Dicaffeoylquinic Acid Isomers Research Articles

Integrated UHPLC-Q-exactive-orbitrap MS/MS and ABTS analyses screen antioxidant and anti‑counterfeiting marker of Eucommiae Cortex (Duzhong) for Pharmacopoeias

Eucommiae Cortex (Duzhong) has been collected in several Pharmacopoeias. However, its Pharmacopoeia quality-marker is outdated and unreasonable. Therefore, the study used database-aided ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry (UHPLC-Q-Exactive-Orbitrap MS/MS) analysis to putatively identify 33 compounds from Eucommiae Cortex. Especially, 14 “unexcavated” compounds were detected in Eucommiae Cortex for the first time; Meanwhile, all monocaffeoylquinic acid isomers (i.e., 1-O-, 3-O-, 4-O-, and 5-O- caffeoylquinic acids) have been strictly distinguished from each other, and three dicaffeoylquinic acid isomers (1,3-O-, 3,5-O-, and 4,5-O- dicaffeoylquinic acids) have been distinguished as well. All identified compounds were further subjected to a semi-quantification analysis, through which cholesteryl acetate (4.11 ± 0.46 %), sucrose (3.53 ± 0.063 %), and (−)-syringaresinol (2.81 ± 1.31 %) were found to be highly abundant. Furthermore, the semi-quantification results were integrated with ABTS+• antioxidant assay to individually calculate the antioxidant contribution of all compounds. (−)-Syringaresinol, neochlorogenic acid, vanillic acid, chlorogenic acid, and (−) pinoresinol were proven to be the top five antioxidant contributors, and their contribution values were 97.50, 46.31, 40.57, 35.05, and 30.90. Of the top contributors, chlorogenic acid was proposed as an additional pharmacopoeia Q-marker for its good testability and traceability. The additional Q-marker not only enhances the pharmacological relevance, but also effectively recognizes Eucommiae Cortex from its counterfeiting via liquid chromatography analysis. Thus, these findings will help Pharmacopoeias to update its Q-markers.

Evaluation of Environmental Factor Effects on the Polyphenol and Flavonoid Content in the Leaves of Chrysanthemum indicum L. and Its Habitat Suitability Prediction Mapping.

The leaves of Chrysanthemum indicum L. are known to have various bioactive compounds; however, industrial use is extremely limited. To overcome this situation by producing high-quality leaves with high bioactive content, this study examined the environmental factors affecting the phytochemical content and antioxidant activity using C. indicum leaves collected from 22 sites in Kochi Prefecture, Japan. Total phenolic and flavonoid content in the dry leaves ranged between 15.0 and 64.1 (mg gallic acid g-1) and 2.3 and 11.4 (mg quercetin g-1), while the antioxidant activity (EC50) of the 50% ethanol extracts ranged between 28.0 and 123.2 (µg mL-1) in 1,1-Diphenyl-2-picrylhydrazyl radical scavenging assay. Among the identified compounds, chlorogenic acid and 1,5-dicaffeoylquinic acid were the main constituents in C. indicum leaves. The antioxidant activity demonstrated a positive correlation with 1,5-dicaffeoylquinic acid (R2 = 0.62) and 3,5-dicaffeoylquinic acid (R2 = 0.77). The content of chlorogenic acid and dicaffeoylquinic acid isomers varied significantly according to the effects of exchangeable magnesium, cation exchange capacity, annual temperature, and precipitation, based on analysis of variance. The habitat suitability map using the geographical information system and the MaxEnt model predicted very high and high regions, comprising 3.2% and 10.1% of the total area, respectively. These findings could be used in future cultivation to produce high-quality leaves of C. indicum.

Use of Multivariate Analysis to Unravel the Differences between Two Chamomile Varieties and Their Anticancer and Antioxidant Activities.

Plants from the Asteraceae family were commonly used to treat various diseases. The metabolomic profile of this family consisted of bioactive flavonoids and other phenolics. Chamomile is a member of the Asteraceae family. Jordanian and European chamomile are two varieties of Matricaria chamomilla (German chamomile), which were grown under different environmental conditions, were studied. Many examples of plant varieties with significant distinction in the secondary metabolite they afford have been described in the literature. Multivariate statistical analysis was employed to measure the depth of this variation in two chamomile varieties. From both types, crude extracts were prepared using solvents of different polarities and tested for their biological activity. The semipolar fraction of the European variety showed anticancer and antioxidant activity. Meanwhile, the semipolar fraction of the Jordanian type exhibited only antioxidant activity. Both extracts were fractionated, and then the biological activity was again assayed. European and Jordanian chamomile fractions produced dicaffeoylquinic acid isomers exhibiting antioxidant capability. Additionally, Z-glucoferulic acid was produced from the European chamomile, demonstrating antioxidant activity. The European samples afforded two major compounds, chrysosplenetin and apigenin, that displayed anticancer activity. Different environmental conditions between Jordanian and European chamomile affected the type of isolated compounds. Structure elucidation was performed with HPLC-MS coupled with dereplication techniques and 2D NMR experiments.

Is acute low-dose gamma irradiation an effective elicitor for secondary metabolism in Leontopodium alpinum (Cass.) callus culture?

Leontopodium alpinum Cass. (Edelweiss) is a protected species in some European countries and has become of growing interest for exploitation by the food supplements and cosmetics industry, due to its valuable composition in bioactive and biocompatible compounds. Small scale field cultivation and in vitro biotechnological approaches are economically viable alternatives to the production via chemical synthesis. In this study, we aimed to compare two performant L. alpinum callus culture lines and to investigate the effect of acute low dose gamma irradiation on biomass accumulation (Gi), total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (DPPH) and secondary metabolites’ profile. A purple, pigmented line (PC) was obtained under lightning conditions and in the absence of light, an unpigmented greenish line (GC) was established. Both lines displayed impressive proliferative capacity after 14 days of culture, with an 18.31 ( ± 1.83) fold mass increase for PC and a 13.27 ( ± 2.42) for GC. In respect to Gi, TPC, TFC and DPPH, the purple callus line significantly outperformed the unpigmented line. According to the UPLC-HRMS analysis of the methanolic extracts, both callus lines displayed similar metabolic profiles, having similar quantities of 3,5-dicaffeoylquinic acid, leontopodic acids A and B, but 60% higher concentration of chlorogenic acid in the GC line. In the methanolic callus extracts, 9 economically important phenylpropanoid derivatives were identified. The two callus lines responded differently to low dose gamma irradiation. Although the control values measured for Gi, TPC, TFC and DPPH were significantly higher in the PC line, this line was generally negatively impacted by the gamma treatments. On the contrary, higher, but not significant, values were registered in some GC variants post irradiation with 25, 30 and 40 Gy. UPLC-HRMS investigations emphasized the overproduction of 7 compounds, post irradiation with all doses, for the PC line, while for the unpigmented line only one compound was stimulated by all irradiation treatments. The highest stimulative effect was obtained for the 35 and 40 Gy experimental variants, in the GC line, where dihydroxybenzoic acid glucoside accumulation was increased by 160%. The results of this study showed, for the first time, the different responses of two distinct L. alpinum callus lines using gamma irradiation elicitation method, which can be successfully applied to stimulate the production of certain industrially important compounds as chlorogenic acid, dicaffeoylquinic acid isomers or characteristic glucaric acids.

A Mixture of Artemisia argyi and Saururus chinensis Improves PM2.5-Induced Cognitive Dysfunction by Regulating Oxidative Stress and Inflammatory Response in the Lung and Brain.

This study was performed to investigate the improving effect of a mixture of Artemisia argyi and Saururus chinensis (AASC) on cognitive dysfunction in mice with long-term exposure to fine particles (particulate matter smaller than 2.5 µm: PM2.5). The main compounds of AASC were identified as dicaffeoylquinic acid isomers of A. argyi and a quercetin-3-glucoside of S. chinesis. As a result of behavioral tests for the evaluation of cognitive function, it was confirmed that cognitive dysfunction was induced in the PM2.5 exposure group, and a tendency to improve in the AASC group was confirmed. Increased oxidative stress and inflammatory response and mitochondrial dysfunction were observed in the brain and lung tissues of the PM group. Damage to the brain and lung affected the accumulation of amyloid beta (Aβ) in the brain. It increased Aβ and induced the cholinergic dysfunction, hyperphosphorylation of the tau protein, and activation of apoptosis, leading to cognitive impairment. However, AASC suppressed brain and lung oxidative stress and inflammation, thereby suppressing brain Aβ expression. Consequently, this study shows the potential that a steady intake of plant resources with antioxidant and anti-inflammatory activity could prevent cognitive impairment caused by PM2.5.

Development and Validation of an On-Line HPLC-DAD-Antioxidant Assay (ORAC)/ESI-HRMS System to Identify Antioxidant Compounds in Complex Mixtures.

An online high-performance liquid-chromatography-diode-array detector coupled with detection of antioxidant compounds using oxygen radical absorbance capacity (ORAC) assay and electrospray ionization-high-resolution mass spectrometer (HPLC-DAD-antioxidant assay (ORAC)/ESI-HRMS) was developed for the identification of antioxidant compounds in complex mixtures. The method was validated using quercetin and a mixture of antioxidant compounds with different antioxidant activities (resveratrol, dihydroxymethoxy-dihydrochalcone, ferulic acid, baicalein and luteolin). Accuracy of the system was established by comparing the results from the developed system with those from ORAC microplate assay determination and reveals the ability of the system to determine the respective contribution of antioxidant compounds to the whole activity of complex mixtures. Application of the system to the identification of antioxidants in a commercial Yerba Mate extract (Ilex paraguariensis St. Hil.) reveals the occurrence of seven actives, which were characterized as chlorogenic acids isomers (3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid), dicaffeoylquinic acid isomers (3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid) and rutin based on UV/Vis spectra, HRMS and MS/MS data. This on-line system is able to generate HPLC-DAD fingerprints, UV/Vis spectra, ORAC activity profile and high-resolution mass spectrometric data.

Natural Deep Eutectic Solvent-Based Matrix Solid Phase Dispersion (MSPD) Extraction for Determination of Bioactive Compounds from Sandy Everlasting (Helichrysum arenarium L.): A Case of Stability Study.

In the present study, vortex-assisted matrix solid-phase dispersion (VA-MSPD) extraction was used to isolate the major bioactive compounds from H. arenarium. To reduce the negative environmental impact of the conventionally used organic solvents, four different choline chloride-based natural deep eutectic solvents (NADES) were investigated as possible eluents. The most influential VA-MSPD extraction parameters: stationary phase (adsorbent), adsorbent/sample ratio, vortex time, and volume of extraction solvent were systematically optimized. Ultrasound-assisted extraction with 80% MeOH was used as the standard method for the comparison of results. The stability of the obtained extracts was studied over a period of 0 to 60 days at three different temperatures (-18 °C, 4 °C, and 25 °C). All extracts were evaluated both spectrophotometrically (determination of total phenolic content (TPC) and antioxidant activity by ABTS and FRAP assay) and chromatographically (HPLC-UV). NADES based on choline chloride and lactic acid (ChCl-LA) was selected as the most effective extractant, with a determined TPC value of its extract of 38.34 ± 0.09 mg GA/g DW (27% higher than the methanolic VA-MSPD extract) and high antioxidant activity. The content of individual phenolic compounds (chlorogenic acid, dicaffeoylquinic acid isomers, naringenin isomers, and chalcones) in the ChCl-LA extract, determined by HPLC-UV, was comparable to that of the conventionally obtained one. Moreover, the stabilization effect of ChCl-LA was confirmed for the studied compounds: chlorogenic acid, naringenin-4'-O-glucoside, tomoroside A, naringenin-5-O-glucoside, isosalipurposide, and naringenin. The optimum VA-MSPD conditions for the extraction of H. arenarium polyphenols were: florisil/sample ratio of 0.5/1, a vortex time of 2 min, and an elution volume of ChCl-LA of 10 mL.

Low concentrations of Ambrosia maritima L. phenolic extract protect endothelial cells from oxidative cell death induced by H2O2 and sera from Crohn's disease patients

Ethnopharmacological relevanceA rising resort to herbal therapies in Crohn's disease (CD) alternative treatments has been recently observed due to their remarkable natural efficiency. In this context, the weed plant Ambrosia maritima L., traditionally known as Hachich el Aouinet in Algeria and as Damsissa in Egypt and Sudan, is widely used in North African folk medicine to treat infections, inflammatory diseases, gastrointestinal and urinary tract disturbances, rheumatic pain, respiratory problems, diabetes, hypertension and cancer. Aim of the studyTo assess an Ambrosia maritima L. phenolic extract for its phenolic profile composition, its potential antioxidant activity in vitro, and its cytoprotective effect on cultured primary human endothelial cells (ECs) stressed with H2O2 and sera from CD patients. Materials and MethodsPhenolic compound extraction was performed with a low-temperature method. Extract chemical profile was attained by HPLC-DAD/ESI-MS. The extract in vitro antioxidant activity was assessed using several methods including cupric ion reducing power, DPPH radical scavenging assay, O-Phenanthroline free radical reducing activity, ABTS cation radical decolourisation assay, Galvinoxyl free radicals scavenging assay. Intracellular reactive oxygen species levels were evaluated in human endothelial cells by H2DCFDA, while cell viability was assessed by MTT. ResultsThe phenolic compounds extraction showed a yield of 17.66% with three di-caffeoylquinic acid isomers detected for the first time in Ambrosia maritima L. Using different analytical methods, a significant in vitro antioxidant activity was reported for the Ambrosia maritima L. extract, with an IC50 value of 14.33 ± 3.86 μg/mL for the Galvinoxyl antioxidant activity method. Challenged with ECs the Ambrosia maritima L. extract showed a biphasic dose-dependent effect on H2O2-treated cells, cytoprotective and antioxidant at low doses, and cytotoxic and prooxidant at high doses, respectively. Viability and ROS levels data also demonstrated a prooxidant and cytotoxic effect of CD sera on cultured ECs. Interestingly, 10 μg/mL of Ambrosia maritima L. extract was able to counteract both CD sera-induced oxidative stress and ECs death. ConclusionOur data indicated Ambrosia maritima L. as a source of bioactive phenolics potentially employable as a natural alternative for CD treatment.

Extraction of Bioactive Metabolites from Achillea millefolium L. with Choline Chloride Based Natural Deep Eutectic Solvents: A Study of the Antioxidant and Antimicrobial Activity

In this study, the extraction efficiency of natural deep eutectic solvents (NADES) based on choline chloride as a hydrogen bond acceptor (HBA) and five different hydrogen bond donors (HBD; lactic acid, 1,4-butanediol, 1,2-propanediol, fructose and urea) was evaluated for the first time for the isolation of valuable bioactive compounds from Achillea millefolium L. The phytochemical profiles of NADES extracts obtained after ultrasound-assisted extraction were evaluated both spectrophotometrically (total phenolic content (TPC) and antioxidant assays) and chromatographically (UHPLC-MS and HPLC-UV). The results were compared with those obtained with 80% ethanol, 80% methanol, and water. The highest TPC value was found in the lactic acid-based NADES (ChCl-LA), which correlated with the highest antioxidant activity determined by the FRAP analysis. On the other hand, the highest antiradical potential against ABTS+• was determined for urea-based NADES. Phenolic acids (chlorogenic acid and dicaffeoylquinic acid isomers), flavones (luteolin and apigenin), and their corresponding glucosides were determined as the dominant individual phenolic compounds in all extracts. The antibacterial and antifungal properties of the extracts obtained against four bacterial cultures and two yeasts were evaluated using two methods: the agar dilution method to obtain the minimum inhibitory concentration (MIC) and the minimum bactericidal or fungicidal concentration (MBC or MFC), and the disc diffusion method. ChCl-LA had the lowest MIC and MBC/MFC with respect to all microorganisms, with an MIC ranging from 0.05 mg mL−1 to 0.8 mg mL−1, while the water extract had the weakest inhibitory activity with MIC and MBC/MFC higher than 3.2 mg mL−1.

A novel basis for monitoring the coffee roasting process: Isomerization reactions of 3-caffeoylquinic and 4-caffeoylquinic acids

The aim of this study was to investigate the changes of individual chlorogenic acids during the coffee roasting process under authentic commercial conditions. Unique chemical reactions of caffeoylquinic acid (CQA) and dicaffeoylquinic acid (diCQA) isomers were shown in different Coffea species (C. arabica vs. C. canephora), batch sizes (1 kg vs. 20 kg) and roaster designs. Temperature determination setups of different roaster machines appeared gave varying results. Isomerization reactions of CQA in C. arabica beans were observed between the end of the drying phase and the crack reaction. Regarding C. canephora beans, the slower degradation rate of 3-CQA and 4-CQA isomers after the crack reaction in contrast to C. arabica species was described for the first time.

3,5-Dicaffeoylquinic Acid Lowers 3T3-L1 Mitotic Clonal Expansion and Adipocyte Differentiation by Enhancing Heme Oxygenase-1 Expression.

Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.

Inhibition of temperature-sensitive TRPV3 channel by two natural isochlorogenic acid isomers for alleviation of dermatitis and chronic pruritus

Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3 (TRPV3) channel cause Olmsted syndrome characterized by severe itching and keratoderma, indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases. However, currently available TRPV3 tool inhibitors are either nonselective or less potent, thus impeding the validation of TRPV3 as therapeutic target. Using whole-cell patch-clamp and single-channel recordings, we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A (IAA) and isochlorogenic acid B (IAB) that selectively inhibit TRPV3 currents with IC50 values of 2.7 ± 1.3 and 0.9 ± 0.3 μmol/L, respectively, and reduce the channel open probability to 3.7 ± 1.2% and 3.2 ± 1.1% from 26.9 ± 5.5%, respectively. In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus. Furthermore, the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol. Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers. Taken together, our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology, but also holds developmental potential for treatment of dermatitis and chronic pruritus.

Ethyl Acetate Fraction of Helianthus tuberosus L. Induces Anti-Diabetic, and Wound-Healing Activities in Insulin-Resistant Human Liver Cancer and Mouse Fibroblast Cells.

Traditional, complementary, and integrative medicine are globally accepted alternative methods for the treatment of diabetes mellitus (DM). However, the mechanism of anti-diabetic effects of Helianthus tuberosus L. remains unproven. In the present study, antioxidant and anti-diabetic activity of the tubers of H. tuberosus were studied in detail. Methanolic extracts of H. tuberosus tubers were subjected to solvent fractionation method by increasing the polarity of the solvent using n-hexane, and ethyl acetate. The obtained methanol extracts and its fractions were subjected to free radical scavenging activity (DPPH and ABTS assay) and in vitro enzyme (α-amylase and α-glucosidase) inhibition assay. Moreover, glucose uptake in insulin-resistant HepG2 cell line was analyzed. The preliminary phytochemical analysis confirmed the presence of phenolic and flavonoid compounds in the active fraction. The radical scavenging and in vitro diabetic related enzyme inhibitory activities were found to be dose dependent. The maximum ABTS+ and DPPH scavenging activity was documented in ethyl acetate fraction of the H. tuberosus followed by methanol extract, hexane fraction, and methanol fraction. We also found that H. tuberosus showed a less toxicity in mouse fibroblast cells and enhance the glucose uptake in insulin-resistant HepG2 cells. Besides, the ethyl acetate fraction of the H. tuberosus analyzed by UPLC-QTOF-MS-MS and GC/MS revealed the presence of phenolic compounds such as neochlorogenic acid, chlorogenic acid, caffeic acid, 5-O-(4-coumaroyl)-quinic acid, feruloylquinic acid, caffeoylquinic acid, isoxazolidine, salicylic acid β-D-glucoside, dicaffeoylquinic acid isomers, salvianolic acid derivative isomers, and 1,4 dicaffeoylquinic acid etc. Among the identified phytochemicals, six were chosen for molecular docking study to explore their its inhibitory interactions with α-amylase and α-glucosidase. Taken together, the findings of the present study suggested that phytocompounds of EAF were responsible for the significant in vitro antioxidant, wound-healing, and anti-diabetic activities.

Ilex paraguariensis extract as an alternative to pain medications.

Pain is a common and distressing symptom of many diseases and its clinical treatment generally involves analgesics and anti-inflammatory drugs. This study evaluated the toxicity of Ilex paraguariensis A. St.-Hil. (Aquifoliaceae) aqueous extract (leaves, petioles and branches) and its performance in a nociceptive response. Hepatotoxicity, psycho-stimulant test and evaluation of enzyme markers for liver damage were also tested. Chromatographic analysis by UPLC-MS demonstrated a series of isomeric monocaffeoylquinic acids, isomers of dicaffeoylquinic acid, flavonol glycosides, and saponins. Phase I and II of nociception were obtained for meloxicam, dexamethasone and aqueous Ilex paraguariensis extract. Ilex paraguariensis extract concentration was negatively correlated (R = -0.887) with alanine aminotransferase (p < 0.05) in acetaminophen-induced hepatotoxicity test, indicating hepatoprotective activity of this extract. Ilex paraguariensis extract also presented analgesic properties equivalent to drugs that already have proven efficacy. Notably, the administration of multiple doses of Ilex paraguariensis extract was considered safe from the therapeutic point of view.

Sunflower (Helianthus annuus L.) Plants at Various Growth Stages Subjected to Extraction-Comparison of the Antioxidant Activity and Phenolic Profile.

The aim of this study was to evaluate the differences in the antioxidant activity and phenolic profile of sunflower (Helianthus annuus L.) extracts obtained from the aerial parts of plants harvested at five growth stages. In vitro assays were used to determine the antioxidant activity, i.e., ABTS•+ and DPPH• scavenging activity, the ferric-reducing antioxidant power (FRAP) and the ability to inhibit β-carotene–linoleic acid emulsion oxidation. Phenolic compounds, such as mono- and dicaffeoylquinic acid isomers and caffeic acid hexose, were identified using the LC–TOF–MS/MS technique. The predominant compound during the growth cycle of the plant was 3,5-di-O-caffeoylquinic acid, whose content was the highest at the mid-flowering stage. The total phenolic content was also the highest in sunflowers at the mid-flowering stage. The main phenolic compound contents were closely correlated with ABTS•+ and DPPH• scavenging activity and FRAP. No significant correlation was found between the total phenolic content and the antioxidant activity in the emulsion system. The highest antiradical activity and FRAP were generally determined in older plants (mid-flowering and late flowering stages). In conclusion, the aerial parts of sunflowers, in particular those harvested at the mid-flowering stage, are a good plant material from which to obtain phenolic compound extracts, albeit mainly of one class (esters of caffeic acid and quinic acid), with high antioxidant activity.

Evidence of noncovalent complexes in some natural extracts: Ceylon tea and mate extracts.

Considering the high complexity of natural extracts, because of the presence of organic molecules of different chemical nature, the possibility of formation of noncovalent complexes should be taken into account. In a previous investigation, the formation of bimolecular complexes between caffeine and catechins in green tea extracts (GTE) has been experimentally proven by means of mass spectrometric and 1 H nuclear magnetic resonance experiments. The same approaches have been employed in the present study to evaluate the presence of bimolecular complexes in Ceylon tea and mate extracts. The obtained results show that in the case of Ceylon tea extracts, protonated theaflavin is detectable, together with theaflavin/caffein complexes, while caffeine/catechin complexes, already detected in green tea, are still present but at lower concentration. This aspect is evidenced by the comparison of precursor ion scans performed on protonated caffeine for the two extracts. The spectra obtained in these conditions for GTE and Ceylon tea show that the complexes of caffeine with epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG), highy abundant in the case of GTE (signal-to-chemical noise ratio in the range 50-100), are negligible (signal-to-chemical noise ratio in the range 2-3) in the case of Ceylon tea. Mate extracts show the formation of bimolecular complexes involving caffeine but not catechins, and chlorogenic acid becomes responsible for other complex formation. Under positive ion and negative ion conditions, accurate mass measurements allow the identification of malealdehyde, chlorogenic acid, caffeine, two isomers of dicaffeoylquinic acid, rutin, and kaempferol-3-O-rutinoside. These data indicate that the formation of complexes in natural extracts is a common behavior, and their presence must be considered in the description of natural extracts and, consequently, in their biological activity.

Quantitative Analysis of the Biologically Active Compounds Present in Leaves of Mexican Sweet Potato Accessions: Phenols, Flavonoids, Anthocyanins, 3,4,5-Tri-Caffeoylquinic Acid and 4-Feruloyl-5-Caffeoylquinic Acid.

Sweet potato is one of the oldest crops cultivated in Mexico, and Mesoamerica is considered as a region with the greatest diversity of this species. Therefore, the present study focused on the evaluation of biologically active compounds, such as caffeoylquinic acid derivatives and flavonoid compounds, in sweet potato leaves of 200 accessions of the main producing regions of Mexico. The analysis of total phenol content (TPC) showed a great variability of concentrations among the examined accessions (54.41 to 284.64 mgTPC/g DW). Likewise, total flavonoid content (TFC) was determined and ranged from 10.01 to 40.17 mgTFC /g DW. Finally, total anthocyanin content (TAC) was evaluated and concentrations obtained varied from 0.05 to 0.98 mgTAC/g DW. Additionally, HPLC analysis of all 200 accessions demonstrated the presence of caffeic acid (CA), 5-caffeoylquinic acid (5-CQA), three isomers of di-caffeoylquinic acid (di-CCA) and 4-feruloyl-5-caffeoylquinic acid (4F-5CQA) in all test samples. Only 21 accessions tested showed the quantitative amount of 3,4,5-tri-caffeoylquinic acid (3,4,5-tri-CQA) with concentrations ranging from 44.73 to 193.22mg/100g DW and high content of 4F-5CQA (139.46 to 419.99mg/100g DW). The gathered data indicate that leaves of Mexican sweet potatoes are a promising source of phenolic compounds with remarkable nutraceutical potential.

Phytochemical composition of wormwood (Artemisia gmelinii) extracts in respect of their antimicrobial activity

BackgroundExtracts from medicinal plants with phytochemicals with known antimicrobial properties can be an effective adjunct in the complex treatment of infectious diseases. This study aimed to evaluate the antimicrobial activity of wormwood extracts collected in Kazakhstan (Artemisia gmelinii Weber ex Stechm.), along with their phytochemical analysis.MethodsThe ethanolic and chloroform extracts were subjected to HPLC combined with quadrupole time-of-flight mass spectrometry method. For quantitative assessment of antimicrobial activity, minimal inhibitory concentration (MIC) of the tested extracts was determined by micro-dilution broth method for the panel of the reference microorganisms. Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) were also determined.ResultsLC/MS analysis showed the presence of 13 compounds in the tested extracts, including flavonoids: apigenin, luteolin, rutin, two O-methylated flavonols (isorhamnetin, rhamnazine), coumarin compounds (umbelliferone, scopoletin and scopolin (scopoletin 7-glucoside), 3-hydroxycoumarin and 4-hydroxycoumarin), chlorogenic acid and two dicaffeoylquinic acid isomers. Quantitative HPLC analysis showed that umbelliferone was dominant in the chloroform extract while chlorogenic acid was identified as a main compound in the ethanolic extract. The antibacterial and antifungal activity of chloroform and ethanolic extracts was comparable. The most sensitive were the Gram-positive bacteria represented by staphylococci, Micrococcus luteus and Bacillus spp. (MIC = 1.25–5 mg/ml) and yeasts represented by Candida spp. (MIC = 2.5–5 mg/ml), irrespective of the assayed extract.ConclusionsExtracts of wormwood Artemisia gmelinii have shown a wide spectrum of antibacterial and antifungal activity. Luteolin, rutin, isorhamnetin and scopolin were identified in A. gmelinii species for the first time. The determining of the most potential compounds of Artemisia gmelinii can be used to develop effective antibacterial and antifungal agents.

Dicaffeoylquinic Acids from the Aerial Parts of Artemisia ciniformis Krasch. & Popov ex Poljakov

Background: Artemisia ciniformis (A. ciniformis) belongs to the genus Artemisia and grows at northeast of Iran. The current phytochemical study was carried out on the most potent extract in cell-free antioxidant assays. Methods: Hydroethanolic extract of the aerial parts was fractionated using vacuum-liquid chromatography (VLC). The selected fraction from the previous cell-free antioxidant study was purified by semi-preparative HPLC. The structures of isolated compounds were identified using one- and two-dimensional NMR and ESIMS techniques. Results: Three identified compounds in this study were the known isomers of dicaffeoylquinic acid (DCQ), including 3,5- DCQ (isochlorogenic acid A), 3,4-DCQ (isochlorogenic acid B) and 4,5-DCQ (isochlorogenic acid C). Conclusion: The outstanding free radical scavenging potential in the hydroethanolic extract of A. ciniformis might be partly related to the presence of isochlorogenic acid derivatives.

Chlorogenic acid isomers directly interact with Keap 1-Nrf2 signaling in Caco-2 cells

Chlorogenic acid (CGA) exists as multiple isomers (e.g., 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in foods such as coffee beverages, fruits and vegetables. This study aimed to investigate relative activities of these six different CGA isomers to modify redox biology in inflamed Caco-2 cells that involved Nrf2 signaling. Caco-2 cells were pre-treated with individual CGA isomers to assess the relative effectiveness to mitigate oxidative stress. Isomer-specific capacity of different CGA isomers for direct free radical scavenging activity and potential endogenous control of oxidative stress were determined using chemical assays and cell-based experiments, respectively. Molecular dynamics simulations of the CGA and Keap1-Nrf2 complex were performed to predict CGA structure-specific interactions. Results demonstrated that dicaffeoylquinic acid (diCQA including 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) isomers had greater (p < 0.05) affinity to ameliorate oxidative stress through direct free radical scavenging activity. This observation corresponded to greater (p < 0.05) capacity to activate Nrf2 signaling compared to caffeoylquinic acid (CQA including 3-CQA, 4-CQA, and 5-CQA) isomers in inflamed differentiated Caco-2 cells. Simulations revealed that differences between the ability of CQA and diCQA to interact with the Keap1-Nrf2 complex may be due to differences in relative orientation within this complex. The observed CGA isomer-specific affinity for CQA to activate Nrf2 signaling was confirmed by nuclear translocation of Nrf2 induced by CGA and greater (p < 0.05) upregulation of genes related to Nrf2 expression.