Authentication of Horsetail Using Thin-Layer Chromatography and DNA Barcoding

Reviewed: Saslis-Lagoudakis CH, Bruun-Lund S, Iwanycki NE, et al. Identification of common horsetail (Equisetum arvense L.; Equisetaceae) using thin layer chromatography versus DNA barcoding. Sci Rep. 2015;5:11942. doi: 10.1038/srep11942.

Horsetail (Equisetum arvense, Equisetaceae) is a popular medicinal herb commonly used as a diuretic and in skin, nail, and hair care products. The cosmetic use is reportedly due to the high silica content in horsetail. The genus Equisetum is composed of 15 species, which are not easily distinguished by morphological criteria, in particular when the strobili are lacking. Of particular concern is the accidental substitution of horsetail with marsh horsetail (E. palustre), which may cohabit with E. arvense, due to the content of potentially toxic spermidine alkaloids (e.g., palustrine). Another complicating factor is the occurrence of hybrids between E. arvense and E. palustre or the water horsetail (E. fluviatile). It is not clear if these hybrids contain the spermidine alkaloids or not.

The authors collected 58 authentic Equisetum samples (among those were 17 samples each of E. arvense and E. palustre), including all 15 Equisetum species from all over the world. One sample of each species was used for thin-layer chromatography (TLC) analysis, according to conditions outlined in the European Pharmacopoeia. In addition, every sample was used to create a phylogenetic tree (using sequences of the plastid regions rps4 and rbcL, the barcoding fragment of matK, the trnH-psbA spacer, and the nuclear ribosomal ITS2 region), but only E. arvense and E. palustre samples were used for the DNA barcoding analysis. Based on previous barcoding studies, the trnH-psbA and matK regions were amplified by polymerase chain reaction (PCR) in order to distinguish the two species. Both analytical methods were able to differentiate E. arvense and E. palustre. The authors contend that only DNA barcoding can distinguish all the Equisetum species, since some of the TLC fingerprints were similar among the 15 species. However, E. arvense has a unique TLC fingerprint and therefore can be authenticated using this method.

The ability to authenticate horsetail by TLC and DNA barcoding was evaluated with three commercial teas and five capsules, originating from the USA (3), UK (3), Bulgaria (1), and Germany (1), sold on the market as E. arvense. Five of the commercial products provided TLC fingerprints characteristic for E. arvense. The tea product from Bulgaria had markers for both E. arvense and E. palustre, indicating admixture of marsh horsetail or the presence of an E. arvense × E. palustre hybrid. The remaining two products were capsules from the UK – one of the capsules showed a TLC fingerprint that did not match any of the Equisetum species, while the other was devoid of any marker compounds. The DNA barcoding approach provided sequences only for two of the commercial products. One tea product could be identified as E. arvense, while in the case of the tea from Bulgaria, only matK was amplified, showing some characters that are typical for E. arvense and some that are typical for E. palustre.

Based on the results, the authors conclude that TLC is the most cost-effective way to authenticate herbal products containing E. arvense. DNA barcoding could be used as a complementary method, but the success rate for amplification of the selected DNA regions was low. In order to avoid the presence of the spermidine alkaloids, a chemical method that directly measures these alkaloids should be developed.

Comment: Much has been written and discussed about the influence of processing on the quality of DNA in the finished product in the aftermath of the New York Attorney General DNA barcoding investigation. This particular study shows the difficulties in obtaining suitable DNA for plant authentication in cases where the plant material has been subject to a number of processing steps. The authors’ conclusion, that a DNA-based assay might be used as a method complementary to a chemical authentication method, is not necessarily supported by the results of the paper. Based on concerns about potentially harmful spermidine alkaloids, the authors’ suggestion to develop a chemical method able to detect the presence of such alkaloids would indeed be a helpful addition to current pharmacopeial monographs.

HPTLC and UHPLC-UV/MS Analysis Show that Many Ginkgo Dietary Supplements are Adulterated with Extraneous Flavonoids

Reviewed: Avula B, Sagi S, Gafner S, et al. Identification of Ginkgo biloba supplements adulteration using high performance thin layer chromatography and ultra high performance liquid chromatography-diode array detector-quadrupole time of flight-mass spectrometry. Anal Bioanal Chem. 2015;407(25):7733-7746.

This study, initiated by the National Center for Natural Products Research (NCNPR), set out to establish appropriate chemical fingerprints for the authentication of ginkgo (Ginkgo biloba, Ginkgoaceae) raw materials and extracts by high-performance thin-layer liquid chromatography (HPTLC) and ultra high-performance liquid chromatography (UHPLC) coupled with ultraviolet (UV) and mass spectrometric (MS) detection. In addition, the usefulness of genistein as a marker compound to detect adulteration with Japanese sophora (Styphnolobium japonicum syn. Sophora japonica, Fabaceae) was evaluated.

In order to establish a reliable chemical fingerprint, eight botanically authenticated ginkgo leaf samples were extracted in methanol under sonication for 30 min., centrifuged, and subsequently analyzed by HPTLC and UHPLC-UV/MS. Additionally, samples of authenticated ginkgo fruit (n=3), stem (n=2), seed (n=2), and one National Institute of Standards and Technology (NIST)-certified leaf extract were analyzed. Also included in the study were authenticated Japanese sophora fruit (n=3) and flower (n=2) samples. The optimized chromatographical conditions were used to evaluate the authenticity of three bulk ginkgo leaf raw materials, two bulk ginkgo extracts, and 25 commercial dietary supplements labelled to contain G. biloba extract. The dietary supplements were purchased online from retailers in the United States.

The authentic ginkgo leaf materials contained the characteristic terpene lactones and flavonol glycosides, with quercetin, kaempferol, and isorhamnetin as aglycones, consistent with previous reports on the constituents of ginkgo. Isoflavones, specifically genistein, and isoflavone glycosides were not detected in any of the authentic ginkgo materials, including leaf, fruit, seed, and stem. However, genistein was present in both Japanese sophora fruit and flower. The fruit of Japanese sophora also contained a number of flavonoids that are absent in ginkgo, e.g., kaempferol-3-O-sophoroside, genistein-4'-O-glucoside, and genistein-4'-O-neohesperidoside (sophorabioside). No flavonoids that would allow distinction from ginkgo leaf, other than genistein, were found in Japanese sophora flowers. The three aglycones quercetin, kaempferol, and isorhamnetin occur at higher concentrations in Japanese sophora flowers compared to ginkgo leaf. However, these aglycones could also be present in larger amounts in a ginkgo extract depending on the manufacturing process and the age of the product (the presence of the flavonols quercetin, kaempferol, and isorhamnetin in authentic ginkgo products may be detected when ginkgo raw material is processed under conditions that allow hydrolysis to occur, or when finished products or raw materials are exposed to heat and humidity, e.g., due to improper storage).

Most of the characteristic ginkgo terpene lactones (i.e., ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide) were present in all of the analyzed ginkgo samples, including the commercial dietary supplements. However, 11 out of the 25 tested supplements contained flavonol glycosides that are typically found in S. japonica fruit. Eight supplements contained genistein, plus quercetin, kaempferol, and isorhamnetin levels inconsistent with authentic ginkgo leaf, suggesting adulteration with Japanese sophora flower or an unknown adulterant. Overall, 19 out of 25 (76%) commercial ginkgo dietary supplements were found to be adulterated.

Comment: The results show a very bleak picture of the quality of ginkgo supplements sold on the market in the United States. Admixture of pure flavonols, or extracts made from Japanese sophora or other flavonol-rich materials have been reported previously, and methods to detect such adulteration are also available, so it is difficult to understand how the adulterated ginkgo extracts still make their way into a finished dietary supplement.

There is some debate as to the occurrence of genistein in genuine ginkgo materials. Although the vast majority of phytochemical investigations did not report genistein to occur in ginkgo leaves, there are a few papers that have described genistein in ginkgo materials. In many of these publications, the alleged ginkgo material in which genistein was detected was never properly authenticated.^1-3 However, Pandey et al. reported genistein from the leaves of a male ginkgo tree and from ginkgo stem and fruit, but not from female ginkgo tree leaves,⁴ the usual source of properly prepared and authenticated commercial ginkgo leaf extract. The plant material analyzed in the paper by Pandey et al. had proper botanical identification, but the results of the HPLC-electrospray ionization (ESI)/MS/MS analysis were unusual, showing the biflavone sciadoptysin as the most abundant flavonoid by far, but finding no rutin in several leaf samples. The authenticated ginkgo leaves analyzed in the paper by Avula et al. include samples from the University of Mississippi campus, a sample certified by NIST, a botanical reference material supplied by ChromaDex, Inc. (Boulder, CO), and an American Herbal Pharmacopoeia (AHP)-verified reference material of ginkgo leaf. Since genistein was not found in any of the authenticated ginkgo leaf materials, even when using the highly sensitive time-of-flight (TOF) MS detection, the results of this paper’s testing suggest that the isoflavone does not occur in ginkgo and, therefore, that genistein can be used as a marker for at least one form of ginkgo leaf extract adulteration.

References

1.     Wang F, Jiang K, Li Z. Purification and identification of genistein in Ginkgo biloba leaf extract. [Article in Chinese]. Se Pu. 2007;25(4):509-513.

2.     Wu J, Xing H, Tang D, et al. Simultaneous determination of nine flavonoids in beagle dog by HPLC with DAD and application of Ginkgo biloba extracts on the pharmacokinetic. Acta Chromatographica. 2012;24(4):627-642. Available at: http://www.akademiai.com/doi/pdf/10.1556/AChrom.24.2012.4.9. Accessed October 6, 2015.

3.     Tang D, Yu Y, Zheng X, et al. Comparative investigation of in vitro biotransformation of 14 components in Ginkgo biloba extract in normal, diabetes and diabetic nephropathy rat intestinal bacteria matrix. J Pharm Biomed Anal. 2014;100:1-10.

4.     Pandey R, Chandra P, Arya KR, Kumar B. Development and validation of an ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method for the simultaneous determination of selected flavonoids in Ginkgo biloba. J Sep Sci. 2014;37(24):3610-3618.