Genes to specialized metabolites: accumulation of scopoletin, umbelliferone and their glycosides in natural populations of Arabidopsis thaliana.

BACKGROUND: Scopoletin and umbelliferone belong to coumarins, which are plant specialized metabolites with potent and wide biological activities, the accumulation of which is induced by various environmental stresses. Coumarins have been detected in various plant species, including medicinal plants and the model organism Arabidopsis thaliana. In recent years, key role of coumarins in maintaining iron (Fe) homeostasis in plants has been demonstrated, as well as their significant impact on the rhizosphere microbiome through exudates secreted into the soil environment. Several mechanisms underlying these processes require clarification. Previously, we demonstrated that Arabidopsis is an excellent model for studying genetic variation and molecular basis of coumarin accumulation in plants. RESULTS: Here, through targeted metabolic profiling and gene expression analysis, the gene-metabolite network of scopoletin and umbelliferone accumulation was examined in more detail in selected Arabidopsis accessions (Col-0, Est-1, Tsu-1) undergoing different culture conditions and characterized by variation in coumarin content. The highest accumulation of coumarins was detected in roots grown in vitro liquid culture. The expression of 10 phenylpropanoid genes (4CL1, 4CL2, 4CL3, CCoAOMT1, C3'H, HCT, F6'H1, F6'H2,CCR1 and CCR2) was assessed by qPCR in three genetic backgrounds, cultured in vitro and in soil, and in two types of tissues (leaves and roots). We not only detected the expected variability in gene expression and coumarin accumulation among Arabidopsis accessions, but also found interesting polymorphisms in the coding sequences of the selected genes through in silico analysis and resequencing. CONCLUSIONS: To the best of our knowledge, this is the first study comparing accumulation of simple coumarins and expression of phenylpropanoid-related genes in Arabidopsis accessions grown in soil and in liquid cultures. The large variations we detected in the content of coumarins and gene expression are genetically determined, but also tissue and culture dependent. It is particularly important considering that growing plants in liquid media is a widely used technology that provides a large amount of root tissue suitable for metabolomics. Research on differential accumulation of coumarins and related gene expression will be useful in future studies aimed at better understanding the physiological role of coumarins in roots and the surrounding environments.

Skimmin ameliorates cardiac function via the regulation of M2 macrophages in a myocardial infarction mouse model.

PURPOSE: Myocardial infarction (MI) is a coronary artery disorder with several complications, such as inflammation, oxidative stress, and cardiac fibrosis. The current study is aimed to explore the protective effect of skimmin (SKI) on impaired heart tissues in MI. METHODS: A mouse model of MI was induced by ligation of the left anterior descending artery. SKI was intragastric administration for 7 days after MI. Masson staining was then conducted to measure the area of fibrosis in the myocardium. The expression levels of collagen I and collagen III were analyzed using Western blot. The levels of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and inflammatory factor were also detected. The expression of M1 polarization markers and M2 polarization markers in mice and LPS-induced RAW264.7 cells were detected using RT-qPCR and Western blot, respectively. Finally, the migration and proliferation of vascular smooth muscle cells (VSMCs) in vitro were analyzed using transwell and EDU, respectively. RESULTS: SKI improved cardiac function and cardiac fibrosis in mice with MI. SKI also decreased collagen I and collagen III expression, and inhibited inflammatory factor TNF-α, IL-1ß, and IL-6 levels. SKI decreased the levels of MDA and increased the levels of GSH and SOD. Meanwhile, SKI could promote M2 macrophage polarization in vivo and in vitro. SKI could also repress the migration and proliferation of VSMCs. CONCLUSIONS: SKI may ameliorate inflammation, oxidative stress, and cardiac fibrosis of MI by promoting M2 polarization.

Simultaneous determination of skimmin, apiosylskimmin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in rat plasma by liquid chromatography-Orbitrap mass spectrometry and its application to pharmacokinetics.

The effective fraction of coumarin glycosides from Hydrangea paniculata Sieb (HP) has been under development for the treatment of chronic kidney disease for years. Skimmin and apiosylskimmin are the main coumarin glycosides of HP, and the major metabolites in rats are 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG). In this study, a sensitive and reliable liquid chromatography-Orbitrap mass spectrometry method was developed for the simultaneous determination of skimmin, apiosylskimmin, 7-HC and 7-HCG in rat plasma. The chromatographic separation was performed on a Zobax SB C18 column (2.1 × 100 mm, 3.5 µm) at a flow rate of 0.3 ml/min with a gradient mobile phase of water and acetonitrile containing 0.2% formic acid. Skimmin, apiosylskimmin and 7-HCG were detected in targeted-selected-ion-monitoring mode at positive ions m/z of 325.0911, 457.1331 and 339.0703, respectively. 7-HC and the internal standard were detected in parallel-reaction-monitoring mode at m/z 163.0387 → 119.0492 and 260.1641 → 116.1071 to overcome the carryover of 7-HC. Linearity was obtained for the analytes within the ranges 20-2,000 ng/ml for skimmin, 5-500 ng/ml for apiosylskimmin and 7-HC and 100-10,000 ng/ml for 7-HCG. Validation parameters were all in line with the criteria of international guidance. The method has been applied to the pharmacokinetic study of HP in rats.

Skimmin protects diabetic cardiomyopathy in streptozotocin-induced diabetic rats.

Skimmin, a natural coumarin derivate, has been showed to be protective against experimental diabetic nephropathy; however, its protective effect on diabetic cardiomyopathy (DCM) is not clarified. By using in vitro and in vivo models, we investigated skimmin's protective effect on impaired heart tissues in DCM. DCM was induced by streptozotocin (STZ, 60 mg/kg) using Sprague Dawley rats, and diabetic rats were treated with either skimmin (15 or 30 mg/kg) or the vehicle for 16 weeks, and normal rats were used as a control. Hematoxylin and eosin and Masson's trichrome staining were performed to evaluate the cardiac histopathology, and the oxidative stress and proinflammation cytokines in heart tissues were measured. The protein levels of key mediators in fibrosis, pyroptosis, and autophagy in heart tissues were investigated using western blotting. In vitro, primary neonatal cardiomyocytes were treated with skimmin (2 and 10 µM) under stimulation by high glucose (30 mM) and low glucose (5 mM) respectively, and the molecular mechanisms on pyroptosis and autophagy were studied. Compared to the vehicle-treated DCM group, skimmin treatment significantly improved the ejection fraction and fractional shortening of the left ventricle and reduced the oxidative stress by increasing the glutathione level and activity of superoxide dismutase and catalase. Skimmin also reduced cardiac fibrosis, and decreased proinflammation cytokines in cardiac tissues. Mechanism studies showed skimmin may enhance the autophagy and ameliorate NLRP3 inflammasome activation to play a protective role in DCM. This study, for the first time, indicates that skimmin might be a promising lead compound for DCM.

Determination and pharmacokinetic study of skimmin by UHPLC-MS/MS in rat plasma.

Skimmin, a major active ingredient derived from Hydrangea paniculata, has been considered to possess various pharmacological activities, including renoprotective activity, anti-inflammatory, anti-cancer, and antiamoebic properties. However, no investigation has reported the quantification and pharmacokinetics of skimmin in biomatrices. In the present study, we established and validated an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the estimation of skimmin in rat plasma, which was successfully applied to explore the oral and intravenous pharmacokinetics of skimmin. All plasma samples were obtained following blood collection from the rat' tail vein and prepared using the protein precipitation method with acetonitrile. Separation of the analyte and internal standard (IS) magnoflorine was achieved by a reversed phase T3 column. The mobile phase consisted of water containing 0.1 % formic acid and acetonitrile with a gradient elution program. The analytical run time was 4 min with a flow rate of 0.3 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI).Multiple reaction monitoring transitions were performed at m/z of 325.34 → 163.00 and 342.24 → 57.98 for skimmin and IS, respectively. The method demonstrated good linearity in the range of 2-2000 ng/mL and was validated by US FDA bioanalytical guidelines. A pharmacokinetic study of skimmin was then successfully conducted using the validated method. Hence, the absolute bioavailability of skimmin was approximately 25.08 % with rapid absorption and elimination. This study will be beneficial in better understanding the pharmacological properties and the further development of skimmin.

human monoamine oxidase (hMAO) A and hMAO B inhibitors from Artemisia dracunculus L. herniarin and skimmin: human mononamine oxidase A and B inhibitors from A. dracunculus L.

The aim of this study was to investigate the effects of extracts and pure Artemisia dracunculus L. (tarragon) metabolites on the antimonoamine oxidase and anticholinesterase activities. The compounds were characterized as stigmasterol (1), herniarin (2), (2E,4E)-1-(piperidin-1-yl)undeca-2,4-diene-8,10-diyn-1-one (3), (2E,4E)-N-isobutylundeca-2,4-dien-8,10-diynamide (4), 3,4-dehydroherniarin (5) and skimmin (6) by 1H-NMR, 13C-NMR, 1D and 2D NMR methods. The compounds 5 and 6 were isolated from tarragon for the first time. The extracts and pure compounds have inhibitory effects on the human monoamine oxidase (hMAO) A and B enzymes, whereas they did not exhibit any anticholinesterase activities. Among the tarragon compounds, only 2 and 6 compounds showed the inhibitory effects against hMAO A (IC50 = 51.76 and 73.47 µM, respectively) and hMAO B (IC50 = 0.84 and 1.63 mM, respectively). In the study, herniarin content in the extracts was also analysed by high-performance liquid chromatography and it was found that there was a relationship between the inhibition effects of the extracts and their herniarin content.

Skimmin Improves Insulin Resistance via Regulating the Metabolism of Glucose: In Vitro and In Vivo Models.

Skimmin is the major pharmacologically active component present in Hydrangea paniculata, in the traditional Chinese medicine as an anti-inflammatory agent, and its anti-inflammation and anti-diabetic effect has had been studied in previous studies. The metabolism of glucose plays an important role in the pathophysiology of diabetes. Therefore, it was identified as an important target for improving diabetic. Herein, we found that skimmin relieved the palmitic acid and high-fat and high sugar-induced insulin resistance. Furthermore, skimmin enhanced the glucose uptake via inhibiting reactive oxygen species (ROS) and reducing the level of inflammatory correlation factor. Meanwhile, skimmin reduced the glucose output by promoting PI3K/Akt signaling pathway and down-regulating the expression of glycogen synthase kinase-3ß (GSK3ß) and glucose-6-phosphatase (G6Pase). In conclusion, skimmin can improve the insulin resistance by increasing glucose uptake and decreasing glucose output in vitro and in vivo.

Chemical constituents and analgesic activity of the aerial parts of skimmia multinervia / 中国药学杂志

OBJECTIVE: To study the chemical constituents and analgesic activity of the aerial parts of Skimmia multinervia. METHODS: The chemical constituents were isolated by column chromatography packed with MCI and silica gel, respectively, and the structures of the purified compounds were elucidated by NMR and MS analysis. The analgesic activities of the extract, fractions and selected compounds from the aerial parts of S. multinervia were evaluated by using acetic acid-induced writhing test in mice model. RESULTS: The structures of compounds 1-13 are identified as skimmin (1), imperatorin (2), auraptene (3), umbelliferon (4), scopoletin (5), limonin (6), limonexic acid (7), nomilin (8), citrusin (9), taraxerol (10), kanalpin (11), skimmianine (12), and methyl vanillate (13). The extract, ethyl acetate fraction, and alkaloid fraction could reduce the times of mice writhing induced by acetic acid, and the analgesic effect of compound 3, with inhibition rate of 88.18%, was much better than that of the positive control, aspirin. CONCLUSION: Compounds 1-13 are isolated from the plant for the first time, while 7, 9, 11, and 13 are firstly isolated from Skimmia genus. The analgesic activities of S. multinervia and compound 3 are reported for the first time.