Part 2 Detection Of Cistanches Herba (Rou Cong Rong) Medicinal Products Using Species-Specific Nucleotide Signatures

By seeking the nucleotide signatures developed in this study, we found that 15 of 31 Chinese patent medicines labeled as containing Cistanches Herba instead contained adulterants, including eight counterfeit ingredients and seven adulterants (Table 2). For example, six batches were replaced with Cy. songaricum, including three batches of Shihu Yeguang pills, one batch of Kangguzhi Zengsheng pills, one batch of Sanbao capsules, and one batch of Wenweishu particles.

Moreover, different batches from the same manufacturer produced somewhat different results. For example, among three batches from one manufacturer (ZCY16, ZCY69, and ZCY71), one batch comprised a mixture of Ci. deserticola and Cy. songaricum, one batch comprised a mixture of Ci. deserticola and Ci. tubulosa and one batch contained only Ci. tubulosa. Two batches from another manufacturer (ZCY44 and ZCY70) also differed: one batch contained Cy. songaricum, whereas the other batch contained Ci. deserticola and Ci. tubulosa.

Cistanches Herba was detected in 23 of the 31 Chinese patent medicines tested (Table 2), and only 16 samples were authentic, e.g., without adulterants or counterfeit ingredients. Ci. tubulosa was detected in 16 batches of Chinese patent medicines, and Ci. deserticola was detected in 10 batches. O. coerulescens was not detected in any of the products (Table 2).

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DISCUSSION

The necessity of Developing a New Method for Monitoring Commercially Available Medicinal Products Containing Cistanches Herba

Cistanches Herba is a tonic that is widely used in restorative Chinese patent medicines and other medicinal products. However, the quality control of Chinese patent medicines presents a great challenge due to the diversity and complexity of the ingredients. Due to the lack of regulatory oversight, there is considerable opportunity for product adulteration or counterfeiting. In addition, all products should be processed in accordance with Pharmacopoeia or other standards; adulterating or counterfeiting is not permitted during processing. Thus, varieties of quality control methods have been established, such as multi-heart-cutting two-dimensional liquid chromatography (Yao et al., 2015), near-infrared reflectance spectroscopy (Zhang and Su, 2014; Zhang et al., 2015), and liquid chromatography-mass spectrometry (Wang et al., 2016b). However, the analytical chemistry methods currently in the Chinese Pharmacopoeia Commission (2015) cannot be used to authenticate all of the ingredients in Chinese patent medicines or to detect the presence of adulterant ingredients. Moreover, studies have shown that targeted metabolites in plants are altered during product processing, resulting in considerable variability in test results or complete failure of test methods (Ananingsih et al., 2013). Thus, molecular tools such as species-specific nucleotide signatures are poised to reinforce quality control systems against the risk of fraudulent product substitution and adulteration and inclusion of unlabeled ingredients.

Although ITS/ITS2 is considered a high-efficiency tool for the identification of herbal medicines, these sequences cannot be amplified from highly processed samples (Newmaster et al., 2013; de Boer et al., 2015). Wang et al. reported that ITS2 could not be amplified from Angelicae Sinensis Radix extract or decoctions boiled for more than 120 min (Wang et al., 2016a). According to traditional technologies and the Chinese Pharmacopoeia Commission (2015), Cistanches Herba is always highly processed to increase its medicinal efficacy; these processes, include oven drying, salting, and steaming with wine (Zou et al., 2017), which lead to DNA degradation. In addition, various excipients are added during processing, such as honey, starch, and dextrin. If these excipients are not removed completely, the purity of DNA will be affected. For example, the following manufacturing process is used to generate Cistanches Herba-containing Sanbao capsules: “Boil the medicinal slices for 1.5 h twice, combine the decoctions and filter the mixture. Concentrate the filtrate to a relative density of 1.20∼1.25 (at 80◦C). Add other ground powders and combine them to obtain a homogeneous mixture. Next, dry the mixture at 60◦C, and then grind it into a fine

powder.” However, after the production process described above, there could be difficulties during the DNA extraction of Chinese patent medicines, and long fragments might not be amplified from the degraded DNA, which would prevent the identification of adulterants. Thus, to ensure the quality and purity of DNA, we added additional steps before the genomic DNA extraction, including washing with prewash buffer and eluting ten parallel tubes into one tube for each batch.

Although all the Chinese patent medicines used in the present study contained 6–25 ingredients, the primer pairs developed could specifically amplify the sequences of the adulterants in these Chinese patent medicines. Direct sequencing of the PCR products showed clean trace files. Thus, this nucleotide signature method is capable of identifying both authentic species ingredients and adulterants and should broaden the application of DNA-based molecular diagnostic tools for market supervision.

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Nucleotide Signatures for the Effective Identification of Cistanches Herba Products

Molecular tools that utilize PCR technology are very promising for medicinal product authentication within quality control systems. The successful application of the primers for identifying DNA-degraded adulterants from Cistanches Herba suggests that a PCR-based detection method could be used widely. In the Chinese herbal medicine market, the price of authentic Cistanches Herba species ingredients is more than five times higher than the prices of its adulterants. Our results showed that Cy. songaricum is the most common adulterant of Cistanches Herba on the market, followed by Ci. Sinensis. Cy. songaricum was added because these medicines share similar morphological characteristics. In addition, the chemical composition of Ci. sinensis is similar to that of Cistanches Herba. As quality control markers for Cistanches Herba extracts, echinacoside and acteoside can be inexpensively extracted from Ci. sinensis. Thus, some pharmaceutical factories use Ci. sinensis as a substitute in the production of Cistanches Herba extracts. Taken together, the results of this study indicate that there is considerable fraud in the market for medicinal products.

Adulteration in Chinese patent medicine is similar to that found in other countries. Similar levels of adulteration have been recorded in North America (Newmaster et al., 2013), Europe (Raclariu et al., 2017), and Asia (Cheng et al., 2014; Shanmughanandhan et al., 2016; Gao et al., 2017). In this study, the adulterated rate of Chinese patent medicines was approximately 48.4%, with only 16 of the 31 samples being authentic Cistanches Herba. In addition, we speculated that the different results produced in products from the same manufacturer could be attributed to differences in the qualities of the different batches of Chinese medicine materials. Therefore, to control the quality of Chinese patent medicines, the raw materials should be authenticated before being processed into products.

Adulteration of Cistanches Herba has traditionally been associated with issues of supply and demand of raw materials. Ci. deserticola and Ci. tubulosa are the two original plants currently used to formulate Cistanches Herba. However, Ci. deserticola is the only original species in traditional authentic Cistanches Herba listed in the Chinese Pharmacopoeia Commission (2000), in which Ci. tubulosa is identified as an adulterant. Owing to the shortage of Ci. deserticola resources, Ci. tubulosa has been listed as a supplement in the Chinese Pharmacopoeia since 2005 (Jiang and Tu, 2009). Until recently, the prices of these herbs have markedly differed; Ci. tubulosa has been much less expensive than Ci. deserticola because there is a much larger supply of the former. Here, our results showed that Ci. tubulosa is more widely used in commercially available Cistanches Herba products.

In conclusion, the nucleotide signatures and PCR-based

methods developed in this study may serve as useful tools for the medicinal product industry to authenticate ingredients and detect adulterants in Cistanches Herba products. In accordance of the sensitivity result, even if the proportion of adulterant was one in ten thousand, it can be detected via qRT-PCR. It means that once a nucleotide signature is detected in Cistanches Herba-containing functional products, it could be identified as an adulterant or counterfeit ingredient. A novel solution for detecting counterfeit ingredients or adulterated Cistanches Herba was provided that was not previously available via chemical detection methods in the Chinese Pharmacopoeia. In addition, this method could be used to validate increasing types of medicine and to broaden the applications of DNA-based molecular diagnostic tools for market supervision.

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AUTHOR CONTRIBUTIONS

JH conceived the study and participated in its design. XW, RX, and JC contributed samples and performed the experiments. XW analyzed the data. XW, JH, ZZ, S-GN, JS, and SC drafted the manuscript. All authors have read and approved the final manuscript.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant number 81673552], the CAMS Innovation

Fund for Medical Sciences [grant number 2016-I2 M- 3-016], and the United Fund Key Project of the National Natural Science Foundation of China [grant number U1403224].

ACKNOWLEDGMENTS

We would like to thank our colleagues who helped with the sample collection, identification, laboratory work, and manuscript preparation, including Chaokui Sun, Dianyun Hou, and Piao Zhang.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 01643/full#supplementary-material

Supplementary Figure S1 | The alignment result of ITS2 sequence from six species.

Supplementary Figure S2 | Agarose gel electrophoresis of all samples.

Supplementary Table S1 | Sampling information of Cistanches Herba and its adulterants.

Supplementary Table S2 | Sequence information of related species downloaded from GenBank.

Supplementary Table S3 | The declared compositions of different Chinese patent medicine samples.

Supplementary Data Sheet S1 | All the sequences obtained in this study.

Cistanche deserticola has many effects

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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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