Discovery and characterization of novel ATP citrate lyase inhibitors from Acanthopanax senticosus (Rupr. & Maxim.) Harms.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism, and abnormally high expression of ACLY occurs in many diseases, including cancers, dyslipidemia and cardiovascular diseases. ACLY inhibitors are prospective treatments for these diseases. However, the scaffolds of ACLY inhibitors are insufficient with weak activity. The discovery of inhibitors with structural novelty and high activity continues to be a research hotpot. Acanthopanax senticosus (Rupr. & Maxim.) Harms is used for cardiovascular disease treatment, from which no ACLY inhibitors have ever been found. In this work, we discovered three novel ACLY inhibitors, and the most potent one was isochlorogenic acid C (ICC) with an IC50 value of 0.14 ± 0.04 µM. We found dicaffeoylquinic acids with ortho-dihydroxyphenyl groups were important features for inhibition by studying ten phenolic acids. We further investigated interactions between the highly active compound ICC and ACLY. Thermal shift assay revealed that ICC could directly bind to ACLY and improve its stability in the heating process. Enzymatic kinetic studies indicated ICC was a noncompetitive inhibitor of ACLY. Our work discovered novel ACLY inhibitors, provided valuable structure-activity patterns and deepened knowledge on the interactions between this targe tand its inhibitors.

Neochlorogenic acid: an anti-HIV active compound identified by screening of Cortex Mori [Morus Alba L. (Moraceae)].

CONTEXT: Chinese herbs such as Cortex Mori [Morus alba L. (Moraceae)] may inhibit human immunodeficiency virus (HIV), but active compounds are unknown. OBJECTIVE: Screening of Cortex Mori and other herbs for anti-HIV active compounds. MATERIALS AND METHODS: HIV-1 virus (multiplicity of infection: 20), and herbs (dissolved in dimethyl sulfoxide, working concentrations: 10, 1, and 0.1 mg/mL) such as Cortex Mori, etc., were added to 786-O cells (105 cell/well). Zidovudine was used as a positive control. Cell survival and viral inhibition rates were measured. The herb that was the closest inactivity to zidovudine was screened. Mass spectrometry identified the active compounds in herbs (mobile phase: 0.05% formic acid aqueous solution and acetonitrile, gradient elution, detection wavelength: 210 nm). The effect of the compounds on reverse transcriptase (RT) products were evaluated by real-time PCR. Gene enrichment was used to analyse underlying mechanisms. RESULTS: With a dose of 1 mg/mL of Cortex Mori, the cell survival rate (57.94%) and viral inhibition rate (74.95%) were closest to the effect of zidovudine (87.87%, 79.81%, respectively). Neochlorogenic acid, one of the active ingredients, was identified by mass spectrometry in Cortex Mori. PCR discovery total RT products of neochlorogenic acid group (mean relative gene expression: 6.01) significantly inhibited (control: 35.42, p < 0.0001). Enrichment analysis showed that neochlorogenic acid may act on haemopoietic cell kinase, epidermal growth factor receptor, sarcoma, etc., thus inhibiting HIV-1 infection. CONCLUSIONS: For people of low socioeconomic status affected by HIV, Chinese medicine (such as Cortex Mori) has many advantages: it is inexpensive and does not easily produce resistance. Drugs based on active ingredients may be developed and could have important value.

Caffeoylquinic Acid Derivatives of Purple Sweet Potato as Modulators of Mitochondrial Function in Mouse Primary Hepatocytes.

Owing to their antioxidant properties, caffeoylquinic acid (CQA)-derivatives could potentially improve the impaired metabolism in hepatic cells, however, their effect on mitochondrial function has not been demonstrated yet. Here, we evaluated the impact of three CQA-derivatives extracted from purple sweet potato, namely 5-CQA, 3,4- and 4,5-diCQA, on mitochondrial activity in primary hepatocytes using an extracellular flux analyzer. Notably, an increase of maximal respiration and spare respiratory capacity were observed when 5-CQA and 3,4-diCQA were added to the system indicating the improved mitochondrial function. Moreover, 3,4-diCQA was shown to considerably increase glycolytic reserve which is a measure of cell capability to respond to an energy demand through glycolysis. Conversely, 4,5-diCQA did not modify mitochondrial activity but increased glycolysis at low concentration in primary hepatocytes. All compounds tested improved cellular capacity to oxidize fatty acids. Overall, our results demonstrated the potential of test CQA-derivatives to modify mitochondrial function in hepatic cells. It is especially relevant in case of dysfunctional mitochondria in hepatocytes linked to hepatic steatosis during obesity, diabetes, and metabolic syndrome.

Bakkenolides and Caffeoylquinic Acids from the Aerial Portion of Petasites japonicus and Their Bacterial Neuraminidase Inhibition Ability.

Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 µM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.

Optimization of an Extraction Solvent for Angiotensin-Converting Enzyme Inhibitors from Hibiscus sabdariffa L. Based on Its UPLC-MS/MS Metabolic Profiling.

Hibiscus species (Malvaceae) have been long used as an antihypertensive folk remedy. The aim of our study was to specify the optimum solvent for extraction of the angiotensin-converting enzyme inhibiting (ACEI) constituents from Hibiscus sabdariffa L. The 80% methanol extract (H2) showed the highest ACEI activity, which exceeds that of the standard captopril (IC50 0.01255 ± 0.00343 and 0.210 ± 0.005 µg/mL, respectively). Additionally, in a comprehensive metabolomics approach, an ultra-performance liquid chromatography (UPLC) coupled to the high resolution tandem mass spectrometry (HRMS) method was used to trace the metabolites from each extraction method. Interestingly, our comprehensive analysis showed that the 80% methanol extract was predominated with secondary metabolites from all classes including flavonoids, anthocyanins, phenolic and organic acids. Among the detected metabolites, phenolic acids such as ferulic and chlorogenic acids, organic acids such as citrate derivatives and flavonoids such as kaempferol have been positively correlated to the antihypertensive potential. These results indicates that these compounds may significantly contribute synergistically to the ACE inhibitory activity of the 80% methanol extract.

Caffeoylquinic Acids, Cytotoxic, Antioxidant, Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria.

Chlorogenic (5-CQA), 1,5-, 3,5-, 4,5- and 3,4-dicaffeoylquinic (DCQA) acids were identified and quantified in the methanol extracts of Inula oculus-christi L., I. bifrons L., I. aschersoniana Janka var. aschersoniana, I. ensifolia L., I. conyza (Griess.) DC. and I. germanica L. by HPLC analysis. The amount of 5-CQA varied from 5.48 to 28.44 mg/g DE and the highest content was detected in I. ensifolia. 1,5-DCQA (4.05-55.25 mg/g DE) was the most abundant dicaffeoyl ester of quinic acid followed by 3,5-DCQA, 4,5-DCQA and 3,4-DCQA. The extract of I. ensifolia showed the highest total phenolic content (119.92±0.95 mg GAE/g DE) and exhibited the strongest DPPH radical scavenging activity (69.41±0.55 %). I. bifrons extract was found to be the most active sample against ABTS.+ (TEAC 0.257±0.012 mg/mL) and the best tyrosinase inhibitor. The studied extracts demonstrated a low inhibitory effect towards acetylcholinesterase and possessed low cytotoxicity in concentration range from 10 to 300 µg/mL toward non-cancer (MDCK II) and cancer (A 549) cells.

Chemical composition of Erycibe schmidtii and antiproliferative activity of scopoletin on immature dendritic cells.

Immature dendritic cells (iDCs) play very important roles in the pathological process of rheumatoid arthritis (RA). Therefore, it is urgent to search for natural products with antiproliferative activity on iDCs for anti-RA drug discovery. Erycibe schmidtii, a traditional Chinese medicine, has been used to treat RA in China. Its bioactive ingredients on RA are still unclear. In this study, twenty compounds including a new caffeoylquinic acid derivative, 3-O-caffeoyl-4-O-syringoylquinic acid methyl ester (16), were isolated from E. schmidtii. Their structures were elucidated by NMR and mass spectroscopic analysis, and comparison with literature data. Seventeen compounds were obtained from this plant for the first time, and ten were first found from the genus Erycibe. Scopoletin (1, 5.0 µM) functionally reduced proliferation level of bone marrow immature dendritic cells (BM-iDCs) more than 50%, relative to vehicle. However, scopoletin (1) exhibited no effect on the phagocytosis or survival of BM-iDCs in vitro.

On-line screening and identification of polyphenolic antioxidant compounds of Convolvulus trabutianus.

Convolvulus trabutianus Schweinf. & Muschl. is an endemic plant from northern Sahara used in folk medicine. Herein we report, the isolation, characterization and evaluation of the radical scavenging properties of twenty three compounds from different extracts of this species by on-line HPLC-ABTS•+ screening. These compounds include nine phenolic acids: 2, 6, 10-16, two phytosterols: 3-4, four coumarins: 5, 7-9, two quinic acids: 21 and 22 and six flavonoids: 1, 17-20 and 23 among which the most active were: 10, 16, 21 and 22. All the extracts showed a significant antioxidant activity on-line. These results were validated off-line by ORAC and TEAC assays. Four compounds: 1, 5, 18 and 19 were described for the first time from the Convolvulaceae family, whereas compounds 2, 6, 8, 10, 13 and 21 were new for the genus Convolvulus.

Exploitation of artichoke byproducts to obtain bioactive extracts enriched in inositols and caffeoylquinic acids by Microwave Assisted Extraction.

Byproducts from artichoke represent the majority of the mass collected from the plant and constitute an interesting source of bioactive compounds such as inositols and caffeoylquinic acids. In this work, a microwave assisted extraction (MAE) methodology was developed for the simultaneous extraction of these compounds from artichoke stalks, leaves, receptacles and external bracts. Optimal MAE conditions to maximize the extraction of these bioactives and the antioxidant activity were 97 °C, 3 min, ethanol:water (50:50, v/v). Moreover, a GC-MS methodology was also developed for the simultaneous determination of these compounds in a single run; optimal derivatization conditions were achieved using hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. Artichoke receptacle extracts were the richest in caffeoylquinic acids (28-35 mg g-1 dry sample), followed by the bracts (9-18 mg g-1 dry sample), while those from leaves showed the highest concentrations of inositols (up to 15 mg g-1 dry sample). Receptacle extracts also had the highest antioxidant activity (123 mg TE g-1 dry sample) and the greatest concentration of total phenolic compounds (47 mg GAE g-1 dry sample). Therefore, the developed methodology could be considered as a valuable procedure to obtain and characterize bioactive ingredients with industrial interest from artichoke byproducts, opening new routes of revalorization of artichoke agro-industrial residues.

Chemical investigation of Hyptis suaveolens seed, a potential antihyperuricemic nutraceutical, with assistance of HPLC-SPE-NMR.

The seed of Hyptis suaveolens, commonly known as wild flour ball (san fen yuan) in Taiwan, serves as a main refreshing drink substance in several regions. This study investigated firstly its secondary metabolites, leading to the isolation of five major caffeoylquinic acid derivatives (1-5) from the ethanol extract. In addition, ten minors, including three caffeoylquinic acid derivatives (12-14), were characterized via assistance of HPLC-SPE-NMR. Of these isolates, sodium 4,5-dicaffeoylquinate (2) and methyl 3,5-dicaffeoylquinate (4) showed moderate inhibitory activity against xanthine oxidase with the respective IC50 values of 69.4 µM and 92.1 µM (c.f. allopurinol IC50 28.4 µM). Quantitative HPLC analysis of the EtOH extract indicates the content of sodium 3,5-dicaffeoylquinate (1) and sodium 4,5-dicaffeoylquinate (2) to be 0.1% and 0.08% (w/w, dry seed), respectively. This study not only discloses the bioactive constituents, but also demonstrates the potential of H. suaveolens seed as an antihyperuricemic nutraceutical.

An Efficient Strategy Based on Liquid-Liquid Extraction With Acid Condition and HSCCC for Rapid Enrichment and Preparative Separation of Three Caffeoylquinic Acid Isomers From Mulberry Leaves.

Morus alba L. is a medicinal plant that contains a high amount of caffeoylquinic acids such as 3-caffeoylquinic acid (3-CQA), 5-caffeoylquinic acid (5-CQA) and 4-caffeoylquinic acid (4-CQA). This study aimed to establish a fast and efficient method for separating caffeoylquinic acids from mulberry leaves by using high-speed countercurrent chromatography coupled with macroporous resin. D101 resin showed better adsorption and desorption capacity for three caffeoylquinic acids among six macroporous resin adsorbents. The contents of 3-CQA, 5-CQA and 4-CQA reached for 4.77%, 18.95% and 9.84% through one cycle of D101 resin, which were 3.13-fold, 4.57-fold and 4.78-fold more than those in crude extracts, respectively. With a two-phase solvent system of ethyl acetate-water (1:1, V/V), >93% purity of target compounds were obtained in one cycle during 150 min with the recovery yields of 80.59%, 99.56% and 94.21% for 3-CQA, 5-CQA and 4-CQA, respectively. The structural identification of target compounds was carried out by ESI-MS, 1H NMR and 13C NMR spectra. The present result represented an easy and efficient separation strategy for the utilization of mulberry resource.

The plant product quinic acid activates Ca2+ -dependent mitochondrial function and promotes insulin secretion from pancreatic beta cells.

BACKGROUND AND PURPOSE: Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. EXPERIMENTAL APPROACH: We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. KEY RESULTS: QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. CONCLUSIONS AND IMPLICATIONS: QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.

Preparative fractionation of 5'-O-caffeoylquinic acid oxidation products using centrifugal partition chromatography and their investigation by mass spectrometry.

When apples are processed into juices or ciders, a great variety of neoformed molecules are generated by enzymatic oxidation of polyphenols. These phenolic oxidation products could be responsible of specific organoleptic properties in apple juices and ciders. 5'-O-Caffeoylquinic acid (CQA) is the major hydroxycinnamic acid in apple and the preferential substrate of apple polyphenoloxidase (PPO). Its main oxidation products were synthetized and purified at the multi-milligrams scale to decipher their structures using mass spectrometry. CQA oxidation products were first synthetized in model solution by oxidizing CQA, in the presence of oxygen and PPO. Then, a specific method involving centrifugal partition chromatography (CPC) was developed to fractionate the main oxidation products corresponding to CQA dehydrodimers (MW 706 Da). For this purpose, CPC was performed in Elution-Extrusion Countercurrent Chromatography (EECCC) mode using a two-phase solvent system precisely selected according to the partition coefficient of the targeted compounds. After a last purification step using semi-preparative reversed phase HPLC, ten CQA dehydrodimers resulting from oxidative coupling were successfully purified, with a UV 280 nm chromatographic purity superior to 85%. Hypothetical structures were formulated for all CQA dehydrodimers based on their UV-vis, MS and MSn spectra. According to this study, centrifugal partition chromatography in combinaison with semi-preparative HPLC was a promising tool to fractionate or purify phenolic oxidation products.

Acidity and Antioxidant Activity of Cold Brew Coffee.

The acidity and antioxidant activity of cold brew coffee were investigated using light roast coffees from Brazil, two regions of Ethiopia, Columbia, Myanmar, and Mexico. The concentrations of three caffeoylquinic acid (CQA) isomers were also determined. Cold brew coffee chemistry was compared to that of hot brew coffee prepared with the same grind-to-coffee ratio. The pH values of the cold and hot brew samples were found to be comparable, ranging from 4.85 to 5.13. The hot brew coffees were found to have higher concentrations of total titratable acids, as well as higher antioxidant activity, than that of their cold brew counterparts. It was also noted that both the concentration of total titratable acids and antioxidant activity correlated poorly with total CQA concentration in hot brew coffee. This work suggests that the hot brew method tends to extract more non-deprotonated acids than the cold brew method. These acids may be responsible for the higher antioxidant activities observed in the hot brew coffee samples.

Preparative separation of caffeoylquinic acid isomers from Lonicerae japonicae Flos by pH-zone-refining counter-current chromatography and a strategy for selection of solvent systems with high sample loading capacities.

Caffeoylquinic acid derivatives exhibit anti-inflammatory, antioxidant, and antibacterial activities. We successfully applied pH-zone-refining counter-current chromatography (pH-ZRCCC) to separation of isomeric caffeoylquinic acids from Lonicerae japonicae Flos using a two-phase solvent system composed of ethyl acetate-n-butanol-acetonitrile-water (3:1:1:5, v/v/v/v). Trifluoroacetic acid (10 mM) was added to the upper phase as a retainer and ammonium hydroxide (10 mM) was added to the lower phase as an eluter. As a result, 167.8 mg of chlorogenic acid, 15.9 mg of isochlorogenic acid B, 103.4 mg of isochlorogenic acid C, and 156.0 mg of isochlorogenic acid A were obtained from 1.2 g of crude extract, and all compound purities were over 96%. In addition, by comparing the numeric values of partition coefficient of compounds, it was found that larger differences between the K values of adjacent compounds in the solvent systems resulted in higher sample loading capacities. pH-ZRCCC method is an efficient preparative separation of isomeric caffeoylquinic acids from natural products.

Homogenate-assisted high-pressure disruption extraction for determination of phenolic acids in Lonicerae Japonicae Flos.

An effective method based on the combined homogenate-assisted high-pressure disruption extraction (HHPDE) was applied to the extraction and determination of the main phenolic acid compounds from Lonicerae Japonicae Flos. The optimized HHPDE showed competitive advantage in yield (The extraction yields of NCA, CA, 3,5DCA and 4,5DCA in HHPDE were 1.21, 1.08, 1.06 and 1.17 fold higher than those in UAE), time-saving (<5 min) and relative low temperature requirement (4-16 °C) compared to HRE and UAE. Furthermore, the HHPDE method behaved a good repeatability and reproducibility according to the HPLC. The mentioned HHPDE method is firstly applied in the extraction and quantification of neochlorogenic acid chlorogenic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid in Lonicerae Japonicae Flos. This work provided an excellent alternative for the extraction and quantification of thermosensitive from plants.

Direct Observation of Hydrangea Blue-Complex Composed of 3-O-Glucosyldelphinidin, Al3+ and 5-O-Acylquinic Acid by ESI-Mass Spectrometry.

The blue sepal color of hydrangea is due to a metal complex anthocyanin composed of 3-O-glucosyldelphinidin (1) and an aluminum ion with the co-pigments 5-O-caffeoylquinic acid (2) and/or 5-O-p-coumaroylquinic acid (3). The three components, namely anthocyanin, Al3+ and 5-O-acylquinic acids, are essential for blue color development, but the complex is unstable and only exists in an aqueous solution. Furthermore, the complex did not give analyzable NMR spectra or crystals. Therefore, many trials to determine the detailed chemical structure of the hydrangea-blue complex have not been successful to date. Instead, via experiments mixing 1, Al3+ and 2 or 3 in a buffered solution at pH 4.0, we obtained the same blue solution derived from the sepals. However, the ratio was not stoichiometric but fluctuated. To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry. In a very low-concentration buffer solution (2.0 mM) at pH 4.0, we reproduced the hydrangea-blue color by mixing 1, 2 and Al3+ in ratios of 1:1:1, 1:2:1 and 1:3:1. All solution gave the same molecular ion peak at m/z = 843, indicating that the blue solution has a ratio of 1:1:1 for the complex. By using 3, the observed mass number was m/z = 827 and the ratio of 1, 3 and Al3+ was also 1:1:1. A mixture of 1, 3-O-caffeoylquinic acid (4) and Al3+ did not give any blue color but instead was purple, and the intensity of the molecular ion peak at m/z = 843 was very low. These results strongly indicate that the hydrangea blue-complex is composed of a ratio of 1:1:1 for 1, Al3+ and 2 or 3.

Chemical constituents from Gnaphalium affine and their xanthine oxidase inhibitory activity.

Gnaphalium affine D. Don, a medicinal and edible plant, has been used to treat gout in traditional Chinese medicine and popularly consumed in China for a long time. A detailed phytochemical investigation on the aerial part of G. affine led to the isolation of two new esters of caffeoylquinic acid named (-) ethyl 1, 4-di-O-caffeoylquinate (1) and (-) methyl 1, 4-di-O-caffeoylquinate (2), together with 35 known compounds (3-37). Their structures were elucidated by spectroscopic data and first-order multiplet analysis. All the isolated compounds were tested for their xanthine oxidase inhibitory activity with an in vitro enzyme inhibitory screening assay. Among the tested compounds, 1 (IC50 11.94 µmol·L-1) and 2 (IC50 15.04 µmol·L-1) showed a good inhibitory activity. The current results supported the medical use of the plant.

Comparison of Polyphenol Profile and Antimutagenic and Antioxidant Activities in Two Species Used as Source of Solidaginis herba - Goldenrod.

European Pharmacopoeia accepts two equivalent species Solidago canadensis L. and S. gigantea Aiton as goldenrod (Solidaginis herba). We compared phytochemical profile of both species from invasive populations in Poland. Further, we compared in vitro antimutagenic and antioxidant activities of solvent extracts from aerial (AP) and underground parts (UP). In S. gigantea, flavonoid profile was dominated by quercetin glycosides, with quercitrin as the major compound. In S. canadensis, quercetin and kaempferol rutinosides were two major constituents. Caffeoylquinic acids (CQAs) were less diverse with 5-CQA as a main compound. In UP, over 20 putative diterpenoids were detected, mostly unidentified. Several CQAs were present in higher amounts than in AP. Antioxidant and antimutagenic activities were different between species and organs, with the strongest inhibition of lipid peroxidation by Et2 O and AcOEt fractions from AP of both species (IC50 13.33 - 16.89 µg/mL) and BuOH fraction from S. gigantea UP (IC50  = 13.32 µg/mL). Chemical mutagenesis was completely inhibited by non-polar fractions, but oxidative mutagenesis was inhibited up to 35% only by S. canadensis. No clear relationship was found between chemical profiles and antimutagenic activity. In conclusion, both species have diverse activity and their phytochemical profiles should be considered in quality evaluation. UP of these weeds can also provide potential chemopreventive substances for further studies.

Semi-Preparative Separation of 10 Caffeoylquinic Acid Derivatives Using High Speed Counter-Current Chromatogaphy Combined with Semi-Preparative HPLC from the Roots of Burdock (Arctium lappa L.).

Burdock roots are healthy dietary supplements and a kind of famous traditional Chinese medicine, which contains large amounts of caffeoylquinic acid derivatives. However, little research has been reported on the preparative separation of these compounds from burdock roots. In the present study, a combinative method of HSCCC and semi-preparative HPLC was developed for the semi-preparative separation of caffeoylquinic acid derivatives from the burdock roots. The ethyl acetate extract of burdock roots was first fractionated by MCI macroporous resin chromatography and give three fractions (Fr. 1-3) from the elution of 40% methanol. Then, these three fractions (120 mg) were separately subjected to HSCCC for purification with the solvent system composed of petroleum ether-ethyl acetate-methanol-water at different volume ratios, and the mixtures were further purified by semi-preparative HPLC. As a result, a total of eight known caffeoylquinic acid derivatives including 3-O-caffeoylquinic acid (32.7 mg, 95.7%), 1,5-O- dicaffeoylquinic acid (4.3 mg, 97.2%), 3-O-caffeoylquinic acid methyl ester (12.1 mg, 93.2%), 1,3-O-dicaffeoylquinic acid (42.9 mg, 91.1%), 1,5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (4.3 mg, 84.5%), 4,5-O-dicaffeoylquinic acid (5.3 mg, 95.5%), 1,5-O-dicaffeoyl-3-O-succinylquinic acid (8.7 mg, 93.4%), and 1,5-O-dicaffeoyl-4-O-succinylquinic acid (1.7 mg, 91.8%), and two new compounds were obtained. The new compounds were 1,4-O-dicaffeoyl-3-succinyl methyl ester quinic acid (14.6 mg, 96.1%) and 1,5-O-dicaffeoyl-3-O-succinyl methyl ester quinic acid (3.1 mg, 92.6%), respectively. The research indicated that the combination of HSCCC and semi-preparative HPLC is a highly efficient approach for preparative separation of the instability and bioactive caffeoylquinic acid derivatives from natural products.