Evaluation of anti-Alzheimer activity of Echinacea purpurea extracts in aluminum chloride-induced neurotoxicity in rat model.

Alzheimer's disease (AD) is one of the neurodegenerative illnesses that impair individual life & increase the demand for caregivers with no available curative medication right now. Therefore, there is a growing concern about employing herbal medicine to limit AD progression & improve patients' life quality, thus potentiating its add-on therapy. In addition, herbs are cost-effective & accessible with nearly no side effects. In the same vein, our study aimed to investigate the potency of Echinacea purpurea (EP) flower extracts to ameliorate the neurodegenerative effect of Aluminum chloride (AlCl3) in a rat model. Moreover, mechanistic studies, including impact on the cholinesterase activity, redox status, inflammatory mediators, behavior performance, glucose level & histopathology, were carried on. Our results showed that 250 mg/kg of Aqueous (AQ) & Alcoholic (AL) extracts of EP inhibited cholinesterase, restored oxidative balance, down-regulated IL-6 & TNF-α cytokines & improved behavior performance in vivo that was reflected in the brain picture by decreasing neuronal degeneration & amyloid plaques in cerebral cortex & hippocampus. The potency of both extracts was compared to reference drugs & AlCl3 positive control group. The AQ extract showed greater potency against COX-1, COX-2 & α-amylase in vitro, while the AL extract was more potent against cholinesterase in vitro, inflammatory cytokines, behavior & pathological improvement in vivo. Conclusively EP overcame AlCl3-induced neurobehavioral toxicity in the rat model via different pathways, which support its regular administration to postpone progressive neural damage in AD patients.

C3H Expression Is Crucial for Methyl Jasmonate Induction of Chicoric Acid Production by Echinacea purpurea (L.) Moench Cell Suspension Cultures.

Echinacea purpurea (L.) Moench is one of the most economically important medicinal plants, cultivated worldwide for its high medicinal value and with several industrial applications in both pharmaceutical and food industries. Thanks to its various phytochemical contents, including caffeic acid derivatives (CADs), E. purpurea extracts have antioxidant, anti-inflammatory, and immuno-stimulating properties. Among CADs, chicoric acid is one of the most important compounds which have shown important pharmacological properties. The present research was aimed at optimizing the production of chicoric acid in E. purpurea cell culture. Methyl jasmonate (MeJa) at different concentrations and for different duration of treatments was utilized as elicitor, and the content of total polyphenols and chicoric acid was measured. Several genes involved in the chicoric acid biosynthetic pathway were selected, and their expression evaluated at different time points of cell culture growth. This was performed with the aim of identifying the most suitable putative molecular markers to be used as a proxy for the early prediction of chicoric acid contents, without the need of expensive quantification methods. A correlation between the production of chicoric acid in response to MeJa and an increased response to oxidative stress was also proposed.

Substrate promiscuity of acyltransferases contributes to the diversity of hydroxycinnamic acid derivatives in purple coneflower.

High pliability and promiscuity are observed widely exist in plant specialized metabolism, especially the hydroxycinnamic acid metabolism. Here, we identified an addition BAHD acyltransferase (EpHMT) that catalyzes phaselic acid biosynthesis and found that the substrate promiscuities of identified BAHD and SCPL acyltransferases are responsible for the diversity of hydroxycinnamic acid derivatives in purple coneflower.

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.

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.

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.

The influence of Echinacea purpurea leaf microbiota on chicoric acid level.

The controversial anti-proliferative effects of Echinacea purpurea (L.) Moench (Asteraceae) might be related to different plant metabolites contained in plant samples, extracts and products. The influence of bacterial endophytes on the synthesis of bioactive compounds in the medicinal plants has been previously demonstrated but there are only few studies addressing anticancer effects and mechanisms of E. purpurea extracts following endophytic colonization. The present study aimed to test and compare the lactate dehydrogenase (LDH) inhibition potential of n-hexane and methanol extracts from in vitro endophyte non-inoculated and inoculated E. purpurea plants. An in vitro model was previously set up to perform the infection of axenic E. purpurea plants with bacterial endophytic strains isolated from E. purpurea aerial part. Only methanol extracts showed LDH5 inhibition, in particular the richest in chicoric acid and most strongly inhibiting extract was obtained from inoculated stem and leaves of E. purpurea (IC50 = 0.9 mg/ml). Chicoric acid showed an IC50 value (66.7 µM) in enzymatic assays better than that of the reference compound galloflavin. Modeling studies were carried out to suggest the putative interaction mode of chicoric acid in the enzyme active site. This in vitro model on plant-bacterial interaction may lead to obtain extracts from plants enriched in bioactive compounds and it is a new approach for the discovery of novel anticancer compounds.

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.

Synthesis and biological evaluation of a series of fatty acid amides from Echinacea.

Alkylamides are lipophilic constituents of Echinacea and possess numerous biological activities. Although significant effort has been focused on the study of crude Echinacea extracts, very little is known regarding the activities of the individual constituents that make up these herbal treatments. Herein we explore the SAR of simple alkylamides found in Echinacea extracts with respect to their ability to decrease the production of the pro-inflammatory mediator TNF-α. Our results have revealed the key structural requirements for activity and provide lead compounds for further investigation of these poorly understood molecules.

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.

Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp.

BACKGROUND: Extracts and products (roots and/or aerial parts) from Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compounds of Echinacea spp. This study aimed at investigating the capacity of selected lactic acid bacteria to enhance the antimicrobial, antioxidant and immune-modulatory features of E. purpurea with the prospect of its application as functional food, dietary supplement or pharmaceutical preparation. RESULTS: Echinacea purpurea suspension (5%, wt/vol) in distilled water, containing 0.4% (wt/vol) yeast extract, was fermented with Lactobacillus plantarum POM1, 1MR20 or C2, previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum, was used as the control to investigate functional features. Echinacea suspension fermented with Lb. plantarum C2 exhibited a marked antimicrobial activity towards Gram-positive and -negative bacteria. Compared to control, the water-soluble extract from Echinacea suspension fermented with Lactobacillus plantarum 1MR20 showed twice time higher radical scavenging activity on DPPH. Almost the same was found for the inhibition of oleic acid peroxidation. The methanol extract from Echinacea suspension had inherent antioxidant features but the activity of extract from the sample fermented with strain 1MR20 was the highest. The antioxidant activities were confirmed on Balb 3T3 mouse fibroblasts. Lactobacillus plantarum C2 and 1MR20 were used in association to ferment Echinacea suspension, and the water-soluble extract was subjected to ultra-filtration and purification through RP-FPLC. The antioxidant activity was distributed in a large number of fractions and proportional to the peptide concentration. The antimicrobial activity was detected only in one fraction, further subjected to nano-LC-ESI-MS/MS. A mixture of eight peptides was identified, corresponding to fragments of plantaricins PlnH or PlnG. Treatments with fermented Echinacea suspension exerted immune-modulatory effects on Caco-2 cells. The fermentation with Lb. plantarum 1MR20 or with the association between strains C2 and 1MR20 had the highest effect on the expression of TNF-α gene. CONCLUSIONS: E. purpurea subjected to lactic acid fermentation could be suitable for novel applications as functional food dietary supplements or pharmaceutical preparations.

Antibodies against Yariv's reagent for immunolocalization of arabinogalactan-proteins in aerial parts of Echinacea purpurea.

Arabinogalactan-proteins are glycoproteins that occur in higher plants and are involved in important processes like cell differentiation and plant growth. In the medicinal plant Echinacea purpurea L., they belong to the putative immunomodulating compounds and are structurally well characterized. For microscopic localization of arabinogalactan-proteins, synthetic (ß-D-Glc)3 Yariv phenylglycoside that specifically binds to most plant arabinogalactan-proteins was used to label arabinogalactan-proteins in fresh cut sections of stems and petioles of Echinacea purpurea. Polyclonal antibodies against (ß-D-Glc)3 Yariv phenylglycoside were used to detect the arabinogalactan-protein-(ß-D-Glc)3 Yariv phenylglycoside complex. After addition of fluorescein isothiocyanate-conjugated secondary antibodies, the sections were analyzed by confocal laser scanning microscopy. Arabinogalactan-proteins are localized mainly in the central cylinder in the collateral vascular bundles, especially in the area of the xylem. In cell walls of fully differentiated vessels and tracheids, arabinogalactan-proteins have been detected mainly at the inner area of the wall close to the cell lumina. Intense labeling occurs around pit canals connecting adjacent vessels. Furthermore, arabinogalactan-proteins are present in the lumina of cells of the sclerenchyma caps and in companion cells of the phloem.

Seasonal variations in the concentrations of lipophilic compounds and phenolic acids in the roots of Echinacea purpurea and Echinacea pallida.

Roots of Echinacea purpurea and Echinacea pallida cultivated for 4 years in a North European climate were analyzed for seasonal variations in the concentrations of lipophilic constituents (alkamides, ketoalkenes, and ketoalkynes) and phenolic acids by harvesting five times during 1 year to establish the optimal time for harvest. A total of 16 alkamides, three ketoalkenes, two ketoalkynes, and four phenolic acids (echinacoside, cichoric acid, caftaric acid, and chlorogenic acid) were identified in aqueous ethanolic (70%) extracts by liquid chromatography-mass spectrometry and quantified by reverse-phase high-performance liquid chromatography. The major alkamides in the roots of E. purpurea were at their lowest concentration in the middle of autumn and early winter, and the total concentration of lipophilic compounds in E. pallida showed the same pattern. Moreover, all of the major phenolic acids in E. purpurea were at their highest concentrations in spring. The optimal harvest time in spring is in contrast to normal growing guidelines; hence, this specific information of seasonal variations in the concentrations of lipophilic and phenolic compounds in E. purpurea and E. pallida is valuable for research, farmers, and producers of medicinal preparations.

[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.

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea.

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.

Carbon nanotube reinforced hollow fiber solid/liquid phase microextraction: a novel extraction technique for the measurement of caffeic acid in Echinacea purpurea herbal extracts combined with high-performance liquid chromatography.

A new design of hollow fiber solid-liquid phase microextraction (HF-SLPME) was developed for the determination of caffeic acid in medicinal plants samples as Echinacea purpure. The membrane extraction with sorbent interface used in this research is a three-phase supported liquid membrane consisting of an aqueous (donor phase), organic solvent/nano sorbent (membrane) and aqueous (acceptor phase) system operated in direct immersion sampling mode. The multi-walled carbon nanotube dispersed in the organic solvent is held in the pores of a porous membrane supported by capillary forces and sonification. It is in contact with two aqueous phases: the donor phase, which is the aqueous sample, and the acceptor phase, usually an aqueous buffer. All microextraction experiments were supported using an Accurel Q3/2 polypropylene hollow fiber membrane (600 microm I.D., 200 microm wall thicknesses, and 0.2 microm pore size). The experimental setup is very simple and highly affordable. The hollow fiber is disposable, so single use of the fiber reduces the risk of cross-contamination and carry-over problems. The proposed method allows the very effective and enriched recuperation of an acidic analyte into one single extract. In order to obtain high enrichment and extraction efficiency of the analyte using this novel technique, the main parameters were optimized. Under the optimized extraction conditions, the method showed good linearity (0.0001-50 microg/L), repeatability, low limits of detection (0.00005 microg/L) and excellent enrichment (EF=2108).

Hairy root cultures for secondary metabolites production.

Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes updated aspects of the hairy root induction, genetics and metabolite production.

Plant cell cultures: bioreactors for industrial production.

The recent biotechnology boom has triggered increased interest in plant cell cultures, since a number of firms and academic institutions investigated intensively to rise the production of very promising bioactive compounds. In alternative to wild collection or plant cultivation, the production of useful and valuable secondary metabolites in large bioreactors is an attractive proposal; it should contribute significantly to future attempts to preserve global biodiversity and alleviate associated ecological problems. The advantages of such processes include the controlled production according to demand and a reduced man work requirement. Plant cells have been grown in different shape bioreactors, however, there are a variety of problems to be solved before this technology can be adopted on a wide scale for the production of useful plant secondary metabolites. There are different factors affecting the culture growth and secondary metabolite production in bioreactors: the gaseous atmosphere, oxygen supply and CO2 exchange, pH, minerals, carbohydrates, growth regulators, the liquid medium rheology and cell density. Moreover agitation systems and sterilization conditions may negatively influence the whole process. Many types ofbioreactors have been successfully used for cultivating transformed root cultures, depending on both different aeration system and nutrient supply. Several examples of medicinal and aromatic plant cultures were here summarized for the scale up cultivation in bioreactors.

Metabolomic analysis of Echinacea spp. by 1H nuclear magnetic resonance spectrometry and multivariate data analysis technique.

INTRODUCTION: The genus Echinacea (Asteraceae) comprises about 10 species originally distributed in North America. Three species are very well known as they are used worldwide as medicinal plants: Echinacea purpurea, E. pallida, E. angustifolia. OBJECTIVE: To discriminate between these three Echinacea species and E. simulata by (1)H NMR-based metabolomics. METHODOLOGY: (1)H NMR and multivariate analysis techniques were applied to diverse Echinacea plants including roots and aerial parts, authentic plants, commercial plants and commercial dry extracts. RESULTS: Using the (1)H NMR metabolomics, it was possible, without previous evaporation or separation steps, to obtain a metabolic fingerprint to distinguish between species. CONCLUSION: A clear distinction between the three pharmaceutical species was possible and some useful metabolites were identified.

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.