Food colorants in St. John’s wort, genistein in ginkgo leaves? A review on new research on quality control of botanicals presented at the Joint Natural Products Conference (JNPC) in Copenhagen, Denmark

 

Keywords: Actaea racemosa, Actaea spp., adulteration, amaranth dye, black cohosh, brilliant blue FCF, cranberry, DNA barcoding, food colorant, genistein, ginkgo, Ginkgo biloba, HPLC-MS, HPLC-UV, HPTLC, Hypericum perforatum, JNPC Copenhagen, proanthocyanidins, St. John’s wort, sunset yellow FCF, tartrazine, Vaccinium macrocarpon

 

The 9th Joint Natural Products Conference (also known as the International Congress on Natural Products Research [ICNPR]) was held from July 24-27, 2016, in Copenhagen, Denmark. With approximately 1100 attendees, this is the largest gathering of scientists with interests in natural products worldwide; it is held every four years, either in North America or Europe. It is hosted by the Association Francophone pour l’Enseignement et la Recherche en Pharmacognosie (AFERP), the American Society of Pharmacognosy (ASP), the Society for Medicinal Plant and Natural Product Research (GA), the Società Italiana di Fitochimica (SIF), the Japanese Society of Pharmacognosy (JSP), and the Phytochemical Society of Europe (PSE).

 

This year’s event had ca. 1100 participants, and included seven plenary lectures, 14 keynote lectures, 49 contributed short lectures, and over 1000 poster presentations. Many presentations focused on drug discovery using biomass from plant, fungal, microbial, and marine sources. Presentations on analytical methods and quality control of botanical ingredients were given in short lectures and in the poster sessions. Below are a number of presentations that may be of interest to the reader of this newsletter.

 

Maged Sharaf, PhD, CSO of the American Herbal Products Association (Silver Spring, Maryland), presented results of an ongoing investigation into the authenticity of materials sold as black cohosh (Actaea racemosa, Ranunculaceae). In collaboration with scientists from the analytical instrument manufacturing company Waters, Inc., a high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (HPLC-qToF-MS) method with subsequent statistical evaluation by principal component analysis (PCA) was developed to distinguish black cohosh from closely related North American Actaea species, and from Asian Actaea species. The method successfully distinguished among black cohosh and the Asian and North American Actaea species. Analysis of 16 commercial products containing crude raw material (which are a subset of the samples that will be investigated as part of the project) showed occurrence of adulteration with Asian Actaea species.

 

There were numerous poster presentations on quality control of botanical ingredients, including topics of adulteration. Kenny Kuchta, PhD, from the National Institute of Health Sciences in Tokyo, Japan, presented the results of the analysis of genistein in crude ginkgo (Ginkgo biloba, Ginkgoaceae) leaves collected from five different provinces in China. The natural occurrence of genistein in ginkgo leaves is controversial – the isoflavone is used as a marker for adulteration with Japanese sophora (Styphnolobium japonicum syn. Sophora japonica, Fabaceae) and is thought by many researchers not to occur in authentic ginkgo leaves. However, other researchers have reported that it is part of the secondary ginkgo metabolites although it is found only in trace amounts.^1,2 Kuchta et al. report genistein from all collected materials at concentrations between 5-28 μg/g dry leaf, with the highest concentrations found in leaves harvested in September and October. The analysis was performed using high-performance liquid chromatography and ultraviolet detection (HPLC-UV) at 350 nm. Genistein was identified by comparison of the retention time with an authentic standard. Based on the trace amounts present in the sample, and the number of ginkgo flavonoids possibly co-eluting at the same time as genistein, verification of the compound identity with mass spectrometry and the UV spectrum would have been beneficial for this project.

 

A poster on extraction efficiency to analyze proanthocyanidins (PACs) and evaluate the quality of cranberry (Vaccinium macrocarpon, Ericaceae) products was presented by Leslie Boudesocque-Delaye, PhD, from the University of Tours; Tours, France. Using a solid/liquid extraction approach, the quality of four commercial cranberry supplements was analyzed by a spectrophotometric method (dimethylacetamide [DMAC]) and by high-performance thin-layer chromatography (HPTLC) analysis. The contents in PACs varied between 5-62 mg with respect to the recommended daily intake. The HPTLC fingerprint showed that only two supplements contained the typical cranberry fingerprint. One contained a grape (Vitis vinifera, Vitaceae) PAC fingerprint, and another showed the presence of two unknown dimeric PACs.

 

Débora Frommenwiler, MSc, research scientist at CAMAG AG (Muttenz, Switzerland), detailed an HPTLC method to authenticate St. John’s wort (Hypericum perforatum, Hypericaceae). The method uses conditions outlined in the United States Pharmacopeia,³ with additional detection under white light. Several of the commercial samples labeled to contain St. John’s wort extracts showed uncommon fingerprints with only four major spots. These spots were identified as a mixture of food colors, including amaranth dye, brilliant blue FCF, sunset yellow FCF, and tartrazine. The addition of food colorants, most likely an attempt to fool the spectrophotometric determination of hypericin contents, has been described previously in issue #3 of the Botanical Adulterants Monitor, but seems to be a quite common occurrence in the marketplace, with eight (22%) out of 37 of the tested commercial products being adulterated with these colorants.

 

Hans Wohlmuth, PhD, scientist at Integria Healthcare (Eight Mile Plains, Queensland, Australia), examined the potential utility of DNA barcoding as a routine test for botanical raw materials, extracts, and finished products, following the same batches from raw material through extraction to finished product. Samples included 17 authentic dried raw materials, and extracts and tablets made from the same raw materials. At the same time, samples were chemically profiled by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) with identification of marker compounds to confirm botanical authenticity. DNA was extracted with the NucleoSpin^® 96 Plant II kit, and barcodes from four genomic regions (matK, rbcL, trnH-psbA, and ITS2) were amplified by polymerase chain reaction (PCR). Sequences were determined by the Sanger method, and results were compared to sequences in GenBank using the BLAST algorithm. For raw plant materials, nine (53%) were correctly identified by at least one barcode. Across all samples, correct species identification was achieved for 18%, 15%, and 5% using trnH-psbA, rbcL, and matK, respectively. None of the ITS2 barcoding experiments allowed species identification. Only one extract yielded amplifiable DNA, but the species from which the DNA originated could not be identified. On the other hand, UHPLC-MS confirmed the identity of all samples. The authors concluded that DNA barcoding, using universal barcode sequences, is not suitable for routine authentication of botanical raw materials, but that the use of shorter, species-specific sequences may be a better way to authenticate these materials by genetic means.

 

References

1.     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.

2.     Wang F, Jiang K, Li Z. Purification and identification of genistein in Ginkgo biloba leaf extract. Chin J Chromatogr. 2007;25(4):509-513.

3.     United States Pharmacopeial Convention. Powdered St. John’s Wort Extract. In: United States Pharmacopeia and National Formulary (USP 39–NF 34). Rockville, MD: United States Pharmacopeial Convention; 2016:6821-6822.