Phytochemical fingerprinting to thwart black cohosh adulteration: a 15 Actaea species analysis

Introduction – The popular use of black cohosh products (Actaea racemosa L., syn. Cimicifuga racemosa L.) is growing as the demand for alternatives to estrogen therapy has increased. Critical to safe use is the assurance of unadulterated, high‐quality products. Questions have been raised about the safety of black cohosh due to cases of liver toxicity in patients who reported taking it; subsequent evaluation found some products to be adulterated with other related herbal species. Correct plant species identification is a key first step for good manufacturing practices of safe black cohosh products. Objectives – To develop analytical methods which distinguish black cohosh from other species (American and Asian) of Actaea increasingly found as adulterants in commercially available black cohosh products. Material and methods – Fifteen species of Actaea were collected from North America and Asia, and the phytochemical fingerprints of these samples were established using HPLC‐PDA and LC‐MS techniques. Results – The HPLC and LC‐MS fingerprints for polyphenols and triterpene glycosides revealed distinct patterns that make black cohosh clearly distinguishable from most other species of Actaea. Two marker compounds, cimifugin and cimiracemoside F, were found to be important to distinguish black cohosh from most Asian species of Actaea. Formononetin was not found from either Asian or American species of Actaea. Conclusions – Phytochemical fingerprinting is a practical, reliable method for authenticating black cohosh and distinguishing it from other species of Actaea increasingly found as adulterants in commercially available black cohosh products. This should facilitate the continued development of high‐quality, unadulterated black cohosh products. Copyright © 2011 John Wiley & Sons, Ltd.     

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.     

Alternative approach to species identification of Actaea racemosa L. (syn. Cimicifuga racemosa (L.) Nutt., black cohosh) herbal starting material: UV spectroscopy coupled with LDA

The North American Actaea racemosa L. (syn. Cimicifuga racemosa (L.) Nutt., commonly known as black cohosh), considered to be a more “natural” alternative to conventional therapies, is used to relieve menopausal symptoms. The high demand for plant material has led to problems with substitution/adulteration of raw material of wholesale origin. The authenticity of the starting material is crucial for the herbal product’s efficacy and safety, and tests on identity and substitution are integral parts of cGMP guidelines. Consequently, there is a need for economical and easy-applicable test procedures. The aim of this study is to reveal the capability of the well-established UV spectroscopy coupled with a multivariate classification procedure to serve as a tool for the identification of A. racemosa. We built a classification model applying linear discriminant analysis (LDA) to distinguish between A. racemosa and its common substitutes. The model showed a high level of accuracy predicting 100% of the samples correctly. Our results indicate that UV spectroscopy shows potential for the development of possible additional authentication methods for this herbal starting material.     

Muscle damage induced by black cohosh (Cimicifuga racemosa)

Extracts of black cohosh (Cimicifuga racemosa) are commonly used for the treatment of symptoms associated with menopause. Adverse events with black cohosh are rare, mild and reversible. A few number of serious adverse events, including hepatic and circulatory conditions, have been also reported, but without a clear causality relationship. We report the case of a woman with severe asthenia and very high blood levels of creatine phosphokinase and lactate dehydrogenase. The patient referred to take a dietary supplement derived from black cohosh for ameliorating menopause vasomotor symptoms. To exclude a possible involvement of this product, the patient was suggested to discontinue this therapy. After suspicion the patient showed a progressive normalization of biochemical parameters and improvement of clinical symptoms. We can hypothesise a causative role for black cohosh in the muscle damage observed in this patient. Factors suggesting an association between black cohosh and the observed myopathy included the temporal relationship between use of herbal product and asthenia and the absence of other identified causative factors. Rechallenge with the suspected agent was inadvisable for ethic reasons because of the risk of a serious relapse. This is the first time that asthenia associated with high muscle enzymes serum levels by black cohosh has been reported. In our opinion, this report is of interest because of the widespread diffusion of use of black cohosh as an alternative medicine for relief from menopausal symptoms.     

Analysis of formononetin from black cohosh (Actaea racemosa)

Black cohosh has been widely used as an herbal medicine for the treatment of symptoms related to menopause in America and Europe during the past several decades, but the bioactive constituents are still unknown. Formononetin is an isoflavone with known estrogen-like activity. This compound was first reported to be isolated from black cohosh in 1985, but subsequent research in 2002 using HPLC-PDA and LC-MS revealed no evidence to show the presence of formononetin in 13 populations of American black cohosh. A more recent report published in 2004 claimed to detect formononetin in an extract of black cohosh rhizomes using a TLC-fluorescent densitometry method. To further resolve these conflicting reports, we analyzed black cohosh roots and rhizomes for the presence of formononetin, using a combined TLC, HPLC-PDA and LC-MS method. We examined both methanolic and aqueous methanolic black cohosh extracts by HPLC-PDA and LC-MS methods, and did not detect formononetin in any extracts. We further determined the limits of detection of formononetin by HPLC-PDA and LC-MS. Our experimental results indicated that the sensitivity and accuracy of the HPLC-PDA and LC-MS methods for the analysis of formononetin were slightly higher than those of the reported fluorescent method, suggesting that the HPLC-PDA and LC-MS methods were reliable for the analysis of formononetin from black cohosh. We also repeated the reported TLC method to concentrate two fractions from a modern black cohosh sample and an 86-year-old black cohosh sample, respectively, and then analyzed these two fractions for formononetin using the HPLC-PDA and LC-MS method instead of the fluorescent method. Formononetin was not detected by HPLC-PDA or LC-MS. From the results of the present study it is not reasonable to attribute the estrogen-like activity of black cohosh extracts to formononetin.     

Analysis of polyphenolic compounds and radical scavenging activity of four American Actaea species

A reversed-phase high-performance liquid chromatography (RP-HPLC) method with diode array detection has been developed for analysis of the major polyphenols in the roots and rhizomes of black cohosh (Actaea racemosa), an important botanical dietary supplement for women's health, and three closely related American Actaea species, A. rubra, A. pachypoda and A. podocarpa. The method was validated with respect to sensitivity, linearity, precision, accuracy and recovery. The total content of eight major polyphenols in the dried root and rhizome of the four species was determined to be from 0.36 to 2.92% (w/w). The antioxidant activities of Actaea extracts and polyphenolic compounds isolated from A. racemosa were evaluated on 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radicals scavenging assay. The radical scavenging activity of the Actaea extracts correlates to their polyphenolic composition. This validated HPLC method can be used to distinguish A. racemosa from the other major American Actaea species based on this study.     

Batch-to-Batch Quality Consistency Evaluation of Botanical Drug Products Using Multivariate Statistical Analysis of the Chromatographic Fingerprint

Botanical drug products have batch-to-batch quality variability due to botanical raw materials and the current manufacturing process. The rational evaluation and control of product quality consistency are essential to ensure the efficacy and safety. Chromatographic fingerprinting is an important and widely used tool to characterize the chemical composition of botanical drug products. Multivariate statistical analysis has showed its efficacy and applicability in the quality evaluation of many kinds of industrial products. In this paper, the combined use of multivariate statistical analysis and chromatographic fingerprinting is presented here to evaluate batch-to-batch quality consistency of botanical drug products. A typical botanical drug product in China, Shenmai injection, was selected as the example to demonstrate the feasibility of this approach. The high-performance liquid chromatographic fingerprint data of historical batches were collected from a traditional Chinese medicine manufacturing factory. Characteristic peaks were weighted by their variability among production batches. A principal component analysis model was established after outliers were modified or removed. Multivariate (Hotelling T ² and DModX) control charts were finally successfully applied to evaluate the quality consistency. The results suggest useful applications for a combination of multivariate statistical analysis with chromatographic fingerprinting in batch-to-batch quality consistency evaluation for the manufacture of botanical drug products.     

Comparison of hormonal activity (estrogen,androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy

Extracts from red clover (Trifolium pratense), soybean (Glycine max.) and black cohosh (Cimicifuga racemosa) are frequently used as alternative compounds for hormone replacement therapy (HRT) to treat menopausal disorders. Fifteen commercially available products made either from red clover, soybean or black cohosh were tested in in vitro assays in this study. The main polycyclic phenolic compounds of soy and red clover products were biochanin A, genistein, daidzein, formononetin, and glycitein. In red clover products glycitein was not abundant. All the compounds showed clear estrogenic activity through estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) and affinity to progesterone receptor (PR) and androgen receptor (AR), whereas the compounds from black cohosh did not. This was corroborated by synthetic isoflavones such as biochanin A, daidzein, genistein and formononetin. They exerted affinity to PR and AR in the range of 0.39-110 mM. Statistical analysis applying principal component analysis (PCA) revealed that all red clover and soy products are grouped in different clusters. Red clover products showed a higher affinity to AR and PR than soy products, which is explained by the higher amount of isoflavones present. In vitro assays and chemical analysis showed that theoretical estrogenic activity expressed as equivalent E2 concentration is in the same range as recommended for synthetic estrogens. Broader spectrum of action and hypothesized lower side effects by action through ERbeta make them suitable for alternative hormone replacement therapy.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80–100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Chemosystematic studies in the saxifragaceae sensu lato. 8. The flavonoids of elmera racemosa (Watson) Rydberg

The flavonoid glycosides of both varieties of Elmera racemosa were isolated and identified. The compounds were the monoglucosides of kaempferol, quercetin and myricetin, the rutinosides of the same aglycones, and the rhamnosylrutinosides of kaempferol and quercetin. All glycosides were linked at position-3 of the flavonols. The two varieties (var. racemosa and var. puberulenta C. L. Hitchcock) were identical. A comparison of the flavonoid chemistry of Elmera, Heuchera, and Tellima supports the existence of Elmera as a genus. A survey of several collections of Tellima grandiflora, Heuchera micrantha, and H. cylindrica showed only minor quantitative differences in the two-dimensional thin layer chromatograms from collection to collection. The possible origin of Tellima and Elmera from ancestral stock having Heuchera-like flavonoid chemistry is discussed.     

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint (Nepeta racemosa) and avocado (Persea americana); evidence for related enzymes with different activities

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYTP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.     

Population genetic analysis of Elliottiaracemosa (Ericaceae), a rare Georgia shrub

Genetic and genotypic diversity found within populations of threatened plant species can have important implications for their conservation and management. In this study we describe genetic and genotypic diversity found within 10 populations of the endemic shrub Elliottiaracemosa (Ericaceae), the Georgia plume. E. racemosa is a threatened species known from fewer than 50 locations, all within the state of Georgia, USA. Seedset is limited to nonexistent in some E. racemosa populations and sexual recruitment has not been documented. However, the species is known to spread vegetatively via root-sprouts. Twenty-one allozyme loci were resolved for E. racemosa, nine of which were polymorphic. Compared with other woody taxa, E. racemosa has low genetic (i.e. allelic) diversity within populations (H_ep = 0.063) and at the species level (H_es = 0.091). Most of the genetic variation (82%) was found within populations, and genetic identities between populations were high (mean I = 0.96). However, genotypic diversity (i.e. the number of multilocus genotypes) differed markedly among populations. Two of the 10 populations consisted almost entirely of single multilocus genotypes, whereas more than 20 multilocus genotypes (in samples of 48 stems) were detected at three sites. Sites in which few multilocus genotypes were detected have low seedset, suggesting that the lack of clonal diversity limits reproduction in some populations of this reportedly self-incompatible species.     

Chemometric Analysis for Identification of Botanical Raw Materials for Pharmaceutical Use: A Case Study Using Panax notoginseng

The overall control of the quality of botanical drugs starts from the botanical raw material, continues through preparation of the botanical drug substance and culminates with the botanical drug product. Chromatographic and spectroscopic fingerprinting has been widely used as a tool for the quality control of herbal/botanical medicines. However, discussions are still on-going on whether a single technique provides adequate information to control the quality of botanical drugs. In this study, high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), capillary electrophoresis (CE) and near infrared spectroscopy (NIR) were used to generate fingerprints of different plant parts of Panax notoginseng. The power of these chromatographic and spectroscopic techniques to evaluate the identity of botanical raw materials were further compared and investigated in light of the capability to distinguishing different parts of Panax notoginseng. Principal component analysis (PCA) and clustering results showed that samples were classified better when UPLC- and HPLC-based fingerprints were employed, which suggested that UPLC- and HPLC-based fingerprinting are superior to CE- and NIR-based fingerprinting. The UPLC- and HPLC- based fingerprinting with PCA were able to correctly distinguish between samples sourced from rhizomes and main root. Using chemometrics and its ability to distinguish between different plant parts could be a powerful tool to help assure the identity and quality of the botanical raw materials and to support the safety and efficacy of the botanical drug products.     

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.     

Plants and traditional knowledge: An ethnobotanical investigation on Monte Ortobene (Nuoro, Sardinia)

Background

Worldwide there is growing research interest in the ethnobiology of mangrove forests. Notwithstanding that, little information has been published about ethnobiology of mangrove forests in Cameroon. The aims of this study were a) to analyze the harvesting methods and the local selling of mangrove wood products by loggers in the vicinity of Wouri estuary and b) to investigate the patterns of subsistence uses of mangrove wood products around the Douala-Edea reserve.

Methods

Semi-structured interviews were conducted with 120 active mangrove loggers in 23 Douala wood markets and 103 households located in three villages (Mbiako, Yoyo I and Yoyo II) close to Douala-Edea reserve. In each of the three densely populated villages, every second household was chosen for sampling while in all markets, mangrove loggers were chosen randomly. In addition, log diameters were measured in each market using a wooden foldable tape measure. A post hoc analysis (Newman-Keuls test) was performed in order to detect the common wood class diameter sold in the Douala wood markets.

Results

The analysis of the loggers' survey data has shown that large logs of Rhizophora with diameter greater than 40 cm were common in the Douala wood markets and were more closely associated with loggers who used chainsaws. In addition to the general mangroves wood products selling, the analysis on a subsistence level (households' survey) suggests the local population's dependence on mangroves, with multiple uses of Rhizophora racemosa Meyer, R. harrisonii Leechman, Avicennia germinans L. Stearn., Laguncularia racemosa Gaertn. f. and Conocarpus erectus L. timbers for furniture, fences, smoking fish, and fuelwood. Finally, Nypa fruticans (Thunb.) Wurmb. leaves were used as thatching material for house walls and roofs.

Conclusion

Our findings revealed that big logs of Rhizophora were commonly sold by the loggers. A majority of loggers (60%) reported that mangrove marketed wood constitute a principal source of income. Most of the villagers (85.83%) often depend on mangroves for subsistence needs and for them there is no substitute for mangrove wood. Therefore, more efforts should be undertaken at the national level to implement conservation, management and sustainable use of these coastal forests.     

Geographical and seasonal patterns of epiphytic diatoms on a subtropical mangrove (Kandelia candel) in southern China

Geographical and seasonal patterns of epiphytic diatoms on a subtropical mangrove (Kandelia candel (L.) Druce) were examined at four sites in southern China. A total of one hundred and three epiphytic diatom species and varieties belonging to 40 genera were identified. Dominant species included Nitzschia fasciculata, Luticola mutica and Achnanthes javanica var. subcontricta. Further analysis using canonical correspondence analysis revealed three diatom associations which could be closely related to environmental variables such as salinity and nutrients resources of nitrogen and phosphate. Epiphytic diatom assemblages on roots would be a good indicator for the evaluation of water quality in mangroves.     

The indirect role of nutrients in enhancing the invasion of Caulerpa racemosa var cylindracea

The effects of nutrients dynamics on biological invasions in marine habitats have not been fully investigated. The present study aimed to evaluate the role of nutrient pollution in determining the successful invasion of the introduced seaweed Caulerpa racemosa var. cylindracea in a Mediterranean subtidal rocky habitat. To this end, a manipulative field experiment was carried out by supplying the water column with nutrients for a one-year period. Afterwards C. racemosa was transplanted in both nutrient-enriched and control macroalgal assemblages. Results showed that the penetration and biomass of C. racemosa in the assemblages previously treated with nutrients were higher than in control ones, indicating that communities stressed by nutrient pollution are more vulnerable to invasion. The nutrient enrichment changed the structure of natural macroalgal assemblages, mainly by encouraging the growth of turf species. No significant effects were detected for the erect and prostrate layers and for the number of species, suggesting that the susceptibility of the community to invasion may depend more on the morpho-functional identity of macroalgae rather than on the diversity of assemblages. Changes in the availability of seawater nutrients may foster the spread of C. racemosa, both by enhancing its growth and eroding the natural resistance of macroalgal assemblages. Thus, the maintenance of good water quality may play an important role in containing both C. racemosa and other introduced seaweeds.     

Genetic diversity and species-specific PCR-based markers from AFLP analyses of Thai bananas

A large amount of banana genetic resource has been found in Thailand which is believed to be one of the centers of its origins. To assess genetic diversity and determine genetic relationships of edible bananas in Thailand, 110 accessions of banana species and cultivars collected from villages and natural locations were investigated. UPGMA clustering of numerical data from Amplified Fragment Length Polymorphism (AFLP) patterns showed two large groups which corresponded to genome designations of Musa acuminata (AA) and Musa balbisiana (BB), the known ancestors of most edible cultivars. The AFLP data suggested that among Thai bananas, AA and AAA cultivars were closely related to M. acuminata subsp. malaccensis, while some of ‘B’ genome contained ones closely related to wild M. balbisiana in Thailand and some may have been imported. Eight species-specific PCR-based primer pairs, generated from the AFLP results clearly identify ‘A’ and ‘B’ genomes within cultivars and hybrids. The analyses were useful to readily and easily infer progenitors of these cultivars and pronounce wide genetic diversity of the bananas in Thailand.