NMR versus HPTLC to Assess the Variability in Crude St. John’s Wort Raw Material

Reviewed: Scotti F, Löbel K, Booker A, Heinrich M. St. John’s wort products – how variable is the primary material? Front Plant Sci. 2019;9:1973.

Keywords: Hypericum perforatum, ¹H NMR, HPTLC

Pharmacopeial test methods to authenticate St. John’s wort (Hypericum perforatum, Hypericaceae) include macroscopic, microscopic, and chemical identification tests. The latter is done by high-performance thin-layer chromatography in both the European and the United States pharmacopeias. However, HPTLC authentication has been challenging in some cases after certain raw materials labeled as St. John’s wort were found to produce HPTLC fingerprints slightly different from those obtained for botanical reference materials. Up to now it has remained unclear if the differences were due to adulteration with closely related Hypericum species, or to natural variability, i.e., differences in the chemistry of St. John’s wort populations depending on the origin of the plant material.

A total of 86 crude raw material samples were obtained for this study from collections in the wild, or from commercial cultivations, markets and pharmacies, composed of 77 St. John’s wort samples and nine samples of other Hypericum species, the latter all originating in China. Wildcrafted St. John’s wort samples were botanically authenticated. The collection included St. John’s wort samples from England, Portugal, Spain, Germany, Switzerland, Italy, Bulgaria, Greece, Lebanon, Tajikistan, China, Chile, Argentina, and Australia. European Pharmacopoeia St. John’s wort extract was used as a reference standard.

Methanol (deuterated methanol for ¹H NMR) extracts of the samples were analyzed by HPTLC and ¹H NMR. HPTLC plates were visually inspected, while ¹H NMR spectra were submitted to multivariate statistical analysis using soft-independent modeling of class analogy (SIMCA) as a classification algorithm. HPTLC data suggested that the fingerprint not only depends on the origin of the plant, but also of the plant part used. Consistent with previous investigations, the lower part of the St. John’s wort plant gave a weaker chromatographic fingerprint, while the flowers and flowering tops showed the highest concentrations of hypericins. The presence of flowers could be established by a yellow-green band at Rf = 0.77. Hypericum perforatum samples originating from China (n = 18) all contained rutin, but uniquely contained a yellow band at Rf = 0.49 corresponding to avicularin. This appears to be a marker compound for St. John’s wort originating in China. Six samples did not provide a sufficiently strong fingerprint to allow evaluation. Rutin was found in only 27 (38%) of the 71 acceptable samples, and was absent in most (81%) of the materials collected in Spain (n = 16).

The ¹H NMR analysis showed a marked difference between the raw material and some of the products obtained from local markets and pharmacies. Six of these samples clustered far from the crude raw material cluster. Discrepancies were explained by differences in the manufacturing process for these products.

Comment: This study is an extension of a prior investigation by the same group into the chemical variability of St. John’s wort.¹ The HPTLC method provides a means to detect the presence of large amounts of material from the lower part of St. John’s wort, which does not comply with the pharmacopeial requirement to use the flowering tops.^2,3 It also allows distinguishing St. John’s wort from other Hypericum species (except H. elodeoides) based on the absence of hypericins in these species. Hypericum elodeoides can be differentiated from H. perforatum by the lack of a characteristic orange flavonoid band at Rf = 0.42.

¹H NMR is useful for classification of the crude raw materials, and gives a better picture of the similarity of the raw material since the NMR spectrum provides a more complete assessment of the compounds present in the plant. However, it does not provide the same ability to differentiate the samples based on their flavonoid and hypericin patterns as does HPTLC. While the samples originating in the Mediterranean countries and in China were generally classified in separate clusters, there was some overlap among these samples. Raw materials from other countries were evenly distributed among the Mediterranean and Chinese samples. Based on the results, NMR is a great tool to distinguish H. perforatum from other Hypericum species, but any additional information obtained is likely more useful in a research setting than in a botanical ingredient quality control laboratory.

References

1. Booker A, Agapouda A, Frommenwiler DA, Scotti F, Reich E, Heinrich M. St. John’s wort (Hypericum perforatum) products – an assessment of their authenticity and quality. Phytomedicine. 2018;40:158-164.
2. St. John's wort, St. John's wort powder, and St. John's wort powdered extract. In: United States Pharmacopoeia 42 and National Formulary 37. Rockville, MD: United States Pharmacopeial Convention, 2019.
3. Hyperici herba, and Hyperici herba extractum siccum quantificatum. In: European Pharmacopoeia 8.0, Volume 2. Strasbourg, France: European Directorate for the Quality of Medicine and Healthcare; 2014:1391.