HerbalEGram: Volume 13, Issue 9, September 2016

By Stefan Gafner, PhD


The ninth Joint Natural Products Conference (also known as the International Congress on Natural Products Research [ICNPR]) was held from July 24-27 in Copenhagen, Denmark. With approximately 1,100 attendees this year, the ICNPR is the largest gathering of scientists with interests in natural products. Held every four years in North America or Europe, the conference is hosted by six organizations: 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 included seven plenary lectures, 14 keynote lectures, 49 contributed short lectures, and more than 1,000 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 session.


Research Highlights

Maged Sharaf, PhD, chief science officer of the American Herbal Products Association, presented results of an ongoing investigation of the authenticity of materials sold as black cohosh (Actaea racemosa, Ranunculaceae). In collaboration with scientists from the analytical instrument manufacturing company Waters Corp., a high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (HPLC-qToF-MS) method with subsequent statistical evaluation by principal component analysis was developed—and used successfully— to distinguish black cohosh from closely-related North American and Asian 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) revealed adulteration of black cohosh with Asian Actaea species.

There were numerous poster presentations on quality control of botanical ingredients and adulteration-related topics. Kenny Kuchta, PhD, from the National Institute of Health Sciences in Tokyo, Japan, presented the results of an analysis of genistein (an isoflavone first isolated from dyer’s broom [Genista tinctoria, Fabaceae]) 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 compound is used as a marker for adulteration of ginkgo 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 genistein is one of ginkgo’s secondary metabolites, although it is found only in trace amounts.^1,2 Kuchta et al. reported genistein concentrations from all collected ginkgo materials of 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 comparing the retention time with an authentic standard. Based on the trace amounts present in the sample and the number of ginkgo flavonoids possibly coeluting* with genistein, verification of the compound’s identity with mass spectrometry and the UV spectrum would have been beneficial for this project.

Leslie Boudesocque-Delaye, PhD, from the University of Tours, France, presented a poster on extraction efficiency to analyze proanthocyanidins (PACs) and to evaluate cranberry (Vaccinium macrocarpon, Ericaceae) product quality. Using a solid/liquid extraction approach, the quality of four commercial cranberry supplements was analyzed by a spectrophotometric method using dimethylacetamide (DMAC) and by high-performance thin layer chromatography (HPTLC). The PAC contents ranged 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 (a dimer is a chemical structure composed of two similar units).

Débora Frommenwiler, MSc, a research scientist at the Swiss company CAMAG Scientific Inc., detailed an HPTLC method to authenticate St. John’s wort (Hypericum perforatum, Hypericaceae). The method uses conditions outlined in the United States Pharmacopeia³ with additional detection under white light. Several of the commercial samples labeled as St. John’s wort extracts showed uncommon fingerprints with only four major spots. These spots were identified as a mixture of food colorants, 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. This type of adulteration appears to be fairly common in the marketplace; Frommenwiler et al. found that eight of the 37 (22%) tested commercial products contained these colorants.

Hans Wohlmuth, PhD, a scientist at Integria Healthcare in 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 the results were compared to sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) algorithm.

For raw plant materials, nine of the 17 (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. UHPLC-MS, however, was able to confirm 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.

* Coelution is a term used in analytical chemistry. In chromatography, it describes instances when two chemical compounds are not being separated (i.e., they elute from a column at the same time).


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.