Pharmacognosy of Black Cohosh: The Phytochemical and Biological Profile of a Major Botanical Dietary Supplement

Abstract

The ethnobotanical, Black Cohosh, a preparation from the roots/rhizomes of Actaea racemosa, has been the subject of extensive investigations. Despite the fact that sales figures place it among the top ten U.S. dietary supplements, the mechanisms of its amelioration of the adverse effects of menstruation and menopause remain largely undefined. The plant is a prodigious “factory” of complex secondary metabolites, with almost 50 cycloartane triterpenes, over 70 alkaloids, and 11 phenolic acids characterized so far, and this chapter discusses the chemistry of the A. racemosa secondary metabolome in detail. While numerous in vitro and in vivo pharmacological and biological studies attribute diverse specific biochemical effects to individual metabolites or defined extracts, an adequate explanation of potencies and drug(-like) concentrations is still lacking. As many of the effects may play a role in the alleviation of menopausal symptoms, the possibility exists that the overall effect may be the result of a combination of both multiple bioactive principles and several synergistic interactions. On careful analysis, a purported hepatotoxicity risk raised has been relegated to “a possible rare idiosyncratic reaction”. More than 50 clinical studies defy definitive analysis, other than confirming that the material is safe, in part because of poor study design and differences in preparations used. The global literature on Black Cohosh is almost unanimous in a call for larger, more well-designed clinical studies, and recent advances in the understanding of the phytoconstituents justify a more thorough secondary metabolomics analysis of this interesting North American plant.

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1 Introduction

The term “Black Cohosh” has been used in both the scientific and commercial literature with a wide variety of meanings including the source plant, portions thereof, and plant extracts. In this chapter, the authors will attempt to use this term and related terms including the Latin binomial rigorously, with the following meanings:

• Actaea racemosa ( A. racemosa ) strictly for the source plant;

• Black Cohosh (BC) for the underground parts (roots and rhizomes) of the plant;

• Black Cohosh Extract (BCE) for an extract of BC.

Difficulties in defining the exact nature of each of these, particularly the last term, will be discussed later in sections covering botany, verification, adulteration, quality control, naming, and pharmacology.

Actaea racemosa L. (syn. Cimicifuga racemosa) [L.] Nutt.) is a species of flowering plant of the family Ranunculaceae (Plate 1). It is an endemic native to North America and distributed mainly from the south of Ontario to central Georgia, and west to Missouri and Arkansas. The roots and rhizomes (Plate 2) have been used medicinally by Native Americans since pre-Columbian times, to treat malaise, gynecological disorders, kidney disorders, malaria, rheumatism, and sore throats (1). Following the appearance of BC as “Black Snakeroot” in the U.S. Pharmacopoeia in 1820 (2), BCEs have gained popularity in contemporary use as herbal dietary supplements to alleviate women’s menopausal symptoms. A recent report released by the American Botanical Council states that BCE is one of the ten top-selling herbal dietary supplements in the food, drug, and mass market channel in the United States in 2011, with sales of over $10 million (3, 4). The widespread use of BC as a dietary supplement has also led to an increasing call for scientific evidence of its quality, safety, and efficacy.

Plate 1

Aerial parts of the title plant, Actaea racemosa L.: entire plant (bottom), inflorescence (middle), flower (top), in front of its leaves

Plate 2

Dried roots and rhizomes of Actaea racemosa L.: entire rootstock (top) and detail (bottom)

In 1999, the University of Illinois at Chicago (UIC)/National Institutes of Health (NIH) Center for Botanical Dietary Supplements Research initiated a systematic study of the entire subject (A. racemosa, BC, and BCEs) and since then has dedicated more than 10 years of research to enhance knowledge of their botany, chemistry, and biology. The present contribution summarizes and analyzes the progress of these studies and those by others, including the phytochemical investigation, fingerprint profiling, biological and pharmacological evaluation, and clinical trials.

Emphasis is placed on the increased recognition of the plant’s chemical diversity, which forms the basis of the understanding of its biological and clinical potential. The phytochemistry of A. racemosa is first reviewed with a focus on the potential bioactive components including cycloartane triterpenes, cimicifugic acids, phenolics, and nitrogen-containing compounds. For each of these major types of constituents, a compilation of the structures and nomenclature of the individual compounds, known to date, is provided. Isolation, purification, and structural characterization methods for these compounds are also discussed. The subsequent section provides an overview of the chemical fingerprinting of A. racemosa, which has evolved as an important approach used for the metabolomic profiling, botanical identification, and quality standardization. The section on pharmacology of BCEs provides a concise yet relatively comprehensive literature survey of the major bioactivities of both crude extracts and pure compounds, such as “estrogenic” activity, anticancer activity, stress relief, and prevention of bone loss. Furthermore, a detailed description is given of the reports of BCE-associated hepatotoxicity with the inherent safety concern. The final section briefly summarizes and discusses the key points of the clinical research of BCEs conducted in the last 20 years, which provides the reader with an overview of the progress and challenges of the safety and efficacy evaluation of BCEs and related botanical preparations in clinical settings.

By assuming an up-to-date perspective of the various aspects of this widely used botanical, it can be demonstrated that the chemical diversity of A. racemosa is far broader than what has typically been considered in experimental (in vitro, in vivo, and clinical) approaches taken to study its biological significance. By placing emphasis on the underlying chemistry and viewing BCE preparations as complex mixtures of pharmacologically active agents with highly diverse biological targets, the present work is intended to inspire future research aimed at recently characterized phytoconstituents and those still to be discovered, and the evaluation of their biological potential.

2 Phytochemistry

2.1 Cycloartane Triterpenes

2.1.1 Structural Diversity

Cycloartane triterpenes are the most well studied components of Actaea racemosa. These compounds possess complex chemical structures as well as many potential bioactivities. Since the 1950s, more than 40 cycloartane triterpenes have been isolated and identified from A. racemosa (5). A few were identified in the aerial parts (6). These triterpenes predominantly occur as 3-O-β-d-xylopyranosides or, less commonly, as 3-O-α-l-arabinopyranosides. Based on the structures of their aglycone side chains, they are classified into several subgroups, as shown in Fig. 1. The structural diversity of Actaea triterpenes is also contributed to by substitution of the core with functional groups such as a double bond at C-7, C-8, hydroxy groups at C-1, C-12 and/or C-15, and acetoxy groups at C-12 and/or C-15.

Fig. 1

The structural types (top) and numbers (bottom) of cycloartane triterpenes identified in A.racemosa. As all structures reported so far exhibit the same configuration alpha at carbon C-17, it is not drawn in the subsequent structural formulas in the present work

Tables 1–9 list, by type, all cycloartane triterpenes identified in A. racemosa prior to 2013. A bar graph of the numbers of the different types of cycloartane triterpenes identified in A. racemosa (see Fig. 1) shows that the cimigenol and acteol types are the major triterpene constituents, in terms of both the number and abundance of these compounds. The hydroshengmanol- and 23-O-acetylshengmanol-type triterpenes are commonly found in A. simplex, and a few of them have also been found in A. racemosa. The other types of Actaea triterpenes, such as the dahurinols, cimicidanols, and cimicidols, have been found only rarely in A. racemosa, possibly due to their non-occurrence or significantly lower abundance. This represents a characteristic biosynthetic profile of triterpenes for A. racemosa.

Table 1 The cimigenol-type triterpenes identified in A. racemosa

Table 2 The 25-dehydrocimigenol-type triterpenes identified in A. racemosa

Table 3 The acteol-type triterpenes identified in A. racemosa

Table 4 The 23-epi-acetol-type triterpenes identified in A. racemosa

Table 5 The 23-O-acetylshengmanol-type triterpenes identified in A. racemosa

Table 6 The cimiracemoside-type triterpenes identified in A. racemosa

Table 7 The hydroshengmanol-type triterpenes identified in A. racemosa

Table 8 The neocimigenol-type triterpenes identified in A. racemosa.

Table 9 The isodahurinol-, cimicidanol-, and cimicidol-type triterpenes identified in A. racemosa

Despite the very interesting and complex structures of many of these triterpenes, especially those containing 3-, 5-, and 6-membered oxygen heterocycles, to the best of our knowledge no synthesis or semisynthesis approaches have been undertaken. The synthesis of these molecules would appear to present a formidable challenge to the synthesis chemistry community.

2.1.2 Naming System

Previously, Actaea/Cimicifuga triterpenes were given a wide variety of trivial names based on the species, traditional Chinese medicinal use, and only occasionally following some structural features. A comprehensive list of the names of aglycones includes acetol, cimiaceroside, cimicidanol, cimicidol, cimifugoside, cimigenol, cimiracemoside, dahurinol, hydroshengmanol, neocimigenols, neocimicigenosides, and 23-O-acetylshengmanol (5, 7). These trivial names, at best, provide clues as to the origin of the compound, but the similarity of names gives no indication of any similarity of the structures. An example of the enormous confusion created by the trivial names given to the Actaea/Cimicifuga triterpenes may be demonstrated by considering cimiracemosides F (38), M (34), and P (27): these compounds have completely different ring systems and also differ from one another in the sites of oxygenation at C-12, C-15, C-16, C-22, C-23, and C-26 (see Fig. 2). The reverse situation occurs in the pair, neocimigenol and neocimicigenoside A (43). While the former has the identical aglycone moiety to the latter, its name varies in a very subtle and rather confusing way (“cimi” vs. “cimici”). Considering that the triterpene structures alone differ in very subtle ways, it becomes essentially impossible to differentiate between subtle structural and subtle naming changes, especially if the naming schemes lack a clear rationale.

Fig. 2

Three Actaea triterpenes that have the same trivial root name but completely different ring systems in their side chains

To address this problem, recently a new systematic naming system was established, which enables the deduction of all known Actaea triterpene structures, as well as those of congeners yet to be discovered. All that is required is a knowledge of the basic cycloartane skeleton (8). As shown in Fig. 1, all of the known cycloartane triterpenes from A. racemosa fall into only a few basic structural skeletons. As far as C-20 to C-27 are concerned, there are acyclic compounds in which these carbons have no connections between themselves other than the basic carbon chain. Then, there are other compounds in which some of these carbons are involved in one or two rings, usually formed by ether or acetal oxygens, often connecting to C-16. On the basis of these structural features, the new system names the acyclic aglycones as actanols, those with a single oxygen bridge forming a further ring as actamonoxols, and those with two oxygen-containing rings actabinoxols. (Note: oxiranes are not counted in determining the mono- or bi-oxonol name; see Fig. 3).

Fig. 3

Naming system for Actaea triterpenes established by Qiu et al. (8). ^aNumber of rings in the aglycone side chain. ^bAglycone. ^cGlycoside

These names all include the 3β-hydroxy group. Where this group is modified to a glycosidic linkage, the suffix would be “-oside”, e.g. actabinoxoside. All of the substituents and other structural modifications need to be fixed using standard chemical nomenclature, with prefixes arranged in alphabetical order. The configuration of these triterpenes is designated via the Cahn-Ingold-Prelog (CIP) system rather than the simpler α/β system used commonly in steroid and terpenoid nomenclature, because the α/β nomenclature fails in bicyclic caged rings that occur in many of the actabinoxols.

2.1.3 Isolation Techniques

Actaea triterpenes are difficult to separate and purify because they occur as mixtures of natural product congeners with highly similar chemical properties. Repeated isolation techniques reported in the literature include gravity and low-pressure liquid chromatography (LPLC), medium-pressure liquid chromatography (MPLC), vacuum-liquid chromatography (VLC), high-speed countercurrent chromatography (HSCCC), and high-performance liquid chromatography (HPLC). Commonly, three to eight steps of fractionation are needed to obtain the compounds with sufficient purity for structural elucidation. It should be noted that from the experience of the authors, the purity level required for the elucidation of structure is relatively low. Moreover, reference materials of Actaea/Cimicifuga triterpenes commonly exhibit significant residual complexity (8) and require thorough analysis, especially prior to being used for any meaningful biological evaluation.

The isolation workflow of Actaea triterpenes usually starts with liquid partitions of the methanol extract of plant materials using a series of organic solvents. This results in triterpene-enriched partitions in phases of medium polarity, such as CHCl₃ and n-BuOH. Subsequent procedures include repetitive fractionation by normal-phase (e.g. silica gel), reversed-phase (e.g. C₈ and C₁₈ silica gel), and, occasionally, Sephadex (e.g. LH-20) column chromatography. Analytical thin-layer chromatography (TLC) has been used to optimize the solvent conditions for the preparative separation on the column chromatography, and using the G.U.E.S.S method (9) to choose an HSCCC solvent system. Isolation of pure compounds can take up to eight fractionation steps even when the conditions are carefully optimized (10). For the resolution of critical pairs of triterpenes, preparative RP-HPLC not only provides optimized resolution, but also reduces sample loss and overall workload. Therefore, HPLC has been used frequently as in the last purification step for these compounds. As the Actaea triterpenes exhibit very weak UV absorption, online ELSD or offline TLC analysis are needed to optimize and monitor the HPLC separation process. Recently, Sezai, Cizek et al. have reported a fast and convenient method for the isolation of triterpenes from A. racemosa by HSCCC/ELSD (11). The optimized solvent system, consisting of n-hexane–acetone–ethyl acetate–iso-propanol–ethanol–water (7:2:4:2:1:4, v/v/v/v/v), enabled a single step of separation of four Actaea triterpenes with high yield and purity from an enriched fraction. This demonstrates the high resolution power of CCC, provided the target analytes elute in the “sweet spot” of the chromatogram (12).

2.1.4 Structural Elucidation

Most modern approaches for the structural elucidation of the Actaea triterpenes involve the use of high-resolution NMR spectroscopy and mass spectrometry techniques, whereby a combination of both has proven to be the most efficient way for the characterization of these molecules. At the same time, X-ray diffraction is vital for the establishment of absolute stereochemical assignments, which, due to the lack of chromophores among these compounds, are otherwise unachievable with the usual chiroptical methods.

2.1.4.1 Mass Spectrometry

High-resolution mass spectrometry (HR-MS) has been commonly used for the determination of the molecular formula of Actaea triterpenes. Soft-ionization techniques, such as fast-atom bombardment (FAB) and electrospray ionization (EI), have made a major contribution. From the early to mid-1990s, positive-mode HR-FAB-MS was frequently employed. This method uses a liquid matrix, which facilitates production of molecular ions of the solutes. In positive HR-FAB-MS, the quasi-molecular cations, such as [M + H]⁺, [M + Na]⁺, and [M − OH]⁺, are often produced. Furthermore, the peaks in FAB-mass spectra have been used occasionally for the determination of the presence of sugar moieties of Actaea triterpenes. For example, the peak [M + H − 132]⁺ indicates the elimination of one pentosyl group (13). Since the late 1990s, positive HR-ESI-MS has been also applied for the structural characterization of Actaea triterpenes. Similarly, the molecular formulas are readily determined based on the molecular ion peaks. MS has a particular advantage in the identification of halogenated compounds, where the characteristic isotope pattern can be observed in the mass spectra, whereas the NMR spectra provide no direct evidence for the presence of chlorine. Using HR-ESI-MS, Chen et al. identified a rare chlorine-containing triterpene, 25-chloro-25-deoxycimigenol-3-O-β-d-xylopyranoside (7), from A. racemosa as an artifact of the isolation procedure, and, thus, established a potential pathway for in vitro/in vivo chemical diversification of the parent triterpenes with oxirane partial structures (14).

2.1.4.2 Nuclear Magnetic Resonance

One-dimensional/two-dimensional (1D/2D) NMR spectroscopic procedures are the most important and useful techniques for the structural elucidation of Actaea triterpenes. Due to its solubility properties and achieved signal dispersion, pyridine-d ₅ is used widely as the “standard” NMR solvent for Actaea triterpenes. Initial ¹H NMR analysis typically reveals five to eight singlet methyl signals, one methyl doublet signal (H-21), and, in the range 0.3–0.9 ppm, a pair of doublet signals of cyclopropane protons (H-19). The presence and type of sugar moieties are also easily identified by the signals at lower field (3.8–5.0 ppm). Owing to the significant peak overlap in the upfield portion of the ¹H NMR spectra, it is difficult to assign some of the methine and methylene protons, e.g. H₂-1, H₂-2, H-5, H₂-6, H₂-7, H-8, H₂-11, H₂-12, and H₂-15. Thus, 2D-NMR spectra are required to aid in the assignment of these proton resonances. The HMBC spectra are particularly useful due to the ^2–3 J _C,H correlations between several methyl protons and nearby methine or methylene carbons. Upon the determination of these δ _C values, the δ _H values of methylenes and methines can be assigned from the HSQC spectra. In addition, the HSQC and COSY spectra may be useful to deduce the J _H,H values. However, significant peak overlap and higher order spin systems often still severely limit the identification of distinct multiplicity patterns. This problem can be solved by using spectroscopic simulation via ¹H iterative Full Spin Analysis (HiFSA) (15, 16). Figure 4 shows an example of such a full spin analysis of an Actaea triterpene, which involves the use of PERCH software. Upon conformational analysis and prediction of basic NMR parameters (δ _H, J _H,H) using a 3D model of the analyte, the ¹H NMR spectrum was simulated using the predicted parameters. Subsequently, iterative and systematic variation of the δ _H/J _H,H values, simulation of the ¹H NMR spectra, and comparison with the experimental spectrum were performed. Finally, the calculated NMR parameters were optimized using Total-Line-Shape (TLS) iteration until the difference between the simulated and experimental spectra (residual) was minimal (typically < 0.1% RMSD).

Fig. 4

Quantum mechanical interpretation of the ¹H NMR spectrum of cimiracemoside F (38) in pyridine-d ₅ at 400 MHz by ¹H iterative Full Spin Analysis (HiFSA) using PERCH software. The difference spectrum (residual) indicates the excellent agreement between the experimental and calculated spectra. The only major residual signals denoted by ↓ belong to EtOAc, which was identified as the residual solvent in this sample

Previously, the relative configuration of Actaea triterpenes has usually been studied by NOESY experiments. With the completion of the full spin analysis (HiFSA), the relative configuration of the protons can be determined readily from the calculated J _H,H values. For example, the axial and equatorial positions of the protons can be determined based on the fact that axial-axial couplings are large (~13 Hz), while axial-equatorial and equatorial-equatorial couplings are much smaller (~3–5 Hz). The availability of a complete set of J values from the HiFSA provides a comprehensive definition of the complex J-coupling network in the triterpenes and, thereby, leads to an unequivocal assignment of the relative stereochemistry of most of the triterpene skeleton from only a 1D ¹H NMR spectrum. Notable exceptions are parts of the O-heterocycles (E/F rings), which can escape this determination by virtue of lack of J-coupling. These situations have to be addressed with NOE measurements or X-ray diffraction.

2.1.4.3 X-Ray Diffraction

Single crystal X-ray diffraction is by far the most powerful experimental method for the characterization of atomic arrangements in molecules. It provides accurate data concerning both the configuration and conformation of compounds, such as precise atomic coordinates, geometries, and crystal packing in the solid phase. However, X-ray diffraction has not been applied as a routine technique for the conformation studies of Actaea triterpenes, because it is challenging to obtain highly pure samples in sufficient amounts for crystal growth. This might change in the future with the recently reported use of porous complexes, which requires no crystallization of the target molecules (17).

The pair, 26-deoxyactein (22) and 23-epi-26-deoxyactein (29), serve as an excellent examples of the few Actaea triterpenes that have been characterized this way (18). X-ray diffraction analyses of these two congeners showed that the torsion angles around atom C-23 are different, revealing the difference in stereochemistry at that chiral center (see Fig. 5), which confirmed the structures and absolute configurations proposed by NMR for 22 and 29. This result clarifies the structure of the compound previously referred to as 27-deoxyactein, as 23-epi-23-deoxyactein (29).

Fig. 5

The 3D structures of 22 and 29 without hydrogens drawn from original X-ray single crystal data (Cambridge Crystallographic Data Center as CCDC 170702 and 170703, respectively) with Mercury (ver. 3.1). The extra oxygen in the vicinity of C-1′ in 23-epi-26-deoxyactein is from a molecule of water

2.1.5 Dereplication

Dereplication is the process of differentiating and identifying one or more natural products present in extracts, fractions, or as isolated materials. Dereplication facilitates rapid identification and quantification of known compounds of interest, but also the identification of unknowns, from complex natural product mixtures. The dereplication of triterpenes from A. racemosa mainly utilizes two techniques: hyphenated LC (LC/ELSD, LC/MS) and NMR. The specifics of these dereplication approaches are described in the following.

2.1.5.1 Dereplication by LC/MS and LC/ELSD

LC-based methods are the most common for the dereplication of Actaea triterpenes. As Actaea triterpenes most commonly lack UV chromophores, UV spectroscopy is an inappropriate method for their LC detection. Instead, MS and ELSD are commonly used. For example, Kan et al. (19) developed an LC/(+)APCIMS method for the direct analysis and identification of four triterpenes, actein (24), 26-deoxyactein (22), cimiracemoside C (2), and cimigenoside (1), in A. racemosa and several commercially available A. racemosa products. An HPLC/MS total ion chromatogram of the triterpene-enriched sample was initially obtained, and identification of each triterpene was based on the presence of the molecular ion and characteristic fragment ions. This is well displayed in Figs. 1 and 2 of their publication (19).

Recently, Avula et al. used UPLC/ELSD for the identification and quantification of three triterpenes, actein (24), 23-epi-26-deoxyactein (29), and cimiracemoside F (38), in A. racemosa and dietary supplements derived from it (20). The marker compounds were readily identified by comparison of the UHPLC/ELSD chromatograms of plant samples and reference materials. This method was capable of giving shorter retention times while maintaining good resolution compared with conventional HPLC. In addition, UHPLC/MS was used for the identification of these three marker compounds. This method involved the use of [M + Na]⁺ ions in the ESI mode for 24, 29, and 38 at retention times of 3.05, 5.10, and 5.40 min, respectively, and corresponding m/z = 699.4, 683.4, and 699.4 in the positive-ion mode with selected-ion monitoring (SIM).

2.1.5.2 Dereplication by NMR

While LC-based methods are used frequently in the dereplication of natural products, they provide limited information for structural identification, especially if performed with nominal mass resolution. The challenge increases further when the analytes exhibit weak UV absorption and/or poor MS ionization, such as the Actaea triterpenes. Moreover, LC-based methods rely on the availability of authentic reference materials for identification and particularly quantification. In contrast, NMR spectroscopy can serve as a more universal and linear detector for biologically complex samples, and, thus, is a highly suitable tool for the dereplication of natural products. Not only does NMR typically provide structural information, but it allows the simultaneous quantification of each detected analyte in a complex matrix, frequently without the need for further separation, and more importantly, without identical calibrants (16, 21–23). Despite these advantages, the complexity of the NMR spectra, such as peak multiplicity and signal overlap, leads to difficulties in the analysis of spectra.

One major challenge with dereplication of the Actaea triterpenes is to determine their structural subtypes, i.e. the partial structures of the aglycone side chains. From a general point of view, in the NMR spectra, any single chemical entity is represented by a unique pattern of NMR signals. Much like fingerprints, a sub-portion of these complex spectroscopic patterns might be sufficient to distinguish the different chemical entities. For Actaea triterpenes, the protons at C-16, C-22, C-23, and C-24 are excellent structural indicators, because they are all elements of the aglycone side chains and have distinct chemical shifts, multiplicities, and coupling constants (Table 10). In addition, their ¹H NMR signals are less overlapped in the region of 3.5–6.0 ppm, making them more easily recognized fingerprints for the identification of the structural subtypes of Actaea triterpenes.

Table 10 Characteristic ¹H NMR fingerprinting signals of the aglycone side chains of the five major classes of Actaea triterpenes

In addition, the skeletal methyl groups of Actaea triterpenes can serve as powerful structural reporter groups. As shown in Table 11, each type of Actaea triterpene has a particular pattern of methyl signals in the ¹H NMR spectra, which encodes the structural characteristics of the triterpene skeletons. Therefore, as the Me ¹H signals are almost all singlets (except that of CH₃-21), they produce patterns that can be used for rapid dereplication in a manner similar to barcodes. Based on this hypothesis, Qiu et al. developed a robust and automatic dereplication system using only the methyl ¹H NMR signals for a group of Actaea triterpenes (8).

Table 11 ¹H NMR “barcoding” of the skeletal methyl groups of the five major classes of Actaea triterpenes

The concept of using methyl ¹H NMR shifts for structural dereplication of the Actaea triterpenes has a particular advantage in that methyl resonances are usually singlets of relatively high intensity. In approximation, comparing a ddd methylene (1H) with a singlet methyl (3H) signal, the individual spectral lines of the former are ~24-fold lower in intensity. Accordingly, minor triterpenes with a content of more than 4% become visible by virtue of their methyl signals even in crowded regions of the spectra.

Two statistical methods have been applied to establish the mathematical relationships between the methyl shifts and the structural characteristics of the Actaea triterpenes. In order to create a more universal dereplication system, an in-house NMR database was initially constructed to include not only the triterpenes identified in A. racemosa, but also those from many other Actaea species. Using a canonical discriminant analysis (CDA), all the Actaea triterpenes included in the in-house database could be classified with an overall correct rate of 86.9% (see Fig. 6).

Fig. 6

Panel a shows that the first three factors account for 97.4% of the total variance in the Me shifts of the Actaea triterpenes. Panel b shows the sub-cluster of all triterpenes with seven skeletal Me groups having CDA-1 scores between 0 and 10, which form further sub-clusters depending on the specific skeleton types [taken from Qiu et al. (8) with permission from the American Chemical Society]

In addition, Classification and Regression Tree Analysis (CART) was used to build a dereplication system for the Actaea triterpenes. CART is a machine-learning technique ideal for large and unbalanced data sets with many descriptors. It generates a tree-like graph or model as a binary-decision support tool to identify the origin or class of the samples being considered. Figure 7 shows the classification binary tree (CBT) generated from CART analysis for the classification of Actaea triterpenes with seven skeletal methyl groups. This CBT is characterized by 13 terminal nodes and 12 non-terminal nodes, with an overall success rate of 94.4%. Leave-one-out cross-validation (LOOCV) indicates that the model has an excellent prediction rate for the majority of the Actaea triterpenes (cimigenols 80.0%, cimiracemosides 91.7%, and hydroshengmanols 100%). This means the CBT model can not only be used for the dereplication of known compounds, but also it has the potential to identify the aglycone type of compounds yet to be discovered. Considering inescapable variations of reported ¹H chemical shift information due to inconsistencies in, e.g. temperature and calibration (TMS vs. residual solvent), the discriminative power of the model could be improved further by future use of a standardized NMR acquisition protocol, such as 1D qHNMR protocols (22, 23).

Fig. 7

The CBT model for classification of Actaea triterpenes with seven skeletal Me groups by using the Me ¹H NMR shifts as descriptors (Me1 > Me2 > … > Me8). Methyls are numbered according to their chemical shift from the most upfield one (8)

The remaining structural elements, such as substituents and sugar moieties, are more readily recognized by their fingerprinting signals in the ¹H NMR spectra. For example, the occurrence of the double bond at C-7 can be deduced from the ¹H chemical shifts of cycloartane protons, e.g. H-19a, a doublet (d) signal at ~1.00 ppm. The sugar moieties, commonly xylose and arabinose, can be differentiated by the multiplicity pattern of their H-5′b signals: a triplet (t) at ~3.75 ppm with J = ~11 Hz for xylose, and a doublet of doublets (dd) at ~3.80 ppm with J ~13 and ~2 Hz for arabinose. The acetoxy substituents at C-12 can be readily identified by the singlet methyl signal at ~2.10 ppm, and confirmed by observing a dd signal (J ~ 9 and ~4 Hz) for H-12 at ~5.10 ppm.

2.2 Cimicifugic Acids

2.2.1 Structural Characteristics

Cimicifugic acids (Table 12) are condensation products of a benzyltartaric acid moiety, such as fukiic acid, and a cinnamoyl moiety, as exemplified by caffeic acid. Several cimicifugic acids were isolated from the aqueous ethanol extract of BC, including cimicifugic acid KC (fukinolic acid (53)), cimicifugic acid KF (cimicifugic acid A, (54)), cimicifugic acid KI (cimicifugic acid B, (55)), cimicifugic acid PF (cimicifugic acid E, (56)), cimicifugic acid PI (cimicifugic acid F, (57)), and cimicifugic acid KS (58). Furthermore, the acyl residues of 53–58, i.e. caffeic (48), ferulic (49), isoferulic (50), piscidic (51), and fukiic acids (52), were isolated as the free acids, also.

Table 12 Structures of (A) the phenylpropanoic acids and their derivatives, including the cimicifugic acids (B)

2.2.2 Naming System

Since the initial discovery of fukinolic acid, historical nomenclature has evolved by giving fukinolic acid congeners isolated from Cimicifuga/Actaea species the names of cimicifugic acids, adding alphabetical letters in the order of their discovery. Structurally, cimicifugic acids are dimeric molecules, consisting of a benzyl tartaric acid moiety and a cinnamoyl moiety, either of which can exhibit variations in the substitution pattern of their respective aromatic rings. In order to directly connect their structural features with the compound names, a new nomenclatural system was established for cimicifugic acids (24). In this systematic nomenclature, two letters are added to the name of the compound class, cimicifugic acid, to indicate the substitution pattern of the two aromatic rings, Ar _A and Ar _B (see Fig. 8). Each letter originates from the abbreviation of the trivial name of the similarly substituted phenylpropanoic moiety (see Sect. 2.2.1).

Fig. 8

The rational naming system for cimicifugic acids. The compounds denoted by asterisks are yet to be found in A. racemosa

2.2.3 Isolation Techniques

Cimicifugic acids are phenolic compounds, which bear several acidic hydroxy groups. Therefore, polyamide column chromatography is an excellent technique for their separation. The selective interaction with the polyamide stationary phase results from the formation of intermolecular hydrogen bonding between the carboxyl groups of the polyamide and the hydroxy groups of the analytes. Common protocols include the use of an aqueous ethanol BCE and the performance of an initial fractionation using polyamide column chromatography, with aqueous methanol for elution. This affords an enriched fraction of cimicifugic acids. Further separation of individual compounds is then performed on repeated RP-MPLC (e.g. C₁₈). Preparative RP-HPLC has usually been employed in the last purification step to resolve the congeners that are otherwise difficult to separate by RP-MPLC (25).

Recently, a protocol for pH-zone refining centrifugal partition chromatography (pHZR-CPC) has been established to separate cimicifugic acids (24). This methodology was developed based on the hypothesis that the cimicifugic acids occur in the BCE in association with bases (alkaloids), some of which are responsible for the observed 5-HT₇ activity. The separation procedures started with ethyl acetate–water partitioning of the methanolic BCE. The aqueous layer was then subjected to column chromatography on Amberlite XAD-2, which was eluted sequentially with water and methanol. The methanolic eluate was further subjected to pHZR-CPC in the RP mode using the solvent system ethyl acetate–butanol–water (1:4:5). The pH was adjusted by adding 0.3% (v/v) of a 28% aqueous ammonia solution and 0.1% (v/v) of trifluoroacetic acid (TFA) in the mobile (lower) and stationary (upper) phases, respectively. This facilitated the separation of the acids from their basic complex-partners. Finally, the enriched fraction of the dissociated acids was separated in a second step of CPC with the solvent system HEMWat +5 (n-hexane–ethyl acetate–methanol–water, 3:7:3:7, with 0.2% of TFA in both phases) to afford the pure compounds.

2.2.4 Structural Elucidation

Combined use of mass spectrometry and NMR spectroscopy is an efficient approach for the structural elucidation of cimicifugic acids. Initially, ESI-MS can be used for the determination of the molecular weight of the compounds. Typically, negative-mode ESI is employed because it has been found to be more sensitive for these phenolic acids. Further structural elucidation is typically aided by 1D- and 2D-NMR methods, including ¹H, ¹³C, COSY, HSQC, and HMBC experiments (24, 25).

2.2.5 Dereplication

One-dimensional ¹H-NMR spectra can serve as highly characteristic fingerprints for the cimicifugic acids. Gödecke et al. (24) reported the use of a structure-based spin-pattern analysis for the dereplication of known cimicifugic acids. This method is based on the ¹H NMR spectroscopic differences resulting from the characteristic substitution patterns on the aromatic rings of the acids (see Fig. 9). For example, the Ar _A rings of monooxygenated acids give rise to an AA′XX′ spin resonance that is distinctly different from the AMX pattern of their 3,4-dioxygenated counterparts. The chemical shifts of the signals of the Ar _B ring are affected by the position of methoxylation, as opposed to hydroxylation. In addition, the signals of the methoxy protons also exhibit shift variations dependent on their position in the Ar _B ring.

Fig. 9

¹H NMR spin pattern of cimicifugic acids: KF, KI, KK, PF, and PI (400 MHz, CD₃OD, chemical shift reference set to 3.21 ppm for residual methanol-d ₃, relative to TMS signal at 0.0 ppm as external standard). According to (24), reprinted with permission from Wiley Ltd

2.3 Nitrogen-Containing Constituents

The first reported occurrence of alkaloids in A. racemosa was reported at the beginning of the last century. Horace Finnemore published in the Pharmaceutical Journal in 1909 (26) that he had detected trace amounts of alkaloids in both basic and acidic extracts of BC, although he did not indicate what kind of alkaloid reagent he used. There was no further report of alkaloids from A. racemosa until 1956, when Gemeinhardt et al. reported the finding of N-methylcytisine (59) from A. racemosa (27, 28).

However, this type of alkaloid has been found usually in the genera Cytisus (Fabaceae) and Caulophyllum (Berberidaceae), which are generally prolific in alkaloids. Cytisine was first isolated from Cytisus laburnum in 1862 (see (29) and references therein). However, from a biosynthesis perspective, it seems unlikely that N-methylcytisine was from Actaea. It is more likely that the plant material in Gemeinhardt’s research was misidentified.

In 1965, Crum et al. screened alkaloids in different parts of seven native Ohio plants (30). The authors used basic and acidic approaches to prepare the extracts. Five different alkaloid reagents were used to detect alkaloids: (1) Dragendorff reagent: bismuth potassium iodide; (2) Mayer reagent: mercuric potassium iodide; (3) Silicotungstic acid reagent: 12% aqueous silicotungstic acid; (4) Sonnenschein reagent: phosphomolybdic acid; and (5) Wagner reagent: iodine-potassium iodide. The criterion was to determine if the investigated plant or plant part tested positive for alkaloids was that the reaction “gave, as a minimum, a distinct turbidity with four of the five reagents in either the acid or base extraction method”. The use of five different reagents eliminated the commonly observed “false-positive” results when only using one reagent. For instance, the most widely used alkaloid reagent, Dragendorff reagent, gave a false-positive color reaction when 2-pyrones were present in the extract (31). Based on Crum et al.’s screening procedure, no alkaloids were detected in acidic and basic extracts of leaves and stems or roots/rhizomes, but occurred in an acidic extract of the seeds. While the seeds of A. racemosa have not been used as a drug or for medicinal purposes, this finding was the first indication of the occurrence of alkaloids in A. racemosa.

In 2005, Fabricant et al. were the first to report a new type of guanidine alkaloid, cimipronidine (60), as a major constituent from an n-BuOH soluble fraction of a methanolic BCE (32). This finding clarified the zwitterionic nature of some of the Actaea alkaloids and subsequently changed the phytochemical protocols used in the same laboratory to further investigate the plant.

Successively more alkaloids were isolated and/or identified from BC. In 2008, the serotonergic active principle, N _ω-methylserotonin (61) (33), was identified from the highly retained fraction of a pH-zone refining fast centrifugal partition chromatography (FCPC) procedure separating the tertiary subfraction from a methanolic crude extract. In addition to two cimipronidine derivatives (62 and 63), Gödecke et al. isolated and identified salsolinol (64) and dopargine (65) (34), which represent another class of alkaloids in BC. These alkaloids can be explained as representing products of dopamine with an aldehyde (acetaldehyde or γ-guanidinobutraldehyde) as substrates through a Pictet-Spengler reaction with an enzyme, such as strictosidine or norcoclaurine synthase.

In 2012, Nikolić et al. published a detailed high-resolution LC-MS analysis of the nitrogen-containing components in a standardized 75% EtOH BCE. The extract was the same material that was used in Phase I and Phase II-b clinical trials conducted by the University of Illinois at Chicago (UIC)/National Institutes of Health (NIH) Botanical Center (35). Using MS² accurate mass measurements, a combination of database searches, known biosynthesis pathways of alkaloids, and comparison with authentic standards and their fragmentation patterns, the authors identified 73 nitrogen-containing compounds with varying degrees of confidence. These results revealed that BC contains a large number of remarkably diverse and previously unreported nitrogen-containing metabolites. The observed compounds covered both primary and secondary metabolites and included derivatives of amino acids, nucleobases, aporphines, betaines, β-carbolines, cholines, cinnamides, guanidines, isoquinolines, and protoberberines (Fig. 10).

Fig. 10

XAD-2 water fraction (top) and the last fraction (bottom) of pH-zone refining FCPC separation of the MeOH fraction of XAD-2 chromatography from the extract (from Nikolić et al. (35) with permission of the Elsevier B.V.) The compounds discovered in this study were, for the most part, characterized by mass spectrometry. Hence no stereochemistry was shown, even in cases where the stereochemistry could be assumed with reasonable confidence

2.3.1 Structural Diversity

2.3.1.1 Primary Metabolites

2.3.1.1.1 Amino Acid Derivatives

This group consists of many amino acids. As well as two nonessential amino acids, pipecolic (66) and pyroglutamic (67) acid, others are derivatives of essential amino acids with a feruloyl or isoferuloyl group attached to the α-amino group. Eight arginine (68–75), two phenylalanine (75, 76) and one histidine (78) derivatives were identified in a BCE.

2.3.1.1.2 Nucleobase Derivatives

As basic units of genetics, nucleobases are found within DNA, RNA, nucleotides, and nucleosides in all organisms. In this group, four adenine derivatives (79–82) were identified from a 75% alcoholic BCE. The two other nucleobases found were cytidine (83) and guanosine (84).

2.3.1.2 Secondary Metabolites

2.3.1.2.1 Aporphines

Aporphines are a class of isoquinoline alkaloids. More than 500 aporphine alkaloids have been isolated from various plant families, and many of these compounds display potent cytotoxic activities, which may be exploited for the design of anticancer agents (36). From this group, six known compounds (85–90) were identified from BC.

2.3.1.2.2 Betaines

As compatible solutes or osmoprotectants, betaines are an important class of naturally occurring compounds to balance the osmotic difference between the cell’s surroundings and the cytosol. Five BC metabolites were identified as belonging to this group (91–95) (37).

2.3.1.2.3 β-Carbolines

β-Carboline alkaloids are one structural sub-type of the indole alkaloids. Previously, most research on the β-carbolines alkaloids focused on effects on the central nervous system, such as being inverse agonists of benzodiazepine receptors, and having an affinity for 5-HT_2A and 5-HT_2C receptors (38–40). Recently, research has revealed many new pharmacological effects relevant to antitumor (41), antiviral (42), antimicrobial (43), and antiparasitic activities (44). Four of these alkaloids (96–99) were identified by LC-MS/MS, and the structure of cimitrypazepine (96) was confirmed by synthesis.

2.3.1.2.4 Cinnamides

Amides of ferulic and isoferulic acids were detected in a BCE. This class of compounds has previously shown broad biological properties, including central nervous system depressant, anticonvulsant, muscle relaxant, antiallergic, antineoplastic, and anti-infective activities. The three cinnamides (100–102) were identified in a BCE.

2.3.1.2.5 Cholines

Cholines generally refer to the various quaternary ammonium salts that contain the partial structures of derivatives of N,N,N-trimethylethanol-ammonium cation. Choline itself is a water-soluble essential nutrient. Egg and fatty meats are high choline-containing foods. Despite the perceived benefits of cholines, dietary recommendations have discouraged eating high-choline foods. However, in 2005, a National Health and Nutrition Examination Survey stated that only 2% of postmenopausal women consume the recommended intake for choline. So far, a total of five cholines (103–107) have been identified from a BCE.

2.3.1.2.6 Guanidines

Besides the above-mentioned three guanidines, 60, 62 and 63, a related structure to this type of alkaloid, 108, was identified using LC-MS. It has a similar fragment pattern to that of cyclocimipronidine (62) and cimipronidine methyl ester (63), except for having 14 Da more and 18 Da less in its molecule ion than do 62 and 63, respectively, which led to the assignment of the methyl group to the amino group at the C-7 position, forming a methylamine group.

2.3.1.2.7 Isoquinolines

Since the first isoquinoline derivative was isolated from coal tar in 1885, more than 400 simple isoquinoline derivatives have been reported. This class of alkaloids shows many different bioactivities, from antimalarial, anti-HIV, antineoplastic, antimicrobial, and antibacterial to insect growth retardation. Several tetrahydroisoquinoline derivatives have been found to act as neurotoxin precursors linked to Parkinson’s disease. In addition to dopargine (63), six hydrogenated isoquinoline alkaloids, including (109–110) and benzyl substituted (111–114) derivatives, were found in BCE.

2.3.1.2.8 Protoberberines

The protoberberine alkaloids are derived biogenetically from tyrosine. The quaternary protoberberine alkaloids represent approximately 25% of all currently known alkaloids with a protoberberine skeleton isolated from natural sources. The protoberberines are mainly distributed in the Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, and Annonaceae. More than 100 berberine alkaloids, including tetrahydroprotoberberines, and quaternary protoberberines were reported. The bioactivities of protoberberines (45, 46) include the inhibition of DNA synthesis, protein biosynthesis, membrane permeability, and the uncoupling of oxidative phosphorylation. From a BCE, two protoberberine alkaloids were identified tentatively by LC-MS/MS, but their unambiguous identification could not be achieved due to the lack of authentic standards. As a result, the structures of phellodendrine (115a) or cyclanoline (115b), and N-methyltetrahydrocolumbamine or its isomer (116) could be proposed by comparison of their fragmentation patterns with those of structural analogs.

2.3.1.2.9 Protopines

Protopine is a benzylisoquinoline alkaloid occurring in the genera Corydalis and Fumaria belonging to the family Papaveraceae. The title compound has been found to inhibit histamine H1 receptors and platelet aggregation, and can act as an analgesic. Two protopine alkaloids (117, 118) were identified on the basis of MassBank database searches and were confirmed by comparison with authentic standards (47).

2.3.1.2.10 Pyridoxines

Vitamin B₆ is the classic pyridoxine, and analogs of this simple compound have been described as helpful to balance hormonal changes in women and aid the immune system. Two compounds belonging to the pyridoxines (119, 120) were identified on the basis of MassBank database searches and confirmed by comparison with authentic standards.

2.3.1.2.11 Miscellaneous

Several other nitrogen-containing components were also identified from BCE including 121–134. The guanidines 121–127 share the highly basic group with the cimipronidines (60, 62, and 63) as well as the salsolinol derivative 65. A general observation is that the BC alkaloids include a variety of guanidines. Similarly, the dopamides 128–131 share certain structural similarities with cimicifugic acid (Sect. 2.2), and it is not unlikely that hybrid molecules are present in BC.

2.3.2 Structural Elucidation

Although alkaloids and other nitrogen-containing compounds are often present at relatively low abundance in plant extracts, this is offset by the fact that they may have potent bioactivities. Accordingly, small quantity changes can lead to large differences in the observed activities of crude extracts. For example, N _ω-methylserotonin (61) shows 5-HT₇ receptor binding activity (IC ₅₀ = 23 pM), induces cAMP (EC ₅₀ = 22 nM), and blocks serotonin re-uptake (IC ₅₀ = 490 nM) (33). Small variations of the absolute content of this marker compound in a BCE preparation would cause major changes in their biological potency and greatly affect biological standardization, while requiring high-sensitivity chemical analysis for quantitation. LC-MS/MS plays a crucial role in the identification of such minor components. High resolution LC-MS/MS using a Q-TOF instrument, analysis of characteristic fragmentation patterns, and database searches (e.g. MassBank) have been demonstrated to be a viable approach to identify a major portion of the alkaloids (~70%, based on LC-MS full-scan peak intensity) in a BCE with the highest confidence level 1, which is established by comparing the retention time and fragmentation pattern of an unknown with those of an authentic standard (48). Of the remaining substances from a total of 73 alkaloids, 26% were identified with confidence level 2, i.e. by comparing their tandem mass spectra either with published spectra or with tandem mass spectra of close structural analogs. Only 4% of the alkaloids described in this study could be identified at no better than confidence level 3. At this level, characterization can only ascertain the chemical class of an unknown based on the similarity of tandem mass spectra with known compounds of the same class. A summary of general LC-MS/MS identification strategies and the characteristic fragmentation patterns of key alkaloids found in BC has been reported recently (35).

2.3.2.1 Nucleobases and Their Derivatives

Most nucleobases and nucleosides from BC were identified by spectral database searching and comparison with authentic standards. The shared elemental composition of methylated adenosine 81 and 82 (C₁₁H₁₅N₅O₄) was of particular interest. Besides a few product ions, the compounds had different product ion tandem mass spectra. The product ion tandem mass spectrum of 81 exhibited only one fragment ion (m/z 150, [MH − 133]⁺), which is protonated after the loss of a sugar moiety. Its elemental composition corresponds to methylated adenine (C₆H₈N₅). Database searches revealed that methylation most likely occurred on the amino group, which led to tentative identification of this compound as N-methyladenosine (confidence level 3). In contrast, two fragment ions were observed in the tandem mass spectrum of 82, one was protonated adenine (m/z 136 [MH − 147]⁺) after loss of a sugar moiety, and another corresponded to loss of ammonia from adenine (m/z 119, [MH − 147 − 17]⁺). Comparing its fragments with those of adenosine, this compound has an extra CH₂ unit in the sugar moiety. Based on literature searches, 82 is the most likely 2′-O-methyladenosine (confidence level 3).

2.3.2.2 Guanidino Alkaloids

Guanidino alkaloids, either acyclic or cyclic, produce a characteristic loss during collision-induced dissociation (CID) in the MS. For acyclic guanidino alkaloids or arginine derivatives (including 68–75, 121–127), the characteristic loss is a neutral guanidine (59 Da; CH₅N₃). To distinguish this neutral guanidine loss from other equal losses of 59 Da, an accurate mass measurement of the fragment is necessary. For example, acetamide (C₂H₅NO) is equal to guanidine with a MW of 59 Da. However, both can be distinguished readily by the elemental compositions. In addition to the loss of neutral guanidine, protonated guanidine [(NH₂)₃C]⁺ of m/z 60 can usually be observed in the acyclic alkaloids. It has been proposed that guanidine is protonated via an ion-neutral complex (Scheme 1), and that its abundance is inversely proportional to the applied collision energy. Although the collision energy was applied as low as 5 eV, this ion was still not abundant, at only ~35% (49).

Scheme 1

Proposed fragmentation pathway of guanidino alkaloids

Representing one group of the BC guanidino alkaloids, the fragmentation pattern of the arginine derivatives, 69–75, is very similar to that of arginine (68) itself (see Fig. 11). The presence of an ion at m/z 60 indicated that there was no substitution on the guanidino group, and that acetylation occurred on the amino group. Further, the identification of α-N-acetylarginine was confirmed by comparison with an authentic standard.

Fig. 11

Product ion tandem mass spectra of (a) arginine (68), (b) N-acetylarginine (70), and (c) N-formyl-arginine (69). From Nikolić et al. (35), with permission of Elsevier B.V.

For another group of guanidino alkaloids, γ-guanidinobutanol and its analogs (122‒127), a characteristic neutral loss of 59 Da was observed at m/z 87 for 125, m/z 71 for 124, and m/z 73 for 123, indicating that they all contain free guanidine groups (see Fig. 12).

Fig. 12

Product ion tandem mass spectra of (a) γ-guanidinobutyric acid (125), (b) γ-guanidino-butyraldehyde (124), and (c) γ-guanidinobutanol (123). From Nikolić et al. (35), with permission of Elsevier B.V.

The compound, γ-guanidinobutyric acid (GBA, 125), was identified by database searches and confirmed with an authentic standard. The fragmentation pathway was proposed as shown in Scheme 2. The product ion of m/z 87 is a protonated butyrolactone formed through an S_N2 attack of the carbonyl oxygen on the carbon atom bearing the guanidino group (see Scheme 2a). This is supported by observation of protonated guanidine at m/z 60. At higher collision energies, protonated guanidine is not observed due to insufficient survival time of the ion-neutral complex. An additional minor pathway for formation of the ion of m/z 87 is by elimination of ammonia from protonated γ-aminobutyric acid (GABA; m/z 104), as determined in separate ion-trap experiments. Similarly, the product ion of m/z 86 has an elemental composition of C₄H₈NO corresponding to protonated butyrolactam. Ion-trap experiments indicated that the main pathways of this ion are a loss of carbon diimine (−42 amu, NH = C = NH) from the ion of m/z 128 [MH − H₂O]⁺ and a loss of water from protonated GABA (see Fig. 12a).

Scheme 2

Proposed fragmentation pathways for γ-guanidinobutyric acid (125) (35)

In contrast to acyclic guanidines, the loss of carbon diimine is a characteristic fragmentation reaction. The two compounds, cimipronidine (60) and cyclocimipronidine (62), were identified and their structures confirmed with extensive NMR data. In addition, a methyl ester of cimipronidine (63) as well as N-methylcyclocimipronidine (108) were identified by analogy with the fragmentation patterns of 60 and 62.

2.3.2.3 Hydroxycinnamic Amides

Caffeic, ferulic, and isoferulic acids have been reported as major hydroxycinnamic acids in BC. The identification of amides and esters of ferulic/isoferulic acid (72–75, 100–102, 105–106, 128–131) was enabled by their characteristic fragmentation pattern, which is dominated by the product ions of m/z 177, 149, 145, 117, and 89 originating from the ferulic/isoferulic acid moieties of the amide. Caffeic acid amides such as 71 produce a similar ion series at m/z 163, 145, 135, 117, and 89 (see Fig. 13c). The presence of a low abundance fragment ion of m/z 163 with the elemental composition of C₉H₇O₃ can be used to distinguish isoferulic acid from ferulic acid (50).

Fig. 13

Product ion tandem mass spectra of amides of hydroxycinnamic acids with amino acids: (a) feruloyl arginine (72), (b) isoferuloyl arginine (73), (c) caffeoyl arginine (71), and (d) isoferuloyl histidine (78). Ion series corresponding to the acid portion are labeled “*” for ferulic and caffeic acid in (a) and (c), respectively, while those corresponding to the amine portion are labeled “◇” for arginine and histidine in (a) and (d), respectively. From Nikolić et al. (35), with permission of Elsevier B.V.

Once the diagnostic ions from ferulic/isoferulic acid are observed in the product ion spectrum of an unknown compound, the amine portion of the amide can be deduced based on a database search of the elemental composition of the remainder of the molecule. As an example of this identification strategy, Fig. 13 shows the product ion spectra of compounds 72 and 73, both with an elemental composition of C₁₆H₂₂N₄O₃. Both spectra show a typical ferulic/isoferulic acid amide fragmentation pattern, with 72 showing an additional peak at m/z 163. This suggested that 72 is an amide of ferulic acid, while 73 is an amide of isoferulic acid. The database search for the composition of the remainder of the molecule (C₆H₁₄N₄O₂) suggested that the amine portion is the amino acid arginine. Protonated arginine was observed at m/z 175, along with other ions originating from fragmentation of arginine such as ions of m/z 158, m/z 70 and m/z 60 (see Fig. 13a). The presence of the latter two ions indicated a free guanidino group and confirmed that the carboxylic acid was attached to the α-amino group rather than a guanidino nitrogen.

2.3.2.4 Choline and Betaine Alkaloids

Both choline and betaine alkaloids contain the quaternary nitrogen group, trimethylammonium. The characteristic fragment is a loss trimethylamine (−59 Da; Me₃N), which can be used to detect, specifically these two classes of alkaloids. For example, 104 displayed a characteristic loss of trimethylamine from the precursor ion of m/z 208 to form an ion at m/z 149, which can further fragment to lose CO₂ and produce an ion of m/z 105.

2.3.2.5 Pictet-Spengler Adducts with Tryptamine Derivatives

Compounds 96 and 99 eluted at 3.3 and 3.8 min, respectively, during the LC-MS and had identical elemental composition (C₁₂H₁₄N₂O) but very different fragmentation patterns (see Fig. 14a, b). The elemental compositions of several key fragment ions such as m/z 160, 159, 132, and 117 were the same as those observed in the product ion tandem mass spectra of N _ω-methylserotonin and serotonin (33), suggesting that 96 and 98 may have the same biosynthetic pathway through a Pictet-Spengler reaction. Accordingly, condensation of serotonin and N _ω-methylserotonin with formaldehyde was carried out under acidic conditions. By comparing the fragmentation patterns of these adduct ions with those of 96 and 99, identical fragment patterns demonstrated that 96 and 99 indeed resulted from Pictet-Spengler addition of N _ω-methylserotonin with formaldehyde. Compound 96 was identified as 6-hydroxy-2-methyl-1,2,3,4-tetrahydro-β-carboline, while the structure of 99 was elucidated as 3,4,5,6-tetrahydro-7-hydroxy-5-methyl-1H-azepino[5,4,3-cd]indole by a combination of its NMR and MS data.

Fig. 14

Product ion tandem mass spectra of the Pictet-Spengler adducts of N _ω-methylserotonin and formaldehyde. From Nikolić et al. (35),with permission of Elsevier B.V.

Fragmentation of 96 is dominated by retro Diels-Alder fragmentation to form an ion at m/z 160 (see Fig. 14a). As retro-Diels-Alder fragmentation is not possible for 99, fragmentation occurs by opening of the azepine ring, followed by elimination of methylene imine to form a base peak of m/z 174.0938 (see Fig. 14b). Both 96 and 99 originate from the same precursor species that cyclizes into either a six- or seven-membered ring (see Scheme 3), as has been demonstrated in studies of the reactions of serotonin and N _ω-methylserotonin with various aldehydes (50, 51).

Scheme 3

Proposed mechanism of formation of 96, 98, and 99 (143)

The elemental composition of 98 (C₁₂H₁₂N₂O) is two hydrogens less than that of 96 and 99 (C₁₂H₁₄N₂O), suggesting a dihydro-β-carboline structure. The loss of a methyl radical (m/z 186), along with the fragment ion of m/z 170, [MH-CH₃NH₂]⁺, indicated that the N(2) nitrogen on the β-carboline ring is methylated. Based on biosynthetic considerations, the most likely position of the double bond should be between 1 and 2. This structure was confirmed by comparison of retention time and fragmentation pattern with an authentic sample of N(2)-methyl-6-hydroxy-3,4-dihydro-β-carboline.

The product ion spectrum of compound 97 was dominated by an ion of m/z 144 with the elemental composition (C₁₀H₁₃N₂), corresponding to protonated tryptamine. The fragmentation of the product ion m/z 144 showed an identical pattern to authentic tryptamine, suggesting that this compound is a tryptamine derivative. The neutral loss of iminoacetic acid (C₂H₃NO₂) combined with database searching suggested that 97 might be a tetrahydro-β-carboline carboxylic acid. As both 1- and 3-substituted isomers are known, both analogs 97a and 97b were synthesized and compared with 97 in order to confirm the correct regioisomer as 97b.

2.3.2.6 Other Alkaloids

The fragment patterns of other alkaloids, including benzylisoquinoline (52), aporphine (53, 54), protoberberine (55, 56), and protopine alkaloids (47) as well as other miscellaneous classes (57–60) have been discussed in detail elsewhere. Verifying their presence in A. racemosa (35) was mostly based on spectral database searching and comparison with appropriate authentic standards.

3 Fingerprinting

Recently, the concept of “fingerprinting” has been used increasingly in herbal medicines for their chemical/biological profiling, botanical identification, and quality standardization. The basic theory is that the individual plant samples have unique genetic and metabolomic profiles, which can be converted into “fingerprints” and used for identification. Commonly used techniques for depicting these characteristic profiles include DNA sequencing, chromatographic, and spectroscopic analysis, and a combination of these. As the resulting profiles are usually comprised of large and complex datasets, chemometric approaches such as classification, pattern recognition, and clustering can be employed to enable or facilitate data interpretation and comparison.

Fingerprinting of A. racemosa has been reported using a variety of chemical and biological techniques. These have focused mainly on two classes of compounds, the triterpenes and the phenolic constituents, which are frequently considered as the major bioactive substances in A. racemosa. Verbitski et al. developed an efficient and economical approach using thin-layer chromatography (TLC) combined with bioluminescence, which provided characteristic patterns corresponding to toxicity profiles for A. racemosa as well as many other Actaea species (61). This technique may not only be used to examine A. racemosa adulterants, such as blue cohosh (Caulophyllum thalictroides), but also to detect unknown contaminants. However, TLC is still less frequently used for chemical fingerprinting due to its limited dynamic range and/or resolution, relatively low sensitivity, and limited specificity. In comparison, HPLC-based methods are more popular, but not necessarily always more specific or of higher resolution. UV, ELSD, and MS detection are commonly used in online hyphenated HPLC separation methods. Of these three detection methods, UV is very appropriate for the detection of phenolic constituents in A. racemosa (62, 63). However, due to the lack of UV pharmacophores in most of the triterpenes, they are better detected by ELSD (64–66) or MS. Compared to UV and ELSD, MS is much more powerful for the identification and quantification of both triterpene and phenolic constituents, due to higher sensitivity and selectivity, but also by providing more structural information. A number of LC-MS techniques have been employed for the fingerprint profiling of A. racemosa. Some of the examples include LC/APCI-MS (19, 63, 65), LC/TIS-MS (67), and LC/EI-MS/MS (68). Instead of using a single detection method, He et al. reported a comprehensive approach based on HPLC-PDA/APCI-MS/ELSD for the fingerprinting of 10 Actaea species, specifically, A. acerina, A. americana, A. biternata, A. dahurica, A. foetida, A. heracleifolia, A. japonica, A. racemosa, A. rubifolia, and A. simplex (62). The combined use of three spectroscopic methods provides increased reliability and versatility for the detection, identification, and quantification of both triterpenes and phenolic constituents in these plants. As a result, the HPLC profiles from multiple detectors provide more detailed fingerprints of the plant samples, and thus improves the overall accuracy of plant identification by chemical fingerprinting.

In addition to chemical methods, a few biological methods have been described for the fingerprinting of A. racemosa. Zerega et al. first reported the use of a DNA fingerprinting technique, Amplified Fragment Length Polymorphism (AFLP), for the differentiation of A. racemosa from three other closely related species, A. pachypoda, A. cordifolia, and A. podocarpa (69). In using four AFLP primer combinations, from these four species altogether 262 unambiguous DNA fragments were generated, of which one was unique to A. racemosa. This characteristic DNA marker was used as a fingerprint to authenticate two commercial A. racemosa products. In a later report, Motley et al. (70) optimized the AFPL analysis and demonstrated that the use of only two-primer combinations was sufficient to identify each species. Principal component analysis (PCA) of the AFPL results enabled the identification of the geographical region from which an A. racemosa specimen was collected. Furthermore, Baker et al. adapted a DNA barcoding methodology to identify unambiguously A. racemosa in dietary supplements (71). According to the Consortium for the Barcode of Life (CBOL), two protein coding regions, matK and rbcL, can be used as core plant DNA barcodes, and two non-coding regions, nrITS and psbA-trnH, can be utilized as supplemental markers. Four regions from the DNA samples extracted from Actaea plants were sequenced after PCR amplification, and two matK nucleotide sequences were found to be specific for A. racemosa. Using these two markers, A. racemosa was correctly distinguished from other Actaea species. Further application of these markers was carried out to examine the adulteration of A. racemosa dietary supplements by other Actaea species. It should be noted that in mainland China, several other species are sold as “Black Cohosh”. Some of these species do not belong to the genus Actaea, but include Vernonia aspera, which is known locally as hei-sheng-ma and is sometimes translated as “Black Cohosh” (4).

4 Names and Origin

As alluded to in the Introduction, “Black Cohosh” has been used with several meanings implied. Today, perhaps the most generally intended meaning of “Black Cohosh” is that of an extract of the underground parts (roots and rhizomes) of A. racemosa—but this is often only implied and not specified (note the definitions for this work, made in the Introduction). However, the term is also used to mean the dried roots and rhizomes, themselves, and occasionally even for the whole plant. The plant was first described by Europeans as Christopheriana facie in 1690 in Leonard Plukenet’s Phytographia, and later was given the name Actaea racemosa by Linnaeus in 1751. Despite this unassailable authority, the name was changed to Macrotrys racemosa, which morphed into Macrotys racemosa and was subsequently changed to Cimicifuga racemosa. The last name was widely used until recently when on the basis of 26SrDNA comparison, botanists reorganized the genera, Actaea, Cimicifuga, and Souliea, and the last two genera disappeared with all of their species being incorporated into Actaea (72). There are now 28 different species of Actaea spread across the entire Northern Hemisphere, with eight endemic to North America and the other 20 occurring mainly in Asia, with three also found in Europe. Despite the predominant use of “Black Cohosh” in the form of root/rhizome extract, even this assignment is not definitive as various types of extracts are sold; 60–80% ethanolic as well as 40% iso-propanolic and other extracts have been used in the search for metabolites or in pharmacological studies. This makes comparison of results from studies, and particularly meta-analysis of clinical trials very difficult and sometimes even impossible. Notably, one of the most widely used commercial preparations, Remifemin^®, has been reformulated from an ethanolic extract to a 40% iso-propanolic extract.

Most plant harvesting (>90%) is from native wild growth (4), and this leads to some concern for sustainability, and to both accidental and deliberate adulteration. A. podocarpa plants resemble those of A. racemosa, and the two species share a fairly common habitat. The traditional Chinese medicine, Sheng-Ma, described as Rhizoma Cimicifugae, includes an extract of the roots and rhizomes of A. heracleifolia, A. dahurica, A. foetida and, less frequently, A. simplex and A. yunnanensis.

One example of highly probable adulteration is the recent discovery of two new triterpenes (135 and 136), which were named “cimipodocarpaside” and “isocimipodocarpaside”, respectively, from a purchased, unauthenticated¹ sample of “Black Cohosh”. Prior to this report, all triterpenes isolated from Actaea having the 9,10 bond cleaved had been described from A. podocarpa. It seems likely that this purported sample of Black Cohosh contained extracts of species of Actaea other than racemosa (73, 74).

Many commercial preparations containing BC are mixtures with extracts of other medicinal herbs, such as red clover, soy bean, magnolia bark, ginkgo, hops, damiana leaf, St. John’s wort, Scutellaria lateriflora, Valeriana officinalis, Passiflora incarnata, chaste tree berry, China root, Chinese bupleurum root, codonopsis root, ginger root, lovage root, rehmannia root, white peony root, horse chestnut seed, and licorice root. However, a number of products purport to be an extract of Actaea racemosa roots/rhizomes, unadulterated with other herbs. These include Remifemin^®, Natrol^®, Spring Valley^®, and CR BNO 1055.

Jiang et al. (75) analyzed the air-dried roots/rhizomes of 15 different species of Actaea, eight endemic to North America and seven to Asia, sampling from 1 to 15 samples per species. Their analysis established some clear marker compounds: cimiracemoside F (38) (76, 77) was present in all samples (10) of A. racemosa, and only in one other sample allegedly from A. pachypoda.

Cimifugin (137) (78) was present in all 27 samples from an Asian species, but only in trace quantities in the four samples from three North American species, two of which are endemic to the Pacific Northwest. Hence, the lack of 38 and the presence of 137 in a commercial sample is compelling evidence that the purported “Black Cohosh” is, in fact, from an Asian species and is not authentic material. The same group (75) had previously carried out an investigation of 11 commercial products available over-the-counter in the New England area and concluded that four were from Asian species, although one of these may have contained some A. racemosa.

Perhaps one of the more unfortunate findings was that (12R)-12-acetoxy-(24R,25S)-24,25-epoxy-(26R/S)-26-hydroxy-3-O-β-d-xylopyranosylacta-(16S,23R)-16,23;23,26-binoxoside (actein) (24) and (12R)-12-acetoxy-(24R,25R)-24,25-epoxy-3-O-β-d-xylopyranosylacta-(16S,23R)-16,23;23,26-binoxoside (23-epi-26-deoxyactein) (29) were found in good quantity in all species examined, with the exception of A. cordifolia, A. podocarpa, and A. laciniata. These two compounds have been, and continue to be, common markers used to calibrate the potency and supposed authenticity of commercial BCEs. Commercial preparations occur as capsules, tablets or solutions, most commonly derived from BCEs, but some are commonly found to contain ground dried BC. Reported extraction solvents vary from 40% isopropanol to 58–79% ethanol and 80% methanol, and all these products/materials have very different triterpene contents (75) and different potencies with respect to binding to the 5-HT₇ subtype of the serotonin receptor (79). The latter can be explained readily with differences in solubilities of the serotonergic in vitro-active principle, N _ω-methylserotonin (61) in the different extraction solvents.

5 Pharmacology

5.1 Estrogenic Activity

A major impediment to the rigorous comparison of results from different studies evaluating potential estrogenic effects of BCEs is the fact that, although it is usually possible to ascertain that “Black Cohosh” is being used to describe the roots and rhizomes of A. racemosa or an extract thereof, the nature of the extract varies widely through the use of different alcohols, methanol, ethanol, and isopropanol, with or without up to 80% water, and made from room temperature to under reflux. In some cases, a commercial preparation such as Remifemin^® has been used, but as mentioned earlier even this product has changed its formulation from an ethanolic extract to an iso-propanolic one.

It may seem very logical, as BCEs have been used to alleviate the adverse symptoms of menopause and menstruation, and as this extract is rich in triterpenes with a clear structural relationship to steroidal hormones, that one or more constituents of BCE probably has phytoestrogenic activity. However, attempts to define such a mode of action have been inconclusive. Estrogenic activity was assessed in a number of different plant extracts, including BCEs using several assays by Liu et al. (80). These assays included binding to the α- and β-estrogen receptors, estrogenic activity as evidenced by induction of alkaline phosphatase, and/or up-regulation of progesterone receptor mRNA in Ishikawa cells (endometrial), as well as up-regulation of presenelin-2 in S30 breast cancer cells. Whereas hops, red clover and chasteberry extracts showed significant activity in the estrogen binding and the Ishikawa cell assays, and although extracts of Angelica sinensis, licorice, Asian and American ginseng showed modest activity in the S30 cell assay, a methanolic BCE showed no activity in any of these assays at 20 μg/cm³. Subsequently, Einbond et al. (81) reported that a BCE inhibited the growth of MCF7 human breast cancer cells. However, the most potent compound was 25-acetoxy-7,8-didehydro-(12R)-12-hydroxy-3-O-β-d-xylopyranosylacta-(16S,23R,24S)-16,23;16,24-binoxoside (138), obtained from C. acerina. This compound inhibited the growth of MCF7 (ER + Her2) human breast cancer cells with an IC ₅₀ of 3.2 μg/cm³, whereas actein showed an IC ₅₀ of 5.7 μg/cm³. For both compounds, this inhibition was increased by further transfection of the cells with Her2.

In addition to the triterpenes, the isoflavonoid, formononetin, a known phytoestrogen, has been reported as a constituent of BC. Initially reported in 1985 (82), its presence was disputed by two groups (64, 83), both in 2002. The 2002 results were refuted in 2004 (84) with the claim that a more accurate analysis could detect formononetin in dried BC at levels of 3.1–3.5 μg/g. In 2006, the Kennelly group, after extending the limits of detection of formononetin to 60 ng/g dry weight, was still unable to detect the compound in any of 13 samples of BC (65). In summary, presently there is no chemical evidence, and also no chemotaxonomic indication, for direct α- or β-estrogenic activity caused by any BCE or components thereof.

5.2 Prevention of Bone Loss

Seidlová-Wuttke et al. (85) have made the case that BCE (BNO 1054, a cold 50% ethanol/water extract) is actually a SERM (selective estrogen receptor modulator), having little or no effect on the uterus in comparison with estradiol-17β (E-17), but inhibiting LH secretion in acute treatment of ovariectomized rats. On chronic treatment (3 months as a food additive), both E-17 and BCE counteracted the metaphyseal bone loss of the femur seen in control untreated ovariectomized animals, although BCE at 400 mg · kg⁻¹ · day⁻¹ was less effective than E-17 at 0.5 mg · kg⁻¹ · day⁻¹. Some correlation of these effects was seen in gene expression for the Type 1 procollagen α-chain, osteoprotogenin, osteocalcin, and tartrate-resistant-α-phosphatase Type 5 genes. A fat deposit seen in the CT scans of the tibias of chronically treated rats was quantified and significantly increased in untreated animals. This increase was almost equally reduced (>50%) in rats treated with E-17 at 0.5 mg · kg⁻¹ · day⁻¹ and BCE at 100 and 400 mg · kg⁻¹ · day⁻¹. It was shown that Remifemin^® at 4.5 mg of triterpene content · kg · day⁻¹ and raloxifene at 3 mg · kg⁻¹ · day⁻¹ when administered orally to ovariectomized rats were both equally effective at lowering urinary levels of the known markers of bone loss, pyridoline, and deoxypyridoline. Moreover, the authors were able to maintain a high level of resistance to bone fracture in these rats.

A mechanistic basis for these beneficial effects on bone structure was provided by Qiu et al. (86) who showed that 25-acetoxy-(15R)-15-hydroxy-3-O-β-d-xylopyranosylacta-(16S,23R,24S)-16,23;16,24-binoxoside (25-O-acetylcimigenol xylopyranoside) (5) inhibited the RANKL or TNF-α pathways to osteoclastogenesis with an IC ₅₀ of approximately 5 μM, while other triterpenes in BCE [cimigenol (16), actein (24), and cimiaceroside B (40)] were inactive or less potent with IC ₅₀ values of 25, 42, and 45 μM, respectively.

Ruhlen et al. (87) found that 23-epi-26-deoxyactein (29) decreased cytokine-induced NO production in murine microglial cells. However, whole CimiPure^® (a BCE containing 2.5% triterpenes) increased this production.

Kim et al. (88) showed that a cold methanol BCE had a strong anti-allergenic effect in several assays, including a murine skin sensitization assay (the local lymph node assay) in which the extract was not an allergen itself. When the extract was administered to rats twice daily for three days, it showed a strong dose-dependent inhibition of anti-IgE-induced passive cutaneous anaphylaxis. In vitro, the extract inhibited the histamine release induced by compound 48/80 in rat peritoneal mast cells. In human leukemia mast cells the mRNA for the inflammatory cytokines, IL-4, IL-5 and TNF-α were less induced by PMA and A23187 in the presence of the extract, but, however, this effect had an inconsistent dose-dependency.

Lee et al. (89), when working with extracts of A. heracleifolia, showed that 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (5) was a strong inhibitor (IC ₅₀ 7.7 μM) of the complement pathway. This compound is present in BC at about two-thirds the concentration of 23-epi-26-deoxyactein (29) (81).

5.3 Potential Anticancer Activity

Burdette et al. (90) have shown that a BCE showed efficient free radical (DPPH) scavenging effects, and with bioassay-directed fractionation ascribed this activity to nine phenolic compounds, methyl caffeate, ferulic acid, isoferulic acid, fukinolic acid, cimicifugic acid A, cimicifugic acid B, cimicifugic acid F, cimiracemate A, and cimiracemate B. These authors went on to show that five of these compounds plus caffeic acid were able to reduce menadione-induced DNA damage in S30 human breast cancer cells, damage that is known to be initiated by reactive oxygen species. Actein (24) inhibited the growth of p53 positive HepG2 liver cells in vitro (MTT assay) with an IC ₅₀ of 27 μg/cm³ (81).

Mimaki et al. (7) isolated from a hot methanolic BCE, (24S)-24-acetoxy-3-O-α-l-arabinopyranosyl-(15R)-15-hydroxyacta-(16S,23R)-16,23;16,25-binoxoside (44) and its xylopyranosyl analog. These compounds had no effect on the ATCH secreted from AtT20 cells (mouse anterior pituitary tumor cells) by themselves, but each significantly increased the effect of corticotrophin-releasing factor (CRF) on the ATCH release by these cells.

5.4 Stress Relief

Using single doses of 7.14 and 35.7 mg · kg⁻¹, Einbond et al. (91) have studied the effect of orally administered actein (24) on stress and statin-associated responses in Sprague–Dawley rats. The study also included measurements of the blood levels, which peaked at 2.4 μg/cm³ at 6 h for the high dose and fell to 0.1 μg/cm³ at 24 h. The authors saw statistical differences in the transcription of 297 and 1325 genes at 6 and 24 h, respectively, compared with control animals (gavage with water). The genetic effects included the down-regulation of erythropoietin, CYP2C, and ATP synthase genes at 6 h, and suggested that the primary effects of actein (24) “may be on hypoxia and the stress response and mitochondrial phosphorylation.” There was a weak signature match to genes involved in the effects of statins on cholesterol biosynthesis, and sections of the fatty acid biosynthesis pathway were significantly down-regulated. The study confirmed the effects on cholesterol biosynthetic pathways by showing that actein (24) inhibited the proliferation of p53 positive HepG2 liver cells. Interestingly, the reported IC ₅₀ values were identical with those determined by Burdette et al. 7 years earlier (90).

Nadaoka et al. (92) extended their earlier studies on the effect of BCE on stress in mice by a small, but well designed, placebo controlled, randomized, double blinded, cross-over study in healthy adult humans. In this, they monitored both physiological and psychological responses to stress induced by mental arithmetic tasks beyond capable completion in a limited time period. Salivary cortisol and chromogranin-A were measured before administering the drug, and during and immediately after the task, and then following a rest period. Both indicators rose significantly during the task and returned to near normal values by the end of the task. There was no significant difference between placebo and treated groups in the cortisol levels, but there was a major amelioration in the rise of chromogranin-A levels in the treated group compared with the controls. Psychological evaluation was done with a visual analog scale and use of the Japanese version of the State-Trait Anxiety Inventory (93).

In a separate, but similar, EEG monitored experiment, topographical changes in the alpha waveband from both the left and right occipital areas were strongly decreased during the task, and only the treated groups showed a tendency to recover after the rest period, although these failed to reach normally accepted statistical significance (P = 0.06 for left and 0.07 for right occipital areas). The authors proposed that BCE could “be useful for the prevention and treatment of stress related disorders.”

More recently, mode of action studies on BCEs have concentrated on CNS effects and this emphasis owes its origin to the isolation and characterization of N _ω-methylserotonin from a cold methanolic BCE and its very potent 5-HT₇ receptor binding (IC ₅₀ = 23 pM), ability to induce cAMP (EC ₅₀ = 22 nM), and to block serotonin uptake (IC ₅₀ = 490 nM). These findings created a compelling argument that at least some of the effects of BCE may be via the CNS. The authors estimated that a 120 mg dose of BCE would contain 3.7 μg of N _ω-methylserotonin (33).

Following up on the CNS-active constituents (11), Cicek and associates carried out a bioassay-guided fractionation of an exhaustive Soxhlet methanolic BCE using an assay to identify synergistic modulators of GABA in the induction of chloride currents. The authors isolated 11 cycloartane glycosides, of which only actein (24), 23-O-acetylshengmanol-3-O-β-d-xylopyranoside (32), cimigenol-3-O-β-d-xylopyranoside (1), and 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside (6) increased the GABA-induced chloride currents, with compound 32 doing so by 700%. This compound was able to increase chloride currents by 150% in the absence of GABA. Enzymatic hydrolysis of 1, 24, and 32 yielded the corresponding aglycones, which were only capable of weak enhancement of GABA-induced chloride currents.

5.5 Hepatotoxicity

In 2002, the United States Pharmacopeia, Inc. (USP) Dietary Supplements Information Expert Committee (DSIEC) recommended a GRAS classification for BCEs. However, by the end of that year, the first report of association of BCE with hepatotoxicity occurred when an article entitled “Black Cohosh and other Herbal Remedies Associated with Acute Hepatitis” described six instances occurring from 1996 through 2001 in patients taking some herbal complementary medicine, of which only two included a BCE. One of these two patients had used a BCE for only one week as a sole treatment for menopausal symptoms. Her hepatic failure was acute and severe, and she underwent a liver transplant a week after presentation. The other was also taking skullcap and valerian, both had been reported previously to be hepatotoxic. This report was immediately criticized by Vitetta et al. (94) on the grounds that there was no data on the source or authentication of the BCE, nor was viral hepatitis adequately excluded as the cause. Nonetheless, another report by Lontos et al. (95) appeared almost concurrently with Vitetta’s criticism and cited a single case of a woman who had taken a BCE for three months and developed severe chronic hepatitis, which worsened over the month following cessation and required a liver transplant. In this case, the source of the BCE was given, but no authentication was supplied. These and other reports worldwide led the Australian Therapeutic Goods Administration to review 47 worldwide instances of BCE-associated liver toxicity reported by 2005 and require a label containing a cautionary message (“Warning: Black cohosh may harm the liver in some individuals. Use under supervision of a healthcare professional”; http://www.tga.gov.au/safety/alerts-medicine-black-cohosh-070529.htm), on all BCE-containing commercial preparations.

Health Canada-Santé Canada (HC-SC) issued a statement on BCEs in which they advised users to consult a healthcare practitioner if any liver trouble developed (http://www.hc-sc.gc.ca/dhpmps/alt_formats/hpfb-dgpsa/pdf/prodnatur/mono_cohosh-grappes-eng.pdf). By April 2009, HC-SC had received six case reports of purported BCE/hepatotoxicity association. On examination, four of these were associated with preparations from a single supplier, who withdrew the product as it actually did not contain any A. racemosa. No analysis of the product was available in the other two cases.

The Committee on Herbal Medicinal Products (HMPC) of the European Medicines Agency, after studying 31 European cases, 11 world-wide case reports, and 15 clinical trials concluded that almost all cases reporting hepatotoxicity and BCE use were poorly documented, and that three cases could be classified with causal relationship as “possible” and two as “probable”. Most cases were judged as unlikely to have any relationship between BCE consumption and hepatotoxicity, or were simply so poorly documented that they had to be excluded from consideration. The 2007 HMCP report recommended that any patient taking BCE should contact their physician immediately on developing any signs of liver injury (http://www.emea.europa.eu.int/pdfs/human/hmpc/26925806en.pdf).

The same year, the USP DSIEC, after reviewing 30 reports of liver damage following use of products containing BCE, decided that preparations containing BCE should be classified as Class 2 hepatotoxic agents (i.e. a possible but not probable cause) and carry a label advising users to discontinue taking the preparation and consult a healthcare practitioner upon any signs of liver trouble (96).

In the same year, Borrelli et al. (97) published a “systematic review of adverse events” associated with BCEs, updating an earlier review. They surveyed clinical trials involving 1,522 patients, post-marketing surveillance of 2,691 patients, and case reports covering 18 patients for adverse effects. Women from 24 to 84 years of age were involved, and they were treated for periods ranging from 1 week to 1 year. There was no hepatotoxicity reported in either the clinical trials or the post-marketing surveillance patients. Among the case reports, seven involved liver problems, but, on examination, only one of these could be assigned probable cause from a BCE and two others, a possible cause. The relative safety of BCEs, specifically with respect to breast cancer patients, has been emphasized in a 2010 review (98).

In 2011, Teschke et al. (99) reviewed the literature on case reports of BCE-associated hepatotoxicity, with careful reference to the definition of herb-induced liver injury as defined by the CIOMS (Council for International Organizations of Medical Sciences), and also for the identification, quality, and authentication of the particular herbal preparation used. Their conclusion was that there is currently no evidence for a causal relationship between BCE and any of the case reports of purported hepatotoxicity.

Virtually simultaneous with Teschke’s review, Naser et al. (100) published the results of a meta-analysis of randomized controlled clinical trials of Remifemin^® or Remifemin^® plus (i.e. a 40% iso-propanol extract of A. racemosa roots/rhizomes without or with St. John’s wort extract, respectively) in healthy pre- and post-menopausal women treated with 40 mg (conventional dose) to 128 mg (high dose) of extract daily for three or six months. Five different trials comprising 1,108 women (557 treated and 551 on placebo) met the inclusion criteria. Eighty-eight women dropped out of the trials, none as a result of liver problems, and all except one of whom had enzyme level data collected as they left the study. Liver function was assessed by measuring aspartate aminotransferase, alanine aminotransferase, and β-glutamyltranspeptidase on entering and at the completion of the trial. There was no significant difference between treated and placebo values or other suggestion of liver toxicity as a result of BCE treatment.

The generally accepted conclusion is that there is no convincing evidence that BCEs cause hepatotoxicity; however, the possibility exists that it may do so in rare idiosyncratic cases. Hence the U.S., Australia, Canada, and the U.K. all require cautionary warnings on all preparations containing a BCE to the effect that patients using these preparations should consult a healthcare provider in the event of any symptoms suggestive of liver malfunction.

Animal studies, predominantly in rats, investigating the hepatotoxic potential of BCE have given mixed results. Lüde et al. (101) observed microvesicular steatosis in rats following doses of 1000 mg · kg⁻¹ (several orders of magnitude above the normal clinical dose). In vitro cytotoxicity with HepG2 cells was observed at 75 μg/cm³, and mitochondrial β-oxidation was impaired at 10 μg/cm³. The authors concluded that their studies were “compatible with idiosyncratic hepatotoxicity as observed in patients”. Mazzanti et al. (102) found a reduction in liver GSH levels in male rats given 300 mg · kg⁻¹ · day⁻¹ for 30 days. However, they concluded that this dose was “quite safe in rats”.

Campos et al. (103) reported a multidimensional study with female Wistar rats comparing groups aimed at mimicking conditions in post-menopausal women, i.e. ovariectomized and ovariectomized with hypertension induced via the 2 kidney, 1 clip model. A subsection of the latter group was treated daily with 0.6 mg/kg with an unspecified BCE. The treatment increased the reactive oxygen species (ROS) generated by mitochondria as measured by DCF generated from DCFH-DA. The GSH levels were significantly decreased in all experimental groups compared with levels in normal rats. The glucose-6-phosphate dehydrogenase activity was significantly less in the two untreated ovariectomized groups than in normal rats, but this effect was significantly moderated by treatment with BCE. Lipid peroxidase levels, as assessed by levels of thiobarbituric acid reactive substances, was significantly increased only in the BCE treated group. The authors concluded from this study that BCE may make women more susceptible to toxic effects of other drugs as GSH levels may be depressed to a greater extent by high doses or prolonged treatment. However, they admitted that these effects were not seen in their study.

6 Clinical Trials

As this contribution demonstrates, BCEs have been the subject of extensive research due in part to a long history of use, which can be traced to centuries-old Native American healing traditions. However, even as new technologies and analytical techniques have become available, teasing out the active principles and mechanism of action as applied to BCE’s clinical benefits remains a challenge to investigators. To date, there have been more than forty clinical studies of BCE, used to address menopausal symptoms, reported in the medical literature. A comprehensive review and summary of BCE studies dating from 1957 to 2009 by Fabricant et al. has been published in the Encyclopedia of Dietary Supplements (104). The early studies dating from the 1950s appear in the medical literature of Germany, where botanical medicines were well established as part of pharmaceutical armamentarium available to physicians. The majority of these early interventions were open studies in the style of serial case reports, most having a study length of about 12 weeks. Physicians demonstrated their patients’ responses to the BCE intervention, generally reporting improvements in menopausal symptoms with few and mild adverse events.

Reviewers of the earlier studies—those published in early 2000 and before—generally concluded that BCE was a safe intervention for short-term treatment of menopausal symptoms (105–108). During the early to mid-2000s, case reports began to appear in the medical literature impugning the safety of BCE, suggesting a link between its use and liver toxicity (see also Sect. 5) (95, 109–111). Reviewers of these adverse responses note that both the evidence for causality and the case review methodology have been lacking (112, 113). The peers emphasize the importance of thorough clinical evaluations in association with adverse event reporting that adhere to inclusive evaluation guidelines. Nevertheless, the placement of warning labels on BCE botanical dietary supplements has been both proposed and challenged (114, 115). Interestingly, results of two separate year-long randomized, double-blinded, placebo-controlled clinical trials, each of which included at least one BCE arm, revealed no liver toxicity during the course of these trials, which were conducted in the mid-2000s. One is the University of Illinois at Chicago (UIC)/National Institutes of Health (NIH) Botanical Center trial, in which post-menopausal women took 246 mg of a BCE (75% ethanol extract) daily for one year (116). The investigators reported that there was no difference between BCE and placebo for any of the monitored safety parameters; significantly, no evidence of hepatotoxicity was uncovered. Similarly, participants in the BCE arm of the Group Health trial took 160 mg of a BCE (a 70% ethanol extract) daily for the one-year duration of the trial (117). In a second arm of the same trial, participants took 200 mg daily of a BCE (a 4:1 water and alcohol extract), which was combined in a multi-botanical mixture. The authors note that there were no statistically significant differences between the four arms of the trial (two of the four arms contained BCE) and placebo. Any severe adverse responses were too few to make meaningful comparisons between groups, but importantly hepatotoxicity was not included among them.

Since the late 1980s, clinical interventions of BCE have evolved to randomized, placebo-controlled clinical trials with mixed results, which can be found in the Fabricant et al. summary (104). Some trials have shown benefit in decreasing menopausal symptoms, while others have shown that BCE is not statistically different from placebo. Different theories have been proposed for these dichotomous results. Some of the key points of discussion and related insights are addressed in the following.

The first aspect relates to clinical methodology. In examining the clinical trial design of these and other menopausal intervention studies, it should be noted that assessing the benefit of drug or botanical interventions has included the use of questionnaires. Often referred to as climacteric scales, these subjective instruments are used to rate menopausal symptom severity using a numerical score. Examples used in BCE clinical trials are the Kupperman Index, Menopausal Rating Scale (MRS), and Greene Climacteric Scale. In the same manner that diaries of hot flashes are often employed in clinical trials, where patients are asked to report the frequency and severity, the climacteric scales rely on self-assessment and self-reporting. Additionally, clinical investigators have developed and increasingly emphasized objective measures to identify the impact of BCEs on physiology, including measuring hormone levels (e.g. serum estradiol, follicle-stimulating hormone, luteinizing hormone, steroid hormone-binding globulin), vaginal cytology, etc. Also of interest is a technological innovation for measuring hot flashes objectively, the hot flash monitor, which has been introduced in recent clinical trials (118).

The second broader aspect, which might contribute to the dichotomous clinical outcome with BCE preparations, relates to the underlying biological targets. Coincident with the utilization of broader physiological assessment tools used in BCE studies, a consensus has developed among many investigators that BCE does not act as a classic phytoestrogen as presumed earlier, but rather may mimic selective estrogen receptor modulator effects on the bones and/or act on the CNS (79, 119–121). Subsequent in vitro and human studies have signaled the activity of BCE on serotonin, mu, as well as GABA_A receptors, with the suggestion that one or all of these biological interactions may function to bring about the diminution of menopausal symptoms, as reflected in many BCE clinical studies (11, 33, 122, 123).

While assessing the biological impact of BCE botanical interventions remains a challenge, so does identifying the active principles, including any and all active components or metabolites as well as dosing concentrations responsible for the botanical’s reported positive clinical outcomes. For those trials of BCE that have not shown benefit, such results may be a reflection of the wide variety of the clinical trial settings, BCE formulations, differences in their standardization schemes, and dosing strengths that researchers have used to investigate the clinical benefit of this botanical in assessing menopausal symptom relief. These four parameters alone represent a four-dimensional set of parameters, which in practice are really impossible to align when comparing different studies. While this alignment is theoretically possible, many studies and/or reports lack the level of detail to enable such comparison.

The third group of factors with major impact on clinical outcome relates to the basic pharmacology of the intervention materials. Research studies have employed a variety of BCE formulations, which have variously been described by all of the following terms: (a) “Black Cohosh” 20 mg/day standardized to 1 mg triterpene glycosides as 27-deoxyactein (124); (b) 6.5 mg of dry rhizome extract, from 60% ethanol extraction with a drug extract ratio: 4.5–8.5:1 (125, 126); (c) “Black Cohosh” dried aqueous/ethanolic 58% v/v (127); (d) “Black Cohosh” standardized to 1 mg triterpene glycosides calculated as 27-deoxyactein, corresponding to 3.75 mg Cimicifuga Rhizoma ² (128); (e) dried aqueous/ethanolic (58% v/v) extract of rhizome (129); (f) a 70% ethanol extract of roots and rhizomes (119). These are just examples and this list could be extended. Some studies reveal more about the formulation of their BCE interventions than others. However, no investigator has thus far been in a position to define the active principle(s) at work in those trials that have demonstrated that BCE relieves menopausal symptoms. Accordingly, a challenge remains to fill the gap between the several known in vitro- and in vivo-active principles in BCEs, the numerous recently discovered phytoconstituents of the plant that have not been studied biologically, the sophistication of standardization schemes required to capture this chemical diversity, and the complex nature of the clinical endpoints associated with menopausal symptom management. This again opens a four-dimensional set of complex parameters, which yield a hyper-complex correlation space for BCEs altogether.

Interestingly, clinical trial reporting guidelines began to emerge in the late 1990s, emphasizing increased transparency. The consolidated standards of reporting trials, or CONSORT guidelines, continue to be revised periodically, with a 2010 publication providing a checklist of 25 elements that should be included in parallel, randomized trial reports (130). Gagnier et al. have focused on specific recommendations on the reporting of clinical trials of herbal interventions (131) The elements emphasized by this group include the herbal intervention’s Latin name, proprietary product name and country of registration, parts of the plant used, type of product (fresh or dry), solvent used, method of authentication, dosage, duration of administration, content (excipients), standardization, quantity of marker constituents, chemical fingerprint, any special testing, rationale for placebo and description of practitioners. This information complements the Product Integrity specifications for research funded by NIH’s NCCAM (National Center for Complementary and Alternative Medicine; see NOT-AT-10-006 and at http://www.nccam.nih.gov/research/policies/naturalproduct.htm). A robust example of botanical intervention descriptions can be found in the UIC/NIH Botanical Center study of BCE and red clover used to ameliorate menopausal vasomotor symptoms (116). The information provided represents a transparent summary of a contiguous step-by-step process for developing a botanical dietary supplement, which begins with the acquisition of raw material and appropriate placement of associated voucher specimens, and extends to details of the final dosage form with comprehensive analytical testing information presented. Providing this information enables reviewers and investigators to focus on trial results, thereby obviating questions of product integrity that could challenge those results. It is also reflective of thoughtful advanced planning in a clinical trial design process that acknowledges the complexity of testing botanical interventions.

At the same time, it is important to note that concurrent progress in the chemical and pharmacological description of a botanical intervention material like BCE produces a constant paradigmatic shift with regard to the assignment of botanical marker compound(s) and botanical standardization. The case of BCE is particularly interesting, as the very recent discovery of several classes of previously unidentified alkaloids in a BCE has the potential to significantly change the integrated view of the clinical and in vivo potential and targets of this plant extract.

With the variety and scope of trial designs, BCE formulations, dosages and intervention outcome measures, as well as conflicting trial results claiming both effective as well as non-effective outcomes, it is not surprising that some BCE clinical trial reviewers have called for more extensive and rigorous clinical trials (97, 132, 133). Two meta-analyses of BCE trials have been published; the authors of each study separately suggest that as a group these trials are too heterogeneous, thus limiting the studies that could be grouped for evaluation by meta-analysis. Shams et al. in their meta-analysis of nine randomized clinical trials found that BCE improved vasomotor symptoms by 26% (134).

In contrast, Leach et al. from the Cochrane Collaboration examined 16 randomized clinical trials, finding no significant difference in the frequency of hot flashes between BCE treated and placebo groups, adding that the quality of the studies examined is uncertain enough that improved reporting methods would be highly beneficial (135). The authors not only conclude that more research on BCE safety and efficacy is warranted, but also that there is adequate justification for conducting further trials. Considering the recent findings of new chemical diversity in BCEs (see Sect. 2.3), it is likely that new active principles in the plant are yet to be discovered, and that phytochemical and other basic research has significant potential to widen the perspective of clinical research on this widely used botanical.

Additionally, meeting the challenge of devising clinical trial designs that adequately capture the impact of botanical interventions on human health is vitally important. Research in this area continues at an exciting time, in which the study of human genomics and drug and/or botanical interactions is at its nascence. Investigators may find that botanicals like some ethical drugs produce a wide continuum of responses in various populations, reinforcing the need to investigate the pharmacokinetic and pharmacodynamic activities of both known and newly recognized A. racemosa chemical constituents. In accordance with the views of so many investigators, the BCE clinical research horizon must be broadened as researchers continue to understand this botanical in light of its long history of use, which continues into the twenty-first century.

7 Concluding Remarks

The last 30 years of research of A. racemosa as a medicinal plant or an herbal dietary supplement have produced much valuable knowledge of its botany, chemistry, and biology. Particularly, the ten-year research project led by the University of Illinois at Chicago (UIC)/National Institutes of Health (NIH) Botanical Center has for the first time carried out systematic and comprehensive studies of this plant in a unified interdisciplinary setting and significantly improved the understanding and interrelationship of its phytochemical constituents and pharmacological activities.

Recent efforts have revealed a much more detailed profile of the characteristic secondary metabolites in A. racemosa, which include almost 50 known cycloartane triterpenes, 10 cimicifugic acids, and more than 70 nitrogen-containing constituents. This information is not only useful in the fingerprint profiling for the botanical identification and authentication, but it also provides the chemical evidence and inspiration for the discovery of the pharmacological activities of this plant. The use of advanced spectroscopic techniques, such as MS and NMR, and computational approaches enables the rapid and reliable identification of these compounds directly from their mixtures (8, 35), making them a great resource for the future studies on the phytoconstituents and quality standardization of A. racemosa and its related botanical preparations. Furthermore, new nomenclature has been established to standardize the naming systems for the Actaea cycloartane triterpenes and cimicifugic acids, both of which are comprised of a number of congeneric compounds (8, 24) These naming systems provide scientists with some basic knowledge of structural characteristics of the compounds concerned.

A. racemosa has a well-established traditional use for the alleviation of women’s menopausal symptoms such as hot flashes, insomnia, and depression. This has been interpreted frequently as being due to the presence of phytoestrogens in A. racemosa, which would contribute to an estrogenic activity of the plant. However, pharmacological evaluation of either the crude extracts or isolated phytoconstituents has not yet provided the required evidence for this mode of action, but has instead pointed to the lack of this type of pharmacological activity for BCEs.

Stress-related conditions, such as anxiety and depression, are among the major symptoms at the menopausal stage. The ability of A. racemosa to attenuate the psychological and physiological stress has been evaluated extensively and confirmed in animals. This activity, at least on the in vitro level, is found to be partly ascribed to the CNS effects of the alkaloid N _ω-methylserotonin, which has potent 5-HT₇ receptor binding ability (33). In addition, four cycloartane triterpenes, including actein (24), 23-O-acetylshengmanol-3-O-β-d-xylopyranoside (32), cimigenol-3-O-β-d-xylopyranoside (1), and 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside (6), are also found to be CNS active, showing GABAergic activity (11). Other studies have found A. racemosa extract effectively counteracts the metaphyseal bone loss of the femur in control untreated ovariectomized animals (85). Further investigations have ascribed this bioactivity to two cycloartane triterpenes, namely, 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (5) and 23-epi-26-deoxyactein (29) (86, 87). The preliminary studies on the anticancer activity of A. racemosa have demonstrated that both cimicifugic acids and cycloartane triterpenes are effective in inhibiting the growth of breast and liver cancer cells (7, 90, 91). Hepatotoxicity has been the only safety concern on A. racemosa consumption, and this topic has been considered in detail. Thus, the vast majority of cases of reported acute hepatitis did not stand up to further investigation, and none met the rigorous requirements of causality. Moreover, in clinical studies, there has been no convincing evidence that A. racemosa causes hepatotoxicity while the possibility exists of rare idiosyncratic cases. This has also raised the concern about possible adulteration of A. racemosa either intentionally or accidentally, especially by related species, some of which are similar superficially.

As seen in previous studies, the pharmacological evaluation was mainly concentrated on the individual compounds. Research in this way continues as long as other new compounds are identified. The empirical knowledge of complementary and alternative medicine suggests the therapeutic effects of herbal remedies possibly result from the synergistic or additive interactions of multiple components (136). Based on this concept, it can be hypothesized that this situation may also exist in A. racemosa. Therefore, in future studies, it is absolutely necessary to explore the potential synergism of the phytoconstituents of A. racemosa. To ascribe the chemical components to this mode of action, it requires a detailed profile of phytochemistry to furnish both qualitative and quantitative information, which can be obtained only from large-scale metabolomic profiling of the plant. As a result of the chemical complexity, and of current knowledge of this profile, the process will be challenging but possible by the use of advanced chromatographic and spectroscopic techniques as well as sophisticated data mining tools (137). Establishment of such a metabolomic profile will provide a deeper understanding of the secondary metabolism in A. racemosa and help map the biosynthesis pathways, identification and understanding of the active secondary metabolites.

Although almost 50 clinical studies of the effects of BCEs on menopausal symptoms have been reported, only a small minority of these have been randomized double-blinded controlled studies and virtually all of those have been underpowered. Attempts at meta-analyses of these have been complicated by the use of different preparations, and result parameters. Investigators are unanimous in only one aspect: the need for larger more rigorously controlled studies (97, 132–135).

In conclusion, while recent advances in basic (phyto)chemical and biological as well as clinical knowledge of A. racemosa have not been able to provide the ultimate rationale for the safe and efficacious use of this popular botanical, there is an increasingly solid foundation to justify further exploration of the metabolomic diversity and biological impact of A. racemosa phytoconstituents, and to conduct clinical trials with rigorously defined intervention materials, possibly targeting previously underexplored clinical endpoints.