Working with the natural complexity: Selection and characterization of black cohosh root extract for use in toxicology testing

Abstract

Black cohosh (Actaea racemosa L.) is a botanical supplement marketed to women of all ages. Due to paucity of data to assess the safe use, the National Toxicology Program (NTP) is evaluating the toxicity of black cohosh. The use of an authentic, quality material is imperative to generate robust data. Because botanical materials are complex mixtures with variable composition, the selection of a material is challenging. We describe selection and phytochemical characterization of an unformulated black cohosh root extract (i.e., an extract that serves as source material for a formulated product) to be used in the NTP assessments. A material was selected using a combination of non-targeted and targeted chemical analyses, including confirmation of authenticity, absence of contaminants and adulterants, and similarity to a popular black cohosh product used by consumers. Thirty-nine constituents covering three major classes, triterpene glycosides, phenolic acids, and alkaloids were identified. Among constituents quantified, triterpene glycosides made up approximately 4.7% (w/w) with total constituents quantified making up 5.8% (w/w) of the extract. Non-targeted chemical analysis followed by chemometric analysis of various materials sold as black cohosh, and reference materials for black cohosh and other Actaea species further confirmed the suitability of the selected extract for use.

Keywords: Black cohosh; Botanical dietary supplements; Constituents; Phytochemical composition.

Figure 1.

High performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) chromatograms: A) commercially available standards of potential black cohosh constituents B) unformulated materials procured as black cohosh root extract, and C) formulated black cohosh products. The key for sample and constituent identification can be found in Tables 1 and 2, respectively.

Figure 2.

High performance thin layer chromatography (HPTLC) analysis of black cohosh root extract sample 5 by three laboratories^a. A) Lab 1 (top panel: untreated plate, UV detection at 365 nm; bottom panel: treated plate, visible light). B) Lab 2 (top panel: untreated plate, UV detection at 366 nm; bottom panel: treated plate, visible). C) Lab 3 (UV detection at 366 nm). ^a Lab 1 lane designation relevant to data reported: 1, actein; 4 and 5, 0.5 or 2 μL sample 5; 12, A. racemosa (rhizome) Lab 1 standard; 13, A. racemosa (root) Lab 1 standard; 14, A. cimicifuga (rhizome) Lab 1 standard; 15, A. podocarpa (root and rhizome) Lab 1 standard; 16, A. heracleifolia/Sheng Ma (root) Lab 1 standard; 17, actein; 18, cimifugin. Lab 2 lane designation: 1, actein; 2, A. racemosa (root) botanical reference material (confirmed specimen through macroscopic and microscopic techniques while the plant is physically intact, before it was dried, grounded, and extracted); 3 and 4, 2 μL sample 5; 5 and 6, Test lab vouchered reference material 2.0 and 6 μL, respectively (an expertly identified pressed vouchered material that was prepared for analysis). Lab 3 lane designation: Left, A. racemosa root Lab 3 standard; Right, sample 5

Figure 2.

High performance thin layer chromatography (HPTLC) analysis of black cohosh root extract sample 5 by three laboratories^a. A) Lab 1 (top panel: untreated plate, UV detection at 365 nm; bottom panel: treated plate, visible light). B) Lab 2 (top panel: untreated plate, UV detection at 366 nm; bottom panel: treated plate, visible). C) Lab 3 (UV detection at 366 nm). ^a Lab 1 lane designation relevant to data reported: 1, actein; 4 and 5, 0.5 or 2 μL sample 5; 12, A. racemosa (rhizome) Lab 1 standard; 13, A. racemosa (root) Lab 1 standard; 14, A. cimicifuga (rhizome) Lab 1 standard; 15, A. podocarpa (root and rhizome) Lab 1 standard; 16, A. heracleifolia/Sheng Ma (root) Lab 1 standard; 17, actein; 18, cimifugin. Lab 2 lane designation: 1, actein; 2, A. racemosa (root) botanical reference material (confirmed specimen through macroscopic and microscopic techniques while the plant is physically intact, before it was dried, grounded, and extracted); 3 and 4, 2 μL sample 5; 5 and 6, Test lab vouchered reference material 2.0 and 6 μL, respectively (an expertly identified pressed vouchered material that was prepared for analysis). Lab 3 lane designation: Left, A. racemosa root Lab 3 standard; Right, sample 5

Figure 3.

Fourier transform infrared spectrum of black cohosh root extract sample 5.

Figure 4.

Total ion chromatograms from A) liquid chromatography coupled with quadrupole-time of flight- (QTOF) and B) quadrupole-ion trap (QTrap)-mass spectrometry analyses of black cohosh extract sample 5.

Figure 5. Structures of constituents identified in black cohosh root extract sample 5: A) triterpene glycosides B) phenolic acids C) alkamides. *Denotes definitively identified constituents. ** Denotes constituents classified as tentatively identified.

Figure 5. Structures of constituents identified in black cohosh root extract sample 5: A) triterpene glycosides B) phenolic acids C) alkamides. *Denotes definitively identified constituents. ** Denotes constituents classified as tentatively identified.

Figure 6.

Phytochemical profile of black cohosh extract sample 5 stored at different conditions. A) High performance liquid chromatography (HPLC)-ultraviolet detection (UV) (317 nm) chromatograms of sample stored for 14 days at −20 °C, 5 °C, ambient temperature, and 60 °C. B) HPLC-UV (317 nm) chromatograms of sample stored for 13 years at −20 °C and ambient temperature C) HPLC-charge aerosol detection (CAD) chromatograms of sample stored for 13 years at −20 °C and ambient temperature.

Figure 7.

High performance liquid chromatography-charged aerosol detection (HPLC-CAD) aligned chromatograms. A) Standards of potential black cohosh constituents. B) Cohosh reference materials (samples 1–4). C) Unformulated materials procured as black cohosh over multiple years (samples 5–21). D) Formulated products procured as black cohosh (samples 22–31). The key for samples and standards can be found in Tables 1 and 2.

Figure 7.

High performance liquid chromatography-charged aerosol detection (HPLC-CAD) aligned chromatograms. A) Standards of potential black cohosh constituents. B) Cohosh reference materials (samples 1–4). C) Unformulated materials procured as black cohosh over multiple years (samples 5–21). D) Formulated products procured as black cohosh (samples 22–31). The key for samples and standards can be found in Tables 1 and 2.

Figure 8.

Heatmap of cohosh samples. SpecAlign software was used to generate a heatmap of correlations between the different samples. Black cohosh root extract reference material (XRM) (Sample 1, Table 1) is at the upper left and the rest of the samples are in order based on similarity with the XRM. The correlations were formatted as percentages and color represents the degree of correlation (green ≥80%; yellow 60–79%; red <60%). The key for sample numbers can be found in Tables 1.

Figure 9.

Principal component (PC) analysis 2-dimensional score plots A) PC1 versus PC2 B) PC1 versus PC3 of data from high performance liquid chromatography-charged aerosol detection (HPLC-CAD) analysis of cohosh samples. Peaks were aligned using SpecAlign software (University of Oxford, England) and the plot was generated using Solo v8.5.1 software (Eigenvector Research, Manson, WA). Each point represents a single sample. Samples include cohosh reference materials (1–4), unformulated materials (5–21), and formulated products (22–31). For sample 22 and 26, half the amount of the product was used inadvertently. The color code represents the year the samples were procured as indicated in the color bar to the right of the graph. The key for sample numbers can be found in Table 1.

Figure 9.

Principal component (PC) analysis 2-dimensional score plots A) PC1 versus PC2 B) PC1 versus PC3 of data from high performance liquid chromatography-charged aerosol detection (HPLC-CAD) analysis of cohosh samples. Peaks were aligned using SpecAlign software (University of Oxford, England) and the plot was generated using Solo v8.5.1 software (Eigenvector Research, Manson, WA). Each point represents a single sample. Samples include cohosh reference materials (1–4), unformulated materials (5–21), and formulated products (22–31). For sample 22 and 26, half the amount of the product was used inadvertently. The color code represents the year the samples were procured as indicated in the color bar to the right of the graph. The key for sample numbers can be found in Table 1.

Figure 10.

Comparison of HPLC-CAD profiles of black cohosh root extract sample 5 with cohosh reference materials and a finished product of black cohosh root extract (Remifemin®).