Validation and optimization of qPCR method for identification of Actaea Racemosa (Black cohosh) NHPs

Abstract 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.

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Validated Identity Test Method for Ginkgo biloba NHPs Using DNA-Based Species-Specific Hydrolysis PCR Probe

Journal of AOAC International ◽

10.1093/jaoac/102.6.1779 ◽

2019 ◽

Vol 102 (6) ◽

pp. 1779-1786 ◽

Cited By ~ 1

Author(s):

Shanmughanandhan Dhivya ◽

Subramanyam Ragupathy ◽

Prasad Kesanakurti ◽

Shanmughanandhan Jeevitha ◽

Isabella Della Noce ◽

...

Keyword(s):

Ginkgo Biloba ◽

Raw Materials ◽

Target Material ◽

Test Method ◽

Target Species ◽

Herbal Products ◽

Hydrolysis Probe ◽

Identity Test ◽

Probe Assay ◽

Species Specific

Abstract Background: There is considerable risk of adulteration of Ginkgo biloba herbal products in the natural health product (NHP) industry. Authentication of G. biloba products is challenging because of the standard, complex, analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. Objective: We sought to develop and validate an alternative method to authenticate G. biloba herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. Methods: A species-specific hydrolysis probe assay was developed, validated, and evaluated for the performance of the assay in accurately identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in identifying the target species ingredient, while not identifying other nontarget 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 species-specific hydrolysis probe assay was successfully developed for raw materials of G. biloba. The specificity of the assay was 100% to the target species. Efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting materials. Conclusions and Highlights: The method developed in this study is simple, rapid, and easy for supplement manufacturers to perform in their laboratories to ensure that their G. biloba supplements are authentic.

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Validated Identity Test Method for Ginkgo biloba NHPs Using DNA-Based Species-Specific Hydrolysis PCR Probe

Journal of AOAC International ◽

10.5740/jaoacint.18-0319 ◽

2019 ◽

Vol 102 (6) ◽

pp. 1779-1786 ◽

Cited By ~ 1

Author(s):

Shanmughanandhan Dhivya ◽

Subramanyam Ragupathy ◽

Prasad Kesanakurti ◽

Shanmughanandhan Jeevitha ◽

Isabella Della Noce ◽

...

Keyword(s):

Ginkgo Biloba ◽

Raw Materials ◽

Target Material ◽

Test Method ◽

Target Species ◽

Herbal Products ◽

Hydrolysis Probe ◽

Identity Test ◽

Probe Assay ◽

Species Specific

Background: There is considerable risk of adulteration of Ginkgo biloba herbal products in the natural health product (NHP) industry. Authentication of G. biloba products is challenging because of the standard, complex, analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. Objective: We sought to develop and validate an alternative method to authenticate G. biloba herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. Methods: A species-specific hydrolysis probe assay was developed, validated, and evaluated for the performance of the assay in accurately identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in identifying the target species ingredient, while not identifying other nontarget 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 species-specific hydrolysis probe assay was successfully developed for raw materials of G. biloba. The specificity of the assay was 100% to the target species. Efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting materials. Conclusions and Highlights: The method developed in this study is simple, rapid, and easy for supplement manufacturers to perform in their laboratories to ensure that their G. biloba supplements are authentic.

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DNA Barcode Identification of Black Cohosh Herbal Dietary Supplements

Journal of AOAC International ◽

10.5740/jaoacint.11-261 ◽

2012 ◽

Vol 95 (4) ◽

pp. 1023-1034 ◽

Cited By ~ 42

Author(s):

David A Baker ◽

Dennis Wm Stevenson ◽

Damon P LittLe

Keyword(s):

Adverse Events ◽

Dietary Supplements ◽

Related Species ◽

Plant Material ◽

Dna Barcode ◽

The Other ◽

Black Cohosh ◽

Test Plant ◽

Actaea Racemosa ◽

Validation Set

Abstract 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.

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

Scientific Reports ◽

10.1038/s41598-020-80465-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jeevitha Shanmughanandhan ◽

Dhivya Shanmughanandhan ◽

Subramanyam Ragupathy ◽

Thomas A. Henry ◽

Steven G. Newmaster

Keyword(s):

Quantitative Pcr ◽

Digital Pcr ◽

Molecular Techniques ◽

Black Cohosh ◽

Natural Health Products ◽

Real Time Quantitative Pcr ◽

Actaea Racemosa ◽

Potential Applicability ◽

Species Specific ◽

Pathogen Testing

AbstractThe 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.

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Quantitative Determination of Triterpenoids and Formononetin in Rhizomes of Black Cohosh (Actaea racemosa) and Dietary Supplements by Using UPLC-UV/ELS Detection and Identification by UPLC-MS

Planta Medica ◽

10.1055/s-0028-1088384 ◽

2008 ◽

Vol 75 (04) ◽

pp. 381-386 ◽

Cited By ~ 27

Author(s):

Bharathi Avula ◽

Yan-Hong Wang ◽

Troy Smillie ◽

Ikhlas Khan

Keyword(s):

Quantitative Determination ◽

Dietary Supplements ◽

Black Cohosh ◽

Detection And Identification ◽

Actaea Racemosa

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Profiles of Phenolic Acids and Triterpene Glycosides in Commercial and Cultivated Black Cohosh

Planta Medica ◽

10.1055/a-0981-4287 ◽

2019 ◽

Vol 85 (14/15) ◽

pp. 1160-1167

Author(s):

Marian Bittner ◽

Regina Schenk ◽

Andreas Springer ◽

Matthias F. Melzig

Keyword(s):

Phenolic Acids ◽

Triterpene Glycoside ◽

Triterpene Glycosides ◽

Black Cohosh ◽

Herbal Products ◽

Chromatographic Fingerprints ◽

Actaea Racemosa ◽

Wild Harvesting ◽

Cultivated Plant

AbstractThe medicinal plant Actaea racemosa is a perennial, whose rhizome (black cohosh rhizome) is usually wild harvested on a multiton scale to meet market requirements. Since this North American species is increasingly endangered, cultivation is needed. Even though studies prove that cultivation is possible, it has not been widely established. This may be due to a different quality of cultivated material, which does not comply with current pharmacopoeial requirements. This study compares contents and chromatographic fingerprints of phenolic acids and triterpene glycosides in different types of black cohosh rhizomes. Commercial batches from wild harvests were compared to individual plants from the wild and from cultivation. Phenolic acidsʼ contents and profiles were generally comparable between wild harvesting and cultivation. On the contrary, the total triterpene glycoside content was significantly lower in cultivation (p ≤ 0.001). In individual plants, different profiles of triterpene glycosides occurred. Possibly, specimen or chemotype selection for cultivation would cause a shift of the triterpene glycoside profile of cultivation batches away from the common pattern found in batches from wild harvesting. Potentially, such differences have an impact on the efficacy of black cohosh herbal products, if cultivated plant material is used for manufacturing.

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Overview of the Russian market of herbal products

Remedium Journal about the Russian market of medicines and medical equipment ◽

10.21518/1561-5936-2021-3-11-22 ◽

2021 ◽

pp. 11-22

Author(s):

Natalia V. Safonova ◽

Elena O. Trofimova

Keyword(s):

Dietary Supplements ◽

Raw Materials ◽

Dosage Forms ◽

Annual Growth Rate ◽

Sources Of Information ◽

Herbal Products ◽

Preparation Methods ◽

Plant Raw Materials ◽

Foreign Products ◽

Scope Of Application

Introduction. In the Russian pharmaceutical market, there is the following situation: herbal medicinal products (HMP) and dietary supplements based on plant raw materials (DS) have similar compositions, dosage forms and scope of application. They’re often considered analogues by consumers and specialists, which indicates the existence of common market for these products.Research aim is to investigate the state and development trends of the Russian HMP and DS market, assessing positions of foreign and domestic companies and general prospects for this market as well.Methodology. The study was conducted using IQVIA retail audit database, official sources of information on HMP and DS registration. In the market analysis the products were classified depending on the scope of application, composition and preparation methods, and dosage forms as well.Results. In 2020 pharmacy of HMP and DS sales amounted to 435.4 million units valued at 51.3 billion rubles in wholesale prices. Beginning with 2016, the average annual growth rate corresponded to 4.2%, in value terms. However, in volume terms, the sales were reduced in the average by 3.4% per year (due to cheap domestic medicines). The main part of the entire market comprises HMP (80% in units and 74% in value terms in 2020). The products, affecting respiratory system, genitourinary sphere, and digestion, provide more than three-quarters of all sales in value terms. More than half of the market is occupied by total extractive drugs. Solid dosage forms (tablets and capsules) account for half of the sales, plant raw materials and fees constitute more than one third. In 2020, Russian companies provided 28% of the sales of HMP and 67% of DS in value terms. In the context of the main segments, the market structure in 2016-2020 remained fairly stable.Conclusion. In the future, the market dynamics in value terms will be determined considering by household income and the epidemiological situation. In units, HMP sales will continue to decline through the unprofitable positions of the traditional domestic drug assortment. The increasing strictness of legislation requirements for the market of dietary supplements may contribute to investment in the development of HMP that are competitive to foreign products in terms of quality and consumer characteristics.

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