[High-throughput screening of 54 alternative plasticizers in sesame oil using gas chromatography-quadrupole time-of-flight mass spectrometry].

Restrictions on the use of phthalates have led to the wide use of alternative plasticizers (APs) such as organophosphate, adipate, citrate, and sebacate. However, because plasticizers combine with polymers in plastic products via unstable noncovalent bonds, they can easily migrate out of these products, causing environmental pollution. In particular, their migration out of food packaging, containers, and other food-contact materials and into food has raised great concerns. Toxicological studies have shown that APs contain potentially toxic substances that can affect endocrine functions and cause neurotoxicity, genotoxicity, and other adverse effects. Thus, their potential risks to food should not be underestimated. Sesame oil is a necessity in daily cooking. The results of risk monitoring in recent years have indicated that sesame oil often contains phthalates in excess of the standard limits. However, the potential risks of APs in sesame oil have not yet been reported. Some common detection methods for APs include gas chromatography-mass spectrometry, gas chromatography-triple quadrupole mass spectrometry, and liquid chromatography-triple quadrupole mass spectrometry. Unfortunately, these methods use low-resolution mass spectrometry and are limited by the resolution, scan rate, and analysis mode. Gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS) has the advantages of high resolution, sensitivity, and analysis speed. In full-scan mode, GC-Q-TOF/MS can accurately collect the full-spectrum mass number of target compounds with low content levels in complex substrates, thereby realizing efficient screening and quantitative analysis. It shows outstanding advantages in the trace analysis of pesticide residues and pollutants. Furthermore, it features strong qualitative and high screening abilities. Establishment of a personal compound database and library (PCDL) addresses limitations in the number of compounds that can be measured and enables the rapid identification of targets without the use of standard products. In addition, increasing the number of targets for synchronous screening enables the retrospective analysis of new targets. In this study, a method based on GC-Q-TOF/MS was developed for the determination of 54 APs in sesame oil. The samples were extracted with acetonitrile and purified using a PSA/silica solid-phase extraction column. The mass-spectral information of the samples was then collected by GC-Q-TOF/MS in full-scan mode, and the 54 APs were searched using an established high-resolution mass-spectrum database to simultaneously achieve the broad-spectrum screening, qualitative identification, and quantitative analysis of multiple targets. The effects of different extraction solvents and purification methods on sample extraction and purification were compared. The accuracy of the screening results was improved by optimizing the GC-separation conditions, quality-extraction window, retention-time deviation, and other screening parameters. The screening detection limits (SDLs) of the 54 APs ranged from 0.01 to 0.02 mg/kg; specifically, the SDL of 41 compounds was 0.01 mg/kg and that of 13 compounds were 0.02 mg/kg. The limits of quantification were in the range of 0.02-0.04 mg/kg. A total of 80 sesame-oil samples were rapidly screened using this method under optimal conditions. Five APs were identified from the 80 sesame-oil samples and quantitatively analyzed using the matrix-matched external-standard method. The results of this quantitative methodology showed that the five APs had good linear relationships in the range of 0.01-0.2 mg/L, with all correlation coefficients greater than 0.99. The accuracy and precision of the method were verified using a standard recovery test with blank sesame-oil samples. Under the three standard levels of 0.04, 0.08, and 0.2 mg/kg, the recoveries of the five APs ranged from 71.3% to 97.8%, and the relative standard deviations (RSDs) ranged from 0.4% to 6.1%(n=6). The developed method is fast, accurate, sensitive, and has high throughput. Thus, it can realize the efficient screening, qualitative identification, and quantitative analysis of the 54 APs in sesame oil and provides a potential solution for the monitoring of other contaminants in food.

Isolation and identification of 3,4-seco-labdane diterpenoids from Callicarpa nudiflora and investigation of their cytotoxicity against HepG2 cells.

Twenty-one previously undescribed compounds, including nineteen 3,4-seco-labdanes (nudiflopenes P-W, Y, AI-JI), one 3,4-seco-pimarane (nudiflopene X), and one labdane (nudiflopene Z), along with nine known compounds (one 3,4-seco-pimarane and eight 3,4-seco-labdanes) were isolated from the leaves of Callicarpa nudiflora Hook. Et Arn. The structures of these compounds were elucidated by high-resolution electrospray ionization mass spectrometry and one- and two-dimensional nuclear magnetic resonance spectroscopy. In addition, configurations of the isolated compounds were determined by electronic circular dichroism, DP4+ probability analysis, and single-crystal X-ray diffraction experiments. All undescribed compounds were evaluated for their cytotoxicity against HepG2 cells in vitro, among which compound 12 exhibited a moderate activity with an IC50 value of 27.8 µM.

Mesenchymal stem cell transplantation worsens intestinal inflammation and microenvironment in PI3Kγ-knockout mice.

Considering the possible interaction between mesenchymal stem cells (MSCs) and PI3Kγ-associated drugs, we evaluated the efficacy and action mechanism of MSCs in the treatment of colitis in PI3Kγ-/- mice. Trinitro-benzene-sulfonic acid enema was used to create a colitis model, and MSCs were transplanted through the caudal vein to treat colitis in wild-type and PI3Kγ-/- mice. We sequenced microbial 16S rRNA genes in the colonic mucosa of PI3Kγ-/- and wild-type mice and quantified colonic IgA, IL-2, IL-10, IL-17A, occludin, and serum IgA. MSC transplantation led to a more serious reduction in the weight of trinitro-benzene-sulfonic acid-administered PI3Kγ-/- mice than that in wild-type mice. The disease activity index, pathological scoring, number of taxa in the colon, Berger-Parker index, I-index, proportion of Proteobacteria, and IgA level in the blood were higher in PI3Kγ-/- mice than in wild-type mice after MSC transplantation. The occludin and IL-10 levels in the colon tissues decreased before and after MSC transplantation in PI3Kγ-/- mice, whereas they were increased in wild-type mice The IL-17 level decreased in both wild-type and PI3Kγ-/- mice, with knockout mice showing a greater decrease. Therefore, MSC transplantation in PI3Kγ-/- mice led to increased numbers of exogenous pathogenic microorganisms and enhanced colitis that was difficult to relieve.

Evansella halocellulosilytica sp. nov., an alkali-halotolerant and cellulose-dissolving bacterium isolated from bauxite residue.

An alkali and salt-tolerating strain FJAT-44876T was isolated from the bauxite residue sample. The 16S rRNA gene sequence and phylogenetic analysis suggest that strain FJAT-44876T was a member of the genus Evansella. It grew at 15-45 â„ƒ (optimum 20-25 â„ƒ) and pH 6.5-11.0 (optimum pH 8.0-9.0) with 0-20% (w/v) NaCl (optimum 6-8%). The major fatty acids were anteiso-C15:0, iso-C15:0, anteiso-C17:0, iso-C17:0, and C16:0. The cell wall peptidoglycan contained meso-diaminopimelic acid and MK-7 as the menaquinone. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G+C content was 38.2%. The average nucleotide identity values between strain FJAT-44876T and closely related members were below the cutoff level for species delineation. Thus, based on the above results, strain FJAT-44876T represents a novel species of the genus Evansella, for which the name Evansella halocellulosilytica sp. nov., is proposed. The type strain is FJAT-44876T (=CCTCC AB 2016264T = DSM 104633T).

Neuraminidase 1 and its Inhibitors from Chinese Herbal Medicines: An Emerging Role for Cardiovascular Diseases.

Neuraminidase, also known as sialidase, is ubiquitous in animals and microorganisms. It is predominantly distributed in the cell membrane, cytoplasmic vesicles, and lysosomes. Neuraminidase generally recognizes the sialic acid glycosidic bonds at the ends of glycoproteins or glycolipids and enzymatically removes sialic acid. There are four types of neuraminidases, named as Neu1, Neu2, Neu3, and Neu4. Among them, Neu1 is the most abundant in mammals. Recent studies have revealed the involvement of Neu1 in several diseases, including cardiovascular diseases, diabetes, cancers, and neurological disorders. In this review, we center the attention to the role of Neu1 in cardiovascular diseases, including atherosclerosis, ischemic myocardial injury, cerebrovascular disease, congenital heart disease, and pulmonary embolism. We also summarize inhibitors from Chinese herbal medicines (CHMs) in inhibiting virus neuraminidase or human Neu1. Many Chinese herbs and Chinese herb preparations, such as Lonicerae Japonicae Flos, Scutellariae Radix, Yupingfeng San, and Huanglian Jiedu Decoction, have neuraminidase inhibitory activity. We hope to highlight the emerging role of Neu1 in humans and potentially titillate interest for further studies in this area.

Untargeted metabolomics and lipidomics uncovering the cardioprotective effects of Huanglian Jiedu Decoction on pathological cardiac hypertrophy and remodeling.

ETHNOPHARMACOLOGICAL RELEVANCE: As a classic herbal prescription, Huanglian Jiedu Decoction (HLJDD) exhibits positive effects against cardiac dysfunction. However, its cardioprotective effects and potential mechanism(s) of action still need to be systematically investigated. AIM OF THE STUDY: This study aimed to reveal the underlying therapeutic mechanism of HLJDD on transverse aortic constriction (TAC)-induced pathological cardiac hypertrophy and remodeling. MATERIALS AND METHODS: TAC-induced cardiac hypertrophy and remodeling mice model was established to evaluate the therapeutic effects of HLJDD. Serum untargeted metabolomics and lipidomic profiling were performed using ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry coupled with multivariate statistical analyses. RESULTS: Oral administration of HLJDD (2.5 g/kg/day, 5.0 g/kg/day) significantly improved the heart morphology, enhanced the heart function, and alleviated the accumulation of fibrosis in the interstitial space and the infiltration of inflammatory cells in TAC-stimulated mice. Serum untargeted metabolomics analysis showed that significant alterations were observed in metabolic signatures between the TAC-model and sham group. Principal component analysis and orthogonal partial least-squares discriminant analysis screened 59 differential metabolic features and 13 metabolites were identified. The disturbed metabolic pathways in TAC group mainly related to lipid metabolism. Further serum lipidomic profiling showed that most lipids including cholesterol esters, ceramides, glycerides, fatty acids and phospholipids were decreased in TAC group and these alterations were reversed after HLJDD intervention. CONCLUSION: HLJDD alleviates TAC-induced pathological cardiac hypertrophy and remodeling, and its potential therapeutic mechanism involves the regulation of lipid metabolism.

Berberine induces apoptosis in non-small-cell lung cancer cells by upregulating miR-19a targeting tissue factor.

BACKGROUND: Berberine (BBR) from the widely used Chinese herbal medicine Huanglian has an array of pharmacological and biochemical properties, including anti-neoplastic activity. However, the specific mechanisms underlying these properties are unknown. The aim of this study was to explore the anti-tumor mechanisms of BBR in non-small cell lung cancer (NSCLC). METHODS: The effects of BBR on NSCLC tumor development and programmed cell death were investigated both in vivo and in vitro. Luciferase reporter assays were used to determine whether tissue factor (TF) was a target of miR-19a. RESULTS: BBR suppressed NSCLC growth and promoted apoptosis in NSCLC cells by modulating miR-19a and TF expression. Luciferase assays showed that TF was a direct inhibitory target of miR-19a in NSCLC cells. BBR induced apoptosis through the miR-19a/TF/MAPK axis. CONCLUSION: The results suggest that BBR induces apoptosis of NSCLC cells via the miR-19a/TF/MAPK signaling pathway.

Investigation into perturbed nucleoside metabolism and cell cycle for elucidating the cytotoxicity effect of resveratrol on human lung adenocarcinoma epithelial cells.

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.

Diterpene synthases facilitating production of the kaurane skeleton of eriocalyxin B in the medicinal plant Isodon eriocalyx.

The Isodon plants (Lamiaceae) have been used in traditional Chinese medicine to alleviate sufferings from inflammations and cancers. This feature has been attributed to the presence of pharmacologically active ent-kaurane diterpenoids such as eriocalyxin B and oridonin. The Isodon eriocalyx (Dunn) Kudô species native to southwest China can accumulate a particularly high content of ent-kaurane diterpenoids (∼1.5% w/w of dried leaves). We previously identified diterpene synthases IeCPS1 and IeCPS2 as ent-copalyl diphosphate synthases (ent-CPS) potentially involved in Isodon ent-kaurane diterpenoids biosynthesis. In this study, analysis of RNA-seq transcriptome of the I. eriocalyx plant revealed three other diterpene synthase genes (IeCPS3, IeKS1, and IeKSL1). Their functional characterization through coupled in vitro enzyme assays has confirmed that IeCPS3 is an ent-CPS specifically producing ent-copalyl diphosphate (ent-CPP). IeKS1 accepted ent-CPP to produce exclusively ent-kaurene and may thus be defined as an ent-kaurene synthase (ent-KS). When IeKSL1 was combined with IeCPS2 or IeCPS3, no product was detected. Based on tissue-specific expression and metabolic localization studies, the IeCPS3 and IeKS1 transcripts were significantly accumulated in leaves where the ent-kaurane diterpenoid eriocalyxin B dominates, whereas weak expression of both were observed in germinating seeds in which gibberellin biosynthetic pathway is normally active. Our findings suggest that both IeCPS3 and IeKS1 possess dual roles in general (gibberellins) and specialized diterpenoid metabolism, such as that of the Isodon ent-kaurane diterpenoids.

Mechanism of Yanghe Pingchuan granules treatment for airway remodeling in asthma.

PURPOSE: Yanghe Pingchuan granules (YPG), a hospital preparation developed by The First Affiliated Hospital, Anhui University of Chinese Medicine, has been used for the clinical treatment of bronchial asthma (BA) for several decades. This study aimed to explore the mechanism of action of YPG in the treatment of BA. MATERIALS AND METHODS: Male Sprague Dawley rats (n=60) were randomly divided into six groups (n=10 per group): control, a BA model, positive drug control (Guilong Kechuanning capsules; a proven effective treatment for BA), and model rats treated with a high, medium, or low dose of YPG. H&E staining was used to detect pathological changes in the bronchial tubes. The mRNA expression levels of PI3K, PKB, PCNA, and AR were determined by real-time PCR, and the protein levels of phospho- (p-)PI3K, p-PKB, p-PCNA, and p-AR were detected by Western blotting. ELISAs were used to detect the expression of PIP2, PIP3 IL-6, IL-8, IL-1ß, and epinephrine (EPI). RESULTS: H&E staining demonstrated that BA can be ameliorated using YPG. Real-time PCR, Western blotting, and ELISA indicated that use of YPG decreased expression of the phosphoinositide 3-kinase (PI3K) signaling pathway and PCNA, and can also ameliorate the condition kidney Yang deficiency, which is associated with BA in Chinese traditional medicine. CONCLUSION: YPG can attenuate BA therapeutically in a dose-dependent manner. The mechanism underlying its therapeutic effect comprises influences on three features that contribute to BA: the PI3K signaling pathway, cell proliferation, and "kidney-Yang deficiency".

Quantitation of camellianin A in HepG2 cells using a high performance liquid chromatography-electrospray ionization tandem mass spectrometric method.

The present study was designed to develop a sensitive and selective high performance liquid chromatography-tandem mass spectrometric method for the determination of Camellianin A in HepG2 cells. The extraction of Camellianin A was achieved using 15% trichloroacetic acid and then separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The mobile phase was consisted of methanol-water (0.1% formic acid) (55 : 45, V/V). The total run time was 5.0 min. The method was linear in the concentration range of 0.25-250.0 ng·mL-1. The lower limit of quantification was 0.25 ng·mL-1. The intra- and inter-day relative standard deviations of entire concentration range were less than 9.3%. The proposed HPLC-MS/MS method was successfully applied to detect the intracellular concentration of Camellianin A in HepG2 cells.

Effect of Phyllanthus amarus Extract on 5-Fluorouracil-Induced Perturbations in Ribonucleotide and Deoxyribonucleotide Pools in HepG2 Cell Line.

The aim of this study was to investigate the antitumor activities of Phyllanthus amarus (PHA) and its potential of herb-drug interactions with 5-Fluorouracil (5-FU). Cell viability, ribonucleotides (RNs) and deoxyribonucleotides (dRNs) levels, cell cycle distribution, and expression of thymidylate synthase (TS) and ribonucleotide reductase (RR) proteins were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) method, flow cytometry and Western blot analysis, respectively. Our standardized PHA extract showed toxicity to HepG2 cells at high concentrations after 72 h exposure and induced G2/M cell cycle arrest. Combined use of 5-FU with PHA resulted in significant decreases in ATP, CTP, GTP, UTP and dTTP levels, while AMP, CMP, GMP and dUMP levels increased significantly compared with use of 5-FU alone. Further, PHA could increase the role of cell cycle arrest at S phase induced by 5-FU. Although PHA alone had no direct impact on TS and RR, PHA could change the levels of RNs and dRNs when combined with 5-FU. This may be due to cell cycle arrest or regulation of key enzyme steps in intracellular RNs and dRNs metabolism.

Quantitation of ligupurpurosides B and C in rat plasma using HPLC-MS/MS.

The present study was designed to develop a sensitive and selective specific high performance liquid chromatography (HPLC)-tandem mass spectrometric method (MS/MS) for the determination of ligupurpurosides B and C in rat plasma. The samples were prepared after protein precipitation and analyzed by liquid chromatography equipped with a C18 column interfaced with a triple quadrupole tandem mass spectrometer using ESI as the ionization source in the negative ion mode. The mobile phase consisted of water (0.01 % formic acid)-methanol (57 : 43, V/V) at the flow rate of 0.3 mL·min(-1). The analytes and internal standard acteoside were both detected by use of multiple reaction monitoring mode. The total run time was 6.0 min. The method was linear in the concentration range of 2.5-500.0 ng·mL(-1) and the lower limit of quantifiation (LLOQ) was 2.5 ng·mL(-1). The intra-day and inter-day relative standard deviations across three validation runs over the entire concentration range were less than 9.8 %. The accuracy determined at three concentrations was within ± 6.1% in terms of relative error. In conclusion, this assay offers advantages in terms of expediency and suitability for the analysis of ligupurpuroside B and ligupurpuroside C in various biological fluids.

Insight into the short- and long-term effects of Cu(II) on denitrifying biogranules.

This study aimed to investigate the short- and long-term effects of Cu(2+) on the activity and performance of denitrifying bacteria. The short-term effects of various concentrations of Cu(2+) on the denitrifying bacteria were evaluated using batch assays. The specific denitrifying activity (SDA) decreased from 14.3 ± 2.2 (without Cu(2+)) to 6.1 ± 0.1 mg N h(-1)g(-1) VSS (100 mg Cu(2+)L(-1)) when Cu(2+) increased from 0 to 100 mg L(-1) with an increment of 10 mg Cu(2+)L(-1). A non-competitive inhibition model was used to calculate the 50% inhibition concentration (IC50) of Cu(2+) on denitrifying sludge (30.6 ± 2.5 mg L(-1)). Monod and Luong models were applied to investigate the influence of the initial substrate concentration, and the results suggested that the maximum substrate removal rate would be reduced with Cu(2+) supplementation. Pre-exposure to Cu(2+) could lead to an 18.2-46.2% decrease in the SDA and decreasing percentage of the SDA increased with both exposure time and concentration. In the continuous-flow test, Cu(2+) concentration varied from 1 to 75 mg L(-1); however, no clear deterioration was observed in the reactor, and the reactor was kept stable, with the total nitrogen removal efficiency and total organic carbon efficiency greater than 89.0 and 85.0%, respectively. The results demonstrated the short-term inhibition of Cu(2+) upon denitrification, and no notable adversity was observed during the continuous-flow test after long-term acclimation.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

BACKGROUND: We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). RESULTS: MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. CONCLUSION: Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.

Determination of corilagin in rat plasma using a liquid chromatography-electrospray ionization tandem mass spectrometric method.

A sensitive and simple liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol-0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5-1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both <9.8%. The relative error was within ±6.0%. This assay offers advantages in terms of expediency and suitability for the analysis of corilagin in rat plasma. The practical utility of this new HPLC-MS/MS method was confirmed in pilot plasma concentration studies in rats following oral administration.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

BACKGROUND: We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). RESULTS: MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. CONCLUSION: Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.

Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses.

AIM: To investigate the potential therapeutic effects of mesenchymal stem cells (MSCs) in inflammatory bowel disease (IBD), we transplanted MSCs into an experimental model of IBD. METHODS: A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg body weight) was administered to female BALB/c mice. Bone marrow mesenchymal stem cells (BMSCs) were derived from male green fluorescent protein (GFP) transgenic mice and were transplanted intravenously into the experimental animals after disease onset. Clinical activity scores and histological changes were evaluated. GFP and Sex determining region Y gene (SRY) expression were used for cell tracking. Ki67 positive cells and Lgr5-expressing cells were determined to measure proliferative activity. Inflammatory response was determined by measuring the levels of different inflammatory mediators in the colon and serum. The inflammatory cytokines included tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-17, IL-4, IL-10, and transforming growth factor (TGF-ß). Master regulators of Th1 cells (T-box expressed in T cells, T-bet), Th17 cells (retinoid related orphan receptor gamma(t), RORγt), Th2 cells (GATA family of transcription factors 3, GATA3) and regulatory T cells (forkhead box P3, Foxp3) were also determined. RESULTS: Systemic infusion of GFP-BMSCs ameliorated the clinical and histopathologic severity of colitis, including body weight loss, diarrhea and inflammation, and increased survival (P < 0.05). The cell tracking study showed that MSCs homed to the injured colon. MSCs promoted proliferation of intestinal epithelial cells and differentiation of intestinal stem cells (P < 0.01). This therapeutic effect was mainly mediated by down-regulation of both Th1-Th17-driven autoimmune and inflammatory responses (IL-2, TNF-α, IFN-γ, T-bet; IL-6, IL-17, RORγt), and by up-regulation of Th2 activities (IL-4, IL-10, GATA-3) (P < 0.05). MSCs also induced activated CD4(+)CD25(+)Foxp3(+) regulatory T cells (TGF-ß, IL-10, Foxp3) with a suppressive capacity on Th1-Th17 effecter responses and promoted Th2 differentiation in vivo (P < 0.05). CONCLUSION: MSCs are key regulators of immune and inflammatory responses and may be an attractive candidate for cell-based therapy of IBD.

Validation of HPLC method for the determination of polydatin in giant knotweed rhizome.

An HPLC method has been developed to determine polydatin in giant knotweed rhizome. In order to systematically validate the method, specificity, precision, linearity of reference solution and test solution, repeatability, reproducibility, accuracy, stability and robustness were measured. In the robustness test, a one-variable-at-a-time procedure was applied to evaluate the influence of slight variations in method factors, including the flow rate, the column temperature, the extraction time, and etc., on the assay result of polydatin. No significant differences were found when the process parameters changed during the experimental domain. And system suitability test limits were defined based on the robustness test. Results showed that the developed method was accurate, reproducible and robust.

IeCPS2 is potentially involved in the biosynthesis of pharmacologically active Isodon diterpenoids rather than gibberellin.

The traditional Chinese medicinal plant, Isodon L., is remarkably rich in pharmacologically active ent-kaurane diterpenoids of diverse carbon skeletons. In an effort to create a resource for gene discovery and elucidate the biosynthesis of Isodonent-kaurane diterpenoids, three cDNAs (named IeCPS1, IeCPS2 and IeCPS2a) were isolated putatively encoding copalyl diphosphate synthases from Isodoneriocalyx leaves. Recombinant proteins of IeCPS1 and IeCPS2 were expressed, respectively, in Escherichia coli, and were shown to specifically convert geranylgeranyl diphosphate to copalyl diphosphate as demonstrated by GC-MS analyses. Based on tissue-specific expression and metabolic localization studies, the IeCPS2 transcripts were detected in young and mature leaves where the dominant ent-kaurane diterpenoid maoecrystal B accumulates, whereas no detectable expression of IeCPS2 was observed in germinating seeds where the gibberellin biosynthetic pathway is usually active. In addition, no evidence for maoecrystal B was found in germinating seeds. On the other hand, IeCPS1 transcripts significantly accumulated in germinating seeds as well as in leaves. The biochemical and molecular genetic evidence thus indicated that IeCPS2 is a copalyl diphosphate synthase potentially involved in the biosynthesis of Isodon diterpenoids in leaves, while IeCPS1 is more probably relevant to gibberellin formation and may, in addition, participate in Isodonent-kaurane diterpenoid production.