EpMYB2 positively regulates chicoric acid biosynthesis by activating both primary and specialized metabolic genes in purple coneflower.

Chicoric acid is the major active ingredient of the world-popular medicinal plant purple coneflower (Echinacea purpurea (L.) Menoch). It is recognized as the quality index of commercial hot-selling Echinacea products. While the biosynthetic pathway of chicoric acid in purple coneflower has been elucidated recently, its regulatory network remains elusive. Through co-expression and phylogenetic analysis, we found EpMYB2, a typical R2R3-type MYB transcription factor (TF) responsive to methyl jasmonate (MeJA) simulation, is a positive regulator of chicoric acid biosynthesis. In addition to directly regulating chicoric acid biosynthetic genes, EpMYB2 positively regulates genes of the upstream shikimate pathway. We also found that EpMYC2 could activate the expression of EpMYB2 by binding to its G-box site, and the EpMYC2-EpMYB2 module is involved in the MeJA-induced chicoric acid biosynthesis. Overall, we identified an MYB TF that positively regulates the biosynthesis of chicoric acid by activating both primary and specialized metabolic genes. EpMYB2 links the gap between the JA signaling pathway and chicoric acid biosynthesis. This work opens a new direction toward engineering purple coneflower with higher medicinal qualities.

Improving growth properties and phytochemical compounds of Echinacea purpurea (L.) medicinal plant using novel nitrogen slow release fertilizer under greenhouse conditions.

Medicinal plant production is most important than other agricultural plants due to their phytochemical compounds effects on human health. Paying attention to plant nutrition requirement is so important. In order to assess the effect of nitrate (NO3-) dosage supplies from two types of fertilizers on growth and phytochemical properties of Echinacea purpurea rhizomata cum radicibus, an experiment with completely simple design was carried out under greenhouse conditions. Two types of fertilizers (new invented nitrogen (N) slow release fertilizer and urea chemical fertilizer) at three dosages (50, 100, and 150 mM) were applied. Plant growth parameters and total phenolic (TPC), total flavonoids (TFC), polysaccarides content, essential oil content, caffeic acid derivatives, and anti-radical scavenging activities of E. purpurea were assessed. The results showed the significant (p ≤ 0.01) differences among treatments, both in growth and phytochemical properties. Using of N slow release, especially in 150 mM dosage, significantly increased all the plant growth and phytochemical properties. The dried E. purpurea rhizomata cum radicibus contained more caftaric acid (max 12.56 mg g-1 DW) and chicoric acid (max 7.56 mg g-1 DW) than other derivatives. Despite the impact of heavy metals on yield and growth of E. purpurea, the concentration of all heavy metals and micronutrients (boron (B), cadmium (Cd), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), and zinc (Zn)) in studied soil and fertilizer samples was less than United States Environmental Protection Agency (USEPA) limits of contamination. Based on the results, using of N slow release fertilizers can improve phytochemical properties of the plant due to its polymeric structure and can be a suitable substitution of chemical fertilizers, especially in medicinal plants growth.

A strategy for test article selection and phytochemical characterization of Echinacea purpurea extract for safety testing.

Botanical dietary supplements (BDS) are used around the world for many purported therapeutic properties. The selection of an authentic product and it's phytochemical characterization is critical to generate robust safety data. Because botanicals are complex mixtures with variable quality, identification of a representative product for testing has been challenging. Echinacea is used for its purported immune stimulant properties and was listed as the 2nd top-selling BDS in 2018. However, there are limited safety data for Echinacea. Hence, the National Toxicology Program (NTP) has selected Echinacea for safety testing using rodent models. Here, we describe selection and comprehensive characterization of an Echinacea purpurea root extract to be used in the NTP testing program. Using non-targeted chemical analyses combined with chemometric analysis, a potential unfinished product (i.e., an extract that serves as source material for finished products) of Echinacea purpurea was selected. The product was then authenticated using chemical and DNA techniques and characterized, including the phytochemical composition. Among numerous constituents identified, caftaric acid, chicoric acid, chlorogenic acid and dodeca-2(E),4(E),8(Z),10(E/Z)-tetraenoic acid isobutylamide made up a small fraction of the extract. Based on these analyses, an approach is proposed for test article selection for Echinacea research which can be adapted to other botanicals.

Heterologous expression of an acid phosphatase gene and phosphate limitation leads to substantial production of chicoric acid in Echinacea purpurea transgenic hairy roots.

MAIN CONCLUSION: A high level of the secondary metabolite chicoric acid is produced by intracellular Pi supply and extracellular phosphate limiting in Echinacea purpurea hairy roots. Chicoric acid (CA) is a secondary metabolite which is gained from Echinacea purpurea. It has been found to be one of the most potent HIV integrase inhibitors with antioxidant and anti-inflammatory activities. However, the low-biosynthesis level of this valuable compound becomes an inevitable obstacle limiting further commercialization. Environmental stresses, such as phosphorus (Pi) deficiency, stimulate the synthesis of chemical metabolites, but significantly reduce plant growth and biomass production. To overcome the paradox of dual opposite effect of Pi limitation, we examined the hypothesis that the intracellular Pi supply and phosphate-limiting conditions enhance the total CA production in E. purpurea hairy roots. For this purpose, the coding sequence (CDS) of a purple acid phosphatase gene from Arabidopsis thaliana, AtPAP26, under CaMV-35S promoter was overexpressed in E. purpurea using Agrobacterium rhizogenes strain R15834. The transgenic hairy roots were cultured in a Pi-sufficient condition to increase the cellular phosphate metabolism. A short-term Pi starvation treatment of extracellular phosphate was applied to stimulate genes involved in CA biosynthesis pathway. The overexpression of AtPAP26 gene significantly increased the total APase activity in transgenic hairy roots compared to the non-transgenic roots under Pi-sufficient condition. Also, the transgenic hairy roots showed increase in the level of total and free phosphate, and in root fresh and dry weights compared to the controls. In addition, the phosphate limitation led to significant increase in the expression level of the CA biosynthesis genes. Considering the increase of biomass production in transgenic vs. non-transgenic hairy roots, a 16-fold increase was obtained in the final yield of CA for transgenic E. purpurea roots grown under -P condition compared to +P non-transgenic roots. Our results suggested that the expression of phosphatase genes and phosphate limitation were significantly effective in enhancing the final production yield and large-scale production of desired secondary metabolites in medicinal plant hairy roots.

A Comparison of the Immunostimulatory Effects of Polysaccharides from Tetraploid and Diploid Echinacea purpurea.

Polyploidization is an effective means of improving the active components and quality of secondary metabolism in medicinal plants. In the present study, we compared the immunostimulatory effects of crude polysaccharides from tetraploid and diploid Echinacea purpurea. The results showed that the carbohydrate contents of crude polysaccharide of tetraploid E. purpurea (CPE4) and diploid E. purpurea (CPE2) were 85.51% and 44.65%, respectively. 1H-nuclear magnetic resonance (NMR) spectroscopy and gel-permeation chromatography (GPC) analyses showed no major differences in the overall structure and molecular weight of polysaccharides between CPE4 and CPE2. However, some differences in the relative content of the same polysaccharides group were observed between CPE4 and CPE2. In in vitro tests, EP4 could stimulate lymphocyte proliferation and secretion of cytokines maximally at the concentration of 0.0312 mg/mL, and EP2 could stimulate lymphocyte proliferation and secretion of cytokines maximally at the concentration of 0.125 mg/mL. In in vivo tests, EP4 was more effective at promoting the proliferation of lymphocytes and secretion of cytokines in mice immunosuppressed by cyclophosphamide than EP2 at the same concentration. Taken together, these data demonstrated that the relative content of the partial polysaccharides group is increased, and the immunoregulatory effect is enhanced in tetraploid E. purpurea.

What's in the box? Authentication of Echinacea herbal products using DNA metabarcoding and HPTLC.

BACKGROUND: Differences in regulatory policies between countries as well as a lack of appropriate standardized methods for the authentication and quality control of herbal products directly impact their quality and safety. Echinacea products are among the top-selling herbal products in Europe and the United States with indications for a broad range of ailments. The increased use of Echinacea species has led to concerns about adulterated products resulting from challenges in morphology-based identification, due to overlapping morphological variation, frequent hybridization between species, and deliberate adulteration. PURPOSE: This study addressed the need for a novel analytical strategy in the authentication of herbal products. METHODS: A combination of high performance thin layer chromatography (HPTLC) and DNA metabarcoding was employed. Fifty-three Echinacea herbal products marketed across Europe were tested to evaluate the accuracy of these methods in plant identification and their potential for detecting substitutes, adulterants and other unreported plant constituents. RESULTS: HPTLC provides high resolution in the detection of Echinacea phytochemical target compounds, but does not offer information on the other species within the product. Alternatively, we showed that the limitation of HPTLC in detecting non-targeted species can be overcome by the complementary use of DNA metabarcoding. Using DNA metabarcoding, Echinacea species were detected in 34 out of the 38 retained products (89%), but with a lack of discriminatory resolution at the species level due to the low level of molecular divergence within the Echinacea genus. All of the tested herbal products showed considerable discrepancies between ingredients listed on the label and the ones detected using DNA metabarcoding, registering an overall ingredient fidelity of only 43%. CONCLUSION: The results confirm that DNA metabarcoding can be used to test for the presence of Echinacea species and simultaneously to detect other species present in even highly processed and multi-ingredient herbal products.

Plant-endophytes interaction influences the secondary metabolism in Echinacea purpurea (L.) Moench: an in vitro model.

The influence of the interaction(s) between the medicinal plant Echinacea purpurea (L.) Moench and its endophytic communities on the production of alkamides is investigated. To mimic the in vivo conditions, we have set up an infection model of axenic in vitro E. purpurea plants inoculated with a pool of bacterial strains isolated from the E. purpurea stems and leaves. Here we show different alkamide levels between control (not-inoculated) and inoculated plants, suggesting that the alkamide biosynthesis may be modulated by the bacterial infection. Then, we have analysed the branched-chain amino acids (BCCA) decarboxylase gene (GenBank Accession #LT593930; the enzymatic source for the amine moiety formation of the alkamides) expression patterns. The expression profile shows a higher expression level in the inoculated E. purpurea tissues than in the control ones. These results suggest that the plant-endophyte interaction can influence plant secondary metabolism affecting the therapeutic properties of E. purpurea.

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

Subtracted diversity array identifies novel molecular markers including retrotransposons for fingerprinting Echinacea species.

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.

Identifying commercially relevant Echinacea species by AFLP molecular markers.

The rising interest in medicinal plants has brought several species of the genus Echinacea to the attention of many scientists. Echinacea angustifolia, E. pallida, and E. purpurea are the most important for their immunological properties, well known and widely used by the native Americans. The three species are easily distinguishable on the basis of their morphological characteristics, but it would be difficult, if not impossible, to distinguish them in commercial preparations of ground, dry plant parts of E. purpurea (the most valuable species for chemotherapeutic properties) mixed with the other two species. Species-specific molecular markers could be useful to address this issue. In the present work, using fresh material collected from cultivated Echinacea spp., AFLP analysis was used to discriminate the three species and to detect species-specific DNA fragments. By using 14 primer combinations it was possible to detect a total of 994 fragments, of which 565 were polymorphic. Overall, 89 fragments were unique to E. purpurea, 32 to E. angustifolia, and 26 to E. pallida. E+CAC/M+AAT or E+CAC/M+AGC alone provided 13, 9, and 4 or 7, 5, and 5 specific fragments for E. purpurea, E. angustifolia, and E. pallida, respectively. A validation trial to confirm the results was carried out on bulked samples of 23 accessions covering most of the genetic diversity of the three species. The results are discussed in terms of practical applications in the field of popular medicine, detecting frauds, and implications for the genus Echinacea.

Metabolic profiling of echinacea genotypes and a test of alternative taxonomic treatments.

The genus Echinacea is used as an herbal medicine to treat a variety of ailments. To better understand its potential chemical variation, 40 Echinacea accessions encompassing broad geographical and morphological diversity were evaluated under controlled conditions. Metabolites of roots from these accessions were analyzed by HPLC-photo diode array (HPLC-PDA), GC-MS, and multivariate statistical methods. In total, 43 lipophilic metabolites, including 24 unknown compounds, were detected. Weighted principal component analysis (WPCA) and clustering analysis of the levels of these metabolites across Echinacea accessions, based on Canberra distances, allowed us to test two alternative taxonomic treatments of the genus, with the further goal of facilitating accession identification. A widely used system developed by McGregor based primarily on morphological features was more congruent with the dendrogram generated from the lipophilic metabolite data than the system more recently developed by Binns et al. Our data support the hypothesis that Echinacea pallida is a diverse allopolyploid, incorporating the genomes of Echinacea simulata and another taxon, possibly Echinacea sanguinea. Finally, most recognized taxa of Echinacea can be identified by their distinct lipophilic metabolite fingerprints.

Cytokine- and interferon-modulating properties of Echinacea spp. root tinctures stored at -20 degrees C for 2 years.

Echinacea spp. phytomedicines are popular for treating upper respiratory infections. The purpose of this investigation was to examine the immunomodulatory properties of Echinacea tinctures from seven species after being stored at -20 degrees C for 2 years. Two experimental techniques were employed using human peripheral blood mononuclear cells (PBMC). In the first set of experiments, PBMCs were stimulated in vitro with tinctures alone and assayed for proliferation and production of interleukin-10 (IL-10), IL-12, and tumor necrosis factor-alpha (TNF-alpha). In the second set of experiments, subjects were immunized with influenza vaccine. PBMCs from vaccinated individuals were stimulated in vitro with Echinacea tinctures and influenza virus; cytokine production (IL-2, IL-10, and interferon-gamma [IFN-gamma]) was compared prevaccination and postvaccination. In the first experiments, (1) tinctures from E. angustifolia, E. pallida, E. paradoxa, and E. tennesseensis stimulated proliferation and tended to increase IL-10, (2) E. sanguinea and E. simulata stimulated only proliferation, (3) E. purpurea stimulated only IL-10, and (4) none of the extracts influenced IL-12 or TNF-alpha. In the second experiments, (1) tinctures from E. pallida, E. paradoxa, E. sanguinea, and E. simulata diminished influenza-specific IL-2, and (2) none of the extracts influenced influenza-specific IL-10 or IFN-gamma. For in vitro models using Echinacea, immune response may vary based on stimulus (Echinacea alone vs. Echinacea + recall stimulation with virus).

Sequence characterized amplified region (SCAR) analysis on DNA from the three medicinal Echinacea species.

In our previous study, RAPD (Random Amplified Polymorphic DNA) analysis revealed species-specific markers for three medicinal Echinacea species (Asteraceae): E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench. In the present work, we have converted a RAPD marker (750 bp) for E. purpurea into a SCAR (Sequence Characterized Amplified Region) marker. SCAR-PCR, in fact, revealed the expected amplicon (330 bp) only in E. purpurea and not in the other two species, giving further evidence for differences in medicinal Echinacea spp. genome and confirming a greater similarity between E. pallida and angustifolia.

Genetic characterization of the three medicinal Echinacea species using RAPD analysis.

The three medicinal species of the Echinacea genus, E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench were distinguished using the RAPD (random amplified polymorphic DNA) technique. Species-specific markers were identified from amplicons obtained with four of the twenty 10-mer primers contained in the Operon RAPD kit A. In particular, one marker was identified for E. angustifolia (OPA 20, 1800 pb) and E. pallida (OPA 10, 600 pb) and three markers for E. purpurea (OPA 11 : 1250 pb; OPA 17 : 750, 1800 pb). Genetic distance analysis indicated a high degree of difference among the three species with a relative lower difference between E. angustifolia and E. pallida.

Predicting quantitative phytochemical markers in single Echinacea plants or clones from their DNA fingerprints.

Amplified restricted fragment length polymorphism (AFLP) data analysis was found to be a statistically significant predictor of phytochemical markers in cultivated Echinacea purpurea germplasm and some related wild species. Over 50 accessions grown under greenhouse conditions were subjected to AFLP analysis and the same assessed for content of tetraene and cichoric acid by high pressure liquid chromatography. The first and second canonical correlation of DNA variables and the phytochemical variables were significant. Individual regressions of cichoric acid and dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutyl amide predicted by DNA polymorphism analysis against actual HPLC determined values were nearly linear. Mantel's test showed that there was a weak correlation but a strong association of values of the phytochemical variables and the DNA polymorphism data.