Metabolic profiling of Actaea species extracts using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Despite persistent questions about the safety of black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa L.), its products continue to be one of the most popular botanical supplements in the United States market. Black cohosh products have been associated with cases of liver toxicity, but subsequent evaluation found some products to be adulterated with other related plants from the same genus. US FDA regulations require that black cohosh products be unadulterated, and correct identification of different species of Actaea is a key first step for their good manufacturing practice. We have developed a phytochemical method to distinguish four different groups of Actaea, including: species other than A. racemosa, Asian species, A. racemosa, and North American species other than A. racemosa. Using HPLC-TOF-ESI-MS technique and principal component analysis, we identified 15 chemical markers (1-3, 5-6, 8-10, 12, 16-21). Three marker compounds were unambiguously identified using authentic standards, and 12 marker compounds were tentatively identified by comparison of fragmentation patterns with previously reported data. The presence of these marker compounds is critical for discrimination among the four groups of closely related plants. The use of metabolic profiling to distinguish black cohosh from related species of Actaea has broader implications in the identification of markers to help authenticate other important medicinal plants.     

DNA barcode identification of black cohosh herbal dietary supplements

Black cohosh (Actaea racemosa) herbal dietary supplements are commonly consumed to treat menopausal symptoms, but there are reports of adverse events and toxicities associated with their use. Accidental misidentification and/or deliberate adulteration results in harvesting other related species that are then marketed as black cohosh. Some of these species are known to be toxic to humans. We have identified two matK nucleotides that consistently distinguish black cohosh from related species. Using these nucleotides, an assay was able to correctly identify all of the black cohosh samples in the validation set. None of the other Actaea species in the validation set were falsely identified as black cohosh. Of 36 dietary supplements sequenced, 27 (75%) had a sequence that exactly matched black cohosh. The remaining nine samples (25%) had a sequence identical to that of three Asian Actaea species (A. cimicifuga, A. dahurica, and A. simplex). Manufacturers should routinely test plant material using a reliable assay to ensure accurate labeling.     

Authentication of black cohosh (Actaea racemosa) dietary supplements based on chemometric evaluation of hydroxycinnamic acid esters and hydroxycinnamic acid amides

Analytical and Bioanalytical Chemistry - Ester and amide derivatives of hydroxycinnamic acids are found in black cohosh (Actaea racemosa) and other Actaea plants. These two compound groups were...     

Identification of caffeic acid derivatives in Actea racemosa (Cimicifuga racemosa,black cohosh) by liquid chromatography/tandem mass spectrometry

Caffeic acid derivatives occurring in black cohosh [Cimicifuga racemosa (L.) Nutt., Actaea racemosa (Ranunculaceae)], some of which may have pharmacological activity, were analyzed using high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in a complex plant matrix. Based on these studies, structurally characteristic product ions and neutral molecule losses were identified, which were then used during LC/MS/MS with product ion scanning, precursor scanning and constant neutral loss scanning to detect caffeic acid derivatives in a crude extract of black cohosh. Several caffeic acid derivatives were detected, and the identification of six of them were confirmed by comparison with authentic standards including caffeic acid, ferulic acid, isoferulic acid, fukinolic acid, cimicifugic acid A, and cimicifugic acid B. Four other compounds were detected that appeared to be caffeic acid derivatives based on LC/MS/MS retention times, molecular weights, and fragmentation patterns during MS/MS. Since standards were unavailable for these four compounds, they were tentatively identified using LC/MS/MS as cimicifugic acid E, cimicifugic acid F, dehydrocimicifugic acid A, and dehydrocimicifugic acid B. Dehydrocimicifugic acid A and dehydrocimicifugic acid B have not been reported previously to be constituents of black cohosh.     

Cimicifuga species identification by high performance liquid chromatography-photodiode array/mass spectrometric/evaporative light scattering detection for quality control of black cohosh products

Black cohosh has become one of the most important herbal products in the US dietary supplements market. It is manufactured from roots and rhizomes of Cimicifuga racemosa (Ranunculaceae). Botanical identification of the raw starting material is a key step in the quality control of black cohosh preparations. The present report summarizes a fingerprinting approach based on high performance liquid chromatography-photodiode array/mass spectrometric/evaporative light scattering detection (HPLC-PDA/MS/ELSD) that has been developed and validated using a total of 10 Cimicifuga species. These include three North American species, Cimicifuga racemosa, Cimicifuga americana, Cimicifuga rubifolia, and seven Asian species, Cimicifuga acerina, Cimicifuga biternat, Cimicifuga dahurica, Cimicifuga heracleifolia, Cimicifuga japonica, Cimicifuga foetida, and Cimicifuga simplex. The chemotaxonomic distinctiveness of the HPLC fingerprints allows identification of all 10 Cimicifuga species. The triterpene glycoside cimigenol-3-O-arabinoside (3), cimifugin (12), and cimifugin-3-O-glucoside (18) were determined to be suitable species-specific markers for the distinction of C. racemosa from the other Cimicifuga species. In addition to identification, the fingerprint method provided insight into chemical interconversion processes occurring between the diverse triterpene glycosides contained in black cohosh. The reported method has proven its usefulness in the botanical standardization and quality control of black cohosh products.     

A triterpene glycoside from black cohosh that inhibits osteoclastogenesis by modulating RANKL and TNFalpha signaling pathways

Osteoporosis is a major age-related source of morbidity and mortality. Increased bone resorption mediated by osteoclasts is central to its pathogenesis. Cytokines, particularly RANKL and TNFalpha, are often increased under pathologic conditions, leading to enhanced osteoclastogenesis. Black cohosh (Actaea/Cimicifuga racemosa L), a popular herbal supplement for the treatment of menopausal symptoms, was recently shown to have the beneficial effect of preventing bone loss. Here, we demonstrate that 25-acetylcimigenol xylopyranoside (ACCX), a triterpenoid glycoside isolated from black cohosh, potently blocks in vitro osteoclastogenesis induced by either RANKL or TNFalpha. This blockage of osteoclastogenesis elicited by ACCX results from abrogation of the NF-kappaB and ERK pathways induced by either RANKL or TNFalpha, respectively. Importantly, this compound attenuates TNFalpha-induced bone loss in vivo. Therefore, ACCX represents a potential lead for the development of a new class of antiosteoporosis agents.     

Podocarpaside, a triterpenoid possessing a new backbone from Actaea podocarpa

Podocarpaside (1), a novel arabinoside possessing a unique triterpene skeleton was isolated from Actaea podocarpa, a closely related species to black cohosh (dietary supplement used for menopausal disorders). Podocarpaside belongs to a new class of triterpenoids, for which the name "ranunculane" is proposed. Compound 1 possesses anticomplement activity. [structure: see text].     

Chemical fingerprinting of Equisetum arvense L. using HPTLC densitometry and HPLC

Equisetum arvense L. is a herbaceous medicinal plant, commonly known as horsetail, whose extracts have been reported to possess diuretic and haemostatic properties. The aim of this study was to evaluate the use of fingerprint chromatographic methods on commercially available raw materials or preparations of E. arvense L. in order to ascertain their quality and identify possible adulterants using HPLC and HPTLC densitometry. Two chromatographic methods were used to determine the chemical fingerprints of E. arvense and other allied species. The first was based on HPTLC identification followed by densitometric measurement at 350?nm. The second was based on HPLC separation. The ease of sample preparation and the possibility of simultaneous analysis of several samples in a short time make HPTLC a method of choice for the comprehensive quality evaluation of herbal products.     

Synthesis of cimiracemate B, a phenylpropanoid found in Cimicifuga racemosa

The synthesis of the rare bioactive phenylpropanoid ester known as cimiracemate B (1) from black cohosh, Cimicifuga racemosa, is described. The procedure employs a coupling method using 1, l'-carbonyldiimidazole and methyltrifluoromethanesulfonate as coupling reagents in the presence of N-methylmorpholine.     

Metabolism of Nω‐methylserotonin,a serotonergic constituent of black cohosh (Cimicifuga racemosa,L. (Nutt.)), by human liver microsomes

The roots/rhizomes of black cohosh (Cimicifuga racemosa L. (Nutt.) (syn. Actaea racemosa L.) are a popular dietary supplements among women for management of menopausal symptoms. Although not estrogenic, N_ω‐methylserotonin has been identified in black cohosh as a potent agonist of serotonin 5‐HT_1A and 5‐HT₇ receptors. In the present study, in vitro metabolism of N_ω‐methylserotonin was investigated to gain insights into aspects of the bioavailability of this compound. The major metabolic pathway was determined to be conversion into 5‐hydroxyindole acetaldehyde catalyzed by the monoamine oxidase A (MAO‐A). 5‐Hydroxyindole acetaldehyde could be further oxidized to form 5‐hydroxyindole acetic acid by the action of microsomal aldehyde dehydrogenase or reduced to 5‐hydroxy tryptophol by the action of aldehyde reductase. The cytochrome P450 enzymes had only a minor role in the metabolism of N_ω‐methylserotonin and then only when MAO‐A was inhibited. In many aspects, the metabolism of N_ω‐methylserotonin was similar to the metabolism of serotonin, suggesting that this compound is unlikely to elicit CNS effects due to rapid metabolism by the widely distributed MAO‐A. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioautography detection in thin-layer chromatography

Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening.     

Comparison between thin-layer chromatography and overpressured layer chromatography fingerprints of commercial essential oils and accelerated solvent extraction plant extracts

Developing fit-to-purpose analytical screening methods for plant marker constituents by simple and affordable techniques represents a constant and important request from the current transformation chain of plant products. In this work, methods developed on classical thin-layer chromatography (TLC) and overpressured layer chromatography (OPLC) have been applied on some of the most valuable commercial plant products, such as star anise (Illicium verum Hook. f.) essential oil and extracts, common thyme (Thymus vulgaris L.) and sweet fennel (Foeniculum vulgare Mill.) essential oils and acerola fruit (Malpighia punicifolia L.) hydroalcoholic extracts, to evaluate OPLC potentiality in comparison with TLC performances in the detection of some characteristic constituents. OPLC provided higher performance with respect to TLC in all experiments performed due to higher selectivity demonstrated on all the tested marker compounds except for flavonoids in acerola extracts. Considering the usage of planar chromatography in the quality control of plant derivatives, the present paper shows how the OPLC protocols were both highly time- and solvent-saving in comparison with classical TLC.

     

Validation of high-performance thin-layer chromatographic methods for the identification of botanicals in a cGMP environment

Current Good Manufacturing Practices (cGMP) for botanicals stipulates the use of appropriate methods for identification of raw materials. Due to natural variability, chemical analysis of plant material is a great challenge and requires special approaches. This paper presents a comprehensive proposal to the process of validating qualitative high-performance thin-layer chromatographic (HPTLC) methods, proving that such methods are suitable for the purpose. The steps of the validation process are discussed and illustrated with examples taken from a project aiming at validation of methods for identification of green tea leaf, ginseng root, eleuthero root, echinacea root, black cohosh rhizome, licorice root, kava root, milk thistle aerial parts, feverfew aerial parts, and ginger root. The appendix of the paper, which includes complete documentation and method write-up for those plants, is available on the J. AOAC Int. Website (http://www.atypon-link.com/AOAC/loi/jaoi).     

Uncertainty in quantitative thin-layer chromatography

Globalization forces analysts to demand extended control of variability in analytical measurements. A calculation procedure named the "error budget model" following recommendations proposed more than 20 years ago by the Bureau International des Poids et Mesures is established as a rule for evaluating and expressing the measurement uncertainty across a broad spectrum of measurements. This metrological approach common in physical measurement is not applicable in separation techniques and cannot quantitate measurement uncertainty. Our experiments show that it can be used as a planning tool in the validation of thin-layer chromatographic (TLC) methods. A computer program that quantitates uncertainty components associated with potential sources of uncertainty in quantitative TLC is prepared and tested with experimental data. TLC plates with different qualities of stationary phases (TLC and high-performance TLC) spotted with different types of samples are measured. Application is performed manually and automatically. Plates are scanned with UV-vis scanners and a video documentation system in remission and transmission mode and fluorescence. Although the calculated values are close to the values obtained with validation procedures, the error budget approach cannot substitute validation. Calculated results can predict critical points in real quantitative TLC, but they cannot confirm the validity of a selected chromatographic procedure.     

Alkaloids and saponins in dietary supplements of blue cohosh (Caulophyllum thalictroides)

Preparations of blue cohosh (Caulophyllum thalictroides) have been used traditionally by Native Americans for medicinal purposes. Dietary supplements containing dried roots or extracts of blue cohosh rhizomes are available as dietary supplements. The safety and efficacy of these preparations have not been systematically evaluated. Recent studies indicate that ingestion of specific alkaloids in blue cohosh preparations can produce birth defects and neonatal heart failure. Blue cohosh also contains saponins, which may be responsible for uterine-stimulating effects. We determined the amounts of major alkaloids and saponins in preparations of blue cohosh by high-performance liquid chromatography (HPLC). Alkaloids and saponins were monitored with a photodiode array detector and an evaporative light-scattering detector, respectively. Profiles were compared with those of authenticated blue cohosh root extracts. Identities of the alkaloids and saponins were confirmed by HPLC/mass spectrometry and nuclear magnetic resonance spectrometry. Calculations based on the results of analyses of dietary supplements showed that maximum daily intake of alkaloids and saponins will vary with the form (e.g., root, liquid extract) and doses recommended in product labeling. Intakes may vary from < 1 to 75 mg/day for alkaloids and from about 9 to 420 mg/day for saponins.     

High-resolution thin-layer chromatography of gangliosides

In this review an updated overview of current improvements on thin-layer chromatography (TLC) of gangliosides over the past decade is provided. Basic general techniques and special advice is given for successful separation of glycosphingolipids. New approaches concerning continuous and multiple development, and several preparative TLC methods are also included. Emphasis is placed on TLC immunostaining and related techniques, i.e. practical applications of carbohydrate-specific antibodies, toxins and bacteria, viruses, lectins and eukaryotic cells. Thus, this review on ganglioside TLC summarizes its power as an analytical tool for a wide range of purposes.     

A direct and simple method of coupling matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk

Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is "proteomics," there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that "clean" spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract.     

Two-dimensional thin-layer chromatography with adsorbent gradient as a method of chromatographic fingerprinting of furanocoumarins for distinguishing selected varieties and forms of Heracleum spp

There are a lot of taxonomic classifications of the genus Heracleum, and many authors indicate they need revision. Morphological identification is difficult to perform, as there are only few characteristic differences between each Heracleum species, varieties and forms. Furanocoumarins are characteristic compounds for the Apiaceae family, and they can be found in the whole genus in large quantities. Despite this fact, it is difficult to use the furanocoumarin profiles of plants, for their discrimination, as furanocoumarins are difficult to separate, due to their similar chemical structures and physicochemical properties. In this paper, a new, simple method is proposed for the discrimination of selected species, varieties and forms of the genus Heracleum. Thin-layer chromatography (TLC) with an adsorbent gradient (unmodified silica gel+octadecylsilica wettable with water) enables complete separation of the structural analogues. The proposed method gives the possibility to distinguish selected species, varieties and forms of the Heracleum genus, as they produce distinctive furanocoumarin fingerprints. The method is characterised by high specificity, precision, reproducibility and stability values. It is for the first time that graft TLC is used for constructing fingerprints of herbs. The complete separation of ten structural analogues, by combining gradient TLC with the unidimensional multiple development technique, has not been reported yet.     

An improved thin-layer chromatography/mass spectrometry coupling using a surface sampling probe electrospray ion trap system

A combined surface sampling probe/electrospray emitter coupled with an ion trap mass spectrometer was used for the direct read out of unmodified reversed-phase C18 thin-layer chromatography (TLC) plates. The operation of the surface sampling electrospray ionization interface in positive and negative ionization modes was demonstrated through the direct analysis of TLC plates on which a commercial test mix comprised of four dye compounds viz., rhodamine B, fluorescein, naphthol blue black, and fast green FCF, and an extract of the caffeine-containing plant Ilex vomitoria, were spotted and developed. Acquisition of full-scan mass spectra and automated collection of MS/MS product ion spectra while scanning a development lane along the surface of a TLC plate demonstrated the advantages of using an ion trap in this combination. Details of the sampling system, benefits of analyzing a developed lane in both positive ion and negative ion modes, levels of detection while surface scanning, surface scan speed effects, and the utility of three-dimensional data display, are also discussed.