In-vitro antimalarial activity of methanolic leaf- and stem-derived extracts from four Annonaceae plants.

OBJECTIVE: Plants in the Annonaceae family are known for having abundant biologically active secondary metabolites. They have been used in alternative drugs for various diseases in several countries, for instance, the bark of Cananga odorata (Lam.) Hook and Thomson is used for Ophthalmic inflammation and wound healing in Malaysia. Extracts from the leaves and stems of four Annonaceae plants, namely Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Dasymaschalon sp., Artabotrys burmanicus A.DC, and Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders were investigated for growth inhibitory activity against blood-stage Plasmodium falciparum growth in vitro and for non-specific cytotoxicity against normal peripheral blood mononuclear cells (PBMCs). Antimalarial activity was assessed by invasion inhibition assay and the percentage of infected red blood cells on blood smears were determined. Cytotoxicity was tested by culturing PBMCs with the extracts, and viabilities were determined by Annexin V/propidium iodide staining. RESULTS: A. burmanicus stem extract and M. modestum leaf extract were capable of inhibiting growth of P. falciparum when used at 200 µg/mL compared to chloroquine. The extracts at effective concentrations, did not affect the viability of PBMCs. These results support further need for characterization of active compounds from specific Annonaceae plants in order to exploit their components for potential malaria treatment.

Detection and quantification of cashew in commercial tea products using High Resolution Melting (HRM) analysis.

Tea, a popular aromatic infusion and food supplement, prepared from Camellia sinensis (L.) Kuntze leaves, is often subjected to adulteration with various undeclared inorganic and plant-derived materials. Cashew (Anacardium occidentale L.) nut husk is one of the most common plant tea adulterants. To date, there are limited DNA-based technologies for tea authentication and quantitative detection of adulterants. Herein, we used a universal plant DNA barcoding marker coupled with High Resolution Melting (Bar-HRM) analysis to authenticate tea products from cashew ground nut. Additionally, cashew-specific markers coupled with HRM technology were used to detect and quantify adulteration of tea with cashew DNA. This methodology can reliably detect admixtures as low as 1% v/v cashew in commercial tea products. Overall, our results demonstrate that the HRM technology is a strong molecular approach in tea authentication, capable of detecting very low adulterations in DNA admixtures. PRACTICAL APPLICATION: In this study, we established the use of high-resolution DNA-based technologies for the detection of cashew adulteration in tea, even in very low quantities. The technology could be applied to a greater range of plant-based tea adulterants. This work is expected to facilitate the traceability and authenticity of tea products and form the basis for the development of strategies against fraudulent practices.

Cytotoxic and cytostatic effects of four Annonaceae plants on human cancer cell lines.

Several species of the Annonaceae plants have been used as complementary medicine for cancer-associated illnesses in some ethnic groups of northern Thailand. This study investigated the cytotoxic and cytostatic activity of methanolic extracts derived from the stems of these plants, including Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Artabotrys burmanicus A.DC, Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders, and Dasymaschalon sp. Cell death induction of seven human cancer cell lines and cell cycle analyses were assessed by Annexin V and/or propidium iodide (PI) staining and analyzed by flow cytometry. Treatment of cancer cell lines with the extract of four Annonaceae plants resulted in various cytotoxic activities depending on cell type. The extract of U. longipes exhibited the highest cytotoxic activity capable of inducing cell death of several cancer cell lines, particularly against hepatocellular carcinoma cell lines (HepG2 and Hep3B). This extract was capable of inducing cell cycle arrest at the SubG1 phase. Phytochemical screening of all the extracts revealed the presence of alkaloids, sterols, tannins, anthraquinone glycoside, coumarin, and flavonoids. Determination of active compounds by high-performance liquid chromatography standards revealed bullatacin and asiminecin in all the extracts. The extract of Annonaceae stem or its compounds may provide an opportunity for the development of new therapies against cancer.

Bar-HRM for authenticating soursop (Annona muricata) tea.

Drinking soursop (Annona muricata) tea has become popular in Thailand due to recent findings about the medicinal properties of soursop tea regarding anti-cancer in particular. Consequently, numerous A. muricata tea products were found to be sold on markets and relatively expensive. It is almost impossible to identify the plant species component in the tea bag or powder products using traditional methods which are based on morphological characters. Therefore, a main objective of this study is to develop a molecular method called Bar-HRM (DNA barcoding coupled with High Resolution Melting) for authenticating A. muricata products. Three chloroplast regions including matK, rbcL and trnL were selected for in silico analyses. The findings show that rbcL is the most suitable region to be used for species identification in HRM analysis. Eleven A. muricata herbal products were purchased and tested with rbcL primers. Results from melting profile indicated that three out of eleven tested products were adulterated with other Annona species. It is believed that the Annona products are adulterated to increase the quantity and to make more profit. Notably, all of the tested products purchased from local producers were found to contain herbal species that differ from the species indicated by the seller.

Species identification approach for both raw materials and end products of herbal supplements from Tinospora species.

BACKGROUND: Nowadays herbal products used in traditional medicine are sold in processed forms and thus morphological authentication is almost impossible. With herbal industry rapidly growing size, consumer safety becomes an important issue that requires special attention. Identification of herbal species in the products is therefore needed. METHODS: Sequences from the selected regions (matK, rbcL, trnL and ITS1) were retrieved and analysed. Then the most suitable barcode was assessed for discrimination of T. crispa from closely related species by HRM analysis and used in authentication of commercial products. RESULTS: The ITS1 barcode was found to be the suitable primer as melting data from the HRM assay proved to be capable of distinguishing T. crispa from its related species. The developed protocol was then employed to authenticate medicinal products in powdered form. HRM analysis of all tested samples here revealed that five out of eight products contained not only the indicated species T. crispa but also other Tinospora, that have a high level of morphological similarity. CONCLUSION: Misrepresentation, poor packaging and inappropriate labeling of the tested medicinal herbal products are thought to be the reason of the results here. Using Bar-HRM with the ITS marker lead to success in authenticating the tested herbal products.

Evaluation of suitable DNA regions for molecular identification of high value medicinal plants in genus Kaempferia.

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Kaempferia (Zingiberaceae). Four primer pairs were evaluated (rbcL, rpoC, trnL and ITS1). It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Thus, the primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL primer pair gave the lowest resolution. Our Bar-HRM developed here would not only be useful for identification of Kaempferia plant specimens lacking essential parts for morphological identification but will be useful for authenticating products in powdered form of a high value medicinal species Kaempferia parviflora, in particular.

Evaluation of a DNA-based method for spice/herb authentication, so you do not have to worry about what is in your curry, buon appetito!

It is long believed that some spices may help protect against certain chronic conditions. Spices are usually parts of plants that have been powdered into small pieces. Have you ever wondered what the curry powder in your dish is made of? The aim of this work was to develop an appropriate DNA-based method for assessment of spice identity. Selecting the best marker for species recognition in the Zingiberaceae family. Six DNA regions were investigated in silico, including ITS, matK, rbcL, rpoC, trnH-psbA and trnL. Then, only four regions (ITS, matK, rbcL and trnH-psbA) were included in the simulated HRM (High-resolution Melting) analysis as the results from previous analysis showed that rpoC and trnL may not be suitable to be used to identify Zingiberaceae species in HRM analysis based on both the percentage of nucleotide variation and GC content. Simulated HRM analysis was performed to test the feasibility of Bar-HRM. We found that ITS2 is the most effective region to be used for identification of the studied species and thus was used in laboratory HRM analysis. All seven tested Zingiberaceae plants were then able to be distinguished using the ITS2 primers in laboratory HRM. Most importantly the melting curves gained from fresh and dried tissue overlapped, which is a crucial outcome for the applicability of the analysis. The method could be used in an authentication test for dried products. In the authentication test, only one of seven store-sold Zingiberaceae products that were tested contained the species listed on their labels, while we found substitution/contamination of the tested purchased products in the rest.

Should DNA sequence be incorporated with other taxonomical data for routine identifying of plant species?

BACKGROUND: A variety of plants in Acanthaceae have long been used in traditional Thai ailment and commercialised with significant economic value. Nowadays medicinal plants are sold in processed forms and thus morphological authentication is almost impossible. Full identification requires comparison of the specimen with some authoritative sources, such as a full and accurate description and verification of the species deposited in herbarium. Intake of wrong herbals can cause adverse effects. Identification of both raw materials and end products is therefore needed. METHODS: Here, the potential of a DNA-based identification method, called Bar-HRM (DNA barcoding coupled with High Resolution Melting analysis), in raw material species identification is investigated. DNA barcode sequences from five regions (matK, rbcL, trnH-psbA spacer region, trnL and ITS2) of Acanthaceae species were retrieved for in silico analysis. Then the specific primer pairs were used in HRM assay to generate unique melting profiles for each plants species. RESULTS: The method allows identification of samples lacking necessary morphological parts. In silico analyses of all five selected regions suggested that ITS2 is the most suitable marker for Bar-HRM in this study. The HRM analysis on dried samples of 16 Acanthaceae medicinal species was then performed using primer pair derived from ITS2 region. 100% discrimination of the tested samples at both genus and species level was observed. However, two samples documented as Clinacanthus nutans and Clinacanthus siamensis were recognised as the same species from the HRM analysis. Further investigation reveals that C. siamensis is now accepted as C. nutans. CONCLUSIONS: The results here proved that Bar-HRM is a promising technique in species identification of the studied medicinal plants in Acanthaceae. In addition, molecular biological data is currently used in plant taxonomy and increasingly popular in recent years. Here, DNA barcode sequence data should be incorporated with morphological characters in the species identification.

Cell cycle arrest and apoptosis induction by methanolic leaves extracts of four Annonaceae plants.

BACKGROUND: Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Artabotrys burmanicus A.DC, Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders and Dasymaschalon sp. have been used for traditional medicine to treat cancer-like symptoms in some ethnic groups of Thailand and Laos. METHODS: We evaluated the anti-cancer activity of these Annonaceae plants against several human cancer cell lines. The apoptosis induction was detected by Annexin/propidium iodide (PI) staining. Phytochemical screening was tested by standard protocols and bioactive compounds were determined by HPLC. RESULTS: The crude extracts from leaves of U. longipes, Dasymaschalon sp., A. burmanicus, and M. modestum showed particular effects that were found to vary depending on the cancer cell line, suggesting that the effect was in a cell-type specific manner. Interestingly, the induction of apoptotic cell death was prominent by the leaves-derived crude extract of M. modestum. This crude was, therefore, subjected to cell cycle analysis by PI staining. Results showed that this crude extract arrested cell cycle and increased the percentage of cells in the SubG1 phase in some cancer cell lines. The phytochemical screening tests indicated that all crude extracts contained tannins and flavonoids. HPLC of flavonoids using standards identified rutin as an active compound in U. longipes and Dasymaschalon sp., whereas quercetin was found in U. longipes and M. modestum. CONCLUSIONS: These crude extracts provide a new source for rutin and quercetin, which might be capable of inducing cancer cell apoptotic death in a cell-type specific manner. This suggests, by analyzing the major bioactive compounds, the potential use of these crudes for chemotherapy in the future.

Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees.

BACKGROUND: Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. MATERIALS AND METHODS: High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. RESULTS: The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. CONCLUSION: The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. SUMMARY: We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily determine the A. paniculata species in herbal products tested. Abbreviations used: bp: Base pair, Tm: Melting temperature.

Evaluation of DNA barcoding coupled high resolution melting for discrimination of closely related species in phytopharmaceuticals.

BACKGROUND: Phytopharmaceuticals are increasingly popular as alternative medicines, but poorly regulated in many countries. The manufacturers of these products should be subject to strict controls regarding each product's quality and constituents. Routine testing and identification of raw materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. HYPOTHESIS/PURPOSE: We have applied DNA Barcoding - High Resolution Melting (Bar-HRM), an emerging method for identifying of medicinal plant species based on DNA dissociation kinetics and DNA barcoding, for the authentication of medicinal plant species. STUDY DESIGN: Commonly commercialized Thai medicinal plants that are widely used for medicinal purposes were used in this study. Publicly available sequences of four plastid markers were used for universal primer design. Species discrimination efficiency of the designed primers was evaluated as single and multi-locus analyses by using the primers sets. METHODS: HRM analysis was performed in triplicate on each of the 26 taxa to establish the Tm for each primer set (matK, rbcLA, rbcLB, rbcLC, rpoC1, and trnL). The shapes of the melting curves were analyzed to distinguish the different plant species. Bar-HRM species identification success rates were assessed for each single-locus as well as for multi-locus combinations to establish the optimal combination of primer sets. RESULTS: In single locus analysis the rpoC1 primer set gave the highest discrimination (58%), and in multi locus analysis this could be increased from 87% to 99% depending on the total number of regions included. Different combinations proved to be more or less effective at discrimination, depending on the genus or family examined. CONCLUSIONS: Bar-HRM has proven to be a cost-effective and reliable method for the identification of species in this study of Thai medicinal plants, and results show an identification success rate of 99% among species in the test set.

Authenticity analyses of Phyllanthus amarus using barcoding coupled with HRM analysis to control its quality for medicinal plant product.

The Phyllanthus genus, a plant used in traditional Thai medicine, has according to several pharmacopeias hepatoprotective properties. Not only is the anatomical morphology of these species relatively similar but they also share the Thai common names Look-Tai-Bai (ลูกใต้ใบ) and Yah-Tai-Bai (หญ้าใต้ใบ), which might cause confusion for laypersons. This study attempted to develop a method for accurate identification of Phyllanthus species, especially Phyllanthus amarus, and to detect contaminants in P. amarus products by using DNA barcoding coupled with high resolution melting (HRM) analysis (bar-HRM). Two plastid loci (rbcL and trnL) were chosen for DNA barcoding to generate a suitable primer for distinguishing Phyllanthus species by HRM analysis. The five species of Phyllanthus were subjected to amplification for testing the specificity and discrimination power of the designed primers derived from rbcL and trnL regions. Sensitivity of the method (DNA barcoding conjugated with HRM) to detect adulterant in P. amarus samples was evaluated. The commercial P. amarus products obtained from a local market were authenticated. The primer pair derived from trnL DNA barcoding (PhylltrnL) had more specificity and power of discrimination for Phyllanthus species than that derived from rbcL DNA barcoding (PhyllrbcL). The result showed that Tm of P. amarus, Phyllanthus urinaria, Phyllanthus debilis, Phyllanthus airy-shawii, and Phyllanthus virgatus was 74.3±0.08, 73.04±0.07, 73.36±0.05, 72.21±0.06, 72.77±0.15°C, respectively. This method proved to be a very sensitive tool that can be used for rapid detection of contamination as low as 1% of other Phyllanthus species in P. amarus admixtures. All commercial products of P. amarus obtained from a local market in Thailand were found to contain pure raw materials of P. amarus without any substitution or contamination. Our results indicated that the use of DNA barcoding coupled with HRM was an efficient molecular tool for correct species identification. This molecular tool provides a noteworthy benefit for quality control of medicinal plants and industry plants for pharmacological prospects.

Fast and reliable detection of toxic Crotalaria spectabilis Roth. in Thunbergia laurifolia Lindl. herbal products using DNA barcoding coupled with HRM analysis.

BACKGROUND: Nowadays, medicinal plants are used as a popular alternative to synthetic drugs. Many medicinal plant products have now been commercialized throughout various markets. These products are commonly sold in processed or modified forms such as powders, dried material and capsules, making it almost impossible to accurately identify the constituent species. The herbal plant known as 'Rang Chuet' in Thai has been widely used as remedies for various ailments. However, two medicinal plants species, Thunbergia laurifolia and Crotalaria spectabilis share this name. Duo to the similarity in nomenclature, the commercial products labeled as 'Rang Chuet' could be any of them. Recently, the evidence of hepatotoxic effects linked to use of C. spectabilis were reported and is now seriously concern. There is a need to find an approach that could help with species identification of these herbal products to ensure the safety and efficacy of the herbal drug. METHODS: Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate T. laurifolia species. Four DNA barcodes including matK, rbcL, rpoC and trnL were selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Commercial products labeled as 'Rang Chuet' were purchased from Thai markets and authentication by HRM analyses. RESULTS: Melting data from the HRM assay using the designed primers showed that the two 'Rang Chuet' species could easily be distinguished from each other. The melting profiles of the all four region amplicons of each species are clearly separated in all three replicates. The method was then applied to authenticate products in powdered form. HRM curves of all ten test samples indicated that three of the tested products did not only contain the T. laurifolia species. CONCLUSION: The herbal drugs derived from different plants must be distinguished from each other even they share the same vernacular name. The Bar-HRM method developed here proved useful in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products.

Bar-HRM for Authentication of Plant-Based Medicines: Evaluation of Three Medicinal Products Derived from Acanthaceae Species.

Medicinal plants are used as a popular alternative to synthetic drugs, both in developed and developing countries. The economic importance of the herbal and natural supplement industry is increasing every year. As the herbal industry grows, consumer safety is one issue that cannot be overlooked. Herbal products in Thai local markets are commonly sold without packaging or labels. Plant powders are stored in large bags or boxes, and therefore buying local herbal products poses a high risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Here DNA barcoding was used in combination with High Resolution Melting analysis (Bar-HRM) to authenticate three medicinal Acanthaceae species (Acanthus ebracteatus, Andrographis paniculata and Rhinacanthus nasutus) commonly used in Thailand. The rbcL barcode was selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Melting data from the HRM assay using the designed rbcL primers showed that the three chosen species could be distinguished from each other. HRM curves of all fifteen test samples indicated that three of tested products did not contain the indicated species. Two closely related species (A. paniculata and R. nasutus), which have a high level of morphological similarity, were interchanged with one another in three tested products. Incorrect information on packaging and labels of the tested herbal products was the cause of the results shown here. Morphological similarity among the species of interest also hindered the collection process. The Bar-HRM method developed here proved useful in aiding in the identification and authentication of herbal species in processed samples. In the future, species authentication through Bar-HRM could be used to promote consumer trust, as well as raising the quality of herbal products.