Bioprospecting of Artemisia genus: from artemisinin to other potentially bioactive compounds.

Species from genus Artemisia are widely distributed throughout temperate regions of the northern hemisphere and many cultures have a long-standing traditional use of these plants as herbal remedies, liquors, cosmetics, spices, etc. Nowadays, the discovery of new plant-derived products to be used as food supplements or drugs has been pushed by the exploitation of bioprospection approaches. Often driven by the knowledge derived from the ethnobotanical use of plants, bioprospection explores the existing biodiversity through integration of modern omics techniques with targeted bioactivity assays. In this work we set up a bioprospection plan to investigate the phytochemical diversity and the potential bioactivity of five Artemisia species with recognized ethnobotanical tradition (A. absinthium, A. alba, A. annua, A. verlotiorum and A. vulgaris), growing wild in the natural areas of the Verona province. We characterized the specialized metabolomes of the species (including sesquiterpenoids from the artemisinin biosynthesis pathway) through an LC-MS based untargeted approach and, in order to identify potential bioactive metabolites, we correlated their composition with the in vitro antioxidant activity. We propose as potential bioactive compounds several isomers of caffeoyl and feruloyl quinic acid esters (e.g. dicaffeoylquinic acids, feruloylquinic acids and caffeoylferuloylquinic acids), which strongly characterize the most antioxidant species A. verlotiorum and A. annua. Morevoer, in this study we report for the first time the occurrence of sesquiterpenoids from the artemisinin biosynthesis pathway in the species A. alba.

Effects of Dicaffeoylquinic Acids from Ilex kudingcha on Lipid Metabolism and Intestinal Microbiota in High-Fat-Diet-Fed Mice.

Kudingcha made from the leaves of Ilex kudingcha and chlorogenic acid have antiobesity and intestinal microbiota modulating effects. However, the effects of kudingcha dicaffeoylquinic acids (diCQAs) on obesity and intestinal microbiota are still poorly understood. In the present study, the effects of kudingcha diCQAs on adipose accumulation and intestinal microbiota were investigated in high-fat-diet-fed mice. As a result, kudingcha diCQAs decreased the liver and adipose tissue masses, concentrations of serum inflammatory factors, and hepatic expressions of lipid synthesis related genes and increased the expressions of genes involved in lipid degradation in the liver. Kudingcha diCQAs also exhibited considerable effects on intestinal microbiota. They increased the relative abundances of Bifidobacterium and Akkermansia and affected the function of the microbial community including bile acid biosynthesis. Kudingcha diCQAs had antiobesity potential, possibly acting through affecting intestinal microbiota. Furthermore, the effects of kudingcha diCQAs on fat accumulation and intestinal microbiota had a dose-dependent manner.

Simultaneous determination of three di-caffeoylquinic acids by UHPLC-MS/MS in rat plasma and its application to a comparative pharmacokinetic study in normal and acute lung injury rat.

Acute lung injury (ALI) is a severe inflammatory disease with high mortality rates. Di-caffeoylquinic acids (DCQAs), the bioactive components of reduning injection (RDN), may play important roles in the protective effect on acute lung injury (ALI). A selective and rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for the simultaneous determination of 3,4-, 3,5- and 4,5-DCQA in rat plasma. The DCQAs were extracted by liquid-liquid extraction with ethyl acetate-isopropyl alcohol (7:3, v/v). Chromatographic separation was accomplished on a C18 column using gradient elution. Detection was performed in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were all 2.0ng/mL for the three analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -13.8% to 10.0%, and the mean extraction recoveries of analytes from rat plasma were all more than 72.9%. Meanwhile, this method had been successfully applied to compare the pharmacokinetics of the three DCQAs in normal and ALI model rat after RDN was given intravenously administration. The pharmacokinetic parameters of the 3,4-, 3,5- and 4,5- DCQA were remarkably different from those in normal rats. It might result from the effects of the pathological status of ALI. This study presented a meaningful basis for the clinical applications of RDN when used in the treatment of ALI.

Identification of anti-inflammatory constituents from Kalimeris indica with UHPLC-ESI-Q-TOF-MS/MS and GC-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Kalimeris indica is a Miao׳s medicinal plant in Guizhou province of China employing to treat various inflammation-related diseases in clinical. The study aims to determine the active fractions of K. indica for its anti-inflammatory activity and to identify their chemical constituents. MATERIAL AND METHODS: The dried K. indica herb was extracted with 50% aqueous ethanol and then successively separated with macroporous resin and MCI column chromatography to give five fractions (A-E). The anti-inflammatory effects were determined by measuring the NO and TNF-α production in murine macrophage RAW 264.7 cells after exposure to LPS. The chemical constituents of the anti-inflammatory fractions were analyzed by the method of UHPLC-ESI-Q-TOF/MS or GC-MS. RESULTS: Five fractions (A-E) of different polarities were prepared from the 50% ethanol extract. Factions C and E showed significant inhibition of NO and TNF-α production. Six constituents, namely 3,4-dicaffeoylquinic acid (1), 3,5-dicaffeoylquinic acid (2), 1,5-dicaffeoylquinic acid (3), rutin (4), 1-malonyl-3,5-dicaffeoylquinic acid (5), and 4,5-dicaffeoylquinic acid (6) were identified from the active fraction C by UHPLC-ESI-Q-TOF/MS. Four compounds including 13-tetradecenal (7), (Z,Z)-9,12-octadecadienoic acid (8), (3α)-12-oleanen-3-yl acetate (9), and (+)-3-oxo-urs-12-en-24-oic acid methyl ester (10) were identified from the active fraction E by GC-MS. CONCLUSION: K. indica possessed pronounced anti-inflammatory effect. Dicaffeoylquinic acids and their dirivatives, rutin, as well as oleanolic and fatty acid derivatives are the major constituents and possibly the anti-inflammatory principles of the active fractions of K. indica. All the compounds were identified in K. indica for the first time. The work provided evidence for further development and utilization of K. indica and formed a basis for the establishment of quality control methods and standards for K. indica and its pharmaceutical preparations.

Comparative pharmacokinetics and tissue distribution study of mono-, and di-caffeoylquinic acids isomers of Ainsliaea fragrans Champ by a fast UHPLC-MS/MS method.

Ainsliaea fragrans Champ, as a well-known herb in Traditional Chinese Medicine, was often used in the treatment of gynecological diseases. Caffeoylquinic acids (CQAs) were the bioactive constituents of this plant medicine which primarily contains mono-CQAs (MCQA) and di-CQAs (DCQA). The biosynthesis showed that MCQAs were the precursor of DCQAs. Recent literatures manifested some particular features of DCQAs, different from MCQAs. Therefore it is apparent that a complete and scientific assessment of DCQAs and MCQAs should include not only the DCQAs' pharmacokinetics and distribution but also its degradation products. So an efficient, sensitive rapid resolution liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of the active ingredients in rat plasma and different tissues had been developed and validated. Mass spectrometric detection was performed by selected reaction monitoring mode (MRM) via an electrospray ionization source operating in negative ionization mode. The method was validated in plasma and tissue samples, which showed good linearity over a wide concentration range (r(2)>0.99), and obtained lower limit of quantification (LLOQ) was 2.34 ng·mL(-1) for the analytes in biological samples. The intra- and inter-day assay variability was less than 15%, and the accuracy was between -8.8% and 5.7%. This study provided the pharmacokinetic profiles and the tissue regional distribution of MCQAs, DCQAs and caffeic acid. The results indicated that the DCQAs isomers were absorbed quickly after oral administration and degradation products MCQAs were mostly found in tissues, not in plasma. Besides, 1,5-DCQA was the prior configuration for the isomerization phenomenon. The small intestine was the main absorption site for DCQAs. Interestingly, the content of the DCQA and MCQA isomers was all high in the ovary and uterus, and some could pass through the barrier between the blood and brain obviously.

Pretreatment of SH-SY5Y cells with dicaffeoylquinic acids attenuates the reduced expression of nicotinic receptors, elevated level of oxidative stress and enhanced apoptosis caused by ß-amyloid peptide.

OBJECTIVES: This in vitro investigation was designed to examine potential neuroprotection by dicaffeoylquinic acids (diCQAs) extracted from a traditional Chinese medicinal herb herba erigerontis and their effects against the toxicity induced by ß-amyloid peptide (Aß25-35 ). METHODS: The neuroblastoma SH-SY5Y cell line was treated with Aß or 3, 4-diCQA, 3, 5-diCQA or 4, 5-diCQA. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction was assayed by spectrophotometrical method, lipid peroxidation (malondialdehyde) on the basis of the level of thiobarbituric acid-reactive substance, the activity of superoxide dismutase by the xanthine oxidase procedure, the frequency of apoptosis by flow cytometry, and the levels of α3 and α7 nicotinic acetylcholine receptor (nAChR) subunit proteins by Western blotting. KEY FINDINGS: When the cells were exposed to Aß25-35 , MTT reduction declined, oxidative stress and apoptosis were enhanced, and the expression of α3 and α7 nAChR subunit proteins was lowered. Expression of the α7 nAChR subunit protein was increased by all three diCQAs, and the level of α3 was increased by 3, 5-diCQA and 4, 5-diCQA. Significantly, pretreatment with diCQAs attenuated the neurotoxic effects of Aß25-35 , a neuroprotective effect in which the upregulation of α7 and α3 nAChR may be involved. CONCLUSION: The diCQAs exert a protective effect on Aß-induced neurotoxicity in SH-SY5Y cells and a potential underlying mechanism involving stimulation of nAChRs.

Antioxidant and genoprotective effects of spent coffee extracts in human cells.

Spent coffee has been shown as a good source of hydrophilic antioxidant compounds. The ability of two spent coffee extracts rich in caffeoylquinic acids, mainly dicaffeoylquinic acids, and caffeine (Arabica filter and Robusta espresso) to protect against oxidation and DNA damage in human cells (HeLa) was evaluated at short (2 h) and long (24 h) exposure times. Cell viability (MTT) was not affected by spent coffee extracts (>80%) up to 1000 µg/mL after 2 h. Both spent coffee extracts significantly reduced the increase of ROS level and DNA strand breaks (29-73% protection by comet assay) induced by H2O2. Pretreatment of cells with robusta spent coffee extract also decreased Ro photosensitizer-induced oxidative DNA damage after 24 h exposure. The higher effectiveness of Robusta spent coffee extract, with less caffeoylquinic acids and melanoidins, might be due to other antioxidant compounds, such as caffeine and other Maillard reaction products. This work evidences the potential antioxidant and genoprotective properties of spent coffee in human cells.