Columbianetin Acetate Research Articles

Columbianetin acetate inhibits the occurrence and development of pancreatic cancer cells by down-regulating the expression of Meiotic nuclear divisions 1

Columbianetin acetate (CE) is one of the effective components of Angelica pubescens. So far, the specific role and molecular mechanism of CE in pancreatic cancer are not clear. Thus, this study aimed to explore the specific mechanism of CE on pancreatic cancer. The target genes combined with CE were predicted through the PharmMapper database and the 3D molecular structure of CE. Then, the Cancer Genome Atlas (TCGA) and Cistrome data browser (DB) databases were used to screen Meiotic nuclear divisions 1 (MND1)-related genes, transcription factors, and transcription factor data sets, and the intersection of the above data sets. The “limma” package in the R and gene expression profiling interactive analysis (GEPIA) databases were used to analyze the correlation and survival difference between the target genes and MND1 to predict the degree of association between CE and MND1. Western blotting and RT-PCR experiments revealed the regulatory relationship among CE, E2F1, and MND1 at the cellular level. The specific effects of CE on pancreatic cancer cells were explored through CCK8, wound healing, migration, and flow cycle experiments. E2F1, also the predictive transcription factor of MND1, was also the predictive target protein of CE. At the same time, E2F1 and MND1 were closely related in pancreatic tissue. In the cell function experiment, CE and interference with E2F1 expression could reduce the gene and protein expression of MND1, which was closely associated with cell proliferation, migration, and cycle development. Similarly, interfering with the expression of mnd1 can also inhibit the further development of tumor cells. CE may inhibit the development of pancreatic cancer cells by reducing the expression of MND1. This implies that CE may be a potential novel agent for the treatment of pancreatic cancer.

Studies on Chemical Constituents of Radix Angelicae pubescentis

Backgound:Angelicae pubescentis is the root of Angelica pubescens Maxim. f. biserrata Shan et Yuan. The main active ingredients are coumarin and volatile oil, and it has a variety of pharmacological activities such as antibacterial, antioxidant and anti-inflammatory. In this paper, the active components of the CO2 supercritical extract of Angelicae pubescentis were studied. The results of this study will provide a scientific basis for the study of the chemical composition of Angelicae pubescentis. Purpose: To study the chemical constituents of supercritical extract of Radix Angelicae pubescentis. Method: The supercritical extract of Radix Angelicae pubescentis was separated by silica gel column chromatography to obtain monomers. The structures were identified based on physicochemical properties and spectroscopic data. Result: Eight compounds were separated from the supercritical extract of Radix Angelicae pubescentis, six of which were identified.They are Osthol, Columbianedin, Columbianetin, Columbianetin Acetate, Xanthotoxin and Bergapten. Conclusion: Xanthotoxin and Bergapten were isolated, which structure were similar. The standard substance of Osthol and Columbianedin were producted in order to offer the good material base for quality evaluation and pharmacodynamic study of Radix Angelicae pubescentis. Their purification were more than 99%. The results of this study are of great significance for exploring the effective material basis and medicinal value of the supercritical extract of Radix Angelicae pubescentis.

A strategy for qualitative and quantitative profiling of Angelicae Pubescentis Radix and detection of its analgesic and anti-inflammatory components by spectrum-effect relationship and multivariate statistical analysis.

This study established a spectrum-effect relationship method for screening and quantifying the analgesic and anti-inflammatory active ingredients in Angelicae Pubescentis Radix (AP) by ultra-high-performance liquid chromatography-quadrupole mass spectrometry detector analysis (UPLC-QDA). First, the fingerprint of AP was established to determine the common peaks. Next, six batches of AP samples, with significant differences, were selected for evaluation of pharmacological activity. Subsequently, the spectrum-effect relationship was used to screen the active ingredients. Finally, the screened ingredients were quantified using UPLC-QDA. In total, 21 common peaks were identified and four effective compounds (bergapten, columbianetin acetate, osthole and isoimperatorin) were selected using the gray relational analysis and partial least squares regression analysis. Quantitative analysis showed that the content of the four effective compounds was the highest in a randomly selected batch, S7 (Hubei). To our knowledge, this is the first attempt that evaluated the quality and spectrum-effect relationship of AP by quantitative analysis and chemometrics. This study identified the key pharmacologically active components of AP and thereby improved the quality evaluation system of AP. This method has broad application prospects for screening effective components and will be helpful in establishing more reliable, scientific and reasonable quality standards for AP and other traditional Chinese medicines.

Separation and Enrichment of Three Coumarins from Angelicae Pubescentis Radix by Macroporous Resin with Preparative HPLC and Evaluation of Their Anti-Inflammatory Activity.

In order to enrich and separate three coumarins (columbianetin acetate, osthole and columbianadin) from Angelicae Pubescentis Radix (APR), an efficient method was established by combining macroporous resins (MARs) with preparative high-performance liquid chromatography (PHPLC). Five different macroporous resins (D101, AB-8, DA-201, HP-20 and GDX-201) were used to assess the adsorption and desorption characteristics of three coumarins. The result demonstrated that HP-20 resin possessed the best adsorption and desorption capacities for these three coumarins. Moreover, the adsorption dynamics profiles of three coumarins were well fitted to the pseudo second order equation (R2 > 0.99) for the HP-20 resin. The adsorption process was described by the three isotherms models including Langmuir (R2 > 0.98, 0.046 ≤ RL ≤ 0.103), Freundlich (R2 > 0.99, 0.2748 ≤ 1/n ≤ 0.3103) and Dubinin Radushkevich (R2 > 0.97). The contents of columbianetin acetate, osthole and columbianadin in the product were increased 10.69-fold, 19.98-fold and 19.68-fold after enrichment, respectively. Three coumarins were further purified by PHPLC and the purities of them reached above 98%. Additionally, the anti-inflammatory effects of these three coumarins were assessed by Lipopolysaccharide (LPS)-induced RAW 264.7 cells. It was found that the production of NO and MCP-1 was obviously inhibited by three coumarins. Columbianetin acetate, osthole and columbianadin could be used as potentially natural anti-inflammatory ingredients in pharmaceutical products. It was concluded that the new method combining MARs with PHPLC was efficient and economical for enlarging scale separation and enrichment of columbianetin acetate, osthole and columbianadin with anti-inflammatory effect from the APR extract.

The Tissue Distribution of Four Major Coumarins after Oral Administration of Angelicae Pubescentis Radix Extract to Rats Using Ultra-High-Performance Liquid Chromatography.

Angelicae pubescentis radix (APR) is widely applied in treating rheumatoid arthritis in China. Coumarins are the major active compounds of APR extract including columbianetin, columbianetin acetate, osthole, and columbianadin. The in vivo behavior of the four major coumarins of APR has not been systematically reported. A feasible and reliable ultra-performance liquid chromatography (UPLC) method was established and validated for the quantification of the above four coumarins in rat various tissues (including heart, liver, spleen, lung, kidney, uterus, ovary, and muscle) after oral administration of APR extract. The separation was implemented on a Waters ACQUITY BEH C18 column (4.6 mm × 100 mm, 1.7 μm) with gradient mobile phase comprising acetonitrile-water (with 1mM formic acid) at a flow rate of 0.3 mL/min. The tissue homogenate samples were prepared by liquid-liquid extraction with ethyl acetate. The calibration curves were linear in the range of 1.6-20000 ng/mL for four coumarins with the lower limit of quantitation of 1.6 ng/mL in rat tissues. The intraday and interday precisions and recoveries were all within 80-100% with the relative standard deviations (RSDs) which were all less than 10.9%. The method was successfully applied to the tissue distribution research after oral administration of 6.0 g/kg APR extract to rat. The results revealed that the tissues distributions of four coumarins were in the liver, followed by the ovary, uterus, kidney, lung, heart, spleen, and muscle.

Distribution Assessments of Coumarins from Angelicae Pubescentis Radix in Rat Cerebrospinal Fluid and Brain by Liquid Chromatography Tandem Mass Spectrometry Analysis.

Angelicae Pubescentis Radix (APR) is a widely-used traditional Chinese medicine. Pharmacological studies have begun to probe its biological activities on neurological disorders recently. To assess the brain penetration and distribution of APR, a validated ultra-performance liquid chromatography tandem mass spectrometry method was applied to the simultaneous determinations of the main coumarins from APR in the rat cerebrospinal fluid (CSF) and brain after oral administration of APR extract, including psoralen, xanthotoxin, bergapten, isoimperatorin, columbianetin, columbianetin acetate, columbianadin, oxypeucedanin hydrate, angelol B, osthole, meranzin hydrate and nodakenetin. Most of the tested coumarins entered the rat CSF and brain quickly, and double-peak phenomena in concentration-time curves were similar to those of their plasma pharmacokinetics. Columbianetin had the highest concentration in the CSF and brain, while psoralen and columbianetin acetate had the largest percent of CSF/plasma and brain/plasma, indicating that these three coumarins may be worthy of further research on the possible nervous effects. Correlations between the in vivo brain distributions and plasma pharmacokinetics of these coumarins were well verified. These results provided valuable information for the overall in vivo brain distribution characteristics of APR and also for its further studies on the active substances for the central nervous system.

Simultaneous assessment of absorption characteristics of coumarins from Angelicae Pubescentis Radix: In vitro transport across Caco-2 cell and in vivo pharmacokinetics in rats after oral administration.

Angelicae Pubescentis Radix (APR), a well-known traditional Chinese medicine, is widely used for the treatments of rheumatism and headache for centuries. To assess the absorption characteristics of coumarins from APR, a sensitive and reliable UPLC-MS/MS method was established for the simultaneous determination of sixteen coumarins from APR, including psoralen, xanthotoxin, bergapten, bergaptol, isoimperatorin, imperatorin, columbianetin, columbianetin acetate, columbianadin, oxypeucedanin hydrate, angelol B, umbelliferone, scopoletin, osthole, meranzin hydrate and nodakenetin. The specificity, linearity, sensitivity, precision, accuracy, recovery, matrix effect and stability of the method were all validated to be satisfactory. The method was then applied to the in vitro transport of APR extract (APRE) across human intestinal epithelial Caco-2 cell and in vivo pharmacokinetics in rats after oral administration of APRE. All of the tested coumarins were well or moderately absorbed across Caco-2 monolayers, and could be quickly absorbed into rat blood circulation after oral administration. Columbianetin was the most easily absorbed compound across Caco-2 cell, and also had extremely highest plasma concentration in vivo. Excellent correlation between in vitro absorption across Caco-2 cell monolayers and in vivo pharmacokinetics of coumarins from APRE was well verified. The results provided valuable information for the overall absorption characteristics of the coumarins from APR, as well as for its further studies of in vivo active substances in the further.

Transport of Twelve Coumarins from Angelicae Pubescentis Radix across a MDCK-pHaMDR Cell Monolayer-An in Vitro Model for Blood-Brain Barrier Permeability.

Angelicae Pubescentis Radix (APR), a widely used traditional Chinese medicine, is reported to have central nervous system activities. The purpose of this study was to characterize the blood-brain barrier permeability of twelve coumarins from APR including umbelliferone (1), osthol (2), scopoletin (3), peucedanol (4), ulopterol (5), angepubebisin (6), psoralen (7), xanthotoxin (8), bergapten (9), isoimperatorin (10), columbianadin (11), and columbianetin acetate (12) with an in vitro model using a MDCK-pHaMDR cell monolayer. The cell monolayer was validated to be suitable for the permeation experiments. The samples’ transports were analyzed by high performance liquid chromatography and their apparent permeability coefficients (Papp) were calculated. According to the Papp value, most coumarins could be characterized as well-absorbed compounds except for 4, 10 and 11 which were moderately absorbed ones, in concentration-dependent and time-dependent manners. The results of P-glycoprotein (P-gp) inhibitor (verapamil) experiments showed that the transport of coumarin 4 was affected by the transport protein P-gp. Sigmoid functions between permeability log(Papp AP-BL*MW0.5) and log D (at pH 7.4) were established to analyze the structure-activity relationship of coumarins. The results provide useful information for discovering the substance basis for the central nervous system activities of APR, and predicting the permeability of other coumarins through BBB.

The pharmacokinetics, bioavailability and excretion of columbianetin acetate and its metabolite columbianetin were analysed in rat plasma by LC-MS/MS after administration of columbianetin acetate and Angelicae pubescentis radix extract

Angelicae pubescentis radix (APR) has been widely used in the clinic for the treatment of rheumatoid arthritis.

Microwave-assisted extraction coupled with counter-current chromatography and preparative liquid chromatography for the preparation of six furocoumarins from Angelica Pubescentis Radix

An efficient separation and purification method by microwave-assisted extraction coupled with counter-current chromatography and preparative liquid chromatography was developed. The extracts obtained from microwave-assisted extraction were rapid separated with counter-current chromatography, and then the fractions were further purified with preparative liquid chromatography. With this method, six furocoumarins including xanthotoxin, bergapten, columbianetin acetate, osthole, isoimperatorin and columbianadin can be obtained from original Angelica Pubescentis Radix samples within total time of 450min. Their purities were 98%, 98%, 99%, 99%, 99% and 99% respectively, as determined by HPLC. The structures of these compounds were identified by 1H NMR and MS spectroscopy. The proposed method was economic, rapid and effective, and it can be used to prepare and purify targets from natural products.

Simultaneous determination of scopoletin, psoralen, bergapten, xanthotoxin, columbianetin acetate, imperatorin, osthole and isoimperatorin in rat plasma by LC–MS/MS for pharmacokinetic studies following oral administration of Radix Angelicae Pubescentis extract

A rapid and sensitive bioassay based on liquid chromatography tandem mass spectrometry (LC–MS/MS) has been developed and validated for the simultaneous determination of eight coumarins in rat plasma. The liquid–liquid extraction method with ethyl acetate was used to prepare the plasma samples after addition of warfarin as an internal standard (IS). Chromatographic separation was performed on an Eclipse plus C18 column (100mm×4.6mm, 1.8μm) using gradient elution when 1mM ammonium acetate aqueous solution – acetonitrile was used as the mobile phase. The lower limit of quantitation (LLOQ) of each coumarin was lower than 2.16ngmL−1.Intra-day and inter-day precisions were less than 15%. The accuracies were in the range of 88.9–117%. The mean recoveries of coumarins and IS were higher than 84%. The method was successfully applied to a pharmacokinetic study of eight coumarins in rats after oral administration of radix angelicae pubescentis.

Accessing columbianetin-containing natural products via a domino on-water, in-water process

A domino on-water, in-water process has been developed for the rapid and efficient synthesis of (±)-columbianetin. This highlights the operational simplicity of on-water chemistry. The domino process forms the key step in the synthesis of the columbianetin-containing natural products (±)-columbianetin acetate, (±)-zosimin, (±)-libanorin and (±)-angelmarin.

HPLC-based activity profiling of Angelica pubescens roots for new positive GABA A receptor modulators in Xenopus oocytes

A petroleum ether extract of the traditional Chinese herbal drug Duhuo (roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan), showed significant activity in a functional two-microelectrode voltage clamp assay with Xenopus oocytes which expressed recombinant γ-aminobutyric acid type A (GABA A) receptors of the subtype α 1β 2γ 2S. HPLC-based activity profiling of the active extract revealed six compounds responsible for the GABA A receptor modulating activity. They were identified by microprobe NMR and high resolution mass spectrometry as columbianetin acetate ( 1), imperatorin ( 3), cnidilin ( 4), osthol ( 5), and columbianedin ( 6). In concentration-dependent experiments, osthol and cnidilin showed the highest potentiation of the GABA induced chloride current (273.6% ± 39.4% and 204.5% ± 33.2%, respectively at 300 μM). Bisabolangelone ( 2) only showed minor activity at the GABA A receptor. The example demonstrates that HPLC-based activity profiling is a simple and efficient method to rapidly identify GABA A receptor modulators in a bioactive plant extract.

Absorption and transport of 6 coumarins isolated from the roots of Angelica pubescens f. biserrata in human Caco-2 cell monolayer model

To study the absorption and transepithelial transport of six coumarins (umbelliferone, osthole, columbianadin, columbianetin acetate, angelol-A and angelol-B, isolated from the roots of Angelica pubescens f. biserrata) in the human Caco-2 cell monolayer model. The in vitro cultured human colon carcinoma cell line, Caco-2 cell monolayer model, was applied to study the absorption and transport of the six coumarins from apical (AP) to basolateral (BL) side and from BL to AP side. The six coumarins were measured by reversed-phase high-performance liquid chromatography (HPLC) coupled with ultraviolet absorption detector. Transport parameters and apparent permeability coefficients (P(app)) were calculated and compared with those of propranolol as a control substance of high permeability and atenolol as a control substance of poor permeability. The transport mechanism of angelol-B was assayed by using iodoacetamide as a reference standard to inhibit ATP-dependent transport and MK571 as a well-known inhibitor of MRP2. The absorption and transport of six coumarins were passive diffusion as the dominating process. The P(app) values of umbelliferone, osthole, columbianadin, columbianetin acetate, angelol-A and angelol-B from AP to BL side were (2.679+/-0.263) x 10(-5), (1.306+/-0.324) x 10(-5), (0.595+/-0.086) x 10(-6), (2.930+/-0.410) x 10(-6), (1.532+/-0.444) x 10(-5) and (1.413+/-0.243) x 10(-5) cm/s, and from BL to AP side were (3.381+/-0.410) x 10(-5), (0.898+/-0.134) x 10(-5), (0.510+/-0.183) x 10(-6), (0.222+/-0.025) x 10(-6), (1.203+/-0.280) x 10(-5) and (0.754+/-0.092) x 10(-5) cm/s, respectively. In this assay, the P(app) value of propranolol was 2.18 x 10(-5) cm/s and the P(app) value of atenolol was 2.77 x 10(-7) cm/s. Among the 6 coumarins, the P(app) values of umbelliferone, osthole, angelol-A and angelol-B from AP to BL side were identical with that of propranolol, and columbianadin and columbianetin acetate lied between propranolol and atenolol. When replaced the HBSS with EBSS, and iodoacetamide or MK-591 were used in the experiment, the P(app) of angelol-B had no statistical difference as compared with the control group. In the mean total recoveries, umbelliferone was (83.31+/-3.52)%, angelol-A was (77.39+/-7.38)%, osthole, columbianadin and angelol-B were between 50% to 65%, and columbianetin acetate was lower than 10%. The accumulation rates of osthole and columbianadin in the Caco-2 cells were (36.15+/-5.87)% and (53.90+/-4.39)%, respectively. The absorption and transport of umbelliferone, osthole, columbianadin, columbianetin acetate, angelol-A and angelol-B are passive diffusion as the dominating process in Caco-2 cell monolayer model. Umbelliferone, osthole, angelol-A and angelol-B are estimated to be highly absorbed compounds, and columbianadin and columbianetin acetate are estimated to be moderately absorbed compounds. In the Caco-2 cells, osthol and columbianadin appear to accumulate, and columbianetin acetate may be metabolized. The absorption and transport of angelol-B are not influenced by the change of pH and the presence of iodoacetamide or MK571.

Comparison of Radix Angelicae pubescentis and Substitutes - Constituents and Inhibitory Effect on 5-lipoxygenase and Cyclooxygenase

Various samples of Radix Angelicae pubescentis (Duhuo) and substitutes from the genera Angelica, Heracleum (Umbelliferae), and Aralia (Araliaceae) were analyzed for their constituents and inhibitory effect on cyclooxygenase (COX-1) and 5-lipoxygenase (5-LO). HPLC analysis showed that osthol, columbianedin, columbianetin acetate, and angelol-type coumarins are the principal constituents of the n -hexane and dichloromethane extracts of the roots of Angelica pubescens f. biserrata. The constituents of the substitutes from Angelica and Heracleum species were found to be mainly furanocoumarins and falcarindiol. No coumarins were detected in Aralia cordata. The presence of fatty acids (linoleic acid) could be confirmed by GC in Duhuo and the substitutes. All the species showed inhibitory effects on COX-1 and 5-LO. Linoleic acid turned out to be the most active constituent in the extract exerting COX-1 inhibition and also had strong inhibitory activity on 5-LO. Osthol was another major inhibitor of 5-LO in Radix Angelicae pubescentis, while in the substitutes falcarindiol was mainly responsible for the 5-LO inhibitory effect, except in Aralia cordata which also contained other polyacetylene derivatives.

Anti-Inflammatory and Analgesic Activities from Roots ofAngelica pubescens

In the present study, we extracted Angelica pubescens (AP) with various solvents in order to find the bioactive constituents that demonstrated analgesic and anti-inflammatory effects. The results were obtained as follows: (1) Methanol-, chloroform-, and ethyl acetate-extracts effectively reduced the pain that was induced by 1% acetic acid and a hot plate. (2) Methanol-, chloroform-, and ethyl acetate-extracts reduced the edema that was induced by 3% formalin or 1.5% carrageenan. (3) Sixteen compounds have been isolated and identified from the roots of AP. Among these compounds, columbianadin, columbianetin acetate, bergapten, umbelliferone, and caffeic acid significantly demonstrated anti-inflammatory and analgesic activities at 10 mg/kg. However, only osthole and xanthotoxin revealed anti-inflammatory activity. Isoimperatorin only demonstrated an analgesic effect. These results revealed that the anti-inflammatory and analgesic constituents from roots of AP were related to peripheral inhibition of inflammatory substances and to the influence on the central nervous system.