Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant.
A gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content.
The data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters.
The Asteraceae species Cynara cardunculus (2n = 2x = 34) includes the two fully cross-compatible domesticated taxa globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC). As both are out-pollinators and suffer from marked inbreeding depression, linkage analysis has focussed on the use of a two way pseudo-test cross approach.
A set of 172 microsatellite (SSR) loci derived from expressed sequence tag DNA sequence were integrated into the reference C. cardunculus genetic maps, based on segregation among the F1 progeny of a cross between a globe artichoke and a cultivated cardoon. The resulting maps each detected 17 major linkage groups, corresponding to the species’ haploid chromosome number. A consensus map based on 66 co-dominant shared loci (64 SSRs and two SNPs) assembled 694 loci, with a mean inter-marker spacing of 2.5 cM. When the maps were used to elucidate the pattern of inheritance of head production earliness, a key commercial trait, seven regions were shown to harbour relevant quantitative trait loci (QTL). Together, these QTL accounted for up to 74% of the overall phenotypic variance.
The newly developed consensus as well as the parental genetic maps can accelerate the process of tagging and eventually isolating the genes underlying earliness in both the domesticated C. cardunculus forms. The largest single effect mapped to the same linkage group in each parental maps, and explained about one half of the phenotypic variance, thus representing a good candidate for marker assisted selection.
Cynara cardunculus; Linkage map; Microsatellite; QTL; Earliness
Hydroxycinnamates (HCs) are mainly produced in plants. Caffeic acid (CA), p-coumaric acid (PA), ferulic acid (FA) and sinapic acid (SA) are members of the HC family. The consumption of HC by human might prevent cardiovascular disease and some types of cancer. The solubility of HCs is increased through thioester conjugation to various compounds such as quinic acid, shikimic acid, malic acid, anthranilic acid, and glycerol. Although hydroxycinnamate conjugates can be obtained from diverse plant sources such as coffee, tomato, potato, apple, and sweet potato, some parts of the world have limited availability to these compounds. Thus, there is growing interest in producing HC conjugates as nutraceutical supplements.
Hydroxycinnamoyl transferases (HCTs) including hydroxycinnamate-CoA shikimate transferase (HST) and hydroxycinnamate-CoA quinate transferase (HQT) were co-expressed with 4-coumarateCoA:ligase (4CL) in Escherichia coli cultured in media supplemented with HCs. Two hydroxycinnamoyl conjugates, p-coumaroyl shikimates and chlorogenic acid, were thereby synthesized. Total 29.1 mg/L of four different p-coumaroyl shikimates (3-p-coumaroyl shikimate, 4-p-coumaroyl shikimate, 3,4-di-p-coumaroyl shikimate, 3,5-di-p-coumaroyl shikimate, and 4,5-di-p-coumaroyl shikimate) was obtained and 16 mg/L of chlorogenic acid was synthesized in the wild type E. coli strain. To increase the concentration of endogenous acceptor substrates such as shikimate and quinate, the shikimate pathway in E. coli was engineered. A E. coli aroL and aroK gene were mutated and the resulting mutants were used for the production of p-coumaroyl shikimate. An E. coli aroD mutant was used for the production of chlorogenic acid. We also optimized the vector and cell concentration optimization.
To produce p-coumaroyl-shikimates and chlorogenic acid in E. coli, several E. coli mutants (an aroD mutant for chlorogenic acid production; an aroL, aroK, and aroKL mutant for p-coumaroyl-shikimates production) were made and each mutant was tested using an optimized construct. Using this strategy, we produced 235 mg/L of p-coumaroyl-shikimates and 450 mg/L of chlorogenic acid.
Chlorogenic acid; Hydroxycinnamic acid; Hydroxycinnamate-CoA quinate transferase; Hydroxycinnamate-CoA shikimate transferase
The history of domestication of artichoke and leafy cardoon is not yet fully understood and when and where it occurred remains unknown. Evidence supports the hypothesis that wild cardoon is the wild progenitor of both these crops. Selection for large, non-spiny heads resulted in artichoke and selection for non-spiny, large stalked tender leaves resulted in leafy cardoon. The two crops differ in their reproductive system: artichoke is mostly vegetatively propagated and perennial, while leafy cardoon is seed propagated and mostly grown as an annual plant. Here, new trends in artichoke cultivation are analysed, while the consequences of these tendencies on the conservation of artichoke genetic resources are highlighted.
The historical and artistic records, together with recent literature on genetics and biosystematics, are examined with the aim of achieving a better understanding of the present-day knowledge on the domestication of these two crops.
Historical, linguistic and artistic records are consistent with genetic and biosystematic data and indicate that the domestication of artichoke and cardoon diverged at different times and in different places. Apparently, artichoke was domesticated in Roman times, possibly in Sicily, and spread by the Arabs during early Middle Ages. The cardoon was probably domesticated in the western Mediterranean in a later period.
Cynara cardunculus; domestication; artichoke; cardoon; wild progenitor; genetic resources
The globe artichoke (Cynara cardunculus var. scolymus L.) is a significant crop in the Mediterranean basin. Despite its commercial importance and its both dietary and pharmaceutical value, knowledge of its genetics and genomics remains scant. Microsatellite markers have become a key tool in genetic and genomic analysis, and we have exploited recently acquired EST (expressed sequence tag) sequence data (Composite Genome Project - CGP) to develop an extensive set of microsatellite markers.
A unigene assembly was created from over 36,000 globe artichoke EST sequences, containing 6,621 contigs and 12,434 singletons. Over 12,000 of these unigenes were functionally assigned on the basis of homology with Arabidopsis thaliana reference proteins. A total of 4,219 perfect repeats, located within 3,308 unigenes was identified and the gene ontology (GO) analysis highlighted some GO term's enrichments among different classes of microsatellites with respect to their position. Sufficient flanking sequence was available to enable the design of primers to amplify 2,311 of these microsatellites, and a set of 300 was tested against a DNA panel derived from 28 C. cardunculus genotypes. Consistent amplification and polymorphism was obtained from 236 of these assays. Their polymorphic information content (PIC) ranged from 0.04 to 0.90 (mean 0.66). Between 176 and 198 of the assays were informative in at least one of the three available mapping populations.
EST-based microsatellites have provided a large set of de novo genetic markers, which show significant amounts of polymorphism both between and within the three taxa of C. cardunculus. They are thus well suited as assays for phylogenetic analysis, the construction of genetic maps, marker-assisted breeding, transcript mapping and other genomic applications in the species.
The globe artichoke (Cynara cardunculus L. var. scolymus) genome is relatively poorly explored, especially compared to those of the other major Asteraceae crops sunflower and lettuce. No SNP markers are in the public domain. We have combined the recently developed restriction-site associated DNA (RAD) approach with the Illumina DNA sequencing platform to effect the rapid and mass discovery of SNP markers for C. cardunculus.
RAD tags were sequenced from the genomic DNA of three C. cardunculus mapping population parents, generating 9.7 million reads, corresponding to ~1 Gbp of sequence. An assembly based on paired ends produced ~6.0 Mbp of genomic sequence, separated into ~19,000 contigs (mean length 312 bp), of which ~21% were fragments of putative coding sequence. The shared sequences allowed for the discovery of ~34,000 SNPs and nearly 800 indels, equivalent to a SNP frequency of 5.6 per 1,000 nt, and an indel frequency of 0.2 per 1,000 nt. A sample of heterozygous SNP loci was mapped by CAPS assays and this exercise provided validation of our mining criteria. The repetitive fraction of the genome had a high representation of retrotransposon sequence, followed by simple repeats, AT-low complexity regions and mobile DNA elements. The genomic k-mers distribution and CpG rate of C. cardunculus, compared with data derived from three whole genome-sequenced dicots species, provided a further evidence of the random representation of the C. cardunculus genome generated by RAD sampling.
The RAD tag sequencing approach is a cost-effective and rapid method to develop SNP markers in a highly heterozygous species. Our approach permitted to generate a large and robust SNP datasets by the adoption of optimized filtering criteria.
Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllusenabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli.
We first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4′-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3′,4′-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F.
We established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.
Avenanthramide; Tranilast; BAHD; Antioxidant; Anti-inflammatory; Tyrosine; Anthranilate; Hydroxycinnamate; Biological synthesis; Escherichia coli
Extract of globe artichoke (Cynara scolymus) is promoted as a possible preventive or cure for alcohol-induced hangover symptoms. However, few rigorous clinical trials have assessed the effects of artichoke extract, and none has examined the effects in relation to hangovers. We undertook this study to test whether artichoke extract is effective in preventing the signs and symptoms of alcohol-induced hangover.
We recruited healthy adult volunteers between 18 and 65 years of age to participate in a randomized double-blind crossover trial. Participants received either 3 capsules of commercially available standardized artichoke extract or indistinguishable, inert placebo capsules immediately before and after alcohol exposure. After a 1-week washout period the volunteers received the opposite treatment. Participants predefined the type and amount of alcoholic beverage that would give them a hangover and ate the same meal before commencing alcohol consumption on the 2 study days. The primary outcome measure was the difference in hangover severity scores between the artichoke extract and placebo interventions. Secondary outcome measures were differences between the interventions in scores using a mood profile questionnaire and cognitive performance tests administered 1 hour before and 10 hours after alcohol exposure.
Fifteen volunteers participated in the study. The mean number (and standard deviation) of alcohol units (each unit being 7.9 g, or 10 mL, of ethanol) consumed during treatment with artichoke extract and placebo was 10.7 (3.1) and 10.5 (2.4) respectively, equivalent to 1.2 (0.3) and 1.2 (0.2) g of alcohol per kilogram body weight. The volume of nonalcoholic drink consumed and the duration of sleep were similar during the artichoke extract and placebo interventions. None of the outcome measures differed significantly between interventions. Adverse events were rare and were mild and transient.
Our results suggest that artichoke extract is not effective in preventing the signs and symptoms of alcohol-induced hangover. Larger studies are required to confirm these findings.
Technical specifications of solid biofuels are continuously improved towards the development and promotion of their market. Efforts in the Greek market are limited, mainly due to the climate particularity of the region, which hinders the growth of suitable biofuels. Taking also into account the increased oil prices and the high inputs required to grow most annual crops in Greece, cardoon (Cynara cardunculus L.) is now considered the most important and promising sources for solid biofuel production in Greece in the immediate future. The reason is that cardoon is a perennial crop of Mediterranean origin, well adapted to the xerothermic conditions of southern Europe, which can be utilized particularly for solid biofuel production. This is due to its minimum production cost, as this perennial weed may perform high biomass productivity on most soils with modest or without any inputs of irrigation and agrochemicals. Within this framework, the present research work is focused on the planning and analysis of different land use scenarios involving this specific energy crop and the combustion behaviour characterization for the solid products. Such land use scenarios are based on quantitative estimates of the crop'sproduction potential under specific soil-climatic conditions as well as the inputs required for its realization in comparison to existing conventional crops. Concerning its decomposition behaviour, devolatilisation and char combustion tests were performed in a non-isothermal thermogravimetric analyser (TA Q600). A kinetic analysis was applied and accrued results were compared with data already available for other lignocellulosic materials. The thermogravimetric analysis showed that the decomposition process of cardoon follows the degradation of other lignocellulosic fuels, meeting high burnout rates. This research work concludes that Cynara cardunculus, under certain circumstances, can be used as a solid biofuel of acceptable quality.
energy crops; thermogravimetry; devolatilization; combustion; cynara cardunculus
The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.
Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Kmr genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.
Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG), the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean.
We studied the inhibition of DNA methyltransferase 3a by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC50 values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance.
Since Dnnmt3a has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols.
Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.
No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots.
Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1.
The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.
Actions of chlorogenic acid, a major component of coffee, andits constituents, caffeic and quinic acids, on theproliferation and invasion of AH109A, a rat ascites hepatomacell line, were investigated using in vitro assay systems. Allthree components suppressed the AH109A invasion atconcentrations of 5–40 μM without altering the cellproliferation. At the concentration of 10 μM, chlorogenic,caffeic and quinic acids significantly (P < 0.05) suppressedthe invasion by 68%, 36% and 31%, respectively, implying thatthe suppressive effect of chlorogenic acid on the AH109Ainvasion might result from the additive effects of itsconstituents, caffeic and quinic acids. At the concentrationof 10 μM, cinnamic acid and p-coumaric acid (4-hydroxycinnamicacid) exerted no or little influence on the invasion, whereascaffeic acid (3,4-dihydroxycinnamic acid) significantly (P <0.05) suppressed it, suggesting the possible involvement ofthe 3,4-dihydroxy group of caffeic acid in the suppression.Chlorogenic acid was thus demonstrated to be one of thechemical entities in coffee suppressing the hepatoma invasionin vitro, and both of its constituents, caffeic and quinicacids, to be responsible for the anti-invasive activity. Theseresults suggest the existence of nutritionally andpharmacologically important substances in coffee which controltumor cell invasion.
hepatoma; invasion; chlorogenic acid; caffeic acid; quinic acid; coffee
The inhibition of growth by octanoic or decanoic acids, two subproducts of ethanolic fermentation, was evaluated in Saccharomyces cerevisiae and Kluyveromyces marxianus in association with ethanol, the main product of fermentation. In both strains, octanoic and decanoic acids, at concentrations up to 16 and 8 mg/liter, respectively, decreased the maximum specific growth rate and the biomass yield at 30°C as an exponential function of the fatty acid concentration and increased the duration of growth latency. These toxic effects increased with a decrease in pH in the range of 5.4 to 3.0, indicating that the undissociated form is the toxic molecule. Decanoic acid was more toxic than octanoic acid. The concentrations of octanoic and decanoic acids were determined during the ethanolic fermentation (30°C) of two laboratory media (mineral and complex) by S. cerevisiae and of Jerusalem artichoke juice by K. marxianus. Based on the concentrations detected (0.7 to 23 mg/liter) and the kinetics of growth inhibition, the presence of octanoic and decanoic acids cannot be ignored in the evaluation of the overall inhibition of ethanolic fermentation.
In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N′,N′′-dicoumaroylspermidine, N′,N′′-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense.
The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μmax), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h−1, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μmax results and the rates of cell and ethanol production, and the appropriate constants were evaluated.
The objective of this study is to evaluate the lowering of uric acid using Balanophora laxiflora extracts and derived phytochemicals on potassium-oxonate-(PO-) induced hyperuricemia in mice. The results revealed that ethyl acetate (EtOAc) fraction of B. laxiflora extracts exhibited strong xanthine-oxidase-(XOD-) inhibitory activity. In addition, among the 10 subfractions (EA1–10) derived from EtOAc fraction, subfraction 8 (EA8) exhibited the best XOD-inhibitory activity. Four specific phytochemicals, 1-O-(E)-caffeoyl-β-D-glucopyranose (1), 1-O-(E)-p-coumaroyl-β-D-glucopyranose (2), 1,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (3), and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (4), were further isolated and identified from this subfraction. Compounds 3 and 4 exhibited the strongest XOD-inhibitory activity compared with other compounds, and both hydrolyzable tannins were determined to be noncompetitive inhibitors according to the Lineweaver-Burk plot. On the other hand, the in vivo hypouricemic effect in hyperuricemic mice was consistent with XOD-inhibitory activity, indicating that B. laxiflora extracts and derived phytochemicals could be potential candidates as new hypouricemic agents.
Active components of complementary/alternative medicines and natural supplements are often anionic compounds and flavonoids. As such, organic anion transporters (OATs) may play a key role in their pharmacokinetic and pharmacological profiles, and represent sites for adverse drug-drug interactions. Therefore, we assessed the inhibitory effects of nine natural products, including flavonoids (catechin and epicatechin), chlorogenic acids (1,3- and 1,5-dicaffeoylquinic acid), phenolic acids (ginkgolic acids (13 : 0), (15 : 1), and (17 : 1)), and the organic acids ursolic acid and 18β-glycyrrhetinic acid, on the transport activity of the human OATs, hOAT1 (SLC22A6), hOAT3 (SLC22A8), and hOAT4 (SLC22A11). Four compounds, 1,3- and 1,5-dicaffeoylquinic acid, ginkgolic acid (17 : 1), and 18β-glycyrrhetinic acid, significantly inhibited hOAT1-mediated transport (50 μM inhibitor versus 1 μM substrate). Five compounds, 1,3- and 1,5-dicaffeoylquinic acid, ginkgolic acids (15 : 1) and (17 : 1), and epicatechin, significantly inhibited hOAT3 transport under similar conditions. Only catechin inhibited hOAT4. Dose-dependency studies were conducted for 1,3-dicaffeoylquinic acid and 18β-glycyrrhetinic acid on hOAT1, and IC50 values were estimated as 1.2 ± 0.4 μM and 2.7 ± 0.2 μM, respectively. These data suggest that 1,3-dicaffeoylquinic acid and 18β-glycyrrhetinic acid may cause significant hOAT1-mediated DDIs in vivo; potential should be considered for safety issues during use and in future drug development.
Gynura medica leaf extract contains significant amounts of flavonols and phenolic acids and exhibits powerful hypoglycemic activity against diabetic rats in vivo. However, the hypoglycemic active constituents that exist in the plant have not been fully elaborated. The purpose of this study is to isolate and elaborate the hypoglycemic activity compounds against inhibition the yeast α-glucosidase in vitro. Seven phenolic compounds including five flavonols and two phenolic acids were isolated from the leaf of G. medica. Their structures were identified by the extensive NMR and mass spectral analyses as: kaempferol (1), quercetin (2), kaempferol-3-O-β-D-glucopyranoside (3), kaempferol-3-O-rutinoside (4), rutin (5), chlorogenic acid (6) and 3,5-dicaffeoylquinic acid methyl ester (7). All of the compounds except 1 and 3 were isolated for the first time from G. medica. Compounds 1–7 were also assayed for their hypoglycemic activity against yeast α-glucosidase in vitro. All of the compounds except 1 and 6 showed good yeast α-glucosidase inhibitory activity with the IC50 values of 1.67 mg/mL, 1.46 mg/mL, 0.38 mg/mL, 0.10 mg/mL and 0.53 mg/mL, respectively.
Gynura medica; yeast α-glucosidase; flavonols; phenolic acids
Rhagadiolus stellatus Gaertn., a Mediterranean member of the Cichorieae tribe of the Asteraceae family used as a food plant, was analyzed for its spectrum of phenolic compounds. Kaempferol 3-O-β-glucoside 1, kaempferol 3-O-β-rutinoside (nicotiflorin) 2, quercetin 3-O-β-glucoside 3, and luteolin 4 were isolated from the n-butanol layer of a methanolic extract of whole plants of Rh. stellatus of Spanish origin by repeated Sephadex LH-20 column chromatography. Structures were determined based on NMR and MS data as well as by comparison with literature data. Additionally, chlorogenic acid 5 and 3,5-dicaffeoylquinic acid 6 were detected by HPLC/DAD and HPLC/MS. Chemosystematic implications of the presented findings are discussed in comparison with other members of the Cichorieae tribe.
Rhagadiolus stellatus Gaertn.; Asteraceae; Cichorieae; Flavonoids; Chemosystematics
Chlorogenic acid, a pharmacologically important compound, is a phenolic compound that occurs in certain commonly used medicinal herbs. We looked for the presence of this compound in the callus cultures of Varthemia persica DC (var. persica). We have evaluated the conditions for establishment of callus cultures of V. persica and the in vitro production of chlorogenic acid. Callus was initiated by culturing seedling of V. persica on MS basal medium supplemented with different concentrations of kinetin, naphthalene acetic acid and 2,4-diphenoxy acetic acid. Also, the influence of light, and phytohormones on the production of chlorogenic acid was examined. Kinetin stimulated the production of chlorogenic acid. Replacement of 2,4-diphenoxy acetic acid with naphthalene acetic acid did not alter the chlorogenic acid production. The ability to induce the accumulation of chlorogenic acid in the V. persica callus cultures offers an opportunity to produce a phenolic compound with therapeutic value.
Chlorogenic acid; Varthemia persica; Callus culture
Reducing the use of pesticides represents a major challenge of modern agriculture. Plants synthesize secondary metabolites such as polyphenols that participate in the resistance to parasites. The aim of this study was to test: (1) the impact of nitrogen deficiency on tomato (Solanum lycopersicum) leaf composition and more particularly on two phenolic molecules (chlorogenic acid and rutin) as well as on the general plant biomass; and (2) whether this effect continued after a return to normal nitrogen nutrition. Our results showed that plants deprived of nitrogen for 10 or 19 days contained higher levels of chlorogenic acid and rutin than control plants. In addition, this difference persisted when the plants were once again cultivated on a nitrogen-rich medium. These findings offer interesting perspectives on the use of a short period of deprivation to modulate the levels of compounds of interest in a plant.
chlorogenic acid; rutin; nitrogen deprivation; phenolic compound; tomato
The successful use of herbal combinations in managing diseases or conditions over a single herb has lead us to evaluate the anti-dyslipidemic properties of the combination of the artichoke leaves extract, turmeric extract, prickly pear dried leaves (PPL) and garlic extract versus each one alone in two different hyperlipidemic animal models. A two-week treatment of each of the natural extracts, combination 1 (artichoke, turmeric and PPL) or combination 2 (artichoke, turmeric, PPL and garlic) prior to a single intraperitoneal injection of Pluronic F-127 resulted in decreasing significantly serum LDL levels by garlic and PPL extracts and serum LDL/HDL ratios by turmeric, PPL, combination 1 and 2. In a 10-day high fat diet model, only the combination 1 and 2 lowered serum cholesterol, LDL by 8–12%, decreased significantly triglycerides, LDL/HDL ratio; and increased significantly HDL (P < 0.0001). However, a long term treatment of each natural product for 7 weeks resulted in decreasing significantly serum LDL levels and LDL/HDL ratio (P < 0.05–0.0001). Furthermore, only artichoke and PPL inhibited significantly HMG-CoA reductase activity (P < 0.05). In conclusion, short term, as well as long term, treatment using the combination of artichoke, turmeric, PPL and garlic extract prevents dyslipidemia; partially through inhibiting HMG-CoA reductase.
The ability of potentially probiotic strains of Lactobacillus plantarum and Lactobacillus paracasei to survive on artichokes for at least 90 days was shown. The anchorage of bacterial strains to artichokes improved their survival in simulated gastrointestinal digestion. L. paracasei IMPC2.1 was further used in an artichoke human feeding study involving four volunteers, and it was shown that the organism could be recovered from stools.