A Comparison of ¹H NMR, Flow Injection MS, and DNA Sequencing for the Identification of Black Cohosh

Reviewed: Harnly J, Chen P, Sun J, et al. Comparison of flow injection MS, NMR, and DNA sequencing: methods for identification and authentication of black cohosh (Actaea racemosa). Planta Med. 2016;82(3):250-262. doi: 10.1055/s-0035-1558113.

In this study, the strengths and limitations of two chemical fingerprinting (flow injection mass spectrometry [FIMS] and proton nuclear magnetic resonance [¹H NMR]) methods and a DNA barcoding assay (using the ITS and psbA-trnH gene regions followed by Sanger sequencing) were evaluated. The results of the chemical fingerprint analysis were submitted to statistical evaluation by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA).

The sample set consisted of raw materials and finished products from black cohosh (Actaea racemosa, Ranunculaceae) and its known adulterants from a variety of sources (see Table 1). The commercial crude raw materials were purchased from the Internet and local stores in China, while commercial finished products were all from stores in Maryland, USA.

Table 1. Sources and numbers of samples used in the black cohosh authentication study

	A. racemosa	A. cimicifuga	A. dahurica	A. heracleifolia	A. pachypoda	A. podocarpa	A. rubra	Vernonia aspera
AHP	9	8			3	2	2
NIST	4
NCAGR	57
Strategic Sourcing	7
Commercial raw	1	5	3	2				3
Commercial finished	14
Total	92	13	3	2	3	2	2	3

AHP: American Herbal Pharmacopoeia; NIST: National Institute of Standards and Technology; NCAGR: North Carolina Arboretum Germplasm Repository

One of the goals of this investigation was to compare the ability of FIMS and ¹H NMR to discriminate among the various species. However, the PCA plots of the spectra for all samples turned out to be complex and difficult to interpret; therefore, only spectra for A. racemosa, A. cimicifuga, A. pachypoda, A. podocarpa, and A. rubra were included. Crude black cohosh raw material could be distinguished from the three other Actaea spp. by both ¹H NMR and FIMS, and the results of the two methods were generally in agreement. In both cases, one raw material labeled as A. pachypoda clustered with A. racemosa, and one sample labeled as A. racemosa was outside the black cohosh cluster. DNA sequencing supported that these samples were correctly labeled, but seem to consist of a different chemotype. None of the commercial finished products fit into the chemometric model built with authentic black cohosh samples. This is likely due to the processing method, or the addition of excipients that change the chemical profile to the extent that these samples are not recognized as authentic black cohosh.

The DNA testing was performed using the nuclear ITS and psbA-trnH gene regions for authentication of the materials, although not all the samples were submitted to the DNA barcoding assay. The ITS sequence was able to authenticate 20 of the AHP samples, with the remaining four having DNA from species of the fungal genus Eurotium, a mold that grows in humid environments and is occasionally found using DNA barcoding for identification. The psbA-trnH gene resulted in 17 AHP sample identifications. For one sample (BCR14), labeled as A. rubra, the results differed between the ITS sequence (A. rubra) and psbA-trnH sequence (A. pachypoda), possibly due to the presence of a hybrid. Based on DNA barcoding, 11 out of 14 commercial raw material samples (79%) were incorrectly labeled, two samples (14%) were correctly labeled, and one sample did not provide useful DNA. Of the 14 commercial finished products, DNA was obtained in six cases. Of the six samples where DNA was successfully amplified, four contained black cohosh, one contained A. brachycarpa, and for one sample, the amplified DNA indicated the presence of rice (Oryza spp., Poaceae).

Comment: The publication describes some of the most advanced technologies for authentication of black cohosh. Due to the number of samples and the different technologies used, it is not always easy to follow. However, the results and subsequent discussion provide important perspectives about botanical identification. In the case of black cohosh, the occurrence of hybrids and various chemotypes makes genetic and chemical identification challenging. Also, chemometric methods for the authentication of commercial finished products are suitable only when such products are consistently processed and contain the same excipients as the materials upon which the model is based. The success of the genetic approach for finished commercial products (excluding liquid products) was 66% using ITS and 44% with psbA-trnH, though higher than previously reported by Baker et al.,¹ with success rates of 38% and 10%, respectively.

Some of the findings are not entirely clear based on the terminology used to report them. Samples in which a fungus from the genus Eurotium was detected were defined as “mix.” However, Eurotium is a common soil fungus—its presence in root crops specifically, or the presence of microbes in natural products in general, does not constitute a mixture of species as implied, highlighting shortcomings in the interpretation of the DNA data. In addition, there are some questions as to the reliability of the original data on which the identification of the Chinese species was based. There is no species named Actaea foetida, however, Cimicifuga foetida is a synonym of the species with the accepted scientific name A. cimicifuga. Because no original data for the DNA classification of the Chinese species were given, and because some of the method parameters used for the DNA analysis were not reported, it is difficult to verify the accuracy of the results where a discrepancy of identity was reported.

Another limitation in interpreting the data presented is that not all samples were tested in the same manner. For example, no DNA findings are given for three out of four samples that originated from NIST. Additionally, the single NIST sample that was tested was defined as a “mix” despite the fact that both DNA sequence sets used confirmed the species to be A. racemosa. Other samples that were DNA-confirmed to the species were similarly defined as “mix,” without clearly defining the meaning of the term.

Reference

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1.      Baker DA, Stevenson DW, Little DP. DNA barcode identification of black cohosh herbal dietary supplements. J AOAC Int. 2012;95(4):1023-1034.