Production Of Chlorogenic Acid Research Articles

In vitro production of chlorogenic acid in culture of transformed Cichorium intybus L.: Plants

Chicory (Cichorium intybus L.) is traditionally recognized for its antiinflammatory, antimicrobial, anticancer and nutritive properties. Among active secondary metabolites detected in chicory, the most important are sesquiterpene lactones and phenolics, including chlorogenic acid (CA). Hereby we have analyzed the content of CA in previously obtained Agrobacterium rhizogenes - transformed chicory hairy root cultures and transformed regenerants. Among three analyzed hairy root clones, clone 13 had exceptionally high biomass production, so the amount of CA in this culture was the highest, e.g. 400 times higher in comparison to the untransformed root culture. Since the roots spontaneously regenerated, the system was upgraded to allow the comparison of CA content not only among the clones, but also between different developmental phases of the regenerants (vegetative vs. flowering plants) and their organs (roots vs. leaves). It was shown that the CA production varies from clone to clone, and also depends on the phenophase of the clone. In the rosette stage of clones 13 and 36, the highest amount of CA was detected in roots, while the same clones in the flowering stage had significantly lower CA content. The clone 35 in the vegetative phase produced the lowest amounts of CA. However, in the flowering stage the roots of clone 35 produced the highest CA amount. Among the regenerants, the clone 13 had the fastest growth, and hence the best CA production. The current paper presents for the first time the CA content in transformed chicory regenerants. The obtained results suggest that the culture of transformed chicory plants can be equally good source of CA as liquid hairy root culture.

Production of hydroxycinnamoyl-shikimates and chlorogenic acid in Escherichia coli: production of hydroxycinnamic acid conjugates

BackgroundHydroxycinnamates (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.ResultsHydroxycinnamoyl 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.ConclusionsTo 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.

Changes in quality and phenolic antioxidants in dark purple American eggplant ( Solanum melongena L. cv. Lucía) as affected by storage at 0 °C and 10 °C

Many beneficial effects associated with fruit and vegetable consumption are related to the presence of antioxidants, which could be greatly affected by postharvest storage conditions. Eggplants or aubergines ( Solanum melongena L.) are among the top vegetables in terms of antioxidant content. In this work, we evaluated the effect of two postharvest temperature regimes on deterioration and antioxidants of dark purple American eggplants (cv. Lucía). Fruit were stored at 0 or 10 °C for 0, 3, 5, 10 or 14 d and weight loss, electrolyte leakage, chilling injury, and pulp browning were evaluated. We also followed DPPH and Folin–Ciocalteu reacting substances and the content of chlorogenic and quinic acid by HPLC. Although weight loss was reduced in fruit held at 0 °C, higher electrolyte leakage and chilling injury manifested as surface scalds and pulp browning were found. Antioxidants (AOX) measured with the DPPH radical and with the Folin–Ciocalteu reagent increased during the first 3 d of storage at 0 °C, but afterwards significant degradation was found. In contrast, a gradual but continuous accumulation of AOX was detected in fruit stored at 10 °C. The slow rate in the reaction between DPPH and eggplant samples suggested that the main changes during postharvest storage were due to modifications in phenolic compounds. The major phenolic detected by HPLC was chlorogenic acid (ChA), an ester between caffeic (CA) and quinic acids (QA), which accumulated in fruit maintained at 10 °C, increasing by 60% after 14 d of storage. No free CA was found at any storage temperature or time, suggesting that its biosynthesis is activated simultaneously with the production of ChA. Free QA showed minor changes at 0 °C as pulp lightness decreased, indicating that ChA rather than CA may be the main substrate for browning reactions. Changes in eggplant fruit antioxidants during storage at chilling and non-chilling temperatures are discussed.

Production of chlorogenic acid and isoorientin hypoglycemic compounds in Cecropia obtusifolia calli and in cell suspension cultures with nitrate deficiency

The antidiabetic properties of Cecropia obtusifolia are attributed to chlorogenic acid (CGA) and isoorientin (ISO) phenolic compounds; both compounds possess hypoglycemic, hypolipidemic, and antioxidant properties. As a potential strategy for an adequate supply of authentic plant raw material, the aim of this study was to establish in vitro conditions for the development of cell suspension cultures that produce these bioactive compounds. Callus cultures of leaf explants from acclimatized tree and in vitro plantlets were set up using different auxin levels; treatments with 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthalene acetic acid (NAA) to 8.92 µM with 6-benzylaminopurine (BAP) at 2.22 μM stimulate highest callus production. Seedling cotyledon, hypocotyl, leaf, and stem explants developed calli bearing roots with 2,4-D. With NAA, hypocotyl, cotyledon, and leaf explants developed morphogenic calli; 75% of stem explants formed calli, and the remaining calli developed shoots. Determined CGA concentrations in calli were similar to those detected in the leaves of wild trees, and ISO was not produced. Cell suspension cultures were established from leaf explants friable calli with 8.92 μM 2,4-D in combination with 2.22 μM BAP, employing 4 and 5% inocula in fresh weight; CGA levels were maintained and ISO was produced only at the end of logarithmic growth. On diminishing nitrate content in Murashige and Skoog (MS) medium to 8.0 mM, maximum cell biomasses diminished, CGA production is increased and twice with 16.0 and, instead of CGA production is tripled and quadrupled with 16.0 and 8.0 mM nitrates, respectively, and ISO synthesis was induced earlier and for a longer time period, increasing its levels at the end of culture. Two compounds with ultraviolet spectra similar to those of caffeic and ferulic acids were formed. Our results offer a protocol of cell suspension cultures for C. obtusifolia bioactive production and hypoglycemic property conservation.

Production of adventitious root biomass and secondary metabolites of Hypericum perforatum L. in a balloon type airlift reactor

The effects of inoculum density, aeration volume and culture period on accumulation of biomass and secondary metabolites in adventitious roots of Hypericum perforatum in balloon type airlift bioreactors (3l capacity) were investigated. The greatest increment of biomass as well as metabolite content occurred at an inoculum density of 3gl−1 and an aeration volume of 0.1vvm. After 6weeks of culture, an approximately 50-fold increase in biomass was recorded containing 60.11mgg−1 dry weight (DW) of phenolics, 42.7mgg−1 DW of flavonoids and 0.80mgg−1 DW of chlorogenic acid. Liquid chromatography–mass spectroscopy/mass spectroscopy demonstrated that the presence of quercetin and hyperoside in adventitious roots at a level of 1.33 and 14.01μgg−1 DW, respectively after 6weeks of culture. The results suggest scale-up of adventitious root culture of H. perforatum for the production of chlorogenic acid, quercetin and hyperoside is feasible.

Chlorogenic acid participates in the regulation of shoot, root and root hair development in Hypericum perforatum

Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, is one of the most widespread soluble phenolic compounds in the plant kingdom. Although CGA is known to have important roles in plant function, its relevance in plant de novo organogenesis is not yet understood. With a series of experiments, here we show that CGA has a potential role in shoot, root and root hair development. In the first phase of our investigation, we developed an efficient and novel thin cell layer (TCL) regeneration protocol for Hypericum perforatum which could bridge all the in vitro morphogenetic stages between single cell and complete plant. Tissues at different morphogenetic states were analysed for their phenolic profile which revealed that shoot differentiation from callus tissues of H. perforatum is accompanied by the onset of CGA production. Further, the relevance of CGA in de novo organogenesis was deciphered by culturing highly organogenic root explants on media augmented with various concentrations of CGA. Results of this experiment showed that CGA concentrations lower than 10.0 mg l⁻¹ did not affect shoot organogenesis, whereas, higher concentrations significantly reduced this process in a concentration-dependent manner. In spite of the differential concentration-dependent effects of CGA on shoot regeneration, supplementation of CGA did not have any effect on the production of lateral roots and root hairs. Interestingly, CGA showed a concentration-dependent positive correlation with lateral roots and root hairs production in the presence of α-naphthaleneacetic acid (NAA). When the culture medium was augmented with 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), induction of shoots, lateral roots and root hairs from the explants was significantly affected. Addition of an optimum concentration of CGA in these cultures partially restored all these organogenic processes.

Studies on a chlorogenic acid-producing endophytic fungi isolated from Eucommia ulmoides Oliver

Eucommia ulmoides Oliver is a traditional medicinal plant of China, and it is one of the main sources of chlorogenic acid. Chlorogenic acid is an ester of caffeic acid, quinic acid, and a phenolic compound that has antibacterial, antifungal, antioxidant, and antitumor activities. The purpose of this study was to determine whether endophytic fungi isolated from Eucommia ulmoides Oliver had the same ability to produce chlorogenic acid. Primary screening was done by antibacterial and antifungal reactions, and the strain reselection was done with high-performance liquid chromatography (HPLC) to identify the fermentation products of the selected strains. Extracts of the leaf and cortex of Eucommia ulmoides Oliver were also deteted by HPLC, then positive results of HPLC were analyzed by GC-MS and LC-MS. In this study, 29 strains were isolated from Eucommia ulmoides Oliver. Most of them had antibacterial activity, and a few of them had antifungal activity. One ingredient of the B5 extract had a retention time identical to that of authentic chlorogenic acid. With GC-MS, other ingredients, isocoumarin and p-chlorocinnamide, were found. With LC-MS, chlorogenic acid and geniposide related to Eucommia ulmoides Oliver were found. The strain B5 was identified as Sordariomycete sp. Thus, endophytic fungi may produce the bioactive compound chlorogenic acid, as their host plant does, and could be used for the production of chlorogenic acid by fermentation in the future.

Belowground ABA boosts aboveground production of DIMBOA and primes induction of chlorogenic acid in maize

Plants are important mediators between above- and belowground herbivores. Consequently, interactions between root and shoot defences can have far-reaching impacts on entire food webs. We recently reported that infestation of maize roots by the root feeding larvae of the beetle Diabrotica virgifera virgifera boosts shoot resistance against herbivores and pathogens. Root herbivory also induced DIMBOA levels and primed for enhanced induction of chlorogenic acid, two secondary metabolites that have been associated with biotic stress resistance. Interestingly, ABA emerged as a putative long-distance signal, possibly responsible for this effect. In this addendum, we investigate the role of root-derived ABA in the systemic regulation of aboveground DIMBOA, and the phenolic compounds chlorogenic acid, caffeic and ferulic acid. We discuss the relevance of the plant hormone in relation to defence against the leaf herbivore Spodoptera littoralis. Soil-drench treatment with ABA mimicked root herbivore-induced accumulation of DIMBOA in the leaves. Similarly, ABA mimicked aboveground priming of chlorogenic acid production, resulting in augmented accumulation of this compound upon subsequent shoot attack by S. littoralis. These findings confirm our notion that ABA acts as an important signal in the regulation of aboveground defence upon belowground herbivory. However, based on our previous finding that ABA alone is not sufficient to trigger aboveground resistance against S. littoralis caterpillars, the results suggest that the ABA-inducible effects on DIMBOA and chlorogenic acid are not solely responsible for root herbivore-induced resistance against S. littoralis.

The use of hairy root cultures to screen for bio-active principles in Cichorium intybus

Hairy roots, formed by plant cells after transformation with the soil bacterium Agrobacterium rhizogenes, have been found to be a suitable technology for the production of secondary metabolites. The aerial part of Cichorium intybus L. (Asteraceae) mainly contains derivatives of hydroxycinnamic acid, including chlorogenic acid and dicaffeoylquinic acids [1, 2]. These acids were identified in the roots by UPLCTM and showed radical-scavenging properties toward the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in a TLC autographic assay. Their biological effects are of interest for the pharmaceutical, food and cosmetic industries. For this reason hairy root culture of Cichorium intybus L. was developed. In order to increase the production of these compounds, different elicitors and stress factors like methyl jasmonate, UV light, Bion® and iron deficiency were used. We could show that methyl jasmonate at a concentration of 1000µM increases the production of chlorogenic acid and dicaffeoylquinic acid at least up to 400% of the control. Hairy root cultures optimised in this work showed a strong potential for the production of secondary metabolites, especially for chlorogenic and dicaffeoylquinic acid. They provide the advantages of a rapid growth and easy culture conditions; moreover, they give the possibility to enhance the synthesis of secondary metabolites by elicitors or stress factors.

가시오갈피 생물반응기 배양에서 질소급원이 세포생장과 이차대사 생산에 미치는 영향

The effects of inorganic nitrogen sources such as KNO<TEX>$_3$</TEX> and NH<TEX>$_4$</TEX> NO<TEX>$_3$</TEX> on cell growth and production of chlorogenic acid and eleutheroside E derivative were investigated in 5L bioreactor cultures of Eleutherococcus senticosus. The cell growth in the 1/2MS medium containing 15mMKNO<TEX>$_{3}$</TEX>. The fresh weight of cells harvested from bioreactor was affected by the concentration ratio of NO<TEX>$_3$</TEX><TEX>$^{[-10]}$</TEX> and NH<TEX>$_4$</TEX><TEX>$^{+}$</TEX> in culture medium. At the viewpoint of secondary metabolite production, the production of chlorogenic acid was affected by the concentration of NH<TEX>$_4$</TEX><TEX>$^{+}$</TEX> in the culture medium, but not by the total concentration of nitrogen sources in the culture medium. Futhermore, eleutheroside E derivative production was also affected by the concentration ratio of NO<TEX>$_3$</TEX><TEX>$^{[-10]}$</TEX> and NH<TEX>$_4$</TEX><TEX>$^{+}$</TEX> in the culture medium. Base on those results, it is suggested that cell growth and production of secondary metabolite(chlorogenic acid and eleutheroside E derivative) could be manipulated by controlling the total concentration of nitrogen sources and the concentration ratio of NO<TEX>$_3$</TEX><TEX>$^{[-10]}$</TEX> and NH<TEX>$_4$</TEX><TEX>$^{+}$</TEX> in the culture medium. medium.

Further studies relating chlorogenic acid concentration in carrots to carrot fly damage

SUMMARYThe effects of first generation carrot fly larval damage on chlorogenic acid concentration in carrots was investigated in a field experiment at Wellesbourne in 1985. In a separate experiment carrots grown in the absence of a resident population of carrot fly were also analysed for chlorogenic acid; these carrots maintained low concentrations of chlorogenic acid through the summer and autumn until low ground temperatures occurred from November to January. The relationship between chlorogenic acid concentration and damage by the first generation of carrot fly was described by a similar model to the one derived previously for late‐generation damage but without the cultivar dependence. This may have been because first generation damage takes place in mid‐summer when soil temperature is not sufficiently low for differential chlorogenic acid production by carrot cultivars. The model supports the hypothesis that carrot fly damage increases chlorogenic acid production which subsequently encourages further attack. The increase in acid production due to the low winter temperature may be the mechanism which, in turn, induced a differential cultivar response in carrots harvested during the winter.

Photocontrol of Growth, and of Anthocyanin and Chlorogenic Acid Production in Cultured Callus Tissues of Haplopappus gracilis

Photocontrol of Growth, and of Anthocyanin and Chlorogenic Acid Production in Cultured Callus Tissues of Haplopappus gracilis

Effects of Abscisic Acid on Phenolic Content and Lignin Biosynthesis in Tobacco Tissue Culture

AbstractA significant depression of callus growth resulted from low concentrations of abscisic acid (ABA) added to the medium recommended by Linsmaier and Skoog. Low concentrations also decreased the chlorogenic acid and lignin content of the callus, and generally decreased amounts of scopolin and scopoletin in the tissue. Gibberellic acid (GA3) stimulated callus growth in a low concentration (0.1 mg/1) and inhibited growth at a high concentration (10.0 mg/1). Both levels of GA3 increased scopoletin accumulation in tobacco callus. A high concentration of GA3 increased the accumulation of scopolin and chlorogenic acids, whereas a low concentration decreased the amounts of these two phenolic compounds. In comparison with the control, lignin synthesis was stimulated by a low GA3 concentration, but a high GA3 concentration did not have a significant effect. Both low and high concentrations of GA3 overcame ABA inhibition of growth and lignin synthesis, and partially reversed ABA inhibition of scopoletin production. However, GA3 did not reverse the inhibitory effect of ABA on scopolin production. The low concentration of GA3 overcame the inhibition of chlorogenic acid production resulting from a 0.01 mg/1 concentration of ABA, but this was the only reversal of chlorogenic acid inhibition resulting from addition of GA3 to the medium.