HMRCF/HMRCR and GHRCF/GHRCR for Ci. deserticola and Ci. tubulosa, respectively—were designed to amplify the short regions of the degraded samples(Table 4). The lengths of the amplicons were 132 and 134 bp, respectively.

Validation of the Nucleotide Signature and Species-Specific Primer Method Based on Artificial Mixtures and Decoction

The amplification efficiencies of the new primer pairs were validated from the mixture. PCR products were obtained via each primer pair for each targeted species, as shown

cq values of B. rossica or O. coerulescens could be obtained even the proportion of samples was 30000:1 or 40000:1, and the amplification results of Ci. sinensis, Ci. tubulosa, and Ci deserticola could be detected when the ratio was 20000:1. But no detectable results could be obtained for Cy. songaricum when it constituted the proportion of 20000:1.

To verify whether the nucleotide signature method functions with processed materials, decoctions of Ci. deserticola and Cy. songaricum were prepared. The results showed that the short barcode from Ci. deserticola could be amplified, even after the samples were boiled for 210 min. In addition, the nucleotide signature of Cy. songaricum could be amplified after the samples were boiled for 150 min, while no PCR products were detected after the samples were boiled for 210 or 240 min (Figure 4). The sequencing results demonstrated that the short nucleotide signature was successfully obtained from the decoction.

Market Survey of Adulteration via Nucleotide Signature and Specific Primers

The above method was applied for the detection of Cistanches Herba products on the market. Thirty-five batches of Cistanches Herba slices, powders, and extracts were amplified and sequenced by using six designed specific primers (the agarose gel electrophoresis results are shown in Supplementary Figure S2 and the sequences are listed in Supplementary Data Sheet 1). Analysis of the sequences via their nucleotide signatures revealed that five batches of slices were authentic. One slice batch and one powder batch were substituted with Cy. songaricum and one extract were substituted with Ci. sinensis. The other six batches of powders were mixtures: five were adulterated with Cy. songaricum and Ci. sinensis and one were adulterated with Cy. songaricum (Table 1). In addition, one slice batch was substituted with Salvia miltiorrhiza.

The Cistanches Herba in Chinese patent medicines is subjected to various processes that make authentication difficult. The ability of the six primer pairs to amplify the species-specific nucleotide signature regions from the Chinese patent medicines was tested (the agarose gel electrophoresis results are shown in Supplementary Figure S2). Most Chinese patent medicines contain components of different types of species. For example, Shihu Yeguang pills (ZCY34) contain 25 ingredients, including Cistanches Herba (Rou Cong Rong), Dendrobii Caulis (Shi Hu), and Ginseng Radix et Rhizoma (Ren Shen). From these ingredients, Cistanches Herba could be amplified specifically via the primer pair GHRCF/GHRCR. Direct sequencing of the PCR products revealed very clean traces. However, a visible band could not be obtained with the primer pair HMRCF/HMRCR.

Another example is Kangguzhi Zengsheng pills (ZCY44). There are eight ingredients in addition to Cistanches Herba in this Chinese patent medicine, but there were no visible bands obtained by either GHRCF/GHRCR or HMRCF/HMRCR, which meant that no Cistanches Herba was present. However, the adulterant region of Cy. songaricum was successfully amplified by the specific primer pair SYF1/SYR1. The sequencing results demonstrated the short nucleotide signature of Cy. songaricum was successfully obtained.

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