An analysis of Echinacea chloroplast genomes: Implications for future botanical identification.

Echinacea is a common botanical used in dietary supplements, primarily to treat upper respiratory tract infections and to support immune function. There are currently thought to be nine species in the genus Echinacea. Due to very low molecular divergence among sister species, traditional DNA barcoding has not been successful for differentiation of Echinacea species. Here, we present the use of full chloroplast genomes to distinguish between all 9 reported species. Total DNA was extracted from specimens stored at the National Museum of Natural History, Smithsonian Institution, which had been collected from the wild with species identification documented by experts in the field. We used Next Generation Sequencing (NGS) and CLC Genomics Workbench to assemble complete chloroplast genomes for all nine species. Full chloroplasts unambiguously differentiated all nine species, compared with the very few single nucleotide polymorphisms (SNPs) available with core DNA barcoding markers. SNPs for any two Echinacea chloroplast genomes ranged from 181 to 910, and provided robust data for unambiguous species delimitation. Implications for DNA-based species identification assays derived from chloroplast genome sequences are discussed in light of product safety, adulteration and quality issues.

An uncommon plant growth regulator, diethyl aminoethyl hexanoate, is highly effective in tissue cultures of the important medicinal plant purple coneflower (Echinacea purpurea L.).

We investigated the effects of various concentrations of diethyl aminoethyl hexanoate (DA-6) on the regeneration and growth of adventitious buds in in vitro purple coneflower cultures. Among the 3 types of explants tested, leaf explants required higher concentrations of DA-6 than petiole and root explants in order to obtain high regeneration rates, while root explants required the lowest concentration of DA-6. Additionally, explants with higher ploidy levels were more sensitive to the addition of DA-6, while explants with lower ploidy levels required higher concentrations of DA-6 to achieve its maximal regeneration rate. Interestingly, the application of a concentration that was conducive to the regeneration of explants with lower ploidy levels was inhibitory to the regeneration of explants with higher ploidy levels. Moreover, during the growth of regenerated buds, DA-6 application significantly improved plant height and weight, root weight, root thickness, root number, primary root length, total root length, and root/top ratio. Differences in the responses of explants to supplementation with DA-6 were also observed among explants with different ploidy levels, with buds having lower ploidy levels responding to lower concentrations of DA-6. Taken together, the results of the present experiments showed that proper application of DA-6 could increase in vitro culture efficiency in purple coneflower.

Gibberellic acid increases secondary metabolite production in Echinacea purpurea hairy roots.

Gibberellic acid (GA(3)) is reported to have diverse effects on hairy root cultures of many plant species; therefore, the effects of GA(3) on the growth, secondary metabolite production (caffeic acid derivatives and lignin), phenylalanine ammonia lyase (PAL) activity, and free radical scavenging activity of light-grown Echinacea purpurea L. hairy roots were investigated. Eight concentrations of GA(3), ranging from 0.005 to 1.0 µM, were added to shake flask cultures. The moderate GA(3) concentration, 0.025 µM, resulted in the highest concentrations of cichoric acid, caftaric acid, and chlorogenic acid, as well as increased PAL activity, cell viability, and free radical scavenging activity, while higher and lower GA(3) concentrations resulted in reduced levels compared to the control (lacking GA(3)). The moderate GA(3) concentration also affected root morphogenesis; supplementation with 0.025 µM GA(3) resulted in the development of thick, dense, purple-colored roots, while roots exposed to the higher and lower concentrations of GA(3) were thin and off-white. This study demonstrates that supplementation with GA(3) may be an excellent strategy to optimize the production of secondary metabolites from E. purpurea hairy root cultures; however, the GA(3) concentration is a critical factor.

Phenylpropanoid glycosides from plant cell cultures induce heme oxygenase 1 gene expression in a human keratinocyte cell line by affecting the balance of NRF2 and BACH1 transcription factors.

Phenylpropanoids have several highly significant biological properties in both plants and animals. Four phenylpropanoid glycosides (PPGs), verbascoside (VB), forsythoside B (FB), echinacoside (EC) and campneoside I (CP), were purified and tested for their capability to activate NRF2 and induce phase II cytoprotective enzymes in a human keratinocyte cell line (HaCaT). All four substances showed similar strong antioxidant and radical-scavenging activities as determined by diphenylpicrylhydrazyl assay. Furthermore, in HaCaT cells, FB and EC are strong activators of NRF2, the nuclear transcription factor regulating many phase II detoxifying and cytoprotective enzymes, such as heme oxygenase 1 (HMOX1). In HaCaT cells, FB and EC (200 µM) induced nuclear translocation of NRF2 protein after 24 h and reduced nuclear protein levels of BACH1, a repressor of the antioxidant response element. FB and EC greatly HMOX1 mRNA levels by more than 40-fold in 72 h. Cytoplasmic HMOX1 protein levels were also increased at 48 h after treatment. VB was less active compared to FB and EC, and CP was slightly active only at later times of treatment. We suggest that hydroxytyrosol (HYD) could be a potential bioactive metabolite of PPGs since HYD, in equimolar amounts to PGGs, is able to both activate HO-1 transcription and modify Nrf2/Bach1 nuclear protein levels. This is in agreement with the poor activity of CP, which contains a HYD moiety modified by an O-methyl group. In conclusion, FB and EC from plant cell cultures may provide long-lasting skin protection by induction of phase II cytoprotective capabilities.

Metabolomic analysis reveals that the accumulation of specific secondary metabolites in Echinacea angustifolia cells cultured in vitro can be controlled by light.

Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14 days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography-mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds.

Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L.

Petiole explants were obtained from in vitro grown diploid (2x = 22) Echinacea purpurea plantlets. Shoots were regenerated by culturing the explants on MS basal medium containing 0.3 mg/L benzyladenine (BA), 0.01 mg/L naphthaleneacetic acid (NAA) and four concentrations (30, 60, 120, and 240 mg/L) of colchicine for 30 days, or 120 mg/L of colchicine for various durations (7, 14, 21, and 28 days). The regenerated shoots were induced to root on MS basal medium with 0.01 mg/L NAA, and then the root-tips of the regenerated shoots were sampled for count of chromosome number. It was found that a treatment duration of >7 days was necessary for induction of tetraploid (4x = 44) shoots, and treatment with 120 mg/L colchicine for 28 days was the most efficient for induction of tetraploids, yielding 23.5% of tetraploids among all the regenerated shoots. Chimeras were observed in almost all the treatments. However, the ratio of tetraploid to diploid cells in a chimeric plant was usually low. In comparison with diploid plants, tetraploid plants in vitro had larger stomata and thicker roots with more root branches, and had prominently shorter inflorescence stalk when mature.

[Microscopic identification study of western herbs. Part I: Three Echinacea-species roots].

OBJECTIVE: To make microscopic identification research of three Echinacea-species roots recorded in the United States Pharmacopeia. METHOD: The root transverse section and powder of E. angustifolia, E. pallida, and E. purpurea were observed. The main microscopic features were photographed. RESULT: The main microscopic features of transverse section and powder in three Echinacea-species roots are basically similar, except for some diagnostic differences. The results provide reliable reference for the authentication of raw materials of western herbal studies.

Structure and accumulation of phenolics in elicited Echinacea purpurea cell cultures.

Echinacea purpurea cell cultures have been established for the production of secondary and possibly bioactive compounds using elicitation techniques. Different elicitors including yeast elicitor, methyl jasmonate, glutathione, and manganese ions were used and compared for their effects on the formation of phenolics in the cell cultures. The accumulation of phenolics in the medium after elicitation with glutathione and manganese ions showed similar patterns as detected by HPLC in control medium. However, in yeast elicitor- and methyl jasmonate-treated cell suspension cultures, other phenolics were formed and accumulated in the medium. In contrast, both control and elicited cells contained the same pattern of phenolics. The main phenolics both in the medium and cells, 19 in total, were isolated and identified on the basis of chromatographic, chemical (derivatisation), enzymatic and spectroscopic techniques. The medium contained lignans, neolignans and acetophenone derivatives as main elicitor-enhanced products. The cells mainly contained phenolic glycosides including a new compound, alpha- O-beta-D-glucopyranosylacetovanillone. Detailed collision-induced dissociation electrospray ionisation mass spectrometric fragmentation pathways for 8,4'-oxyneolignan glycosides are discussed.

Protoplast isolation of callus in Echinacea augustifolia.

Preparations from Echinacea are among the most widely used herbal medicines. Most uses of Echinacea are based on the reported immunological properties. In this paper, we used callus of Echinacea augustifolia for isolation and researched the factors influencing the process of protoplasts preparation, the result indicated it was easy to isolate protoplast from buff-green Callus in E. augustifolia which was looked like granule with symmetrical character. The result showed that the best enzyme solution concentration is composed of cellulase 2.0% (w/v), pectinase1.0% (w/v), hemicellulase 0.5% (w/v), 0.7 mol/L mannitol and 50.0 x 10(4) gFW(-1) protoplasts were obtained after 8 h later. The yield of protoplasts was significantly influenced by cellulase concentration and incubation time. Finally, we summarized an integrated approach including callus induction, callus culture, isolation, purification, identification and calculation of protoplasts.

[Studies on pharmacognosy of Echinacea purpurea].

Pharmacognostical studies of Echinacea purpurea were carried out by botanical analysis, microscopic analysis and physicochemical analysis. The detalied pharmacognostical characteristics of Echinacea purpurea were described respectively.