Comparative Contents of Polyphenolic Compounds in Aerial Parts of Cirsium esculentum, C. serratuloides, and Ancathia igniaria (Asteraceae).

Because Ancathia igniaria (Spreng.) DC. (Cirsium igniarium Spreng.) has been segregated as a monotypic genus from the genus Cirsium on the basis of phylogenetic data, chemotaxonomic differences are of interest to detect in the composition of polyphenolic components of aerial plant parts. Phenolic compounds are of chemotaxonomic significance in a number of genera and families. The polyphenolic profile of aerial parts was therefore compared for Cirsium esculentum (Siev.) C.A. Mey., Cirsium serratuloides (L.) Hill, and A. igniaria. The last two species were for the first time examined in this context. The compounds were identified against known standard via high-performance liquid chromatography (HPLC). The species of the genus Cirsium were found to have similar compositions of simple phenols, but differ in the set of flavonoids. Six to eight phenolic compounds were detected in the species, and three simple polyphenols (syringin, chlorogenic acid, and ethyl gallate) proved to be common. The flavonoid profiles of aerial parts included rutin in both Cirsium species. Cymaroside and quercetin-3-O-ß-D-diglucoside-O-α-L-rhamnoside were species specific for C. serratuloides; salipurposide and hyperoside, for C. esculentum. An extract of A. igniaria aerial parts contained cinaroside (like in C. serratuloides), chrysin 7-O-glucoside, and eriodictyol. A greater difference in flavonoid composition was observed between the genera Cirsium and Ancathia. Data on phenolic compound composition are of importance for chemosystematics and use of plants as medicinal raw materials. The total content of coumarins, aglycones, and flavonoid glycosides in the species was determined by a spectrophotometric method. The contents of flavonoids and coumarins in C. esculentum and C. serratuloides were comparable and exceeded their contents in A. igniaria. Thus, A. igniaria proved to differ from the genus Cirsium in the quantitative and qualitative composition of phenolic compounds.

[Chemome profiling of Qijiao Shengbai Capsules by UPLC-IT-TOF-MS].

Qijiao Shengbai Capsules(QJ) are a common Miao medicine serving as an adjuvant cancer therapy in clinical practice.QJ consists of seven medicinal materials such as Astragalus membranaceus and Lespedeza buergeri.Its chemical components have not been clarified and the quality control needs to be improved.In this study, LC-IT-TOF-MS was used to comprehensively collect MS~1 and MS~2 fragment information of QJ and rapidly identify the chemical compositions.The chromatographic separation was performed on the Capcell core ADME column(2.1 mm×150 mm, 2.7 µm) with 0.1% formic acid aqueous solution(A) and acetonitrile(B) as mobile phases for gradient elution.High-resolution mass spectrometric information was obtained by scanning in the positive and negative ion ESI modes.A total of 107 compounds were structurally identified according to the deduced MS fragmentation patterns and comparison with standards and data reported in the literature, including 54 flavonoids, 16 phthalides, 13 alkaloids, 12 phenolic acids, 7 saponins, 2 coumarins, 2 condensed tannins, and 1 purine.This study clarified the chemical composition of QJ and provided references for the improvement of its quality standards and the elucidation of its medicinal substances.

Deciphering the chemical constituents of Shengjiang Xiexin decoction by ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry and the impact of 20 characteristic components on multidrug resistance-associated protein 2 in the vesicular transport assay.

Shengjiang Xiexin decoction, a traditional Chinese medical formula, has been utilized to alleviate the delayed-onset diarrhea induced by irino tecan. However, the chemical constituents of this formula and the activities of its constituents remain unclear. In this study, ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry was employed to comprehensively analyze the chemical constituents of Shengjiang Xiexin decoction. A total of 270 components, including flavonoids, coumarins, triterpenoids, alkaloids, diarylheptanoids and others, were identified or characterized. Multidrug resistance-associated protein 2 is an efflux transporter responsible for regulating drug absorption. A total of 20 characteristic components from the formula were selected to evaluate their effects on the function of multidrug resistance-associated protein 2 using the vesicular transport assay. Glycyrrhizic acid and glycyrrhetinic acid were identified as potential multidrug resistance-associated protein 2 inhibitors, while 9 flavonoid aglycones increased the uptake of the substrate [3 H]-estradiol 17-ß-glucuronide in the vesicles. This was the first systematic investigation of the chemical constituents from Shengjiang Xiexin decoction and the effect of its characteristic components on the transporter. The results offered a basis for further exploring the detoxification mechanisms of this formula and its interactions with other drugs.

Simultaneous Determination of Five Coumarins in Peucedanum Decursivum Radix by UPLC.

OBJECTIVE: To establish an ultra-high performance liquid chromatography (UPLC) method for simultaneous determination of umbelliferonel, nodakenin, psoralen, xanthotoxin and bergapten contents in Peucedanum decursivum Radix. METHODS: The analysis was achieved on a Symmetry®C18 column (4.6 mm × 250 mm, 5 µm), with acetonitrile and water as the mobile phase in gradient elution mode. The column temperature was maintained at 30°C, with flow rate 1.0 mL·min-1. The injection volume of sample was 10 µL. The ultraviolet detection wavelength was set at the maximum absorption wavelength 325 nm for umbelliferonel and nodakenin, 259 nm for psoralen, xanthotoxin and bergapten, respectively. RESULTS: The five kinds of coumarins in Peucedanum decursivum Radix were separated well and the linear relation was obtained (R2 ≥ 0.9998). The average recoveries were 101.31, 105.27, 90.85, 106.42 and 90.19%, respectively, with Relative standard deviation (RSD) 3.07, 3.17, 1.62, 2.53 and 4.54%, respectively. CONCLUSIONS: The established method was accurate and feasible, which could be used as the basis of quality control of Peucedanum decursivum Radix.

Metabolic Profile of C-Prenyl Coumarins Using Mass Spectrometry-Based Metabolomics.

C-prenyl coumarins (C-PYCs) are compounds with similar structures and various bioactivities, which are widely distributed in medicinal plants. Until now, the metabolic characterizations of C-PYCs and the relationship between metabolism and bioactivities remain unclear. In this study, ultra-performance chromatography electrospray ionization quadrupole time-of-flight mass spectrometry-based metabolomics (UPLC-ESI-QTOF-MS) was firstly used to determine the metabolic characterizations of three C-PYCs, including meranzin hydrate (MH), isomeranzin (ISM), and meranzin (MER). In total, 52 metabolites were identified, and all of them were found to be novel metabolites. Among these metabolites, 10 were from MH, 22 were from ISM, and 20 were from MER. The major metabolic pathways of these C-PYCs were hydroxylation, dehydrogenation, demethylation, and conjugation with cysteine, N-acetylcysteine, and glucuronide. The metabolic rate of MH was much lower than ISM and MER, which was only 27.1% in MLM and 8.7% in HLM, respectively. Additionally, recombinant cytochrome P450 (CYP) screening showed that CYP1A1, 2B6, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of metabolites. Further bioactivity assays indicated that all of these three C-PYCs exhibited anti-inflammatory activity, but the effects of ISM and MER were slightly higher than MH, accompanied by a significant decrease in inflammatory cytokines transcription induced by lipopolysaccharide (LPS) in macrophages RAW 264.7. Taken together, the metabolic characterizations of the three C-PYCs suggested that the side chain of the prenyl group may impact the metabolism and biological activity of C-PYCs.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of six compounds in Guizhi Fuling capsule and tablet samples.

A fast and simple ultra-high performance supercritical fluid chromatography method has been developed for the determination of six analytes, namely (paeonol, coumarin, cinnamic alcohol, cinnamic acid, paeoniflorin, and amygdalin) in Guizhi Fuling capsule and tablet samples. The influence of the key chromatographic parameters for the separation purposes was evaluated. The optimal column was Trefoil CEL1 column. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol at flow rate of 1.0 mL/min. The back pressure of the system was set to 1.38 × 107  Pa and the temperature to 45°C. The six compounds were separated within 11 min by the proposed ultra-high performance supercritical fluid chromatography method with satisfactory resolution. Method validation confirmed that the procedure is accurate with the recovery rates from 87.04 to 104.30%, intraday precision values less than 4.81% and interday precision less than 5.22%, and linear with R2 higher than 0.9967. Therefore, this work provides a simple and novel method for the simultaneous analysis of six compounds in Guizhi Fuling capsule and tablet samples.

Preclinical safety assessment of the crude extract from Sida rhombifolia L. aerial parts in experimental models of acute and repeated-dose 28 days toxicity in rats.

Sida rhombifolia (Malvaceae) is popularly used as a treatment for several pathological conditions; however, there is a lack of studies that identify its compounds and that evaluate comprehensively the safety of its consumption. Therefore, the aim of this study was to determinate the phytochemical constitution of the crude extract of Sida rhombifolia (CESR), and its safety in models of acute and repeated doses (28 days) toxicity. The tested dose for the model of acute toxicity was 2000 mg/kg doses for the repeated dose model were 150, 300 e 600 mg/kg. Hematological, biochemical, histopathological and oxidative markers were investigated. HPLC-DAD-MS analysis evidenced the presence of caffeic acid, coumarin, and rutin. In the acute toxicity model the only altered parameters were tissue ROS, and AST and BUN in serum. As for the repeated dose experiment both hematological and biochemical markers remained within the values of reference for the species. Obtained results demonstrate that the CESR did not present significant toxic effects when administrated orally to male and female rats in acute and repeated doses.

De novo transcriptome assembly of Angelica dahurica and characterization of coumarin biosynthesis pathway genes.

Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav (A. dahurica) is a famous Chinese herb known for the production of coumarins, important secondary metabolites with wide-ranging pharmacological activities. In particular, the methoxylated coumarins like those produced by A. dahurica are known for their anti-inflammatory, anti-cancer, and anti-oxidant pharmacological effects. However, the molecular mechanism of coumarin biosynthesis in A. dahurica has not been studied. Such investigation could help scientists harness the biosynthesis potential of methoxylated coumarins. Here we present, three transcriptomes corresponding to leaf, root, and stem tissues of A. dahurica. A total of 114,310 unigenes with an average length of 1118 bp were de novo assembled, and 81,404 (71.21%) of those unigenes were annotated. Then, 181 unigenes encoding the seven key enzymes involved were identified, for which COMT (Caffeic acid 3-O-methyltransferase) was spatially used in a phylogenetic analysis, and some of these key enzyme genes were verified by qRT-PCR. Differentially expressed genes and root-specific-expressed genes were identified, by comparing genes' profile activity between roots and other tissues. Furthermore, multiple genes encoding key enzymes or transcription factors related to coumarin biosynthesis were identified and analyzed. This study is the first to report comprehensive gene information of A. dahurica at the transcriptional level, and to distinguish candidate genes related to its biosynthesis of coumarin, thus laying a foundation for this pathway's further exploration in A. dahurica.

Chemical characterization and in vitro immunomodulatory effects of different extracts of moss Hedwigia ciliata (Hedw.) P. Beauv. from the Vrsacke Planine Mts., Serbia.

Bioactive compounds from natural sources are of great importance because of their potential pharmacological activity and tremendous structural diversity. In this study, the chemical composition of different moss extracts of Hedwigia ciliata P. Beauv. have been examined, as well as their antioxidant, antineurodegenerative/anti-neuroinflammatory, antidiabetic, and antiproliferative potential. The extracts were prepared by Soxhlet extractor using solvents of different polarity. Chemical characterization of the extracts revealed the presence of phenolics and flavonoid compounds, together with triterpenoids as secondary metabolites of high biological activity. Significant antioxidant properties of all the extracts were exhibited using the ß-carotene assay. The highest activities were found for water:ethanol extract (with the highest inhibition rate of 96%), but also significant inhibition was measured for ethanol and ethyl acetate extracts (80% and 70%, respectively). Confirmation of biocompatibility of investigated moss extracts has been performed using normal human fibroblast cell line, MRC-5. The H. ciliata extracts exhibited significant antiproliferative activity (~ 50%) against the MDA-MB-231 (human breast adenocarcinoma cell line), which has not previously been reported elsewhere. The Griess assay confirmed the potential anti-neuroinflammatory activity of the extracts, as significant effects in reducing NO production by LPS-stimulated BV2 (normal murine microglia cell line) was observed. This data is in line with noted antineurodegenerative potential measured by the inhibition of acetylcholinesterase (with the highest inhibition rate of 60% for ethyl acetate extract) and tyrosinase (with the highest inhibition rate of 70% for ethanol extract). Additionally, the H. ciliata extracts exhibited significant antidiabetic effect mediated by α-glucosidase inhibition (with the highest inhibition rate of 80% for ethyl acetate extract). The obtained data suggest the presence of immunomodulatory effects of the moss extracts in vitro, which allows the design of new experiments aimed at detecting and characterizing bioactive compounds of the extracts and additionally elucidate detailed mechanisms of their effects.

Enzyme-Instructed Self-Assembly Enabled Monomer-Excimer Transition to Construct Higher Ordered Luminescent Supramolecular Assembly for Activity-based Bioimaging.

It is challenging to construct high-performing excimer-based luminescent analytic tools at low molecular concentrations. We report that enzyme-instructed self-assembly (EISA) enables the monomer-excimer transition of a coumarin dye (Cou) at low molecular concentrations, and the resulting higher ordered luminescent supramolecular assemblies (i.e., nanofibers) efficiently record the spatiotemporal details of alkaline phosphatase (ALP) activity in vitro and in vivo. Cou was conjugated to short self-assembly peptides with a hydrophilic ALP-responsive group. By ALP triggering, EISA actuated a nanoparticles-nanofibers transition at low peptide concentrations followed by monomer-excimer transition of Cou. Analysis of structure-property relationships revealed that the self-assembly motif was a prerequisite for peptides to induce the monomer-excimer transition of Cou. Luminescent supramolecular nanofibers of pYD (LSN-pYD) illuminated the intercellular bridge of cancer cells and distinguished cancer cells (tissues) from normal cells (tissues) efficiently and rapidly, promising potential use for the early diagnosis of cancer. This work extends the functions of EISA and provides a new application of supramolecular chemistry.

Accurate and sensitive detection of Catechol-O-methyltransferase activity by liquid chromatography with fluorescence detection.

Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.

Dihydropyridine-coumarin-based fluorescent probe for imaging nitric oxide in living cells.

Nitric oxide (NO) is a messenger molecule in organisms, participating in the regulation of many biological processes. The abnormal expression of NO is often observed in a variety of diseases, including cerebral ischemia, atherosclerosis, and cancer. However, a suitable tool that can directly and sensitively detect NO in vitro and in vivo is important for understanding its various biological functions. In this report, a new fluorescent probe for nitric oxide, DHP-4, was prepared, based on dihydropyridine-coumarin. DHP-4 was able to greatly enhance the fluorescence of NO, but did not affect the fluorescence emissions of other reactive oxygen species and nitrogen species, demonstrating its highly selective and sensitive response to NO. The probe generated stable optical signals in a buffer solution at pH values ranging from 3 to 10. In addition, DHP-4 could detect NO directly, showed low cellular toxicity, and was successfully applied to determine NO in Raw 264.7 cells, indicating its great potential as a tool for investigating the biological roles of NO in vivo.

Ultrasound-assisted extraction extracts from Stemona japonica (Blume) Miq. and Cnidium monnieri (L.) Cuss. could be used as potential Rhipicephalus sanguineus control agents.

Nicotiana tabacum, Stemona japonica, and Cnidium monnieri are common plants that are widely used for their anti-parasitic properties. The purpose of this study was to evaluate the acaricidal activity of extracts from these plants against the brown dog tick, Rhipicephalus sanguineus. A composition analysis of crude extracts by GC-MS was conducted to discover compounds with acaricidal effects. The toxicity of extraction against the engorged nymphs of R. sanguineus was evaluated by an immersion test. The results showed that the crude extracts of S. japonica and C. monnieri in varying ratios, concentrations, and from different extraction methods, had a killing effect on R. sanguineus. Lethality reached 76.67% ± 0.04410 when using a 1:1 extract of S. japonica:C. monnieri in 75% ethanol with ultrasonic extraction; the crude extract was determined at a concentration of 0.5 g/mL. GC-MS results showed that osthole and 5-hydroxymethylfurfural (5-HMF) are the main components of the extract. These results suggested that ultrasound-assisted extraction (UAE) extracts contained acaricidal components acting against R. sanguineus, which may result in the development of effective extracts of S. japonica and C. monnieri as a source of low-toxicity, plant-based, natural acaricidal drugs.

A Systematic Study of Coumarin-Tetrazine Light-Up Probes for Bioorthogonal Fluorescence Imaging.

Fluorescent probes that light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications. In this work, a thorough study is presented on a set of seventeen structurally diverse coumarin-tetrazine probes that produce fluorescent dyes with exceptional turn-on ratios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general, formation of the fully aromatic pyridazine-containing dyes resulting from the reaction with BCN was found superior in terms of fluorogenicity. However, evaluation of the probes in cellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability, prevail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experiments and enabled no-wash fluorogenic imaging on a timescale of seconds.

Urolithin A attenuated ox-LDL-induced cholesterol accumulation in macrophages partly through regulating miR-33a and ERK/AMPK/SREBP1 signaling pathways.

Promoting cholesterol efflux from foam cells represents one of the therapeutic strategies for ameliorating atherosclerosis. Urolithin A (UA) has been shown before to attenuate ox-LDL induced endothelial dysfunction in endothelial cells with its anti-inflammatory properties. The aim of this study was to investigate whether UA could promote cholesterol efflux via modulating related microRNA (miR) and signaling pathways. RAW264.7 cells were treated with 50 µg mL-1 ox-LDL to induce foam cell formation. After treatment with UA at different concentrations, intercellular and extracellular cholesterol levels were determined. Expression of Erk1/2, AMPKα and their phosphorylation forms, and SREBP1, was analyzed by western-blotting. The effect of UA on miR-33a expression and the involvement of miR-33a in cholesterol efflux regulation were also investigated. UA reduced ox-LDL induced cholesterol accumulation in macrophage cells and promoted cholesterol efflux from cells. Compared with ox-LDL treated cells, UA treatment reduced the level of phosphorylated ERK1/2, increased the expression of phosphorylated AMPKα and decreased the SREBP1 expression. Moreover, UA decreased the miR-33a expression at the transcriptional level but increased the transcriptional expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1, two genes contributing to reverse cholesterol transport. Furthermore, pre-miR-33a attenuated cholesterol efflux induced by UA. Collectively, UA promoted the reverse cholesterol transport in macrophage-derived foam cells and interfered with cholesterol metabolism possibly through regulating the miRNA-33 expression and interaction with the ERK/AMPKα/SREBP1 signaling pathway.

A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages.

BACKGROUND: Eleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines. PURPOSE: The purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages. STUDY DESIGN AND METHODS: HPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts. RESULTS: Chemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF-α and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization. CONCLUSION: The ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant.

Malva species: Insights on its chemical composition towards pharmacological applications.

Malvaceae family is typical from the Mediterranean region, contains 240 genera and more than 4,200 species. They are most commonly used as ornamental plants, although they can also be conceived as a food resource and remedy for various diseases, such as digestive, respiratory, genitourinary, throat infections, and skeletal and skin disorders, as also injuries where they are profoundly applied for skin care and as antiseptic and demulcent. They also possess diuretic, lenitive, spasmolytic, and laxative effects, besides to be used as antidiarrheal. Thus, the present review provides in-depth data on Malva spp. potential applications and phytochemical composition for food and pharmaceutical industries. Habitat and cultivation conditions and the clinical reports related to its biological effects are also emphasized. Malva spp. possess a wide variety of chemical constituents (such as polysaccharides, coumarins, flavonoids, polyphenols, vitamins, terpenes, and tannins) found in different plant organs, especially in leaves and flowers, connected to their biological activity. In general, Malva spp. have rather moderate antimicrobial activity, high antiinflammatory and wound healing activities, strong antioxidant activity, and anticancer properties. Results from in vitro and in vivo experiments encourage more in-depth studies, namely clinical trials, towards to improve knowledge on the use of Malva spp. for the treatment of various health conditions in humans.

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.

Pomegranate peel polyphenols reduce chronic low-grade inflammatory responses by modulating gut microbiota and decreasing colonic tissue damage in rats fed a high-fat diet.

Recent studies have found that a high-fat diet (HFD) causes gut microbiota imbalance and colon tissue damage, resulting in increased intestinal permeability, which is one of the main reasons for the existence of constantly circulating low-grade inflammatory cytokines. Pomegranate extracts have been shown to protect from HFD-induced metabolic inflammation (e.g., colitis) and to promote the growth of beneficial bacteria in in vitro stool cultures. However, whether the beneficial effects of pomegranate extracts on the HFD-induced metabolic inflammation are achieved by acting on intestinal tissues has not yet been studied. In our present study, we found that pomegranate peel polyphenols (PPPs) alleviated HFD-induced obesity, elevated circulating pro-inflammatory cytokines, colonic tissue damage, and depressed colonic tight junction protein expression level in rats. Moreover, PPPs normalized the HFD-induced gut microbiota imbalance by increasing the abundance of beneficial bacteria in the colon. Furthermore, we also found that PPPs, punicalagin, and urolithin A (the main microbiota metabolites of pomegranate ellagitannins) all increased the LPS-induced decreased tight junction protein expression level and reversed the LPS-induced inflammatory response in Caco-2 cells. Urolithin A exhibited the best effects among the three pomegranate components. Our results suggested that the protective effects of PPPs in HFD-induced metabolic inflammation can be due to the recovery of colonic tissue damage and the regulation of gut microbiota and that urolithin A is the major component that contributes to the in vivo effects of PPPs.

Effects of enzymatic browning reaction on the usability of tobacco leaves and identification of components of reaction products.

The enzyme browning reaction results in grey speckles on tobacco leaves, which impairs the value and industrial usability of tobacco leaves. To demonstrate the influences of different browning degrees (BDs) of tobacco leaves on the usability of different cultivars and positions and identified structure of brown (grey) matter, we selected three flue-cured tobacco cultivars (K326, Yunyan87, and Honghuadajinyuan (Hongda)) and set four different BDs (<25%, 25% to 50%, 50% to 75%, and >75%). Indices related to: economic traits, chemical components, physical properties, and sensory quality of tobacco leaves with different cultivars were evaluated. Moreover, by utilising thin-layer chromatography and high-performance liquid chromatography, we analysed and identified the structure of the grey matter in terms of chemical composition. The experimental results show that the main component of grey speckles on tobacco leaves is 3-acetyl-6,7-dimethoxycoumarin (YC-ZJF). With the increase of BD, the amount of total sugar and reducing sugar, output value, the proportion of superior tobacco, shatter resistance index, and sensory evaluation score of the three cultivars significantly decrease, while the starch content increases significantly. The changes in protein, total nitrogen, and nicotine are insignificant with changing BD. In addition, other indices show different trends for different cultivars of flue-cured tobacco. After separation and identification of the components of grey speckled leaves, it is proved that the substance derived from grey speckles on tobacco leaves is YC-ZJF. The research is important to the study of browning mechanisms in tobacco leaves and provides corresponding targets for strategies to reduce browning thereof.