Increasing medicinal and phytochemical compounds of coneflower (Echinacea purpurea L.) as affected by NO3-/NH4+ ratio and perlite particle size in hydroponics.

Medicinal plants are considered as one of the most important sources of chemical compounds, so preparing a suitable culture media for medicinal plant growth is a critical factor. The present study is aimed to improve the caffeic acid derivatives and alkylamides percentages of Echinacea purpurea root extract in hydroponic culture media with different perlite particle size and NO3-/NH4+ ratios. Perlite particle size in the growing media was varied as very coarse perlite (more than 2 mm), coarse perlite (1.5-2 mm), medium perlite (1-1.5 mm), fine perlite (0.5-1 mm), and very fine perlite (less than 0.5 mm) in different ratios to peat moss (including pure perlite, 50:50 v/v, 30:70 v/v, and pure peat moss). Two NO3-/NH4+ ratios (90:10 and 70:30) were tested in each growing media. All phytochemical analyses were performed according to standard methods using high performance liquid chromatography (HPLC). It was found that the E. purpurea grown in the medium containing very fine-grade perlite with 50:50 v/v perlite to peat moss ratio had the maximum caffeic acid derivatives, including chicoric acid (17 mg g-1 DW), caftaric acid (6.3 mg g-1 DW), chlorogenic acid (0.93 mg g-1 DW), cynarin (0.84 mg g-1 DW), and echinacoside (0.73 mg g-1 DW), as well as, alkylamides (54.21%). The percentages of these phytochemical compounds increased by decreasing perlite particle size and increasing of NO3-/NH4+ ratio. The major alkylamide in the E. purpurea root extract was dodeca-2E, 4E, 8Z-10 (E/Z)-tetraenoic acid isobutylamide in all treatments, ranging from 31.12 to 54.21% of total dry weight. It can be concluded that optimizing hydroponic culture media and nutrient solution has significant effects on E. purpurea chemical compounds.

Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine-Suppressive and Cytokine-Inducing Compounds, Including Some That Originate from Endophytic Bacteria.

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.

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.

[Induction of adventitious roots of Echinacea pallida and accumulation of caffeic acid derivatives].

OBJECTIVE: To investigate the effect of auxins 2,4-D,IAA,IBA,NAA on induction of adventitious roots as well as that of IBA concentrations on the growth of adventitious roots and the accumulation of caffeic acid derivatives, with test-tube seedling leaves Echinacea pallida as the explant, and cultivate adventitious roots in bioreactors. RESULT: 1.0 mg x L(-1) IBA was found the best for the induction of adventitious roots, with the numer of induced adventitious roots up to 22. 5 in each culture dish. Among different concentrations for suspension cultivation of IBA tested, 1.0 mg x L(-1) IBA was found the most suitable for the growth of adventitious roots and the accumulation of caffeic acid derivatives. In a 5 L balloon type bubble bioreactor, 8.98 g x L(-1) dry weight was achieved after one month, which was 2.05 times of 4.38 g x L(-1) dry weight cultivated in a triangular flask. The content of echinacoside cultivated in a bioreactor was 14.08 mg x g(-1) DW, which was 2.4 times of cultivated roots. The contents of chlorogenic acid, chicoric acid and total caffeic acid derivatives were 4.0-25.6 times of ultivated roots. CONCLUSION: The study can provide high-quality biomedical drugs containing such caffeic acid derivatives as echinacoside for mass production of Echinacea purpurea medicines.

Establishment of adventitious root cultures of Echinacea purpurea for the production of caffeic acid derivatives.

Echinacace purpurea (purple cone flower) is an important medicinal plant, and widely used for phytochemical purposes. The roots are traditionally used in herbal medicines and dietary supplements as an immunostimulant in treating inflammatory and viral diseases. Extensive research work has been carried out on both the induction of adventitious roots from E. purpurea as well as established small-scale (shake flask) to large-scale (bioreactor) cultures for the production of adventitious root biomass and caffeic acid derivatives. This chapter describes the methodologies of induction of adventitious roots from explants of E. purpurea, propagation of adventitious roots in suspension cultures, estimation of total phenolics, flavonoids, and antioxidant activities. The detailed methodology for high-performance liquid chromatographic analysis of caffeic acid derivatives present in the adventitious roots is also discussed.

Root colonization by an arbuscular mycorrhizal (AM) fungus increases growth and secondary metabolism of purple coneflower, Echinacea purpurea (L.) Moench.

Purple coneflower, Echinacea purpurea (L.) Moench, is an important phytomedicinal species that contains phenolics and alkamides with antipathogenic properties. This study aimed to examine the effect of arbuscular mycorrhizal (AM) colonization on the physiology and biochemistry of E. purpurea. It was hypothesized that AM colonization enhances the growth and secondary metabolism in E. purpurea. In this regard, a 13-week factorial greenhouse experiment was performed with E. purpurea, inoculated (or not) with the AM fungus Glomus intraradices Schenck & Smith. Overall, the results indicated that AM colonization significantly increased the mass of shoots and roots and the concentrations of proteins and most of the phenolics in the roots. Hence, the selected trait of mycorrhiza could play an important role in optimizing the growth of E. purpurea by inducing the production of secondary phytomedicinal metabolites.

[Rapid propagation of Echinacea angustifolia].

The embryo of Echinacea angustifolia was used for explants to induce shoots and callus on MS medium. The plantlets could produce from embryo directly and the shoots could be induced to form fasciculate buds through the callus differentiation. The green plantlets from embryo or fasciculated buds were used for rooting inducement. The experiment showed that the embryo pre-cultured at 4 degrees C and in dark for 7 days can germinate easier than at other cultural conditions; Treatment with low temperature and dark were favorable for growth of radicle and plumule and development of green plantlets; A single shoot could proliferate rapidly to form fasciculated buds by means of callus induction and differentiation; the optimal medium for growth and proliferate were MS + BA 1.0 mg/L + NAA 0.5 mg/L. With this method, the proliferous coefficient should be achieved 3.05.

Variability of the essential oil from three sorts of Echinacea MOENCH genus during ontogenesis.

Variability of the essential oil from three sorts of Echinacea MOENCH genus during ontogenesis The content and quality of the essential oil in relation to the main ontogenetic stages of plants were studied in three various sorts of Echinacea genus. The comparison included Echinacea purpurea, Echinacea atrorubens, and Echinacea pallida. The differences in the content of the oil in different parts of plants and the abundance of individual oil constituents in oils from the sorts under study at the optimum stage of ripeness for harvest were evaluated as well.

Growing environment and nutrient availability affect the content of some phenolic compounds in Echinacea purpurea and Echinacea angustifolia.

Medicinal plant production is different from other agricultural production systems in that the plants are grown for the production of specific phytochemical(s) for human use. To address this need, a Good Manufacturing Practice (GMP)-compliant, controlled-environment production system was developed for production of Echinacea purpurea and Echinacea angustifolia. Within the prototype facility, the growing systems, nutrient availability, water and physical environment were highly controlled. The current study was designed to evaluate the effects of different hydroponic systems, nutrient solution NO (3)(-)/NH (4)(+) ratios and mild water stress on the content of some phenolic compounds in Echinacea plants. The deep-flow solution culture system in which the plant roots were continuously immersed in the nutrient solutions was optimum for the growth of E. purpurea. Higher concentrations of caftaric acid, cynarin and echinacoside were produced in E. angustifolia plants grown in the soil-based growing media while the plants grown in the deep-flow solution system had higher levels of cichoric acid. Altering the NO (3)(-)/NH (4)(+) ratio or limited water stress did not have any significant effect on the phytochemical content of Echinacea plants. Echinacea plants grown in the controlled environment systems had higher or similar amounts of cynarin, caftaric acid, echinacoside and cichoric acid as previously reported in the literature for both field-cultivated and wild-harvested Echinacea plants. This growing system offers the advantages of year-round crop production with minimal contamination by environmental pollutants and common microbes.

Isolation, culture, and plant regeneration from Echinacea purpurea protoplasts.

A plant regeneration system from the isolated protoplasts of Echinacea purpurea L. using an alginate solid/liquid culture is described in the chapter. Viable protoplasts were isolated rom 100 mg of young leaves of 4-wk-old seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase, and 0.3 mol/L mannitol. After isolation and purification, the mesophyll protoplasts were embedded into 0.6% Na-alginate at the density 1 x 10(-5) mL and cultured in modified Murashige and Skoog (MS) culture medium supplemented with 0.3 mol/L sucrose, 2.5 micromol/L benzylaminopurine (BA), and 5.0 micromol/L 2,4-dichlorophenoxyacetic acid (2,4-D). The visible colonies were present after 4 wk of culture. The protoplast-derived clones were transferred onto gellan gum-solidified basal medium supplemented with 1.0 micromol/L BA and 2.0 micromol/L indole-3-butyric acid (IBA) and formed compact and green calli. Shoot development was achieved by subculturing the calli onto the same basal medium supplemented with 5.0 micromol/L BA and 2.0 micromol/L IBA. Further subculture onto basal medium resulted in the regeneration of complete plantlets.

Human gene expression as a tool to determine horticultural maturity in a bioactive plant (Echinacea purpurea L. Moench).

A phenological study was conducted to determine the impact of harvest maturity on the immune-modulating properties of Echinacea purpurea. The aerial parts of this plant were collected during seven stages of development and were assayed for a common botanical marker for this species, cichoric acid. Plants of selected development stages were also assayed for total polysaccharides and compared for their immune-modulating effects on the THP-1 monocyte/macrophage cell line by means of a gene expression study. Although the concentration of cichoric acid did not change significantly during the course of the study, stage 1 (advanced vegetative) had the highest concentration of total polysaccharides and exhibited the most potent induction activity on immune-modulating cytokines such as interferon-gamma and tumor necrosis factor-alpha. These findings suggest that the use of gene expression may be an effective tool not only to standardize botanical extracts but also to optimize harvest time.

[Applied fundamental research of echinacea species].

Echinacea is a most famous "immune herb" in western countries, and continues to be the best selling herb for many years. For the last five years, our research group has cooperated with Institute of Medicinal Plants in Huairou District of Beijing, carrying out studies on Echinacea purpurea, which involved botany, cultivation, pharmacognosy, phytochemistry, quality control, pharmacology and toxicology of the species. Two other species introduced from Canada, Echinacea angustifolia and Echinacea pallida, were also included in the taxonomic, cultivated and pharmacognostic studies. The results acquired have opened up the path to introduce Echinacea species into Traditional Chinese Medicine, thus established the possibility of developing more promising drugs from them.

Arbuscular mycorrhizal inoculation enhances survival rates and growth of Micropropagated plantlets of Echinacea pallida.

In an attempt to induce positive effects on the acclimatization of in vitro propagated Echinacea pallida, four arbuscular mycorrhizal (AM) fungi, Glomus mosseae, Gigaspora ramisporophora, Scutellospora fulgida and Entrophospora colombiana were selected to aid the soil adaptation process. Fungal inocula affected the survival of E. pallida plantlets ranging from 83 % to 92 %, depending on the AM species and also contrasting with 58 % survival of the non-inoculated plantlets. Growth and development were faster in mycorrhizal treated plantlets than in non-treated ones, especially among those treated with Glomus mossae and Scutellospora fulgida. The presence of well-formed arbuscules and vesicles in Echinacea infested roots was confirmed by microscopic examinations in addition to 90 % success in the survival rate of vigorous plants indicated that mycorrhization is a valuable tool to overcome Echinacea acclimatization shock.

[Qualitative characteristics of selected species of the genus Echinacea Moench]. / Stúdium kvalitatívnych vlastností vybraných druhov rodu Echinacea Moench.

The paper examined the effect of production-technological conditions in relation to the ontogenetic stage of plants in three different species of the genus Echinacea Moench., comparing the species Echinacea purpurea, Echinacea atrorubens, and Echinacea pallida. It also examined the differences in the content of essential oil in different plant organs and evaluated the representation of individual components in the essential oils of the species under study at the stage of the optimal maturity for collection.