An effective detection and quantification method for adulterated Natural Health Products with Actaea racemosa (Black cohosh) as a model, assessed using targeted and non-targeted adulterants.
Natural health products are obtained from various sources like plants, animals, micro-organisms and marine sources, are being consumed for various health benefits. They are in various forms such as tablets, capsules, tinctures, solution, creams, vitamins, minerals. These end products, after manufacturing process are not in their original forms and hence, it is difficult to identify and quantify them macroscopically and using chemical markers. However, it is certain that all these end products have DNA in them. DNA is persistent and hard to destroy under any environmental conditions. The aim of this study is to develop an effective method for the detection and quantification of Natural health products using DNA based molecular methods such as Q-PCR. Model plant species taken in this study is which is commonly known as Black cohosh. It is one of the important herbal medicines used to treat premenstrual discomfort and menopause symptoms. But there are various reports about their detrimental events and noxiousness concomitant with their usage. Misidentification, Intentional or unintentional adulteration and/or substitution results in replacement of original black cohosh with other related species that are known to be toxic when consumed. Therefore, development of specific assays for their specific detection and quantification are needed, which can be specified as a standard to facilitate our/industry use in routine laboratory sample testing.
Modern living in the present century needs ancient wisdom along with worldwide accepted allopathic treatment. Several ancient traditions emphasize healthy living. One among them are the Natural health products (NHP’s). They are a class of health products, which include: vitamin and mineral supplements, herbal preparations, traditional and homeopathic medicines, probiotics and enzymes. Herbal phytopharmaceuticals which have reached US $60 billion currently, with annual growth rates of 5–15 %, is expected to increase up to US$115 billion by 2020, represent a significant share of the total world pharmaceutical market [9, 13]. The current increase may be due to the interest of phytopharmaceuticals in psychosomatic, metabolic and minor disorders. Therefore traditional medicine is highly recommended by the WHO as they are essential for more than 70 % of the world’s population that do not have access to Western medicine .
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An increasing awareness of quality irregularities during the manufacturing process of these NHPs is calling attention to the quality of traded mass-produced herbal products with direct impact on their efficacy and safety . During the manufacturing process, NHP’s are being compromised in both their quality as well as quantity either knowingly or unknowingly (Fig.1). The herbal medicines adulteration in NHPs could cause a more serious problem since it can directly reduce the therapeutic effect/value of the original drug or even pose a serious risk to the health of the consumers . Consequently, authenticity has consequently become a major concern for all parties (farmers, producers, suppliers, retailers and regulators) involved in the herbal industry, which affects all levels of the production and distribution process, from raw materials to finished products .
It is difficult to detect and trace the source of unintentional contamination and related NHP safety concerns. It is even more difficult to detect and trace back instances of intentional product fraud, especially in highly processed NHPs with inputs from multiple suppliers. Authentication of constituents in NHPs using analytical chemistry methods (like HPLC-High Performance Liquid Chromatography, MS- Mass Spectrometry and TLC-ThinLayer Chromatography) can help detect contaminants and toxins, but are often limited or incapable of detecting the source of the contamination . Standardization procedures currently exist and are based on:
1) The quantification of the active principle(s) (when they are known),
2) Detection of chemical marker(s) for assessing the correct botanical origin of the plant material,
3) Acquisition of the complete metabolite profile (metabolome) and
4) A comprehensive estimation of the biological variability of the extracts.
In addition, depending on the nature of the plant material used, various analytical validations are performed to ensure the absence of toxic or allergenic compounds. These procedures ensure the quality of the botanical in terms of its composition and safety for human consumption .
Recent studies focus only on the qualitative analysis of NHPs. For example, the roots of Actaea racemosa (black cohosh) are readily distinguished macroscopically from A. dahurica, A. foetida, and A. heracleifolia. The Chinese Cimicifuga species are almost always sliced, displaying a sinusoid pattern that is not generally observed in authentic black cohosh. The Chinese Cimicifuga adulterant Serratula chinensis can be similarly distinguished since it has no morphological resemblance to either Actaea species . The phytochemical profiles of authentic black cohosh and the various adulterating species are similar. Therefore identification by these profiles alone is difficult.
Molecular (DNA) based qualitative analytical methods are also being used to detect the quality of the NHPs. For example, methods like AFLP (Amplified Fragment Length Polymorphism) fingerprinting has been used to detect black cohosh from its adulterant species like A.cordifolia, A.pachypoda and A.podocarpa . DNA based barcode methods have been proposed to identify black cohosh from its adulterant species . All these above-mentioned methods allows to verify only the quality of NHPs (whether it is adulterated or not).
But it is very important to quantify these NHPs as it determines the quality of a substance (presence or absence) present in them. However, only a very few studies have used PCR based methods to quantify biomaterials like meats or genetically modified products in animals, plants and various genetically modified organisms (GMOs) [6, 15-18]. Therefore, DNA-based molecular biology techniques are gaining popularity for quantitative analysis of biological materials, as DNA is more species specific and less influenced by environmental conditions than other chemical compounds. The use natural health products (NHPs) are consistently increasing and their quality is still at risk. There are not many publications related to quantification of these NHPs.
My study aims to quantify plant based herbal medicines and natural health products that are frequently adulterated or contaminated using species-specific molecular markers. One such industrially and economically very important herbal medicine is Actaea racemosa syn. Cimicifuga racemosa, commonly known as ‘Black cohosh’, is native to North America, where it occurs in Eastern Canada (Ontario, Quebec), and in the Eastern United States from Massachusetts to Georgia, and west to Illinois and Arkansas. Currently, black cohosh is used to alleviate premenstrual discomfort, dysmenorrhea and symptoms of menopause (hot flashes, excessive sweating, sleep disorders, irritability). Traditionally, black cohosh is used for colds, dyspepsia, rheumatoid arthritis, sciatica, snakebites, tinnitus, and whooping cough, but such use is not supported by experimental or clinical data. Bulk black cohosh, which can be described as a- crude raw material is sold as whole rhizome and roots, in the form of a teabag, cut, or as powdered rhizome and roots. Extracts such as fluid extracts or dry extracts in the form of tablet or capsule are being used. The dry extracts are often standardized to contain 2.5% triterpene glycosides.
Literature Review on Actaea racemosa
Foster has published the most extensive review on the adulteration of Black cohosh. Since its publication in 2012, there have been three new studies that used combinations of genetic and chemical methods to authenticate commercial black cohosh products. These studies provided additional evidence of continuing adulteration of purported A. racemosa products with Actaea species indigenous to Asia. Two of the studies used genetic authentication techniques (restriction fragment length polymorphism [RFLP] and amplification refractory mutation system [ARMS] or direct sequencing of the ITS1-ITS2, ITS1, and trnL-F regions) combined with high-performance liquid chromatography (HPLC-ELSD or HPLC-MS) to establish botanical identity. These results had significantly different phytochemical profiles. Therefore, in the absence of a sufficiently large number of finished product samples with well-established provenance, the chemometric methods were considered unsuitable for adequate authentication of the ingredients in dietary supplements . There are only a very few DNA based methods (DNA barcoding) of identifying black cohosh supplements which only detects the presence of target species and its adulterants .
We do not yet understand molecular authentication issues. For example, fresh botanical materials are processed effortlessly as DNA will be intact, in large enough pieces, sufficient to be amplified and render enough sequence information for identification at the species level . During NHP production, as previously discussed, botanical ingredients undergo various processes such as heat treatments, ultraviolet exposure, filtration and fluid extraction. and differential PCR biased mechanisms which makes it difficult to quantify botanicals used in NHP industry . All these treatments will eventually lead to DNA degradation and be an hindrance to DNA authentication given the ability to amplify and sequence DNA targets.
In addition to molecular authentication, various PCR biased mechanisms (DNA amplification) also makes it difficult to quantify botanicals. DNA amplification can be a hindrance in development of robust quantitative analytical assays . Numerous PCR based studies have reported that primer affinity to a corresponding DNA target gets affected by aspects like GC content .
Hence the focus of my research is to address the gap between detection and quantification of these adulterated botanical mixtures of NHPs and to develop a qualitative and quantitative PCR assay suitable for identification and quantification of adulterated NHPs.
Materials and Methods
Target species selection (model plant):
- Due to the current socio economic and environmental stress, women experiencing greater premenstrual discomfort and menopause. Hence this black cohosh is been extensively used for medication.
- All other Actaea species such as Actaea podocarpa, Actaea pachypoda are poisonous.
- It is industrially very important and has high commercial value.
- The higher price of authentic black cohosh compared to Chinese cimicifuga has created an incentive for economically-motivated adulteration. While accidental adulteration of domestically wild-crafted black cohosh with North American species of Actaea may occur, it is rarely reported. Most often black cohosh is adulterated with or substituted by Chinese species of Actaea .
- From 2012-2014, black cohosh has consistently been one of the 10 top-selling herbs in the mainstream market and has ranked within the 30 top- selling herbs of the natural foods sector in the United States .
- To my knowledge, this study is the first to quantify Actaea racemosa from its adulterant species.
Non-target species selection:
- There are 28 closely related species of A. racemosa. Out of these 28, some of the closely related species (e.g. A.pachypoda, A.podocarpa, A.rubra, Cimicifuga rubifolia, C.simplex).
- Sometimes, Actaea products are believed to contain other plant species as added/additional ingredients (e.g. Medicago sativa, Avena sativa, Vitex agnus-castus, Angelica sinensis ).
- And finally, in the process of preparing of an Actaea product, medicinal and non-medicinal ingredients are added as fillers (e.g. Oryza sativa, Glycine max, Zea mays, Trifolium ).
In order to quantify Actaea racemosa, I am going to choose 45 samples containing three species of Actaea- Actaea racemosa, A.podocarpa, A.pachypoda (36 industrial samples and 9 Biological Reference Material samples) will be included in the experiment. All the experiments and sampling would be in triplicates.At first, DNA will be extracted from these samples and will be mixed in different ratios of non-targets and target respectively such as 5:95, 10:90, 15:85. 20:80, 25:75, 30:70.Then the original target and non-target samples will be mixed in the same ratios as mentioned above and then DNA extraction will be carried out.
DNA extraction and Quantification
DNA will be extracted using the Macherey-Nagel NucleoSpin® Plant II “Genomic DNA from Plant” Kit, from industry samples as well as BRM samples. DNA metrics will then be assessed by Fluorometric quantification using a QubitTM 3.0 Fluorometer with a Qubit® dsDNA HS assay kit, according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA).
Real time PCR via Probes
First, suitable probes for the three species of Actaea will be deisgned and it will be tested for it’s
- Specificity– refers to the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample .
- Repeatability– (short-term precision or intraassay variance) refers to the precision and robustness of the assay with the same samples repeatedly analyzed in the same assay .
- Reproducibility- (long-term precision or intraassay variance) refers to the variation in results between runs or between different laboratories .
- Sensitivity– limit of detection/ linit of quantification of DNA from highest to lowest quantity.
Real time PCR analysis will be carried out on a LightCycler® 480 Instrument II (Roche Diagnostics, USA) using the SensiFASTTM Probe No-ROX Kit (Bioline, UK). In real-time quantitative PCR (often shortened to real-time PCR or qPCR), PCR product is measured at each cycle. By monitoring reactions during the exponential amplification phase of the reaction, we can determine the initial quantity of the target with great precision via fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product molecules (amplicons) generated. Data collected in the exponential phase of the reaction yield quantitative information on the starting quantity of the amplification target. In Q-PCR, amplification and detection occur in a single tube, eliminating post-PCR manipulations. [Fig.2].
Quantification will be performed to quantify the amount of targets and non-targets [Fig.2] in a given mixture by:
- Absolute Quantification: Unknown quantity of a known mixture will be calculated by extrapolating a value and comparing the standard curve of known sample [Fig.3a].
- Relative Quantification: Unknown quantity of an unknown mixture will be calculated by using the comparative Quantification cycle (Cq)/ Threshold cycle (Ct) method [Fig.3b].
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Fig 1: Risk factors present in Natural health products.
Fig. 2: Types of quantifications
Fig.3a:Absolute quantification -using standard curve method.
Fig.3b: Relative quantification- using comparative Ct/Cq method.
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