Lateral Flow Dipstick to Detect Adulteration of Cordyceps

Reviewed: Wong Y-L, Wong K-L, Shaw P-C. Rapid authentication of cordyceps by lateral flow dipstick. J Pharm Biomed Anal. 2015;111:306-310.

Cordyceps, a valuable ingredient in traditional Chinese medicine (TCM), is the fruiting body of the fungus Ophiocordyceps sinensis (syn. Cordyceps sinensis, Ophiocordycipitaceae [formerly Clavicipitaceae]). O. sinensis is a parasitic fungus living on the larvae of butterflies and moths. It attacks and grows on caterpillars, specifically on larvae from the genus Thitarodes. Cordyceps is found in cold, grassy, alpine meadows and shrublands of the Tibet Autonomous Region and the Sichuan, Gansu, Qinghai, and Yunnan provinces of China, from 3000 to 5000 meters above sea level. It is also found in Nepalese, Bhutanese, and Indian Himalayan areas.¹ Due to its scarcity, cordyceps has become one of the most expensive TCM ingredients, and also one of the most frequently adulterated materials. Since cordyceps is sold by weight, and intact fruiting bodies fetch higher prices in traditional markets, collectors have historically inserted a small bit of twig into the caterpillars (which also helps to keep the caterpillar intact). In rare cases, collectors have also inserted lead or other metals in order to boost the weight.² Other adulterants include the related species C. militaris and C. gunnii. There are ongoing conflicts about the control of this important resource, such as in 2014, when a Chinese official who was on his way back from a mission to maintain order at a cordyceps collecting site was shot dead in Sichuan province.³

In this paper, the authors have used a lateral flow dipstick (LFD) to detect species-specific DNA products obtained after polymerase chain reaction (PCR). This stick helps to visualize the particular PCR products. The LFD is similar in appearance to a pH indicator stick; however, the analyte solution is applied at the bottom and moves along the stick due to capillary forces, similar to thin-layer chromatography. The particular LFD in question has three line-shaped test zones: the dipstick control line, the PCR control line, and a species-specific line. The primers for internal control were built so that all of the three species were detected in order to confirm that the amount and quality of DNA extracts were adequate for PCR. In contrast, the species-specific primers were designed based on the elongation factor 1α (EF-1α) region of the three Cordyceps species, where the nucleotide sequence is different among the species. The technology was successful in distinguishing authenticated samples of O. sinensis, C. gunnii, and C. militaris. Based on differences in the species-specific primer region, the authors suggest that the PCR-LFD method will also distinguish O. sinensis from C. cicadae and Metacordyceps liangshanensis, two other known cordyceps adulterants. The ability to distinguish these two additional species will need to be confirmed in future research.

Comment: Organoleptic characteristics may be helpful in some instances to help with authentication of cordyceps. According to Xiong Weiping, associate researcher of the Tibet Academy of Agricultural and Animal Husbandry Service, fine hairs can be found on quality O. sinensis when examined in the sunlight, and fresh O. sinensis has a brighter color than older materials. Fruiting bodies with heavier weight might have been filled with metal powders. Moreover, since cordyceps consists, in part, of an insect’s body, a fishy odor is quite common, and the fresher the material is, the more pronounced this odor is.⁴ Further, Eric Brand, TCM expert from the Chinese Medicine Teaching and Research Division, School of Chinese Medicine, Hong Kong Baptist University, commented: “Macroscopic identification (with the proper training) is highly effective for identifying genuine cordyceps, and microscopy is also effective. While chemical tests and DNA assays may be necessary for the identification of materials in powder form, most cordyceps that is sold as a powder is cultivated rather than wild. The genuine wild product is typically sold whole” (e-mail communication, September 1, 2015). “Powdered O. sinensis is available made from broken pieces or from material where only the fruiting body is traded since the larva is too old” (Daniel Winkler, ethno-mycologist specializing in Tibetan Plateau fungi, e-mail communication, September 1, 2015).

Correct identification of cordyceps by chemical means is challenging due to the lack of species-specific markers. Previous chemical methods have proposed ubiquitous compounds like mannitol or ergosterol as markers, or looked at differences in the nucleoside fingerprint or the sugar ratios in polysaccharides.^5,6 Therefore, the development of a DNA-based method is a meaningful contribution to solve the problem of authenticating cordyceps raw materials and detect adulteration with related species. Since the analysis of DNA is still a relatively new approach for identification of botanical and – in this case – fungal raw materials, it may take some time until such methods are incorporated into an industrial quality control laboratory. Until then, companies interested in this technology will have to rely on contract laboratories or collaborations with academic institutions.

References

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2.     Holliday J, Cleaver M, Tajnik M, Cerecedes JM, Wasser SP. Cordyceps. In: Coates PM, Betz JM, Blackman MR, et al., eds. Encyclopedia of Dietary Supplements. 2nd ed. Boca Raton, FL: CRC Press; 2010:185-192.

3.     Su L. Cordyceps sinensis price rises as purchasing peak season comes. China Tibet Online website. Available at: http://eng.tibet.cn/2010sh/zyy/201408/t20140820_2014703.html. Published June 27, 2014. Accessed October 6, 2015.

4.     Su L. Stories of Cordyceps sinensis. China Tibet Online website. Available at: http://eng.tibet.cn/2010sh/zyy/201408/t20140820_2014702.html. Published June 25, 2014. Accessed October 6, 2015.

5.     Cheung JKH, Li SP, Tsim KWK. Authentication and quality control of Cordyceps sinensis, a traditional Chinese medicine known as summer-grass winter-worm. Orient Pharm Exp Med. 2005;5(4):262-271. Available at: http://koreascience.or.kr/article/ArticleFullRecord.jsp?cn=E1OGB9_2005_v5n4_262. Accessed October 6, 2015.

6.     Li SP, Yang FQ, Tsim KWK. Quality control of Cordyceps sinensis, a valued traditional Chinese medicine. J Pharm Biomed Anal. 2006;41(5):1571-1584.