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Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP
Abstract
The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.
... Recently, many dietary supplements labeled as ''Kwao Keur'' have been sold in Japan, owing to its rejuvenating and anti-aging effects, as well as its potential to improve skin appearance, infertility, and menopausal disorder. However, ''Kwao Keur'' is classified into three types, depending on color: white, red, and black [5,6]. Among them, only white ''Kwao Keur'' is Pueraria candollei var. ...
... mirifica (PM). The others are Butea superba for red and Mucuna collettii for black; these two species do not have an estrogenic effect [5,6]. They are often misidentified and, hence, the discrimination method based on PCR-RFLP utilizing matK sequence differences between them was reported [6]. ...
... The others are Butea superba for red and Mucuna collettii for black; these two species do not have an estrogenic effect [5,6]. They are often misidentified and, hence, the discrimination method based on PCR-RFLP utilizing matK sequence differences between them was reported [6]. In addition to this confusing situation, congeners such as P. montana var. ...
In the course of our study on the quality of dietary supplements in Japan, both the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron sequence of cpDNA of products labeled as "Kwao Keur" were investigated. As a result, the DNA sequence of Pueraria candollei var. mirifica, which is the source plant of Kwao Keur, was observed in only about half of the products. Inferred from the determined sequences, source plants in the other products included Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas, etc. These inferior products are estimated to lack the efficacy implied by their labeling. In order to guarantee the quality of dietary supplements, it is important to identify the source materials exactly; in addition, an infrastructure that can exclude these inferior products from the market is needed for the protection of consumers from potential damage to their health and finances. The DNA analysis performed in this study is useful for this purpose.
... ex Benth. [3][4][5] and P. mirifica Airy Shaw & Suvat. [6][7][8], according to the world flora online database [9,10]. ...
... The nucleotide position 702 serves as a distinctive nucleotide signature that separates the two species. This finding aligns with the research conducted by Wiriyakarun et al. [4]. In case of trnH-psbA, there were many polymorphic sites but the NJ tree was differentiable white Kwao Krua sample to two species. ...
White Kwao Krua, a crucial Thai medicinal plant, contains various phytoestrogen substances used to alleviate menopausal symptoms in estrogen-deficient women. It originates from two species, namely Pueraria candollei Wall. ex Benth. and P. mirifica Airy Shaw & Suvat. However, there exists morphological variation, and the taxonomic status between both species is ambiguous, making discrimination challenging. In this study, we aim to clarify and differentiate the morphological characteristics, palynology, and DNA barcoding of both species. The morphological results showed the stipule size is a phenotypic marker for the differentiation of both species during the vegetative stage. The palynological results, however, exhibited similarity. Through an examination of nucleotide sequences and neighbor-joining tree analysis, it was determined that the DNA barcoding of the matK region has the capability to distinguish between P. candollei and P. mirifica at nucleotide position 702. Specifically, P. candollei manifested a G base, contrasting with the C base observed in P. mirifica. This study concludes that stipule size and the matK gene in DNA barcoding serve as a distinctive characteristic for distinguishing between P. candollei and P. mirifica. These methodologies prove valuable for ensuring the accurate identification of white Kwao Krua for horticulturists.
... superba) was often misidentified as Black Kwao Khruea (M. macrocarpa) because of the misidentification of dried tuberous roots in crude drug markets [17]. Although Red and Black Kwao Khruea are used for male rejuvenation, the former is less effective [1] and less expensive than the latter. ...
... superba) and Black (M. macrocarpa) Kwao Khruea, is a problem in drug markets [17]. In this experiment, multiplex cycleave PCR technique was performed to simultaneously detect B. superba and M. macrocarpa in a DNA admixture. ...
Kwao Khruea, the tuberous roots of Pueraria candollei Graham ex Benth. (White Kwao Khruea), Butea superba Roxb. (Red Kwao Khruea), and Mucuna macrocarpa Wall. (Black Kwao Khruea), are used as rejuvenating herbs in traditional medicine in many tropical countries. Although Kwao Khruea has attracted strong interest because of its rejuvenation properties, each species is used for specific purposes and effects. P. candollei shows estrogenic effects in females. In contrast, B. superba and M. macrocarpa show androgenic effects in males. The potential misidentification of dried tuberous roots of various Kwao Khruea species might cause problems in the drug market, especially when they are reduced into powders. A cycleave PCR, which is based on the sequence of chloroplast matK gene, was developed to differentiate P. candollei, B. superba, and M. macrocarpa. The results showed that cycleave PCR is able to identify specific Kwao Khruea species. A multiplex cycleave PCR was optimized for the simultaneous detection of two different DNA targets in a DNA admixture. The specificity of this technique was confirmed by its ability to distinguish M. macrocarpa from five related Mucuna species. Cycleave PCR can be a specific, sensitive, and rapid method for the identification of medicinal plants and crude plant samples.
... For example, M. pruriens and M. bracteata have been used as green manure or covered crops in agriculture [4,6]. M. macrocarpa has been consumed to boost male potency and cure erectile dysfunction [7]. M. pruriens has long been used to treat Parkinson's disease and utilized for its aphrodisiac properties in Indian traditional medicine as well as Thai folklore [8]. ...
Aphrodisiac herbal products originated from various plants including Mucuna species. In Thai folklore, Mucuna macrocarpa Wall. and M. pruriens (L.) DC. have long been consumed and utilized for their aphrodisiac properties. Consumption of these plants can lead to serious adverse effects caused by l-dopa. The plants have been legally banned for use as foods, dietary supplements, or nutraceuticals by the FDA of several countries. To protect consumers, methods for the identification of illicit plants or herbal products are needed. This study aimed to identify the selected twelve Mucuna species and examine the aphrodisiac herbal products containing M. macrocarpa and M. pruriens by using HPLC analysis of l-dopa coupled with DNA barcoding profiles of ITS, matK, rbcL, and trnH-psbA. The results showed that l-dopa could be found not only in the seeds of M. macrocarpa and M. pruriens but also in associated allied Mucuna species. Then, a DNA barcode was introduced to support in HPLC profiling to identify the plants. DNA barcodes of twelve Mucuna species found in Thailand were established and used to reconstruct a phylogenetic tree. In this study, ITS2 sequences showed the highest interspecific variability and could be used to differentiate all Mucuna species. The results of ITS2 sequence coupled with HPLC analysis revealed that all the purchased aphrodisiac products originated from M. pruriens only. Therefore, the integration of HPLC analysis and DNA barcoding profile was an efficient method for the identification of prohibited Mucuna species for safety monitoring of herbal supplements and protecting customer safety. Regulatory agencies should raise awareness and restrain the use of these commercial products.
... Single barcode-regions for identification have been reported for matK. [50,61,63,[65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] Multi-region approach to the barcoding is supported by most of the literature. [43,[83][84][85][86][87] Such multi-gene (matK and trnH-psbA) approach was explored in the identification of ethnomedicinal plants (Catharanthus roseus (L.) G. Don., Alstonia scholaris (L.) R.Br.., Thevetia peruviana (Pers.) ...
... The tuberous root of this plant has been used by local communities in Thailand for its rejuvenating qualities in menopausal women. Thai people also used it to recover black hair, promote an appetite, and increase their longevity [17]. The ordinary dosage of PM for women is a peppercorn-sized piece, which is equivalent to approximately 250 mg/kg body weight, taken once daily at night [18]. ...
The effects of the phytoestrogen-enriched plant Pueraria mirifica (PM) extract on ovari-ectomy (OVX)-induced cognitive impairment and hippocampal oxidative stress in mice were investigated. Daily treatment with PM and 17β-estradiol (E2) significantly elevated cognitive behavior as evaluated by using the Y maze test, the novel object recognition test (NORT), and the Morris water maze test (MWM), attenuated atrophic changes in the uterus and decreased serum 17β-estradiol levels. The treatments significantly ameliorated ovariectomy-induced oxidative stress in the hippocampus and serum by a decrease in malondialdehyde (MDA), an enhancement of superoxide dismutase, and catalase activity, including significantly down-regulated expression of IL-1β, IL-6 and TNF-α proinflammatory cytokines, while up-regulating expression of PI3K. The present results suggest that PM extract suppresses oxidative brain damage and dysfunctions in the hippocampal antioxidant system, including the neuroinflammatory system in OVX animals, thereby preventing OVX-induced cognitive impairment. The present results indicate that PM exerts beneficial effects on cognitive deficits for which menopause/ovariectomy have been implicated as risk factors.
... Barcode sequences have been applied for the rapid identification of medicinal plants using techniques such as multi-plex PCR (Chiang et al. 2012), PCR-RFLP (Boonsom et al. 2012;Wiriyakarun et al. 2013), LAMP (Sasaki et al. 2008), reversed dot blot (Jaipaew et al. 2018), and cycleave PCR (Wiriyakarun et al. 2014). Recently, next-generation sequencing (NGS) has been used as a tool for obtaining genomic data, which increases the resolution of identification of plant taxa (Hollingsworth et al. 2016). ...
In Thailand, there are three species of Bacopa, namely, B. monnieri, B. caroliniana, and B. floribunda. Among these species of Bacopa, B. monnieri is the only medicinal species, used for the treatment of cognitive impairment and improvement of cognitive abilities because of its bioactive constituents, bacoside A and B. However, because of the similar characteristics of these species, it is difficult to differentiate among related species, resulting in confusion during identification. For this reason, and to ensure therapeutic quality for consumers, authentication is important. In this study, the three abovementioned species of Bacopa were evaluated using barcoding coupled with high-resolution melting (Bar-HRM) analysis based on primers designed for the trnL-F sequences of the three species. The melting profiles of the trnL-F amplicons of B. caroliniana and B. floribunda were clearly different from the melting profile of the trnL-F amplicon from B. monnieri; thus, the species could be discriminated by Bar-HRM analysis. Bar-HRM was then used to authenticate commercial products in various forms. The melting curves of the six commercial samples indicated that all the tested products contained genuine B. monnieri species. This method provides an efficient and reliable authentication system for future commercial herbal products and offers a reference system for quality control.
... The misidentification of raw materials has a greater impact on the efficacy of the marketed product. The conventional method to differentiate these species is polymerase chain reaction restriction fragment length polymorphism (Wiriyakarun et al., 2013). However, this analysis method has difficulties, such as multistep sample preparation and the requirement for advanced technology with a corresponding high analysis cost. ...
Pueraria candollei or White Kwao Krua (Leguminosae) is indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation particularly in elder women. Among bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold‐based detection system for the simultaneous detection of miroestrol and puerarin in a one‐step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limit of miroestrol and puerarin were 0.15 and 4.5 μg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme‐linked immunosorbent assay (ELISA) and presented a good correlation between these two methods. This is the first report for the development of an ICS that can detect miroestrol and puerarin in one‐step. The developed ICS indicates a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.
... Vasomotor symptoms affects 57% of women, begins before menopause and continues for 1 to > 15 y (mean 4.5 y), but is shorter in Asians, and more prolonged in African contained no P. mirifica (Maruyama et al., 2014). Genomic studies have detected sequence differences in batches of P. mirifica (Bunmanop et al., 2011;Wiriyakarun et al., 2013) which may contribute to content inconsistencies. ...
Ethnopharmacological relevance:
Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (commonly termed P. mirifica, PM) growing in upland Thailand has a long history as a postmenopausal rejuvenant therapy for indigenants. Its amelioration of menopause symptoms in clinical trials was assessed.
Materials and methods:
International and Thai databases were searched from inception to February 2017. Clinical trials investigating effects of PM menopausal or postmenopausal women were included. Outcomes were self-reported menopausal symptoms, serum reproductive hormones, urino-genital tract function, and bone surrogates. Methodological quality was assessed by Cochrane risk-of-bias v2.0, and a 22-parameter quality score based on the CONSORT checklist for herbal medicines.
Results:
Eight studies (9 articles) used data from 309 menopausal patients. Five-studies demonstrated that PM was associated with climacteric scores reduced by ~50% compared to baseline. Other PM studies using limited numbers of placebo participants suggested improved vaginal and other urogenital tract symptoms. Bone alkaline phosphatase halved (suggesting lowered bone turnover). Variable serum reproductive hormone levels suggested menopausal status differed between studies. PM active ingredients and sources were not defined. Adverse event rates (mastodynia, vaginal spotting, dizziness) were similar in all groups (PM, conjugated equine estrogen, and placebos) but serum C-reactive protein doubled. These studies had design and reporting deficiencies, high risks of biases, and low quality scores.
Conclusions:
The efficacy of PM on menopausal symptoms remains inconclusive because of methodological short-comings especially placebo effects inherent in self-assessment/recall questionnaires and no PM standardization. PM efficacy and safety need a fundamental re-appraisal by: (i) cohort (retro- and prospective) studies on current users to define its traditional use for rejuvenation; (ii) tightly coupling long-term efficacy to safety of well-defined PM and multiple end-points; (iii) using study design related to current understanding of menopause progression and estrogen pharmacology (iv) robust pharmacovigilance.
... Pueraria Candollei (PC) var. Mirifica , also known as white kwao krua, is used as rejuvenative in many tropical countries (Wiriyakarun et al., 2013). PC is highly estrogenic and has evidence-based regenerative effect on bones. ...
Background. Osteoporosis is a bone metabolic disease affecting a large percentage of aging population, which leads to an increased risk of fractures and has a negative impact on life quality. The available treatments for osteoporosis are effective, but are associated with several severe adverse reactions, hence the interest for alternative treatments devoid of such redoubtable side effects. Medicinal plants represent a viable resource for new therapeutic agents. The purpose of this review is to provide an overview about the medicinal plants that have been reported to have anti-osteoporotic effects in human clinical studies. Materials and Methods. Relevant studies found in PubMed database, pertaining to efficacy in humans, mechanism of action, osteoactive phytochemicals and safety, were selected. For the inquiry, keywords such as “medicinal plant”, “osteoporosis”, “bone”, “fracture”, “osteoclast” and “osteoblast” were used in various combinations. The information extracted was integrated with the traditional knowledge on the correspondent medicinal plants. Results. Eight medicinal plants (Cimicifuga racemosa, Cissus quadrangularis, Eleutherococcus senticosus, Epimedium spp., Glycine max, Pueraria spp., Panax notoginseng, Salvia milthiorriza) were selected. Mechanisms involved include the activation of osteoblasts, inhibition of osteoclastogenesis, estrogen-like activity, anti-inflammatory activity, inhibition of collagen degradation by cathepsin K. Conclusion. Several medicinal plants have been included in clinical studies successfully targeting osteoporosis, thus showing the potential to modulate bone resorption and bone formation.
... The author refers to witnesses having testified that women of seventy and eighty starting to menstruate again after taking these pills. Actually, there are many legends about this plant but, in fact, activities potentially classified as "rejuvenating" are known from Butea superba (Wiriyakarun et al. 2013). The drug leads to hyperandrogenemia (Chaiyasit and Wiwnaitkit 2012;Malaivijitnond et al. 2009) and improves erectile dysfunction (Tocharus et al. 2006(Tocharus et al. , 2012Ho and Tan 2011;Cherdshewasart et al. 2010;Cherdshewasart and Nimsakul 2006). ...
Reports on traditional use of medicinal plants may be used as starting points for phytochemical and pharmacological research. As has recently been shown, publications, letters, diaries and reports of exploring botanists are a valuable source of historical ethnopharmacological information. In this study, the heritage of the British botanist Arthur Francis George Kerr (1877-1942), mainly working in Thailand, was screened for information about traditionally used medicinal plants. Information given was compared to state-of-the-art scientific knowledge about these species. Many historical uses could be confirmed, some did not, while a number of species reported to be traditionally used have not been sufficiently investigated so far. These, strongly suggested for further research, include Kurrimia robusta, Alpinia siamensis, Amomum krervanh (= A. testaceum), Trichosanthes integrifolia (= Gymnopetalum scabrum), Croton cumingii (= C. cascarilloides), Lobelia radicans (= L. chinensis), Willughbeia sp., Nyctanthes arbor-tristis (against “floodings”), Pluchea indica, Heliotropum indicum, and some fungi and woods.
... Leptorhynchoides thecatus [12] Pomphorhynchus tereticollis [13] Acanthocephalus lucii [14] Allolobophora chlorotica [15] Polymorphus brevis [16] Axiothella constricta, Deosergestes corniculum, Caprella andreae, Microcosmus squamiger, Microcosmus squamiger, Nucula sulcata, Leptoplana tremellaris [17] Synecdoche constellate, Bruchomorpha beameri, Cixius nervosus [18] Diopatra neapolitana [19] Andrena humilis, Andrena fulvida [20] Puerariacandollei, Butea superb, Mucunacollettii [30] Galpemia spp. rbcL [31] Dendrobium spp. ...
Global warming is affecting regional climate, ecosystem and diversity array of species by causing physical and biological changes throughout the planet. Therefore, there is a need to develop a technique which can identify organisms and differentiate between very closely related species in order conserve species diversity. Classical taxonomy has accelerated its progress with the adoption of new molecular techniques like DNA barcoding to cope with the huge population of organisms and biodiversity available in this planet. DNA barcoding uses short gene sequences which are well classified portion of the genome. With the advent of high throughput sequencing technology such as Next-Generation Sequencing (NGS) technology the DNA barcoding has become more accurate, fast and reliable in the last decade. The Consortium for the Barcode of Life (CBOL) has given a platform for taxonomists across all the countries to collaborate, identify and preserve the biodiversity across the globe. In this review we summarized the recent advances and developments in the DNA barcoding attempts across animals, plants, bacteria, fungi, viruses and protists. We have also attempted to present the popular tools used in DNA barcoding in a chronological order of their development.
... In an altogether different approach, Wang et al. (2012) employed the methylationsensitive amplified polymorphism (MSAP) marker to appraise cytosine methylation difference in several regenerated plantlets and among organs of Clivia miniata. Wiriyakarun et al. (2013) reported the utilization of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) to recognize plant origin of Pueraria candollei, Butea superb and Mucuna collettii. Nevertheless, to develop marker-assisted selection (MAS) breeding (for example in Catharanthus roseus) Chaudhary et al. (2013) reported the detection and mapping of QTLs, Simmondsia chinensis L. Schneider Detection of genetic variability Amarger and Mercier (1995) Afgekia sericea Craib Genetic diversity within and among Thai populations Prathepha and Baimai (1999) Mentha species Assessment of genetic relationships in 11 accessions from six taxa of Mentha developed by CIMAP Khanuja et al. (2000) Dioscorea floribunda Detection of genetic alteration in the post-cryo regenerated plants Ahuja et al. (2002) Panax sikkimensis, P. pseudoginseng and P. quinquefolium ...
The pain relief formula “Ya Pa Som Kho-Khlan (YPSKK)” or “ยาผสมโคคลาน” in Thai is officially recorded in the Natural List of Essential Medicines (NLEM) of Thailand. The main component is Mallotus repandus (Willd.) Müll. Arg.; however, Anamirta cocculus (L.) Wight & Arn and Croton caudatus Gleiseler share the same common name: “Kho-Khlan”. Confused usage of A. cocculus or C. caudatus can have effects via toxicity or unsuccessful treatment. This study aimed to combine a high-performance thin-layer chromatography (HPTLC) technique and DNA barcoding coupled with high-resolution melting (Bar-HRM) to differentiate M. repandus from the other two species. The M. repandus extract exhibited a distinct HPTLC profile that could be used to differentiate it from the others. DNA barcodes of the rbcL, matK, ITS and psbA-trnH intergenic spacer regions of all the plants were established to assist HPTLC analysis. The rbcL region was selected for Bar-HRM analysis. PCR amplification was performed to obtain 102 bp amplicons encompassing nine polymorphic nucleotides. The amplicons were subjected to HRM analysis to obtain melting curve profiles. The melting temperatures (Tm) of authentic A. cocculus (A), C. caudatus (C) and M. repandus (M) were separated at 82.03±0.09°C, 80.93±0.04°C and 80.05±0.07°C, respectively. The protocol was applied to test crude drugs (CD1-6). The HPTLC profiles of CD2-6 showed distinct bands of M. repandus, while CD1 showed unclear band results. The Bar-HRM method was applied to assist the HPTLC and indicated that CD1 was C. caudatus. While ambiguous melting curves from the laboratory-made formulae were obtained, HPTLC analysis helped reveal distinct patterns for the identification of the plant species. The combination of HPTLC and Bar-HRM analysis could be a tool for confirming the identities of plant species sharing the same name, especially for those whose sources are multiple and difficult to identify by either chemical or DNA techniques.
This chapter will provide an overview of the food fraud challenges that exist within herb and spice commodities. Herbs and spices hold a high economic value while also being vulnerable to fraud. Several examples of herb and spice fraud will be reviewed to show the primary drivers of food fraud within the specific commodity. Other topics covered in this chapter include ongoing analytical research to strengthen methods of detection of adulteration in herbs and spices, as well as various mitigation efforts that can be taken to reduce the vulnerability to fraud within supply chains.
Abstract
Introduction
The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects.
Objectives
This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species.
Methods
A gold nanoparticle‐based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high‐performance liquid chromatography with UV detector (HPLC‐UV). The results of the two methods were compared for consistency checking.
Results
The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC‐UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross‐reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false‐positive and false‐negative results from other plant extracts were not observed.
Conclusion
The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors.
Herbal products, such as dietary supplements, have become a subject of increasing global importance for their health benefits and economic considerations. However, they have also been targets of adulteration practices, being the accurate identification of botanicals in herbal products of utmost importance to protect the health and expectations of consumers. Particularly, in the case of dietary supplements, which can have different types of formulations, the identification of plant material used in their production is often a research challenge. DNA‐based techniques have played a crucial role on the development of a wide range of tools for the authentication of herbal products. Therefore, this review intends to describe their main progresses, critically discussing their advantages and drawbacks when applied to authenticate herbal products, focusing on dietary supplements. DNA barcoding is particularly emphasized because it has provided the highest number of applications, followed by the advances on high‐resolution melting analysis combined with DNA barcodes. A special emphasis is also given to the promising approaches relying on DNA metabarcoding and isothermal amplification.
The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH–psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic “white Kwao Krua” as claimed on their labels.
In this research, near-infrared (NIR) spectroscopy in combination with moving window partial least squares-discrimination analysis (MWPLS-DA) was utilized to discriminate the variety of turmeric based on DNA markers, which correlated to the quantity of curcuminoid. Curcuminoid was used as a marker compound in variety identification due to the most pharmacological properties of turmeric possessed from it. MWPLS-DA optimized informative NIR spectral regions for the fitting and prediction to {–1/1}-coded turmeric varieties, indicating variables in the development of latent variables in discrimination analysis. Consequently, MWPLS-DA benefited in the selection of combined informative NIR spectral regions of 1100 – 1260, 1300 – 1500 and 1880 – 2500 nm for classification modeling of turmeric variety with 148 calibration samples, and yielded the results better than that obtained from a partial least squares-discrimination analysis (PLS-DA) model built by using the whole NIR spectral region. An effective and rapid strategy of using NIR in combination with MWPLS-DA provided the best variety identification results of 100% in both specificity and total accuracy for 48 test samples.
DNA barcoding is a useful technique for diversity analysis whereby a standardized region of DNA is used for the identification of a species or a taxonomic group of organisms. These standard regions used for identification are called the DNA barcode. These are small sequences of the entire genome. In plants, DNA barcoding has application in phylogenetic analysis, authentication, inter- and intraspecific diversity, classification into wild and cultivated genotypes, the study of phylogeographical patterns, and in the detection of adulteration. The barcode loci, i.e., the DNA regions used for the identification are able to discriminate the closely related species and identify new cryptic species as well. Depending on the taxon and complexity of the species, different barcode loci are used for the purpose. In animals, the universal DNA barcode, i.e., mitochondrial cytochrome c oxidase I (COI) gene is used for species discrimination. However, this gene cannot be used for plants due to its limited divergence. Thus, its use is limited only to some algae. Efforts are going on to find suitable universal barcode loci for plants. Since the last decades, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA, and 18S-rRNA candidate regions are being used as DNA barcodes in plants. The article provides an overview of the use of these candidate regions through different approaches which have gained importance due to the challenges in DNA barcoding of plants. The development of multilocus and tiered approaches along with the new frontier areas for application of this technique has been analyzed in detail.
Achyranthis Bidentatae Radix has a long history in China as a commonly used herb that can be used to treat various diseases, including those related to the liver, muscles, bones, and kidneys. Recently, an increase in the number of adulterants has been reported, which affects the clinical safety of Achyranthis Bidentatae Radix. To identify adulterants of Achyranthis Bidentatae Radix, we collected samples from major regions and conducted an in-depth genetic comparison of the herb and its commonly used adulterants. We amplified and sequenced three genomic regions, internal transcribed spacer (ITS), psbA-trnH, and internal transcribed spacer 2 (ITS2), to confirm whether ITS2 is a suitable identifier for Achyranthis Bidentatae Radix. Results showed that the ITS2 sequence length of Achyranthis Bidentatae Radix was 199 bp, with no variation between samples. The inter-specific genetic distance of ITS2 between Achyranthis Bidentatae Radix and its adulterants was 0.390. Neighbor-joining trees showed that Achyranthis Bidentatae Radix and its adulterants are easily differentiated by monophyly. In conclusion, ITS2 regions accurately and effectively distinguished between Achyranthis Bidentatae Radix and its adulterants.
The use of plants for the treatment of illness is a common practice in Mexico. Almost 80% of the population utilizes medicinal plants at some point of their lives. The high demand of these plants generates the production of a diversity of herbal products in some of which different vegetal species are used as adulterants that can generate non-desired effects. This problem has led to the search of different methods for the identification of the vegetal species used in the herbal preparations. However, the path has not been obstacle free, some of these methods requires specific characteristics of the analyzing material, others shows low reproducibility and high costs. In this context, the present review covers diverse aspects of the identification methods in particular, the DNA barcoding. This a simple, low cost methodology used to identify medicinal plant materials in various presentations. The implementation of this methodology for the quality control of medicinal plants products in Mexico, would bring a number of benefits both, the herbal industry and the consumers, since the authentication of medicinal plants by DNA barcoding will guarantee high quality herbal medicinal products, without adulterants and without side effects, which would have a direct impact on users health, in addition to detonate the growth of the national herbalist sector.
"Cat ginseng", the dried root of Actinidia macrosperma, is a famous traditional Chinese medicine against cancers in eastern China. The roots of some other species of the genus Actinidia such as A. valvata and A. melanandra have also been used as fake "cat ginseng", but they have little therapeutic value. However, identification of the original plants used to make the crude drugs is difficult, especially during the vegetation period. In this study we developed molecular markers for the determination and authentication of A. macrosperma. The restriction digestion of the chloroplast trnK region using endonucleases DdeI and DraI produces two unique patterns in A. macrosperma, and in the matK sequences, there are 11 sites unique to A. macrosperma. The molecular markers provide an effective and accurate identification and authentication of A. macrosperma in Actinidia.
A flavonoid (3, 7, 3'-Trihydroxy-4'-methoxyflavone) (1) and a flavonoid glycoside (3, 3'-dihydroxy-4'-methoxyflavone-7-O-β-D-glucopyranoside) (2) were isolated from the tuber root of Butea superba Roxb. The structures were determined on the basis of spectral analysis, including 2D-NMR techniques. These compounds show higher inhibitory effects on cAMP phosphodiesterase than caffeine and theophylline.
Herbal drug technology is used for converting botanical materials into medicines, where standardization and quality control with proper integration of modern scientific techniques and traditional knowledge is important. The use of chromatographic techniques and marker compounds to standardize botanical preparations has limitations because of their variable sources and chemical complexity. DNA-based molecular markers have utility in the fields like taxonomy, physiology, embryology, genetics, etc. DNA-based techniques have been widely used for authentication of plant species of medicinal importance. Pharmacognosy mainly addresses quality-related issues using routine botanical and organoleptic parameters of crude drugs, and chemoprofiling-assisted characterization with chromatographic and spectroscopic techniques. The new pharmacognosy includes all the aspects of drug development and discovery, where biotechnology-driven applications play an important role. Current focus on chemotype-driven fingerprinting and related techniques requires integration with genotype-driven molecular techniques so that an optimal characterization of botanical materials is possible. This review provides a brief account of various DNA-based technologies that are useful in genotyping and quick identification of botanicals with suitable examples.
Dioscorea is an important plant genus in terms of food supply and pharmaceutical applications. However, its classification and identification are controversial. DNA barcoding is a recent aid to taxonomic identification and uses a short standardized DNA region to discriminate plant species. In this study, the applicability of three candidate DNA barcodes (rbcL, matK, and psbA-trnH) to identify species within Dioscorea was tested.
One-hundred and forty-eight individual plant samples of Dioscorea, encompassing 38 species, seven varieties and one subspecies, representing majority species distributed in China of this genus, were collected from its main distributing areas. Samples were assessed by PCR amplification, sequence quality, extent of specific genetic divergence, DNA barcoding gap, and the ability to discriminate between species. matK successfully identified 23.26% of all species, compared with 9.30% for rbcL and 11.63% for psbA-trnH. Therefore, matK is recommended as the best DNA barcoding candidate. We found that the combination of two or three loci achieved a higher success rate of species discrimination than one locus alone. However, experimental cost would be much higher if two or three loci, rather than a single locus, were assessed.
We conclude that matK is a strong, although not perfect, candidate as a DNA barcode for Dioscorea identification. This assessment takes into account both its ability for species discrimination and the cost of experiments.
The genus Phyllanthus (Phyllanthaceae) is distributed in tropical and subtropical regions, and its members are widely used as medicinal plants in many countries. We analyzed the nucleotide sequences of the internal transcribed spacers of ribosomal DNA of 56 plant samples covering 23 Phyllanthus species collected from various habitats in Thailand. Based on the sequence alignment, we constructed phylogenetic trees of all Phyllanthus species distributed in Thailand. Furthermore, a simple protocol to discriminate three important medicinal Phyllanthus species, P. amarus, P. debilis, and P. urinaria, was developed using a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method and successfully applied to the crude drug samples obtained in Thai markets.
In this study, crude extracts of Pueraria mirifica, Butea superba and Mucuna macrocarpa were prepared using sequential extraction with three different solvents (hexane, ethyl acetate and methanol). The extracts obtained were then used to test for their antimicrobial activity by the disc diffusion method, which showed that, against a wide range of Gram-positive and Gram-negative bacteria, only the P. mirifica extract obtained with ethyl acetate exhibited antimicrobial activities. The minimum inhibitory concentration (MIC) of the extract was also determined with values between 15 and 50 mg/ml depending on the microbes tested. Thin layer chromatography (TLC) was subsequently used to separate the chemical constituents of the extract. When tested against B. cereus, there were only two bands which showed anti-B. cereus activity. Additionally, the crude extracts of P. mirifica, B. superba and M. macrocarpa were analysed for some antioxidant compounds using HPLC. Our results showed that all the extracts contained daidzin, genistin, daidzein and genistein, all of which were present in the highest amounts (0.045, 0.037, 0.049 and 0.060 % respectively) in the ethyl acetate extract of P. mirifica.
A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF-atpH, and psbK-psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85-100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK-psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69-71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.
The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.
DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a “DNA barcoding gap” is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes.
• CITES
• Kruger National Park
• Mesoamerica
In this study, crude extracts of Pueraria mirifica, Butea superba and Mucuna macrocarpa were prepared using sequential extraction with three different solvents (hexane, ethyl acetate and methanol). The extracts obtained were then used to test for their antimicrobial activity by the disc diffusion method, which showed that, against a wide range of Gram-positive and Gram-negative bacteria, only the P. mirifica extract obtained with ethyl acetate exhibited antimicrobial activities. The minimum inhibitory concentration (MIC) of the extract was also determined with values between 15 and 50 mg/ml depending on the microbes tested. Thin layer chromatography (TLC) was subsequently used to separate the chemical constituents of the extract. When tested against B. cereus, there were only two bands which showed anti-B. cereus activity. Additionally, the crude extracts of P. mirifica, B. superba and M. macrocarpa were analysed for some antioxidant compounds using HPLC. Our results showed that all the extracts contained daidzin, genistin, daidzein and genistein, all of which were present in the highest amounts (0.045, 0.037, 0.049 and 0.060% respectively) in the ethyl acetate extract of P. mirifica. © 2009 by Maejo University, Sansai, Chiang Mai, 50290 Thailand.
Chinese medicinal materials are widely used in the Orient and have found increased popularity in the West as an alternative source of health care. Traditionally, authentication of these materials relies on organoleptic methods. Progress in pharmacognosy has capitalised on the anatomical and chemical profiles as markers for authentication. The advancement of techniques in molecular biology has provided new means for examining genotypic characteristics. Since 1990, several research groups have employed polymerase chain reaction based methods, restriction fragment length polymorphism and DNA sequencing to authenticate medicinal materials and to define the phylogenetic relationship of related species. This review provides an account of these methods and assesses their value.
This procedure has been used with success on a wide variety of plant groups and even some animals. The method is used to isolate total genomic DNA (nuclear, chloroplast, and mitochondrial). It is a rapid, inexpensive method that is suitabie for use in conjunction with other protocois, such as isolation of DNA enriched for cpDNA. it is also easy to scale down for use in population sampling, using 0.01g or less of fresh tissue. Other applications include isolation of DNA from herbarium specimens (Doyle & Dickson, 1987. Taxon 36:715–722), and isolation of RNA. A brief word on the history of the protocol is in order. This procedure was modified by us (Doyle and Doyle, 1987. Phytochemical Bulletin 19:11–15) for use with fresh plant tissue from a method of Saghai-Maroof et al. (1984, PNAS USA 81:8014–8019) who used lyophilized tissue. They in turn had developed their procedure from earlier protocols. We were recently asked to publish a slightly modified version of our procedure (Doyle and Doyle, 1990 Focus 12:13–15). We recently learned from Brian Taylor (Texas A&M University, USA) that he had published a virtually identical procedure for fresh tissue, also in Focus, in 1982 (Taylor & Powell, Focus 4:4–6) of which we (and apparently the editors of Focus!) were entirely unaware. It is indeed a useful procedure, thus independently confirmed.
Traditionally, the authentication of the traditional Chinese medicines (TCM), Angelica sinensis, is based on slightly different morphological characters and complex compounds. Usually, those methods are simultaneously affected by several factors, leading to subtle and ambiguous results. In this study, the internal transcribed spacer (ITS) regions of A. sinensis and seven other Angelica species used as adulterants were sequenced. A pair of specific primers was designed from the polymorphic ITS regions to distinguish A. sinensis from the adulterants and regional substitutes. These ITS-derived primers amplified approximately 520 bp specific fragments from the adulterants, whereas no products was amplified with the DNA of A. sinensis. We tested eight commercially crude materials purchased in the market by using these specific primers. The result showed that there were four samples adulterating A. sinensis with regional substitutes. This indicated that A. sinensis could be accurately distinguished from the adulterants and regional substitutes. Therefore, the method of molecular authentication based on the ITS sequences may be contributed to raw material production and quality control of A. sinensis.
Herbal medicinal materials have been used worldwide for centuries to maintain health and to treat disease. However, adulteration of herbal medicines remains a major concern of users and industry for reasons of safety and efficacy. Identification of herbal medicinal materials by DNA technology has been widely applied, sarted from the mid-1990s. In recent years, DNA barcoding of global plant species using four standard barcodes (rbcL, matK, trnH. psbA and ITS) has been a major focus in the fields of biodiversity and conservation. These DNA barcodes can also be used as reliable tools to facilitate the identification of herbal medicinal materials for the safe use of herbs, quality control, and forensic investigation. Many studies have applied these DNA barcodes for the identification of herbal medicinal species and their adulterants. The present article reviews efforts in the identification of herbal medicinal materials using the standard DNA barcodes and other DNA sequence-based markers.
Four new species, Cruddasia craibii, Flemingia kradungensis, F. tiliacea and Pueraria maesenii are described and illustrated. The following new combinations are proposed: Cruddasia pinnata; C. laotica, Dolichos grahamianus; D. oxyphyllus; Dolichovigna pilosa; Erythrina stricta var. suberosa; Flemingia macrophylla var. sootepensis; Pueraria candollei var. mirifica; Shuteria suffulta var. sinensis and Vigna radiata var. grandiflora. Pueraria rigens is moved to the tribe Millettieae (=Mellettia rigens).
DNA technology provides a powerful tool to complement chemical analyses for authentication of Chinese medicinal plants and to ensure that herbal materials are not contaminated with ineffective or potentially harmful substitutes or adulterants. In the last two decades molecular biotechnology has provided sophisticated molecular techniques for authentication of botanical materials at the DNA level. This review provides an account of the most commonly used DNA-based technologies (RAPD, RFLP, ARMS, CAPS, AFLP, DAF, ISSR, SSR, sequencing, hybridization and microarrays) including suitable examples of Chinese medical plants. A critical evaluation of all methods is presented concerning sensitivity, reliability, reproducibility, and running costs. Recent achievements in the field of DNA barcoding and DNA chip technology that offer great potentials for screening of DNA and emerging new developments for future identification of species are briefly outlined.
A high-performance liquid chromatography (HPLC) method was developed to determine the contents of miroestrol and deoxymiroestrol in the tubers of Pueraria candollei var. mirifica and P. candollei var. candollei. The linear detection ranges were 0.78-25.00 μg/mL for miroestrol and 1.56-25.00 μg/mL for deoxymiroestrol. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 0.78 μg/mL, respectively, for miroestrol and 0.78 and 1.56 μg/mL, respectively, for deoxymiroestrol. Our results suggest that both varieties of P. candollei can produce miroestrol and deoxymiroestrol and that the developed HPLC method can be applied for quality control of plants and their products.
Stemona sessilifolia, S. japonica and S. tuberosa are the three genuine sources of Stemonae Radix specified in the Chinese Pharmacopoeia (CP) for antitussive and insecticidal remedy. Significant variations in alkaloids composition and content, as well as different degree of antitussive activity were found among them. In order to accurately identify the genuine sources of Stemonae Radix in the genetic level, two polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were developed based on the sequence differences in chloroplast DNA trnL-trnF and petB-petD regions of the species recorded in CP, as well as S. parviflora and a counterfeit of Stemonae Radix, Asparagus cochinchinensis. By using the restriction enzymes MwoI, AciI and XmnI which were able to recognize specific sequence sites in the trnL-trnF region, and BclI, HincII and BslI which can recognize those in the petB-petD region to digest the corresponding PCR products, the specific digestion pattern enabled the discrimination of the botanical sources of Stemonae Radix effectively and efficiently.
As a widely used and expensive herbal medicine, Panax ginseng has many adulterants in the commercial market. PCR-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) based on 5S rDNA sequence analysis were applied to identify two common adulterants of P. ginseng. The sizes of 5S rRNA gene non-transcribed spacers (NTS) sequences in P. ginseng and its adulterants were determined, ranging from 143 to 424 bp. The PCR product of P. ginseng only could be digested among the tested specimens because of its specific SpeI restriction site found in the 5S rDNA sequence. In addition, P. ginseng was successfully identified from compound medicinal preparations and from the Single-Taste medicines. These results suggest that the methods are able to authenticate P. ginseng.
In Thailand, Stemona tuberosa Lour. , S. phyllantha Gagnep. , S. collinsae Craib , S. burkillii Prain , S. aphylla Craib and S. sp. are found. The identification based on morphological characters alone is difficult and can lead to confusion regarding chemical constituents and biological activities. The tuberous roots of S. tuberosa have long been used for treatment of respiratory diseases and as anthelmintics. However, accurate identification of S. tuberosa is needed to ensure efficacy. Sequence comparison indicated that these Stemona spp. could be identified from the sequence of the trnH- psbA locus. As a result of different sequence lengths, the PCR products generated from newly designed primers could be used to preliminarily group the two species, S. tuberosa and S. phyllantha, apart from others. However, these products could be further sequenced to discriminate among Stemona spp.
The trnK gene endocing the tRNALys(UUU) has been located on mustard (Sinapis alba) chloroplast DNA, 263 bp upstream of the psbA gene on the same strand. The nucleotide sequence of the trnK gene and its flanking regions as well as the putative transcription start and termination sites are shown. The 5' end of the transcript lies 121 bp upstream of the 5' tRNA coding region and is preceded by procaryotic-type "-10" and "-35" sequence elements, while the 3' end maps 2.77 kb downstream to a DNA region with possible stemloop secondary structure. The anticodon loop of the tRNALys is interrupted by a 2,574 bp intron containing a long open reading frame, which codes for 524 amino acids. Based on conserved stem and loop structures, this intron has characteristic features of a class II intron. A region near the carboxyl terminus of the derived polypeptide appears structurally related to maturases.
Total DNA was extracted from the fresh underground parts of three Panax separate species. The 18S rRNA regions of extracted DNA were amplified by the polymerase chain reaction (PCR) and their sequences were determined. In each species, the sequences were found to be of 1809 base pairs (bps) but with different gene sequences. Different base substitutions were observed at nucleotide positions 497, 499, 501 and 712. The same procedure was performed on commercial samples of Ginseng Radix, Panacis Japonici Rhizoma and American Ginseng. Each sequence completely corresponded with that of each original plant, namely P. ginseng, P. japonicus and P. quinquefolius, respectively. This is the first time that 18S rRNA gene sequencing on Panax species was carried out. Previously, Ginseng drugs have been identified mainly by their external and internal structure. Thus this method will be useful in identifying Ginseng drugs at the gene level.
In order to develop convenient and reproducible methods for the identification of Ginseng drugs at a DNA level, PCR-Restriction fragment length polymorphism (PCR-RFLP) and Mutant allele specific amplification (MASA) analyses were applied, based on differences of the 18S rRNA gene sequence among three Panax species. The PCR product of each species on the 18S rRNA gene was digested with the restriction enzymes Ban II and Dde I. Each fragment gave unique electrophoretic profiles for each species (PCR-RFLP analysis). The extracted DNA of each species was amplified by PCR using a designed species-specific oligonucleotide primer. The expected size of the fragments corresponding to each species were detected only when the optimum temperature and reaction time for annealing and extension were established (MASA analysis). These two analytical methods were carried out on three Ginseng drugs and the same results as in their original plants were obtained. The results suggest that PCR-RFLP and MASA analyses under the established conditions are convenient for identifying three Ginseng drugs. Moreover, to insure completion of the identification, a partial sequence of the plastid gene matK was determined in addition to the 18S rRNA gene. The gene sequences of three Panax species were of 1259 base pairs and that of P. quinquefolius was different from the other two at nucleotide position 102.
LC-UV-mass spectrometry and bioassay co-directed fractionation of an aqueous acetone extract of the roots of Gentiana macrophylla gave three new chromene derivatives and two novel and six known secoiridoids, along with kurarinone, kushenol I, beta-sitosterol, stigmasterol, daucosterol, beta-sitosterol-3-O-gentiobioside, alpha-amyrin, oleanolic acid, isovitexin, gentiobiose and methyl 2-hydroxy-3-(1-beta-D-glucopyranosyl)oxybenzoate. The structures of the new products were established from spectral and chemical evidence as 2-methoxyanofinic acid and macrophyllosides A-D. The six known secoiridoids were gentiopicroside, sweroside, 6'-O-beta-D-glucosylgentiopicroside, 6'-O-beta-D-glucosylsweroside, trifloroside and rindoside. The new acid (2-methoxyanofinic acid), its methyl ester, kurarinone and kushenol I were shown to be active against the plant pathogenic fungus Cladosporium cucumerinum. The methyl ester and kurarinone inhibited also the growth of the human pathogenic yeast Candida albicans. Structure-activity relationships were studied. Thus, addition of a methoxyl group to the benzene nucleus of anofinic acid (2,2-dimethyl-2H-1-benzopyran-6-carboxylic acid) increased the antifungal activity remarkably whereas glycosylation at the carboxylic moiety was found to remove the activity. Esterification of the new acid induced its activity against C. albicans, but decreased its growth inhibition properties against C. cucumerinum. Hydroxylation of kurarinone at the 3 beta-position removed its activity against C. albicans and decreased the inhibition of C. cucumerinum. In addition, the chemotaxonomic significance of the identified constituents is discussed.
Previously, we have determined marker nucleotides on the chloroplast matK gene to identify Rheum palmatum, R. tanguticum and R. officinale used as Rhei Rhizoma officially. In the present study, we further developed a convenient and efficient identification method on the basis of marker nucleotides with Amplification Refractory Mutation System analysis. On the basis of the nucleotide substitutions at positions 367 and 937 among the three species on the matK gene, at each position two kinds of reverse primers with complementary 3'-terminal nucleotides were designed. Upon PCR amplification using three sets of primers and template DNA from each species, one or two fragments (202 bp or/and 770 bp) were detected. As the resultant three fragment profiles were species-specific, the procedure enabled us to classify the botanic origins of 22 drug samples of Rhei Rhizoma.
The differential anti-proliferation effect of white (Pueraria mirifica), red (Butea superba) and black (Mucuna collettii) Kwao Krua plant extracts on the growth of MCF-7 cells was evaluated after 4 days of incubation. The percent cell growth comparison was based on protein determination of the harvested cells in parallel with the control group and Pueraria lobata treatment group. Pueraria lobata led to no proliferation and a mild anti-proliferation effect on the growth of MCF-7 cells. Pueraria mirifica caused proliferation at 1 microg/mL and an anti-proliferative effect on the growth of MCF-7 cells at 100 and 1000 microg/mL with an ED50 value of 642.83 microg/mL. Butea superba led to no proliferation and an anti-proliferation effect on the growth of MCF-7 cells at 10, 100 and 1000 microg/mL with an ED50 value of 370.91 microg/mL. Mucuna collettii led to no proliferation and an anti-proliferation effect on the growth of MCF-7 cells at 100 and 1000 microg/mL with an ED50 value of 85.36 microg/mL. The results demonstrated that only Pueraria mirifica showed an estrogenic effect on MCF-7 cell growth and a clear antagonistic effect with E2 at high concentration. Butea superba and Mucuna collettii exhibited only anti-proliferation effects on the growth of MCF-7 cells in relation with a possible anti-estrogen mechanism or a potent cytotoxic effect.
Identification of 10 white fish species associated with U.K. food products was achieved using PCR-RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end-point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials.
Fritillaria pallidiflora Schrenk (Liliaceae) is a commonly used antitussive herb. There are 9 species of Fritillaria recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as F. pallidiflora, and thus, the therapeutic effects of F. pallidiflora are not achieved. Methods to distinguish F. pallidiflora from the 8 other species of Fritillaria are limited by the current morphological and chemical methods. In this study, we report two molecular authentication methods based on the sequences of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions. For diagnostic PCR, we designed a pair of species-specific primers to authenticate F. pallidiflora. The PCR program consisted of only two steps for every repeated cycle. For PCR-RFLP, we identified a distinctive site which can be recognized by the restriction endonuclease Eco81I in the nrDNA ITS1 region of F. pallidiflora. PCR-RFLP analysis was established to differentiate F. pallidiflora from the other species of Fritillaria. These methods provide effective and accurate identification of F. pallidiflora.
As a widely used medicinal plant, Alisma orientale is always a possible target for fraudulent labeling. The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) of the six species of genus Alisma were sequenced, and two variant sites were found to be specific for A. orientale. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and amplification refractory mutation system (ARMS) analysis were applied to the ITS region for the identification of A. orientale. A restriction site for PSTI useful for PCR-RFLP analysis was detected and a pair of diagnostic primers DFZX-JB02S and DFZX-JB02X were designed for ARMS.
Bulbus Fritillariae (BF) is the most commonly used antitussive herb in China. There are nine species of Fritillaria recorded as the drug BF in the Chinese Pharmacopoeia. Bulbus Fritillariae cirrhosae (BF cirrhosae) is a group that includes four species of BF; these four species come from wild sources with higher efficiency and lower toxicity compared to the other five species of BF. Due to reasons of carelessness and reduced costs, the other five species are often sold as BF cirrhosae. Analysis through appearance, microscopic and chemical techniques has limitations. Identifying botanical resources is a primary step in the standardization of herbal medicine. In the present article, the internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal DNA (nrDNA) of nine species and one variety of Fritillaria genus have been sequenced. A mutation site in the ITS1 region among BF cirrhosae and other species of BF has been found and can be recognized by the restriction endonuclease SmaI. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the nuclear ribosomal ITS1 region was used to differentiate BF cirrhosae from other species of BF and is a successful method in distinguishing the subgroups.
In Thailand, there are four Mitragyna species; M. speciosa, M. hirsuta, M. diversifolia, and M. rotundifolia. One, M. speciosa, is a narcotic plant and has medicinal importance for its opium-like effect. Since the use of M. speciosa has been forbidden in Thailand, the leaves of M. diversifolia or others are frequently used as substitutes but are not considered as effective. Therefore, accurate authentication of M. speciosa is essential for both medicinal and forensic purposes. The nucleotide sequences of internal transcribed spacers (ITS) and the 5.8S coding region of nuclear ribosomal DNA (rDNA) of the Mitragyna species were analyzed. The whole length of ITS1-5.8S-ITS2 region was 608 bp in M. speciosa, 607 bp in the other species. Nineteen sites of nucleotide substitutions and 3 sites of 1-bp indels were observed, and M. speciosa showed specific sequence differed from the others. Based on the ITS sequences, a distinctive site recognized by a restriction enzyme XmaI in M. speciosa was found and then PCR-restriction fragment length polymorphism (RFLP) analysis was established to differentiate M. speciosa from the others. By the method, a 409-bp PCR fragment of ITS1-5.8S (partial) rDNA region from M. speciosa was cleaved into two fragments of 119 bp and 290 bp while the other species remained undigested. This method provides an effective and accurate identification of M. speciosa.
DNA sequence analysis of the rDNA internal transcribed spacer 1 (ITS1) and TaqMan real-time polymerase chain reaction were exploited for their applications in the differentiation of the traditional Chinese medicinal plants Euphorbia humifusa and E. maculata from three related adulterants E. hypericifolia, E. atoto and E. prostrata. The data demonstrated that variations in the ITS1 regions were very low at the intra-species level but extremely high at the inter-species level, so that they could be easily distinguished at the DNA level. The sequence difference allowed an effective and reliable differentiation of E. humifusa and E. maculata from the adulterants by TaqMan real-time PCR.
Abbreviations
ITS: internal transcribed spacer
DNA protocols for plants-CTAB total DNA isolation Molecular techniques in taxonomy
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Identification of the botanical source of Stemonae Radix based on PCR with specific primers and PCR-RFLP
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The remedy pamphlet of Kwao Krua tuber of Luang Anusarnsuntarakromkranpiset
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Applications of biotechnology and molecular markers Genetic resources, chromosome engineering, and crop improvement series
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Identification of the botanical source of Stemonae Radix based on PCR with specific primers and PCR-RFLP
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