Figure 3 - uploaded by Anita Ankli
Content may be subject to copyright.
High-performance thin-layer chromatogram of different Cimicifuga species, white light; 1-5: C. racemosa; 6-8: C. rubra; 9-16: C. dahurica.
Source publication
Black cohosh (Cimicifuga racemosa) is used to treat discomfort during menopause and as a substitute for synthetic drugs in hormone replacement therapy. The mostly wildcrafted plant is ranked among the top-selling herbs in the United States. There is a risk for adulteration with the similar-looking C. americana, which grows in the same habitats of t...
Citations
... In evaluation of finished product composition, challenges with matrix interference and the detection of herbal extract ingredients may arise. As described in reference to detection of adulteration by HPTLC, ability to detect declines as levels within a mixture drop below 5% (Ankli et al., 2008). Limits of detection by HPTLC may contribute to identity challenges as well. ...
Herbal tea is a mainstay dosage form in practically all systems of traditional medicine and widely used in modern alternative and complementary medicine. Incorporating botanical extracts into herbal tea formulations is of vital interest to manufacturers as it allows for the use of herbal ingredients that would otherwise not be suitable for the dosage form, for instance, dosing requirements, solubility in water, sensory constraints etc. Furthermore, reducing the amount of ingredients in a formula increases compliance with dosing recommendations and thus therapeutic benefit. However, formulating with botanical extracts comes with challenges, ranging from sourcing ingredients of appropriate quality, developing suitable methods for quality control with combinations of (herbal) ingredients, processing constraints such as hygroscopicity, solubility, dispersibility, homogeneity of distribution, and packaging machinability, all the way to stability required for hot-water infusion. We report on experiences with overcoming such challenges in a set of examples and provide guidance to the extract industry on how to tap into the bagged tea sector with better suited or tailor-made solutions for the formulator.
... Therefore, Ankli et al. (2008) proposed another HPTLC test for detection of adulteration in mixtures, which was incorporated into the monograph as part of the tests section under the title "Adulteration with Cimicifuga americana Michx., C. foetida L., C. dahurica (Turcz.) Maxim. ...
Background: Herbal products regulated under different categories were found to be of different quality. This has been demonstrated by the increasing number of reports on the quality of herbal products in the scientific literature. Proper identification is an effective way to address this concerning issue early on in a products’ manufacturing process.
Objectives: To assess the quality of milk thistle, coneflower and black cohosh herbal drugs, preparations and products commercialized under different regulatory categories, and to illustrate the usefulness of HPTLC as a tool for evaluating quality.
Methods: HPTLC methods were adapted from the European Pharmacopeia’s monographs for milk thistle fruits, black cohosh and purple coneflower. Additional detection modes beyond those described in the monographs were employed, and the entire HPTLC fingerprints were used for examination of identity and purity of the investigated samples.
Results: All products regulated as Traditional Herbal Medicinal Products were shown to be of high quality: their fingerprints were consistent and without unexpected zones. A significant number of food supplements show quality issues (mainly adulterations): 52.4% of milk thistle, 33.3% of coneflower, and 45.5% of black cohosh products. The same was observed in 66.6% of black cohosh herbal drugs and preparations.
... Chemical methods always encounter impediments when distinguishing different herbal species that share similar chemical compounds within the complicated prescription (16). One of the chemicalbased TLC profiles designed to differentiate black cohosh from its closely related species was found to be ineffective due to similar banding patterns of A. racemosa and other species such as A. dahurica and A. rubra (17). These intricacies will hinder chemical-based processes from being developed into a rapid authentication method for non-scientist users, such as commercial enterprises and government regulators. ...
BACKGROUND
Actaea racemosa (Black cohosh) herbal dietary supplements are commonly used to treat menopausal symptoms in women. However, there is a considerable risk of contamination of A. racemosa herbal products in the natural health product (NHP) industry, impacting potential efficacy. Authentication of A. racemosa products is challenging because of the standard, multi-part analytical chemistry methods that may be too costly and not appropriate for both raw and finished products.
OBJECTIVE
In this paper, we discuss about developing and validating quick alternative biotechnology methods to authenticate A. racemosa herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay.
METHODS
A qPCR based species-specific hydrolysis probe assay was designed, validated, and optimized for precisely identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in determining the target species ingredient, while not identifying other non-target species, (2) sensitivity in detecting the smallest amount of the target material, and (3) reliability (repeatability and reproducibility) in detecting the target species in raw materials on a real-time PCR platform.
RESULTS
The results show that the species-specific hydrolysis probe assay was successfully developed for the raw materials and powders of A. racemosa. The specificity of the test was 100% to the target species. The efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting and powder materials.
CONCLUSION
The method developed in this study can be used to authenticate and perform qualitative analysis of A. racemosa supplements.
... Numerous high-performance (HP)TLC methods for phenolic acids are reported in the literature. The most frequently used stationary phase is silica gel [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], however, bonded silica gel stationary phases, namely, octadecyl (RP-18) [21], aminopropyl (NH 2 ) [22][23], diol [22,[24][25], and cyanopropyl (CN) [2], as well as non-silica adsorbents, cellulose [26][27][28][29][30][31][32], and polyamide [33], were also reported. The applied developing solvents comprise a wide array of various solvent combinations, wherein the addition of acidic modifier is common with all the developing solvents. ...
... A combination with mass spectrometry, namely, high-performance thin-layer chromatography-mass spectrometry (HPTLC-MS), was also applied [40][41]. Other detections are less sensitive and include post-chromatographic derivatizations, usually with natural product reagent (NP, diphenylboryloxyethylamine solution) [13,17] in combination with polyethylene glycol (PEG) [2,8,18] or with paraffin-n-hexane (1:2, v/v) [9] reagents, but also with iodine vapor [42] and solutions of ferric (III) chloride [14,31] or aluminum chloride [5,10]. ...
High-performance thin-layer chromatography (HPTLC) and HPTLC–mass spectrometry (MS)/(MSⁿ) methods for analyses of phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) were developed. Separation was performed on HPTLC silica gel plates with and without fluorescent indicator (F254) in a saturated twin-trough chamber using n-hexane‒ethyl acetate‒formic acid (12:8:2, v/v) as the developing solvent. The developed HPTLC method is also suitable for the preliminary screening of some flavonoids (flavone, apigenin, luteolin, chrysin, quercetin, myricetin, kaempferide, kaempferol, hesperetin, naringenin, pinocembrin), although some interferences of phenolic acids with flavonoids were observed. The effect of pre-development on the HPTLC analysis of phenolic acids on the detection by densitometry and mass spectrometry was also explored. Pre-development of the plates with chloroform‒methanol (1:1, v/v) decreased the intensity of secondary front like dark band that appeared at RF 0.7 on unpre-developed plates and enabled densitometric evaluation of phenolic acids at 280 and 330 nm. To eliminate severe spectral background observed during HPTLC–MS analysis, caused by the presence of an acidic modifier in optimized developing solvent, two pre-developments of the plates (1st methanol‒formic acid 10:1, v/v and 2nd methanol) were applied. This resulted in a substantial decrease in the intensity of the background signals of sodium formate clusters and considerably improved the analysis of phenolic compounds. The applicability of the developed HPTLC and HPTLC–MS/(MSⁿ) methods was confirmed by analyses of different complex matrix samples, e.g., propolis, roasted coffee, and rose hip crude extracts.
... This established tool offers several advantages; it presents results as colourful images, documents multiple profiles together on one image and it can be fully automated. This technique has gained popularity among various analytical fingerprinting techniques for the quality control of traditional medicines (Fan et al., 2006;Ankli et al., 2008). ...
The leaves, root and bark of the aromatic African indigenous plant, Croton gratissimus Burch. (Euphorbiaceae), are widely used in traditional medicine to treat coughs, chest complaints, rheumatism, abdominal disorders and fever among others. In Afrikaans it is referred to as “Koorsbessie” which alludes to its traditional use as a pyrogenic. The chemical composition of plants is very complex and analysis and quality control can be very challenging due to natural variability. In addition, very few reference standards from plants, especially from Africa, are commercially available. Due to its visual nature and the holistic fingerprint produced, high performance thin layer chromatography (HPTLC) is often recommended for the quality control of plant material. The aim of this study was to develop an HPTLC fingerprint and isolate marker compounds for inclusion on HPTLC plates to enable quality control. Croton gratissimus leaf samples were collected from various parts of South Africa and extraction was optimised. HPTLC fingerprints were developed and optimised and images were captured before and after derivatisation under UV (254 nm, and 366 nm) and white light. Preparative high performance liquid chromatography hyphenated to mass spectrometry (HPLC-MS) was used to isolate marker compounds. Method development and optimisation determined the following: most efficient extraction solvent = methanol:water (8:2 v/v); mobile phase = ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v/v/v); and derivatising agent = natural product reagent. UPLC-MS analysis and 1D NMR spectroscopy were used to characterise and identify compound 1 as isoorientin and compound 2 as kaempferol-3-β-d-(6″-O-trans-p-coumaroyl) glucopyranoside, which correlated well with published spectral data. The final HPTLC fingerprint with biomarkers included showed good separation for profiling purposes and well-defined bands. The biomarkers at retention factor (Rf) 0.30 and Rf 0.69 for isoorientin and kaempferol-3-β-d-(6″-O-trans-p-coumaroyl) glucopyranoside, respectively, were present in all samples but varied quantitatively. The HPTLC method developed provided a good fingerprint for species authentication. Preparative HPLC-MS played a major role in successfully isolating marker compounds to be used for the quality control of C. gratissimus.
... The increasing pharmaceutical interest in A. racemosa demands fast and reliable techniques for authentication of the raw plant material as well as quantification of valuable secondary metabolites for QA. Usually, for authentication of A. racemosa rhizome, according to the pharmacopoeia, HPTLC of the extracts is performed, whilst polyphenols and triterpene glycosides are considered in various LC methods [12,[19][20][21][22][23]. Especially polyphenols are favourably examined to investigate quality and homogeneity of plant materials due to their high abundance and UV activity [24,25]. ...
Rhizomes of Actaea racemosa L. (formerly Cimicifuga racemosa) gained increasing interest as a plant-derived drug due to its hormone-like activity and the absence of estrogenic activity. According to the Current Good Manufacturing Practices guidelines and pharmacopeial standards, quality assessment of herbal starting materials includes tests on identity and substitution, as well as quantification of secondary metabolites, usually by HPTLC and LC methods. To reduce the laboratory effort, we investigated near-infrared spectroscopy for rapid species authentication and quantification of metabolites of interest.
Near-infrared spectroscopy analysis is carried out directly on the milled raw plant material. Spectra were correlated with reference data of polyphenols and triterpene glycosides determined by LC/diode array detection and LC/evaporative light scattering detection, respectively. Quantification models were built and validated by cross-validation procedures. Clone plants, derived by vegetative propagation, and plants of a collection from different geographical origins cultivated in Berlin were analysed together with mixed batches from wild harvests purchased at wholesalers.
Generally, good to excellent correlations were found for the overall content of polyphenols with coefficients of determination of R² > 0.93. For individual polyphenols such as fukinolic acid, only models containing clone plants succeeded (R² > 0.92). For the total content of triterpene glycosides, results were generally worse in comparison to polyphenols and were observed only for the mixed batches (R² = 0.93).
Next to quantitative analysis, near-infrared spectroscopy was proven as a rapid alternative to other, more laborious methods for species authentication. Near-infrared spectroscopy was able to distinguish different Actaea spp. such as the North American Actaea cordifolia and the Asian Actaea cimicifuga, Actaea dahurica, Actaea heracleifolia, and Actaea simplex.
... Thus, the herbal raw material authentication as a part of current Good Manufacturing Practices is essential. Rapid high performance thin layer chromatography (HPTLC) methods are remarkable powerful in the authentication of species and in detection of adulterants (Ankli et al., 2008). Hence, the corresponding monographs dedicated to A. racemosa roots and rhizomes in the United States and the European Pharmacopeia have adopted such methods. ...
Introduction:
The medicinal plant Actaea racemosa L. (Ranunculaceae, aka black cohosh) is widely used to treat climacteric complaints as an alternative to hormone substitution. Recent trials prove efficacy and safety of the approved herbal medicinal products from extracts of pharmaceutical quality. This led to worldwide increasing sales. A higher demand for the plant material results in problems with economically motivated adulteration. Thus, reliable tools for herbal drug authentication are necessary.
Objective:
To develop an economical, plain, and rapid method to distinguish between closely related American and Asian Actaea species, using securely established and resilient analytical methods coupled to a chemometric evaluation of the resulting data.
Methodology:
We developed and validated a RP-PDA-HPLC method including an extraction by ultra-sonication to determine the genuine contents of partly hydrolysis-sensitive polyphenols in Actaea racemosa roots and rhizomes, and applied it to a large number of 203 Actaea samples consisting of seven species.
Results:
We were able to generate reliable data with regards to the polyphenolic esters in the samples. The evaluation of this data by principle component analysis (PCA) made a discrimination between Asian Actaea species (sheng ma), one American Actaea species (Appalachian bugbane), and A. racemosa possible.
Conclusion:
The developed RP-PDA-HPLC method coupled to PCA is an excellent tool for authentication of the Actaea racemosa herbal drug, and can be a powerful addition to the TLC methods used in the dedicated pharmacopoeias, and is a promising alternative to expensive and lots of expertise requiring methods. Copyright © 2016 John Wiley & Sons, Ltd.
... TLC was the principal method used in authentication of these valuable herbs (Upton, 1999a(Upton, ,b,c,d,e,f, 2000a(Upton, ,b,c,d, 2001a(Upton, ,b,c, 2002a(Upton, ,b, 2003a(Upton, ,b, 2004(Upton, , 2007a(Upton, ,b, 2008(Upton, , 2009(Upton, , 2010a(Upton, ,b,c, 2012aRomm and Upton, 2012). Inspired by early work, authentication of various species of famous food supplements such as Chamomile, Ginseng, Ginkgo, black Cohosh, and Radix Puerariae and Scutellaria as well as measurement of the consistency of their preparations became possible (van Beek and Montoro, 2009;Ankli et al., 2008;Ahmad et al., 2009;Wang et al., 2004;Hong et al., 2009;Xie et al., 2006;Chen et al., 2006). p0240 ...
... HPTLC can also be coupled to densitometric detectors to generate quantitative and qualitative data for use in chemometric modeling, although visual inspection is generally used for differentiating between species. HPTLC has been shown to be capable of differentiating between various Actaea, Echinacea, and Panax species (59)(60)(61). Information regarding the validation of HPTLC identity methods has been summarized (62). A considerable amount of experience and skill is required to properly interpret the data, but as new detection and recording techniques are developed, visual interpretation may be replaced by more objective instrumental and statistical interpretation techniques. ...
The American Herbal Products Association estimates that there as many as 3000 plant species in commerce. The FDA estimates that there are about 85,000 dietary supplement products in the marketplace. The pace of product innovation far exceeds that of analytical methods development and validation, with new ingredients, matrixes, and combinations resulting in an analytical community that has been unable to keep up. This has led to a lack of validated analytical methods for dietary supplements and to inappropriate method selection where methods do exist. Only after rigorous validation procedures to ensure that methods are fit for purpose should they be used in a routine setting to verify product authenticity and quality. By following systematic procedures and establishing performance requirements for analytical methods before method development and validation, methods can be developed that are both valid and fit for purpose. This review summarizes advances in method selection, development, and validation regarding herbal supplement analysis and provides several documented examples of inappropriate method selection and application.
... Methods from the following sources were evaluated in this review: Upton, 2 the USP 34, 11 the EP 7.5, 13 Gafner et al., 30 Wagner and Bladt, 49 Zheng et al., 50 Ankli et al., 51 and Verbitski et al. 52 Comments: The HPTLC analysis of Actaea is a rare instance where the majority of authors have relied on the same stationary and mobile phases. The thorough validation, proven ability to detect adulteration, and flexibility to target various phytochemicals depending on the detection approach make the ethyl formate-toluene-formic acid (3:5:2, v/v) mobile phase using HPTLC silica gel 60 F 254 plates the method of choice for HPTLC analysis of black cohosh ( Figure 4). ...
... The thorough validation, proven ability to detect adulteration, and flexibility to target various phytochemicals depending on the detection approach make the ethyl formate-toluene-formic acid (3:5:2, v/v) mobile phase using HPTLC silica gel 60 F 254 plates the method of choice for HPTLC analysis of black cohosh ( Figure 4). 2,11,13,30,[51][52][53] Since method validations were conducted using the sample preparation and detection system described in references 11 and 51, the consensus of authors and expert peer reviewers of this Laboratory Guidance Document is that this procedure is the most suitable in a routine QC lab. While this method is capable of distinguishing various Actaea species based on the chemical fingerprint, the detection of adulterating species -in particular when such species are added to A. racemosa -remains challenging. ...
... While this method is capable of distinguishing various Actaea species based on the chemical fingerprint, the detection of adulterating species -in particular when such species are added to A. racemosa -remains challenging. Some of the related North American species exhibit a constituent profile similar to black cohosh; in addition, the constituent profile may vary depending on the geographic location and manufacturing process, although according to Eike Reich of CAMAG, the chemical composition of black cohosh is rather consistent (Eike Reich e-mail communication, November 19, 2014) To detect adulteration with Chinese Actaea species, the presence of 15 (found in, e.g., A. cimicifuga, A. dahurica, A. heracleifolia, and A. simplex) can be verified using boric acid-oxalic acid reagent reported by Ankli et al. 51 The application of boric acid-oxalic acid reagent leads to a strong fluorescence of 15 under UV light at 366 nm, and allows the detection of as little as 1% of A. cimicifuga and A. simplex in black cohosh ( Figure 5). For obvious reasons, the boric acid-oxalic acid reagent does not allow the detection of adulteration with Actaea species where 15 is absent (e. g., A. pachypoda, A. podocarpa, and A. rubra). ...
In recent years, adulteration of black cohosh roots and rhizomes (Actaea racemosa, Ranunculaceae) has become more apparent. Adulteration predominantly occurs with Chinese species of Actaea such as A. heracleifolia, A. dahurica, and A. cimicifuga (all known by the common name Chinese cimicifuga and by the Chinese name of sheng ma). Additionally, the Chinese cimicifuga (sheng ma) market is commonly adulterated with Serratula chinensis (guang dong sheng ma [Asteraceae]). Adulteration has also been reported with North American Actaea species growing in the same area as black cohosh, such as A. pachypoda, A. rubra, and A. podocarpa. This Laboratory Guidance Document presents a review of the various analytical technologies used to differentiate between authentic A. racemosa and its potentially adulterating species.
