Quantification of Actaea racemosa L. (black cohosh) from some of its potential adulterants using qPCR and dPCR methods.

The demand for popular natural health products (NHPs) such as Black Cohosh is increasing considerably, which in turn challenges quality assurance (QA) throughout the supply chain. To detect and quantify the target species present in a given NHP, DNA-based molecular techniques such as Real-time quantitative PCR (qPCR) and digital PCR (dPCR) are standard tools in the food and pathogen testing industries. There is a gap in the literature concerning validated quantitative PCR methods for botanicals that can be utilized for QA and good manufacturing practices. The objective of this study is to develop an efficient quantification method using qPCR and dPCR techniques for the detection and quantification of Actaea racemosa (Black cohosh) NHPs from its potential adulterants. These developed methods are validated for applicability on commercial NHPs. Species-specific hydrolysis probe assays were designed to analyze the black cohosh NHPs using qPCR and dPCR techniques. The results confirmed that the developed qPCR and dPCR methods are highly precise for identifying and quantifying black cohosh NHPs, indicating their potential applicability in future routine industrial and laboratory testing. This enables a single qPCR test to determine not only the presence of a specific botanical, but also the amount when mixed with an adulterant.

Cimicifuga heracleifolia is therapeutically similar to black cohosh in relieving menopausal symptoms: evidence from pharmacological and metabolomics studies.

In the market of botanical dietary supplements, Cimicifuga heracleifolia (CH) has always been considered as an adulterated species of Cimicifuga racemosa (CR), a conventional American herb with promising benefits to counteract troubles arising from the menopause. However, the detailed comparison of their therapeutic effects is lacking. In present study, the pharmacological and metabolomics studies were comparatively conducted between CH and CR in ovariectomized (OVX) female rats. Specifically, estrogen-like, anti-hyperlipidemia and anti-osteoporosis effects were evaluated through measuring serum biochemical parameters, histopathological examination and micro computed tomography (Micro-CT) scanning. At the same time, a gas chromatography-mass spectrometry (GC-MS)-based serum metabolomics method was employed to profile the metabolite compositional changes. As a result, both CR and CH displayed anti-osteoporosis and anti-hyperlipemia on menopause syndrome. Meanwhile, their potentials in improving the OVX-induced metabolic disorders were discovered. In conclusion, these results demonstrated that CH is therapeutically similar to CR in relieving menopausal symptoms and CH could be considered as a promising alternative to CR instead of an adulterant in the market of botanical dietary supplements.

Comparison of Flow Injection MS, NMR, and DNA Sequencing: Methods for Identification and Authentication of Black Cohosh (Actaea racemosa).

Flow injection mass spectrometry and proton nuclear magnetic resonance spectrometry, two metabolic fingerprinting methods, and DNA sequencing were used to identify and authenticate Actaea species. Initially, samples of Actaea racemosa from a single source were distinguished from other Actaea species based on principal component analysis and soft independent modeling of class analogies of flow injection mass spectrometry and proton nuclear magnetic resonance spectrometry metabolic fingerprints. The chemometric results for flow injection mass spectrometry and proton nuclear magnetic resonance spectrometry agreed well and showed similar agreement throughout the study. DNA sequencing using DNA sequence data from two independent gene regions confirmed the metabolic fingerprinting results. Differences were observed between A. racemosa samples from four different sources, although the variance within species was still significantly less than the variance between species. A model based on the combined A. racemosa samples from the four sources consistently permitted distinction between species. Additionally, the combined A. racemosa samples were distinguishable from commercial root samples and from commercial supplements in tablet, capsule, or liquid form. DNA sequencing verified the lack of authenticity of the commercial roots but was unsuccessful in characterizing many of the supplements due to the lack of available DNA.

[Application of high resolution melting curve to identification of Cimicifugae Rhizoma].

High-resolution-melting analysis (HRM) is a new technology derived from q PCR and is widely used in the study of polymorphism, genotyping, and single nucleotide mutation. Advantages of HRM include cost-effectiveness and time-efficiency over PCR-based genotyping. However, the application of HRM in the authentication of herbal products is still limited with few studies on the classification and identification of herbal products. In this study, Cimicifugae Rhizoma was used as an example to verify the stability and accuracy of HRM technique in identification of Chinese materia medica. HRM assay was established for identification based on ITS2 region of Cimicifugae Rhizomas and its adulterants(including 41 samples). Our findings showed that HRM allows not only the identification of adulteration but also the quantification of the most common admixture. This study is significant for better quality in the verification of the authenticity of herbal medicine. The method is promising for future identification of traditional Chinese medicinal materials.

[Identification of cimicifugae rhizoma and its adulterants using ITS2 sequence].

In order to identify Cimicifugae Rhizoma from its adulterants and to ensure its safe use, the internal transcribed spacer 2 (ITS2) sequence of Cimicifugae Rhizoma and its adulterants were amplified and bidirectionally sequenced by DNA barcoding technology. Sequence assembly and consensus sequence generation were performed by the CodonCode Aligner V3.7.1. The genetic distances were computed by MEGA 5.0. Identification analyses were performed using neighbor-joining (NJ) methods. The length of ITS2 sequence of the three origin plants of Cimicifugae Rhizoma include Cimicifuga heracleifolia, C. foetida, C. dahurica was 217, 219 and 219 bp, respectively. Their intraspecific genetic distance was much lower than the interspecific genetic distance with their closely related species. The NJ tree of ITS2 indicated that the three origin plants of Cimicifugae Rhizoma formed a monophyletic clade, Cimicifugae Rhizoma and its adulterants could be distinguished clearly. The authors proposed that ITS2 sequence was suitable for the authentication of Cimicifugae Rhizoma and its adulterants.

[Classification of Cimicifuga species based on 1H-NMR fingerprint combined with pattern recognition technique].

The metabolomic analysis of three Cimicifuga species was performed using H-NMR spectroscopy and pattern recognition (PR) techniques. A broad range of metabolites could be detected by 'H-NMR spectroscopy without any chromatographic separation. The analysis using principal component analysis (PCA) and discriminant partial least square (DPLS) of the 1H-NMR spectrum showed a clear discrimination between C. foetida and the other two species. The major metabolites responsible for the discrimination were triterpenoid saponins and saccharides. These results indicated that the combination of 1H-NMR and PR provides a useful tool for chemotaxonomic analysis and authentification of Cimicifuga species, and could used for the quality control of plant materials.

[Quick identification and discrimination of sun-dried and sulfur-fumigated cimicifugae rhizoma by Fourier transform infrared spectroscopy].

OBJECTIVE: To develop a method for quick identification of sun-dried and sulfur-fumigated Cimicifugae Rhizoma by Fourier transform infrared spectroscopy (FTIR). METHODS: The alcoholic and aqueous extracts of sun-dried and sulfur-fumigated Cimicifugae Rhizoma were analyzed and compared by FTIR combined with second derivative infrared spectroscopy. RESULTS: FTIR spectra showed that there were some differences in the positions of infrared absorption peaks and the relative intensities in the alcoholic and aqueous extracts of sun-dried and sulfur-fumigated Cimicifugae Rhizoma, and the second derivative IR spectra clearly enhanced the spectral resolution of their differences. FTIR spectra showed that the new absorption peaks of Cimicifugae Rhizoma appeared and a part of original absorption peaks disappeared after sulfur-fumigation in aqueous extracts, while a lot of new absorption peaks appeared and the intensities of almost all absorption peaks significantly decreased after sulfur-fumigation in alcoholic extracts. Second derivative IR spectra showed that both sun-dried and sulfur-fumigated Cimicifugae Rhizoma extracted by water differed significantly from each other ranging from about 3 950 to 3 940 cm(-1), 3 850 to 3 800 cm(-1), 1 800 to 1 750 cm(-1), as well as from 1 400 to 1 350 cm(-1); Differences also existed between sun-dried and sulfur-fumigated Cimicifugae Rhizoma extracted by ethanol ranging from about 3 980 to 3 960 cm(-1), 3 850 to 3 800 cm(-1), and 1 500 to 1 460 cm(-1). CONCLUSION: The FTIR method combined with the second derivative IR spectrum can be used to analyze and distinguish sun-dried and sulfur-fumigated Cimicifugae Rhizoma quickly and accurately. The developed method provides an efficient approach for the quality control of Chinese herbal medicines with its simplicity and strong specificity.

Triterpenoids from Cimicifugae rhizoma, a novel class of inhibitors on bone resorption and ovariectomy-induced bone loss.

OBJECTIVE: Increasing research suggested that Cimicifugae rhizoma might be protective against osteoporosis. In this study, we investigated the effects of three cycloartane-type triterpenoids isolated from Cimicifugae rhizoma, cimicidol-3-O-beta-D-xyloside (1), cimicidanol-3-O-beta-D-xyloside (2) and acetylacteol-3-O-beta-d-xyloside (3) on bone resorption in vitro and bone loss in ovariectomized (OVX) mice. METHODS: The activities of the tested compounds on bone resorption were evaluated using three assays, neonatal mouse parietal bone organ culture, osteoclast-like cells (OCLs) formation and pit formation. The effects on bone mineral density (BMD) and uterine weight were examined using OVX mice. Using LC-MS/MS method, the serum concentrations of the triterpenoids were measured in mice serum collected at 0.5, 1, 3, 6 and 12h following its oral administration. RESULTS: All of the tested compounds exerted the inhibitory effects on bone resorption in bone organ culture, suppressed both of the formation and the resorbing activity of OCLs. Furthermore, a synergistic effect was observed among those compounds. In vivo studies revealed that compounds 1-3 and the mixture of compounds 1-3 prevented the bone loss in OVX mice without affecting uterine weight, and each compound was detected in the mice serum after single oral administration. CONCLUSIONS: The triterpenoids exerted the inhibitory effects on osteoclastic bone resorption through the suppression of both OCLs formation and the resorbing activity of OCLs, and also showed a significant protective effect on BMD in OVX mice. The present results might provide a new pharmacological potential for the treatment of osteoporosis.

[Comparison of activities of various species of Rhizoma Cimicifugae and their honey processed products].

OBJECTIVE: To compare the analgesic and sedative effects of various species of Rhizoma Cimicifugae and their processed products with honey. METHODS: The three kinds of analgesic experiments of formalin, hot plate and writhing tests, and the number of spontaneous activities and raise forelimbs were adopted in mice. RESULTS: All samples showed antinociceptive activities in the two phases of formalin, tail immersion and writhing tests and sedative properties in the spontaneous locomotor activity test. The analgesic and sedative effects of processed Rhizoma Cimicifugae were higher than crude drugs. CONCLUSION: The present results demonstrate that the antinociceptive and sedative activity of samples processed with honey are more potent than crude drugs.

Cimicifugae rhizoma: from origins, bioactive constituents to clinical outcomes.

Cimicifugae Rhizoma, mainly originated from C. acerina, C. dahurica, C. foetida, C. heracleifolia, C. racemosa and C. simplex, has long been used in traditional medicine system. During the past 45 years, a lot of efforts have been dedicated to the studies on their bioactive constituents, pharmacological effects and clinical uses, and a variety of biological activities including relief of hot flash, anti-osteoporosis, anti-human immunodeficiency virus (HIV), antiinflammatory, antidiabetes, antimalaria and vasoactive property have been discovered. Although C. racemosa is widely applied to relieve menopause symptoms in clinic for its hormonal-like action, meaningfully, no estrogenic effect was confirmed up to date. The purpose of this paper is to systematically highlight these achievements and the further therapeutic potential. The origins and distributions of the rhizome are simply listed and phytochemical aspects including over 200 compounds mainly belonging to cycloartane-type triterpenoids have been summarized. The pharmacological characterizations, especially, as a clinically effective phytomedicine, the effects of the rhizoma on menopause symptoms and the clinical applications including possible mechanism have been reviewed in detail. Various in vivo, in vitro studies on the anti-bone resorption effects of the triterpenoids, together with structure-activity relationships are also incorporated to explore the therapeutic potential on osteoporosis, a major public health threat for postmenopausal women.

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.

[Direct determination of rhizoma cimicifuga by FTIR spectroscopy].

Fifteen samples of original plants of Rhizoma Cimicifuga were determined quickly and directly with the method of FTIR and comparing software. The results showed that the spectra of these samples were characteristic relying on the difference of chemical components and their content of related components. The difference of spectra among samples belonging to different families was distinct, and in different species with same genus too. To the same species, between two samples with different producing areas, or with same producing area but different collection date, the spectra were not obvious. This approach provide a quickly and accurately determination method of crude drug without extraction and separation.