Two Methods Published to Distinguish between Cynanchum wilfordii and Cynanchum auriculatum Root tubers

Reviewed: Kim Y, Choi H, Shin J, Jo A, Lee KE, Cho SS, Hwang YP, Choi C. Molecular discrimination of Cynanchum wilfordii and Cynanchum auriculatum by InDel markers of chloroplast DNA. Molecules. 2018;23(6):pii:E1337.

Ham WS, Kim J, Park DJ, Ryu HC, Jang, YP. Discrimination of Cynanchum wilfordii and Cynanchum auriculatum by terahertz spectroscopic analysis. Phytochem Anal. 2018;29(5):472-475.

Keywords: Adulteration, Cynanchum auriculatum, Cynanchum wilfordii, DNA barcoding, indel marker, terahertz spectroscopy

In 2014, the Korean Consumer Agency (KCA) launched an investigation into products labeled to contain Cynanchum wilfordii (Apocynaceae), after initial results showed that EstroG-100^®, a product manufactured by Naturalendo Tech Co Ltd. (Seongnam, South Korea), contained C. auriculatum instead, a related species not authorized for medicinal use.¹ EstroG-100, which is composed of root extracts from C. wilfordii, Angelica gigas (Apiaceae), and Phlomis umbrosa (Lamiaceae), is sold not only in South Korea, but also in the United States, Canada, Japan, and several European countries. On June 16, 2015, the KCA released results of the analysis of 32 commercial products labeled to contain C. wilfordii using a DNA-based authentication method. DNA from C. wilfordii was found in only three (9.4%) products, while nine (28.1%) contained mixtures of C. wilfordii and C. auriculatum, and twelve (37.5%) contained only C. auriculatum. In eight products, no suitable DNA for analysis could be obtained. In order to prevent safety accidents involving fake baeksuo or baekshuoh (the Korean name for C. wilfordii roots) products, the KCA recommended that business operators voluntarily recall and discard the products.

Given the fact that the roots of C. wilfordii and C. auriculatum are morphologically very similar, distinction by macroscopic or microscopic means is difficult. In the years since the investigation, a number of new methods to differentiate these species have been published, mainly using genetic markers,^2-5 although Jang et al. developed a ¹H NMR method to quantitatively measure conduritol F ([1R,2R,3R,4S]- cyclohex-5-ene-1,2,3,4-tetrol), a cyclitol that reportedly occurs only in C. wilfordii.⁶

Using the whole chloroplast sequences of C. wilfordii and C. auriculatum, Kim et al. searched for indel markers that would allow distinguishing the two species. Contrary to single nucleotide polymorphisms (SNPs), which are mutations in a single nucleotide, indels are either insertions or deletions of one or many nucleotides in the genome of an organism. Three indel sequences were chosen to discriminate between the two species, and were found to be consistent in the 20 verified market samples of C. wilfordii (n = 10) and C. auriculatum (n = 10) that were analyzed. After polymerase chain reaction (PCR) amplification of 30 ng of genomic DNA, the resulting DNA products were separated on an agarose gel and visually compared to those of authenticated plant material.

The ability of this method to determine the identity of the Cynanchum species was confirmed by analyzing 10 commercial samples of baekshuoh roots obtained from markets in North Gyeongsang, South Gyeongsang, and Gangwon provinces of South Korea. Eight of these 10 samples were made from C. wilfordii, while one was a mixture of C. wilfordii with C. auriculatum, and one contained only C. auriculatum.

The paper by Ham et al. used for botanical ingredients a hitherto obscure analytical method, terahertz spectroscopy, to distinguish between C. wilfordii and C. auriculatum roots. Terahertz spectroscopy is a non-destructive approach that uses electromagnetic radiation in the frequency range from 0.1 to 4 terahertz (THz), which places it between extremely high frequency microwaves and far infrared wavelengths. Absorption resulting from exposure to terahertz pulses produces a unique and reproducible spectral pattern that may help identify a material.

In the first experiment, the amplitude and phase of a terahertz during its travel through a Cynanchum sample pellet was measured, and a small difference was observed between C. wilfordii and C. auriculatum. Then the pulse amplitude and phase as a function of the terahertz frequency were calculated by Fourier transformation. Again, a small variation was noticed in the data of the two species, which was linked to a difference in permittivity (the ability of a sample to store electric energy in an electric field) of the C. wilfordii and C. auriculatum roots. The authors conclude that “it is possible to easily distinguish these species using non‐contact and non‐destructive methods using terahertz waves.” 

Comment: According to the KCA report,¹ products containing roots of C. wilfordii have become rapidly popular as a remedy for menopausal symptoms, strengthening immunity, and having antioxidant effects. The roots of C. auriculatum are sold at a markedly lower cost, and take only one year to grow rather than two to three years required for C. wilfordii roots. At the same time, these closely-related species are phytochemically similar⁷ and have historically been used interchangeably. While only C. wilfordii is allowed in the Korean Herbal Pharmacopoeia, the use of C. auriculatum has been used in other traditional healing systems and may not present a health risk as suggested. (Roy Upton, e-mail communication, August 25, 2015)

Nevertheless, the problem of C. wilfordii adulteration has received a lot of media coverage, particularly in South Korea. The Korean Ministry of Food and Drug Safety has made efforts to develop suitable methods for the authentication of crude C. wilfordii root tubers using genetic methods.^4,5 Such methods may not be helpful for materials that were exposed to high heat or to extensive processing, making the availability of additional methods based on the chemical composition desirable. However, the chemical composition of roots of the two species is reportedly similar. While conduritol F has been proposed as a marker compound for C. wilfordii,^6,8 the presence of this compound in a baekshuoh root powder or extract does not preclude admixture of C. auriculatum. Methods which are based on a chemical fingerprint, or pattern, such as the HPLC-UV method by Li et al,⁷ or the terahertz spectroscopy assay presented above provide a more robust approach to the identity determination of C. wilfordii. The results from the latter need to be confirmed with more than one sample of each Cynanchum species to ensure that terahertz spectroscopy is a robust test for species identification, especially since it is a relatively new approach that has not yet been embraced by the dietary supplement and herbal medicine industry or the natural products research community.

Concerns have been raised about the possible toxicity of C. auriculatum, since it is the subject of a publication listed in the US FDA poisonous plants database⁹ based on research showing miscarriages in pigs that ingested at least 56.7 g of cooked C. auriculatum.¹⁰ However, the same database also contains a paper on the isolation and structure elucidation of saponins from C. wilfordii without any data on its potential toxicity.¹¹ Therefore, the inclusion of a publication reporting scientific research on a plant species in the FDA poisonous plants database is not in itself evidence for the plant having toxic effects in humans.

References

1. Korea Consumer Agency press release. Baeksuo (medicinal root) products sold in the market mostly bogus – cheaper root found in more than 60% of products. June 16, 2015. http://english.kca.go.kr/brd/m_11/view.do?seq=278. Accessed August 13, 2015.
2. Han EH, Cho K, Goo Y, Kim M, Shin YW, Kim YH, Lee SW. Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum. Mol Biol Rep. 2016;43(4):323-332.
3. Ryuk JA, Lee HW, Ju YS, Ko BS. Monitoring and identification of Cynanchum wilfordii and Cynanchum auriculatum by using molecular markers and real-time polymerase chain reaction. J Korean Soc Appl Biol Chem. 2014;57(2):245-251.
4. Kim KH, Kim YS, Kim M-R, Lee HY, Lee KH, Kim JH, Seong RS, Kang TS, Lee JH, Jang YM. Development of primer sets for the detection of Polygonum multiflorum, Cynanchum wilfordii, and C. auriculatum. J Food Hyg Saf. 2015;30(3):289-294.
5. Kim JH, Moon JC, Kang TS, Kwon K, Jang CS. Development of cpDNA markers for discrimination between Cynanchum wilfordii and Cynanchum auriculatum and their application in commercial C. wilfordii food products. Appl Biol Chem. 2017;60(1):79-86.
6. Jang HS, Jeong B, Choi SY, Jang GH, Park, KC, Kwon YS, Yang H. Conduritol F, the discriminant marker between C. wilfordii and C. auriculatum by ¹H NMR spectroscopy. Microchem J. 2017;135:153-157.
7. Li Y, Piao D, Zhang H, Woo MH, Lee JH, Moon DC, Lee SH, Chang HW, Son JK. Quality assessment and discrimination of the roots of Cynanchum auriculatum and Cynanchum wilfordii by HPLC–UV analysis. Arch Pharm Res. 2013;36(3):335-344.
8. Jiang Y, Choi HG, Li Y, Park YM, Lee JH, Kim DH, Lee JH, Son JK, Na M, Lee SH. Chemical constituents of Cynanchum wilfordii and the chemotaxonomy of two species of the family Asclepiadaceae, C. wilfordii and C. auriculatum. Arch Pharm Res. 2011;34(12):2021-2027.
9. US Department of Health & Human Services. FDA Poisonous plants database. Silver Spring, MD: US Food and Drug Administration. Available at: https://www.accessdata.fda.gov/scripts/plantox/index.cfm. Accessed January 29, 2019.
10. Han J, Luan DH. Sow abortion caused by feeding Cynanchum auriculatum [Chinese]. Xumu Yu Shouyi. 1984;16(6):266.
11. Tsukamoto S, Hayashi K, Mitsuhashi H. Studies on the constituents of Asclepiadaceae plants. LX. Further studies of glycosides with a novel sugar chain containing a pair of optically isomeric sugars, D- and L-cymarose, from Cynanchum wilfordi [sic.]. Chem Pharm Bull. 1985;33(6):2294-2304.