Production Of Chlorogenic Acid Research Articles

Analysis of the influence of technological parameters of the chlorogenic acid extraction process from sunflower meal on the degree of its extraction

The object of research is the efficiency of extraction of chlorogenic acid ‑ the natural antioxidant, depending on the conditions of its extraction from sunflower meal. The main problem of this issue is the fact that the extraction degree of the specified phenolic compound is influenced by many factors, such as the degree of grinding of the product, the type of raw material, the method of extraction, the nature of the solvent-extractant, the temperature and duration of the extraction process, the hydromodule in the system «raw – extractant» etc. The impact of each of these parameters requires careful consideration and appropriate research. This will determine the optimal values of the specified parameters of the extraction process and increase the efficiency of extraction of chlorogenic acid.In this work, the raw material for the production of chlorogenic acid is a meal made from sunflower seeds – a cheap second raw material of oil and fat production. Previous studies have found that a high-efficiency extractant of the specified antioxidant is a solution of ethyl alcohol with a concentration of 60 %, and the optimum temperature of the process of extracting chlorogenic acid from sunflower meal is the boiling point of the extractant. As a result of this study, the regularity of the influence on the extraction degree of chlorogenic acid of such technological parameters as the hydromodule in the system «meal ‑ ethyl alcohol solution 60 %» (hereinafter referred to as «meal – extractant») and the duration of the extraction process were studied. Experiments to determine the dependence of the extraction degree of chlorogenic acid on the hydromodule in the system «meal – extractant» and the duration of extraction were carried out in accordance with the plan of the full factor experiment. Mathematical methods using the Microsoft Office Excel 2003 (USA) and Stat Soft Statistica v6.0 (USA) software packages have been applied for experiment planning and data processing.The dependence indicated in the paper is a quadratic function that predicts an increase in chlorogenic acid content during extraction, with an increase in the hydromodule in the «meal‑extractant» system from 1:5 to 1:10 and a decrease in the extraction time from 60 minutes up to 30 minutes. It is established that for the maximum possible increase in the extraction degree of chlorogenic acid from the meal of sunflower seeds, the optimal value of the hydromodule extraction ‑ 1:10, the duration of extraction ‑ 30 minutes. The obtained results allow to increase the extraction degree of chlorogenic acid from sunflower meal from 2.46 % to 5.58 %. This indicates the possibility of increasing the efficiency of extraction of antioxidant more than 2 times.

Enhancement of chlorogenic content of the eggplant fruit with eggplant hydroxycinnamoyl CoA-quinate transferase gene via novel agroinfiltration protocol

Background: Eggplant (Solanum melongena L.) is rich in health-promoting phenolic acids, primarily in the chlorogenic acid. For the production of chlorogenic acid in the eggplant hydroxycinnamoyl CoA-quinate transferase (SmHQT), is a central enzyme that catalyzes the reaction to the chlorogenic acid production. Objective: The function of SmHQT is not well determined in the eggplant fruit, and the fruit agroinfiltration procedure is not standardized for eggplant. Materials and Methods: Here, the overexpression of SmHQT in the eggplant fruit´s flesh was studied using the agroinfiltration technique. In our gene construct, we also co-expressed the P19 protein for overexpression, and the results were validated with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography (HPLC). Results: Due to the overexpression of the SmHQT gene, higher chlorogenic content was exhibited by the eggplant fruits, which was further validated by HPLC. The chlorogenic acid content after following the agroinfiltration procedure was more two times in the agroinfiltrated fruit. To identify the optimal target for increasing chlorogenic pathway flux post-SmHQT activity, expression patterns were analyzed with qRT-PCR, and the results showed the changes in the expression level of the other chlorogenic acid pathway genes. Furthermore, the cis regulating elements and protein-protein interaction (PPI) analyses supported the HPLC results. Conclusion: Overall, here, insights into the eggplant chlorogenic content increment at the molecular level and the opportunities for the improvement of chlorogenic content as nutrition in crops are provided.

Influence of exogenous cinnamic acid on the production of chlorogenic acid in Cichorium intybus L cell culture

Cichorium intybusL(C. intybus) is a source for several important secondary metabolites (SM), including 3-O-caffeoylquinic acid (CGA). Isolation of SM from field plants is difficult due to their batch to batch variation in response to various environmental factors. The study aims to circumvent this problem in C. intybus by increasing CGA concentration through optimization of media conditions and elicitors using plant cell culture. An increased callus growth was observed in leaf explant inoculated on MS solid medium supplemented with 2 mg/l 1-napthaleneacetic acid (NAA), 2 mg/l kinetin (Kn), 30 g/l sucrose and 0.7% agar. Optimum callus growth (13 g/l) was obtained with 5% callus (w/v) sub-cultured in MS suspension medium containing 2 mg/l NAA, 2 mg/l Kn, and 50 g/l glucose at pH 5.5. After optimization of culture conditions, elicitors (cinnamic acid, jasmonic acid, chitosan and methyl jasmonate) were added towards the end of log phase to increase CGA content in callus. Among the various concentrations of elicitors, 0.5 mg/l cinnamic acid was found to reduce cell death and increase phenol content in callus as well as the culture media. These results correlated with the TLC-densitometry study, which showed cinnamic acid (0.5 mg/l) treatment to increase CGA content in callus by 0.39- fold in comparison to control callus. The elicitation was confirmed when 3T3-L1 adipocytes treated with 1 ng/ml methanol extract of callus exposed to 0.5 mg/l cinnamic acid showed a significant 0.3-fold increase in glucose uptake compared to 1 ng/ml control callus and field plant methanol extracts. The elicited callus showed a decrease in the levels of hydrogen peroxide, nitric oxide, and phenylalanine ammonia lyase activity, demonstrating the role of cinnamic acid as a precursor for CGA synthesis.

Chlorogenic acid-mediated chemical defence of plants against insect herbivores.

Chlorogenic acid is one of the most abundant beneficial polyphenols in plants and is well known as a nutritional antioxidant in plant-based foods. Apart from its dietary antioxidant activity, it has been proved to be an efficient defence molecule against a broad range of insect herbivores. In the last two decades, several reports have shown the effectiveness of chlorogenic acid in insect growth deterrence. The pathway for chlorogenic acid biosynthesis in plants was previously elucidated, and metabolic engineering of the principal pathway showed high chlorogenic acid production in tomato plants. Herbivore-mediated induction of chlorogenic acid biosynthesis was also demonstrated both at metabolite and transcript level, although herbivore-mediated molecular regulation of chlorogenic acid biosynthesis is not yet fully elucidated. In this communication, we present our views on the efficacy of chlorogenic acid as an anti-herbivore defence molecule in plants and also discuss its future outlook.

Identification and Quantification of Allelochemicals from Selected Sweet Potato (<i>Ipomoea batatas</i> (L.) Lam.) Cultivars

Allelopathic compounds have the potential to inhibit the growth and development of other organisms in a diverse manner ranging from shifting nutrients and enhancing their growth to inflicting diseases. In addition, these compounds influence seedling growth and seed germination of various crops. The goal of this study was to identify and quantify different allelochemicals in various sweet potato cultivars through high-performance liquid chromatography techniques. Selected sweet potato slips (weight: 2.0 - 2.5 grams/slip) were propagated in separate glass tubes filled with 10.0 mL distilled water. Water extract from each glass tube was collected after 2, 4, and 6 weeks after transplanting (WAP) to identify and quantify allelochemical compounds by comparing their peaks with the retention time of standards. Results show that the concentration of allelochemicals in water extract was increased from 2 to 4 WAP but remained constant in the sixth week. Quantitative analysis revealed that the amount of chlorogenic acid was higher in all sweet potato cultivars compared to other allelochemicals. Some sweet potato cultivars, A5 and A39, exhibited higher allelopathy (18.28 - 19.37 ppm/slip) and reduced the height and biomass of Palmer amaranth the most due to the presence of increased concentration of combined allelochemicals, while other cultivars produced lesser allelochemicals (10.90 ppm/slip) and did not reduce the growth of the weed species. Allelopathic sweet potato cultivars high in chlorogenic acid production can effectively suppress Palmer amaranth with minimal dependence on chemicals to manage weeds and harmful pests under sustainable agricultural system.

Molecular Identification and Characterization of Hydroxycinnamoyl Transferase in Tea Plants (Camellia sinensis L.).

Tea (Camellia sinensis L.) contains abundant secondary metabolites, which are regulated by numerous enzymes. Hydroxycinnamoyl transferase (HCT) is involved in the biosynthesis pathways of polyphenols and flavonoids, and it can catalyze the transfer of hydroxyconnamoyl coenzyme A to substrates such as quinate, flavanol glycoside, or anthocyanins, thus resulting in the production of chlorogenic acid or acylated flavonol glycoside. In this study, the CsHCT gene was cloned from the Chin-Shin Oolong tea plant, and its protein functions and characteristics were analyzed. The full-length cDNA of CsHCT contains 1311 base pairs and encodes 436 amino acid sequences. Amino acid sequence was highly conserved with other HCTs from Arabidopsis thaliana, Populus trichocarpa, Hibiscus cannabinus, and Coffea canephora. Quantitative real-time polymerase chain reaction analysis showed that CsHCT is highly expressed in the stem tissues of both tea plants and seedlings. The CsHCT expression level was relatively high at high altitudes. The abiotic stress experiment suggested that low temperature, drought, and high salinity induced CsHCT transcription. Furthermore, the results of hormone treatments indicated that abscisic acid (ABA) induced a considerable increase in the CsHCT expression level. This may be attributed to CsHCT involvement in abiotic stress and ABA signaling pathways.

Chlorogenic acid, anthocyanin and flavan-3-ol biosynthesis in flesh and skin of Andean potato tubers (Solanum tuberosum subsp. andigena).

Natural variation of Andean potato was used to study the biosynthesis of phenolic compounds. Levels of phenolic compounds and corresponding structural gene transcripts were examined in flesh and skin of tubers. Phenolic acids, mainly chlorogenic acid (CGA), represent the major compounds, followed by anthocyanins and flavan-3-ols. High-anthocyanin varieties have high levels of CGA. Both metabolite and transcript levels were higher in skin than in flesh and showed a good correspondence. Two hydroxycinnamoyl-CoA transferases (HCT/HQT) have been involved in CGA production, of which HCT reflects CGA levels. Catechin was found in pigmented tissues whereas epicatechin was restricted to tuber skin. Transcripts of leucoanthocyanidin reductase (LCR), which generates catechin, could not be detected. Anthocyanidin reductase (ANR) transcripts, the enzyme responsible for epicatechin production, showed similar levels among samples. These data suggest that the biosynthesis of flavan-3-ols in potato tuber would require ANR but not LCR and that an epimerization process is involved.

Responses of Crepis japonica induced by supplemental blue light and UV-A radiation

Crepis japonica (L.) D.C. (Asteraceae), a weed with antioxidant, antiallergenic, antiviral and antitumor properties displays both medicinal properties and nutritional value. This study aims to assess the effects of a supplementation of blue light and UV-A radiation on the growth, leaf anatomical structure and phenolic profile of the aerial parts of Crepis japonica. Plants were grown under two light treatments: W (control - white light), W + B (white light supplemented with blue light) and W + UV-A (white light supplemented with UV-A radiation). We recorded the length, width, and weight of fresh and dry leaves, the thickness of the epidermis and mesophyll, and stomata density. The phenolic profiles of the aqueous extracts of the aerial parts were analyzed by HPLC-DAD. There was an increase in the leaf size, stomatal density, and phenolic production, and a thickening of the mesophyll and epidermis. UV-A radiation increased the phenolic production more than blue light. Blue light and UV-A radiation both improved the production of caffeic acid by about 6 and 3 times, respectively, in comparison to control. This compound was first reported as a constituent of the extract from the aerial parts together with caftaric acid. UV-A also promoted the production of chlorogenic acid (about 1.5 times in comparison to the control). We observed that the morphological and chemical parameters of C. japonica are modified in response to blue light and UV-A radiation, which can be used as tools in the cultivation of this species in order to improve its medicinal properties and nutritional value.

Transcriptional regulation of chlorogenic acid biosynthesis in carrot root slices exposed to UV-B light

Orange carrots are well known for their nutritional value as producers of β-carotene, a vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, flavonols and isoflavonoids, the phenylpropanoid pathway. This pathway can be induced by UV-B light and wounding to produce large amounts of chlorogenic acid. We identified a number of the carrot structural genes in this pathway and their proposed regulators in carrot root slices and analyzed their expression in response to UV-B light exposure using real-time PCR. Our results indicate that not only does the expression of the structural genes correlate with their proposed regulators, including DcHY5 and others, but also correlate with chlorogenic acid production. Our results indicate that the HY5 — UVR8 signaling network may exist and function in carrot roots.

Improved production of chlorogenic acid from cell suspension cultures of <i>Lonicera macranthoids</i>

Purpose: To evaluate the potential of Lonicera macranthoides Hand. -Mazz. Yulei1 suspension culture system for enhanced production of the main secondary metabolite, chlorogenic acid.Methods: The callus of L. macranthoides Hand.-Mazz. “Yulei1” was suspension cultured in B5 liquid medium supplemented with different plant growth regulators. Biomass accumulation was calculated by weight method and chlorogenic acid production was measured using high performance liquid chromatography (HPLC). HPLC was carried out on C18 analytical column at 35 °C and the detection wavelength was set at 324 nm.Results: The results showed that maximum accumulation of biomass and chlorogenic acid were achieved 15 days after culture growth. The optimized conditions for biomass accumulation and chlorogenic acid production were 50 g/L of inoculum on fresh weight basis, B5 medium supplemented with plant growth regulators, 30 - 40 g/L sucrose and initial medium pH of 5.5. Maximum accumulation of chlorogenic acid and biomass were observed when the culture medium was supplemented with 2.0 mg/L6-BA. Optimal accumulation of chlorogenic acid was observed using combination of hormones 2.0 mg/L 6-Benzyladenine (BA) + 0.5 mg/L2, 4-Dichlorophenoxyacetic acid (2,4-D), while optimal accumulation of biomass was observed with 2.0 mg/L 6-BA + 2.0 mg/L2, 4-D. In addition, phenylalanine also contributed to the synthesis of chlorogenic acid at a concentration > 50 mg/L.Conclusion: Cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei1” have successfully been established. The findings provide a potential basis for large scale production of chlorogenic acid using cell suspension cultures of L. macranthoides.Keywords: Lonicera macranthoides, Cell suspension culture, Chlorogenic acid, Phenylalanine, Optimization

Influence of LED light spectra on in vitro somatic embryogenesis and LC-MS analysis of chlorogenic acid and rutin in Peucedanum japonicum Thunb.: a medicinal herb.

Background Peucedanum japonicum Thunb, an important medicinal herb is reported to possess pharmacological properties such as anti-obesity, anti-oxidant, anti-inflammatory, anti-bacterial, anti-diabetic and anti-platelet aggregation. The present study aimed to develop an in vitro plant regeneration system of P. japonicum via somatic embryogenesis and to analyse chlorogenic acid and rutin contents in a few commercially available plant products of P. japonicum in Japan and Taiwan markets, and tissue culture plants derived from somatic embryos.ResultsInduction of somatic embryogenesis could be achieved when root derived calli after three subcultures were transferred from Murashige Skoog’s salts and vitamins (MS basal) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1–5 mg/L) to a medium with abscisic acid (ABA) (0.5–4 mg/L), or exposed to eight different light spectra provided by light-emitting diode (LED) sources. Concentrations of ABA and LED light spectra had an influence on number of somatic embryos induced and proliferation of callus. Development of secondary somatic embryos and conversion of embryos to plantlets was achieved on a medium with ABA, or their exposure to red or blue lights in a special incubation chamber. Four months old tissue culture plants derived from somatic embryos showed significantly higher levels of chlorogenic acid (10.5 mg/g dw) compared to commercial product sold in Japanese market (0.55 mg/g dw). However, rutin was absent in tissue culture plants in contrast to commercial sample (0.33 mg/g dw).ConclusionIn this report, we describe in vitro plant regeneration system in P. japonicum via somatic embryogenesis and production of chlorogenic acid in tissue culture plants. The present study has application in further tissue culture propagation of elite plant material with high chlorogenic acid content, and identification of high yielding plants with the LC–MS method.

Insecticidal potential of an endophytic Cladosporium velox against Spodoptera litura mediated through inhibition of alpha glycosidases

Alpha glucosidase inhibitory activity was exhibited by partially purified fractions obtained from an endophytic Cladosporium velox, isolated from Tinospora cordifolia. Taking into account the increasing importance of digestive enzyme inhibitors as insecticidal agents, the entomopathogenic potential of the fractions obtained was evaluated against Spodoptera litura (Fab.), a polyphagous pest. Considerable mortality was obtained when the larvae were fed on diet supplemented with the partially purified extract. All the concentrations of the extract significantly prolonged the overall developmental period of S. litura. At higher concentrations, the extract influenced the longevity of females as well as their reproductive potential. Phytochemical analysis revealed the presence of phenolic compounds in the active fraction. The phenolic compound responsible for the bioactivities was purified and identified to be chlorogenic acid using HPLC and MS analysis. The content of chlorogenic acid in the extract was quantified to be 250μg/ml. The purified compound also demonstrated inhibition of alpha glycosidases in vivo. The present study indicates that the endophyte imparted resistance to the insects in the plants could be mediated through chlorogenic acid targeting the alpha glycosidases present in the gut of the insect. The isolate obtained can be exploited for the production of chlorogenic acid, which has the potential to be exploited as a biocontrol agent against S. litura.

The role of silicon in physiology of the medicinal plant (Lonicera japonica L.) under salt stress.

Silicon(Si) is the only element which can enhance the resistance to multiple stresses. However, the role of silicon in medicinal plants under salt stress is not yet understood. This experiment was conducted to study the effects of silicon addition on the growth, osmotic adjustments, photosynthetic characteristics, chloroplast ultrastructure and Chlorogenic acid (CGA) production of Honeysuckle plant (Lonicera japonica L.) under salt-stressed conditions. Salinity exerted an adverse effect on the plant fresh weight and dry weight, whilst 0.5 g L−1 K2SiO3·nH2O addition obviously improved the plant growth. Although Na+ concentration in plant organs was drastically increased with increasing salinity, higher levels of K+/Na+ ratio was obtained after K2SiO3·nH2O addition. Salinity stress induced the destruction of the chloroplast envelope; however, K2SiO3·nH2O addition counteracted the adverse effect by salinity on the structure of the photosynthetic apparatus. K2SiO3·nH2O addition also enhanced the activities of superoxide dismutase and catalase. To sum up, exogenous Si plays a key role in enhancing its resistance to salt stresses in physiological base, thereby improving the growth and CGA production of Honeysuckle plant.

Production of chlorogenic acid and its derivatives in hairy root cultures of Stevia rebaudiana.

Chlorogenic acid and its derivatives (CADs) are valuable bioactive plant secondary metabolites with many health benefits. In the present study, Stevia rebaudiana hairy root cultures were established, and the culture conditions for the production of CADs were optimized. The hairy roots were induced by coculture of S. rebaudiana leaves and Agrobacterium rhizogenes (C58C1) after infection, which were further verified by PCR detection of rolB and rolC genes. HPLC-MS and HPLC analysis showed that chlorogenic acid (3-caffeoylquinic acid, 3-CQA), 3,5-dicaffeoylquinic acid (3,5-CQA), and 4,5-dicaffeoylquinic acid (4,5-CQA) were the major CADs in the hairy roots. Eight single roots with rapid growth rate were selected. Among them, T3 had the highest yield of CADs. B5 medium supplemented with 40 g/L sucrose was more suitable for the production of CADs than others. Under optimal culture conditions, the total content of these three compounds reached 105.58 mg/g and total yield was 234.40 mg/100 mL.

Effect of Shading on Anthocyanin and Non-flavonoid Polyphenol Biosynthesis of Gynura bicolor Leaves in Midsummer

The effect of shading in midsummer on anthocyanin and non-flavonoid polyphenol biosynthesis of Gynura bicolor DC leaves was examined using a control (full solar radiation) and a shade treatment (50% shading of full solar radiation). Leaf temperature in the shade plot remained ≈40 °C in the daytime, ≈6 °C lower than in the control. Plants in the shade plot grew better than the control. The content of chlorogenic acid (CGA) in leaves decreased with leaf maturation for both treatments, and a larger amount of CGA was detected in leaves from the control than the shade treatment. The profiles of reactive oxygen species (ROS) scavenging activity exhibited an identical pattern to the content of CGA. Although there was an abrupt increase in the content of anthocyanin in the early stage of leaf expansion, the content decreased rapidly as the leaves matured. The increase in anthocyanin early during leaf expansion was much more limited in control leaves than shaded leaves. There were no correlation between the profiles of anthocyanin and gene expression such as GbPAL, GbC4H, Gb4CL, GbCHS, GbCHI, GbF3H, and GbUFGT. However, the profiles of expression of genes such as GbMYB2, GbF3′H, GbDFR, and GbANS were similar to the anthocyanin profiles. These results suggest that artificial shading in midsummer is an effective method to promote anthocyanin accumulation but reduces ROS scavenging capacity as a result of lowered CGA production.

Plants as biofactories: Stress-induced production of chlorogenic acid isomers in potato tubers as affected by wounding intensity and storage time

In the present study, the feasibility of using wounded-potato tubers as biofactories of antioxidant phenolic compounds (PC) was evaluated. Potato tubers were wounded to obtain different wounding intensities (slices, pie-cuts, and shreds) and stored for 144h at 10°C. The accumulation of total and individual PC was determined during storage of the wounded-tissue. Results indicated that the highest accumulation of total PC was obtained at 96h of storage for slices and pie-cuts. At this storage time, slices and pie-cuts showed 100% and 65% higher total PC content, respectively, whereas shredded-potatoes showed 40% lower PC content as compared with wholes before storage. The main PCs identified in wounded-potatoes were chlorogenic acid (CGA), neo-chlorogenic acid (neo-CGA), and crypto-chlorogenic acid (crypto-CGA). Results also indicated that selecting the wounding intensity to apply and storage time could manipulate the accumulation of specific PC in potato tubers. For instance, if the production of CGA or crypto-CGA were desirable, potato tubers should be wounded to obtain slices and pie-cuts. Likewise, to produce neo-CGA the appropriate wounding intensity should be pie-cuts. Furthermore, the highest accumulation of CGA and neo-CGA in wounded-potatoes was obtained at 96h of storage, whereas for the crypto-CGA its highest accumulation was observed in wounded-potatoes stored for 144h at 10°C. The stressed-potato tubers with increased concentrations of chlorogenic acids could be used as a starting material for the extraction of high value antioxidant PC with potential applications in the pharmaceutical and dietary supplements industries.

Enhancement of chlorogenic acid production in hairy roots of Platycodon grandiflorum by over-expression of an Arabidopsis thaliana transcription factor AtPAP1.

To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA.

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.