A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle.

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

[Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography].

Brazilein is among the main chemical constituents of Caesalpinia sappan. It has diverse pharmacological activities. Modern pharmacological studies have shown that the compound has antitumor, anti-inflammatory, antibacterial, antioxidant, immunomodulatory, and other pharmacological activities. Brazilein is often used as a stain in various industries. The separation of brazilein by traditional column chromatography will not only result in contamination of the chromatographic column materials, but also lead to loss of the active ingredient. Countercurrent chromatography is an advanced liquid-liquid chromatographic separation technique. It has been widely used for natural product separation and isolation as it offers several advantages, such as low solvent consumption, a highly selective solvent system, and high recoveries. Typical countercurrent chromatography techniques include centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and high performance countercurrent chromatography (HPCCC). It is well known that choosing a suitable solvent system is vital in countercurrent separation. Therefore, two methods were introduced for choosing a suitable solvent system. One is the generally useful estimation of solvent systems (GUESS) method, which employs thin-layer chromatography (TLC) to identify a suitable solvent system with minimal labor for the rapid purification of target compounds, and another is the Shake-Flash method. The solvent system could be determined by observing the distribution of the sample in the upper and lower phases. Two kinds of solvent systems were screened using the TLC-GUESS and Shake-Flash methods, and tested through the analysis mode of the HPCCC instrument. The results showed that chloroform-methanol-water (4:3:2, v/v/v) was the optimal solvent system for HPCCC separation. A total of 15.2 mg of brazilein and 5.7 mg of caesappanin C were obtained from an ethyl acetate extract with high purities (95.6% and 89.0%, analyzed by HPLC) in one step using the preparation mode of HPCCC, the reversed-phase liquid chromatography mode with the apparatus rotated at 1600 r/min, a flow rate of 10 mL/min, separation temperature of 25℃, and detection wavelength of 285 nm. Their structures were determined by spectroscopic and spectrometric analyses. Brazilein stained the solid packing material in the column and was difficult to elute. The results showed that the use of HPCCC for the separation of brazilein can not only prevent the loss of target active ingredients in Caesalpinia sappan, but also shorten the separation and purification times and improve the operating efficiency. Therefore, HPCCC can be used for the separation and preparation of other pigment compounds in Caesalpinia sappan and other dye plants.

Sordarin Diterpene Glycosides with an Unusual 1,3-Dioxolan-4-one Ring from the Zoanthid-Derived Fungus Curvularia hawaiiensis TA26-15.

Six new sordarin tetracyclic diterpene glycosides, moriniafungins B-G (1-6), and a new sordaricin tetracyclic diterpene, sordaricin B (8), together with two known analogues, moriniafungin (7) and sordaricin (9), were isolated from the zoanthid-derived fungus Curvularia hawaiiensis TA26-15. The structures of the new compounds were elucidated by comprehensive analyses of spectroscopic data, including 1D and 2D NMR and MS data. Compounds 1-6 represent the first case of sordarins from marine-derived fungi possessing a sordarose with a spiro 1,3-dioxolan-4-one ring, which is rare in the nature. Compound 4 showed antifungal activity against Candida albicans ATCC10231 with an MIC value of 2.9 µM.

Enantioseparation of 5-chloro-2-{2-[3,4-dihydroisoquinoline-2(1H)-yl]ethyl}-2-methyl-2,3-dihydro-1H-inden-1-one (SYA 40247), a high-affinity 5-HT7 receptor ligand, by HPLC-PDA using amylose tris-(3, 5- dimethylphenylcarbamate) as a chiral stationary phase.

In previous structure-activity relationship studies to identify new and selective 5-HT7 receptor (5-HT7 R) ligands, we identified the chiral compound, 5-chloro-2-{2-[3,4-dihydroisoquinoline-2(1H)-yl]ethyl}-2-methyl-2,3-dihydro-1H-inden-1-one (SYA 40247), with high-affinity binding to the 5-HT7 R. Thus, it was of interest to separate the enantiomers in order to evaluate their affinity at the 5-HT7 R. To achieve this separation, a normal-phase analytical method using HPLC-PDA and a 4.6 × 250 mm Chiralpak AD-H column was developed. Optimized isocratic conditions of 1.00 mL/min 95:5:0.1 v/v/v hexane-ethanol-diethylamine and a 254 nm analysis wavelength yielded a 6.07 min baseline separation. The method was scaled up to a 10 × 250 mm Chiralpak AD-H column, allowing 3 mg of racemate to be separated with a single injection, and 6 mg for an overlapping double injection in the same run. The separated enantiomers were reinjected into the analytical HPLC system, peak identities confirmed by retention time and PDA UV spectra, and the enantiomeric purities determined to be 100% for peak 1 and 100% for peak 2. A Jasco P-1020 polarimeter was used to determine the specific rotation [α] of the enantiomers of peaks 1 and 2, which were -86.2 and +93.3 (deg mL)/(g dm) respectively. No racemization was observed, and the enantiomeric purity remained at 100% for each peak.

Isolation and structure determination of a new indene derivative from endophytic fungus Aspergillus flavipes Y-62.

As part of the search for naturally derived secondary metabolites, a novel indene derivative, compound 1, together with nine known metabolites (2-10) have been purified from an ethyl acetate extract of the plant-associated fungus Aspergillus flavipes Y-62, isolated from Suaeda glauca (Bunge) Bunge which was collected along Zhoushan coast, Zhejiang province, East China. The structure of the new compound 1 was elucidated by extensive use of spectroscopic techniques like 1D, 2D NMR, and HR-TOF-MS, while the known metabolites were established based on both spectral methods as well as by comparison with the previous literature. Compound 1 exhibited antimicrobial activities against the gram-negative pathogen Pseudomonas aeruginosa and Klebsiella pneumoniae with equal MIC values of 32 µg/ml.

Method Development and Validation of Indaziflam and Its Five Metabolites in Soil, Water, and Fruits by Modified QuEChERS and UHPLC-MS/MS.

A method for simultaneously determining indaziflam and its five metabolites in soil, water, and fruits using ultraperformance liquid chromatography/tandem mass spectrometry was established. The analytes were eluted in <4.5 min. Positive electrospray ionization mode was used. The analytes were extracted using acetonitrile containing 1% ammonium hydroxide, and then the extracts were purified using octadecylsilane and PRiME HLB cartridges. The quantification limits were 0.01-1.01 µg kg-1. The linearities of the calibrations for all analytes were excellent ( R2 > 0.9952). The recoveries at spike concentrations of 0.01, 0.1, and 1 mg kg-1 were 81.3-112.1%. The intraday and interday relative standard deviations were <13.5% and <12.3%, respectively. The method was successfully used to determine indaziflam and its five metabolites in samples from markets and fields. The results confirmed that the method is an effective and robust procedure for routinely determining indaziflam and its metabolites in soil, water, and fruit samples.

Structural Characterization and Assessment of the Cytotoxicity of 2,3-Dihydro-1H-indene Derivatives and Coumarin Glucosides from the Bark of Streblus indicus.

A pair of enantiomers and a pair of 2,3-dihydro-1H-indene epimers, rac-indidene A (rac-1), indidenes B and C (2, 3); four new coumarin glucosides (4-7); and four known coumarin glucosides (8-11) were isolated from the bark of Streblus indicus (Bur.) Corner. The structures of 1-11 were defined by physical data analyses, including MS, NMR, and single-crystal X-ray diffraction. The absolute configurations of the 2,3-dihydro-1H-indene derivatives were defined via experimental and calculated ECD data. rac-Indidene A and indidenes B and C showed inhibitory activity against A549 and MCF-7 tumor cells with IC50 values in the range of 2.2 ± 0.1 to 7.2 ± 0.9 µM.

Impact of Altitudes and Habitats on Valerenic Acid, Total Phenolics, Flavonoids, Tannins, and Antioxidant Activity of Valeriana jatamansi.

The changes in total phenolics, flavonoids, tannins, valerenic acid, and antioxidant activity were assessed in 25 populations of Valeriana jatamansi sampled from 1200 to 2775 m asl and four habitat types of Uttarakhand, West Himalaya. Significant (p < 0.05) variations in total phenolics, flavonoids, valerenic acid, and antioxidant activity in aerial and root portions and across the populations were observed. Antioxidant activity measured by three in vitro antioxidant assays, i.e., 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) (ABTS) radical scavenging, 2,2'-diphenyl-1-picryylhydrazyl (DPPH) free radical scavenging, and ferric-reducing antioxidant power (FRAP) assays, showed significant (p < 0.05) differences across the populations. However, no clear pattern was found in phytochemicals across the altitudinal range. Among habitat types, (pine, oak, mixed forest, and grassy land), variation in phytochemical content and antioxidant activity were observed. Equal class ranking, neighbor-joining cluster analysis, and principal component analysis (PCA) identified Talwari, Jaberkhet, Manjkhali, and Khirshu populations as promising sources with higher phytochemicals and antioxidant activity. The results recommended that the identified populations with higher value of phytochemicals and antioxidants can be utilized for mass multiplication and breeding program to meet the domestic as well as commercial demand.

Brazilwood, sappanwood, brazilin and the red dye brazilein: from textile dyeing and folk medicine to biological staining and musical instruments.

Brazilin is a nearly colorless dye precursor obtained from the heartwood of several species of trees including brazilwood from Brazil, sappanwood from Asia and the Pacific islands, and to a minor extent from two other species in Central America, northern South America and the Caribbean islands. Its use as a dyeing agent and medicinal in Asia was recorded in the 2(nd) century BC, but was little known in Europe until the 12(th) century AD. Asian supplies were replaced in the 16(th) century AD after the Portuguese discovered vast quantities of trees in what is now Brazil. Overexploitation decimated the brazilwood population to the extent that it never fully recovered. Extensive environmental efforts currently are underway to re-create a viable, sustainable population. Brazilin is structurally similar to the better known hematoxylin, thus is readily oxidized to a colored dye, brazilein, which behaves like hematein. Attachment of the dye to fabric is by hydrogen bonding or in conjunction with certain metallic mordants by coordinative bonding. For histology, most staining procedures involve aluminum (brazalum) for staining nuclei. In addition to textile dyeing and histological staining, brazilin and brazilein have been and still are used extensively in Asian folk medicine to treat a wide variety of disorders. Recent pharmacological studies for the most part have established a scientific basis for these uses and in many cases have elucidated the biochemical pathways involved. The principal use of brazilwood today is for the manufacture of bows for violins and other stringed musical instruments. The dye and other physical properties of the wood combine to produce bows of unsurpassed tonal quality.

Characterization of the Coronatine-Like Phytotoxins Produced by the Common Scab Pathogen Streptomyces scabies.

Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America.

Anmindenols A and B, inducible nitric oxide synthase inhibitors from a marine-derived Streptomyces sp.

Anmindenols A (1) and B (2), inhibitors of inducible nitric oxide synthase (iNOS), were isolated from a marine-derived bacterium Streptomyces sp. Their chemical structures were elucidated by interpreting various spectroscopic data, including IR, MS, and NMR. Anmindenols A and B are sesquiterpenoids possessing an indene moiety with five- and six-membered rings derived from isoprenyl units. The absolute configuration of C-4 in anmindenol B was determined by electronic circular dichroism (ECD) of a dimolybdenum complex. Anmindenols A (1) and B (2) inhibited nitric oxide production in stimulated RAW 264.7 macrophage cells with IC50 values of 23 and 19 µM, respectively.

Brazilein, a compound isolated from Caesalpinia sappan Linn., induced growth inhibition in breast cancer cells via involvement of GSK-3ß/ß-Catenin/cyclin D1 pathway.

Caesalpinia sappan Linn. has long been used in traditional medicine in China. Here, the anticancer activity of brazilein, a compound isolated from C. sappan Linn. was investigated. MTT assay showed that the IC50 value of brazilein against human breast cancer MCF-7 cells was 7.23 ± 0.24 µmol/L. PI staining and flow cytometry analysis indicated that brazilein caused cell cycle arrest in G1 phase. Western blot and RT-PCR assay demonstrated that cyclin D1, a key factor of the G1 to S phase progression, was downregulated in a concentration-dependent manner by brazilein treatment. Further Western blot and RNA interference assay showed that brazilein treatment activated GSK-3ß and following reduced ß-Catenin protein, which accounted for the downregulation of cyclin D1 and blockage of cell cycle at G1 phase. Together, all these results illustrated that brazilein induced growth inhibition of breast cancer cells and downregulation of GSK-3ß/ß-Catenin pathway was involved in its action mechanism.

Manzamenone O, new trimeric fatty acid derivative from a marine sponge Plakortis sp.

A new structurally unique trimeric fatty acid derivative, manzamenone O (1), was isolated from a marine sponge Plakortis sp. Manzamenone O (1) has a novel skeleton consisting of C-C bonded octahydroindenone and dioxabicyclo[3.3.0]octane moieties and three long aliphatic chains. The structure of 1 was elucidated on the basis of spectroscopic data and conformational analysis. Manzamenone O (1) exhibited antimicrobial activity against Micrococcus luteus, Aspergillis niger, and Trichophyton mentagrophytes.

Antioxidant effect and study of bioactive components of Valeriana sisymbriifolia and Nardostachys jatamansii in comparison to Valeriana officinalis.

The roots of Nardostachys jatamansi have been used as a substitute for valerian in Iranian traditions. Moreover, six species from Valeriana genus such as V. sisymbriifolia grow in Iran which has not been studied yet. We aimed to study of antioxidant effect of Valeriana officinalis, Nardostachys jatamansi and Valeriana sisymbriifolia and comparing their content of valerenic acid and valepotriate. Antioxidant effect was evaluated using diphenylpicrylhydrazyl (DPPH) inhibition and beta carotene-bleaching assays. Identification of valepotriates was achieved using chemical and TLC method. Qualitative and quantitative analysis of valerenic acid was performed using TLC and spectrophotometry methods. Among the tested samples, V. Officinalis showed the highest DPPH inhibition effect with IC(50) value of 38mg/mL. All of the tested plants potentially inhibited beta-carotene oxidation. The calibration curve of authentic valerenic acid was linear in the range of 2-51 mg L(-1). The most and least amount of valepotraites was detectable in V. officinalis and V. sisymbriifolia respectively. Total valerenic acid in different plant species ranged from 0.02% in V. sisymbriifolia to 0.07% (w/w) in V. Officinalis. Our results indicated that all three tested plants contain different amount of valepotriates and valerenic acid. The highest percentage of valepotriates and valerenic acid was detectable in V. officinalis. Overall can conclude that N. jatamansii and V. sisymbriifolia would be a good candidate for substitutation of V. officinalis with noticeable antioxidant effect.

LC-MS-based method for the qualitative and quantitative analysis of the novel PPARγ agonist KR-62980.

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for a novel peroxisome proliferator-activated receptor γ (PPARγ) agonist, KR-62980, in rat plasma. It involves liquid-liquid extraction (LLE) followed by HPLC separation and electrospray ionization tandem mass spectrometry. The linear ranges of the assay were 0.01-10 µg/mL with a correlation coefficient (R (2)) greater than 0.99 and the lower limit of quantification was 0.01 µg/mL. The average recovery was 90.1 and 98.4% from rat plasma for KR-62980 and imipramine, respectively. The coefficients of variation of intra- and inter-assay were 1.2-10.6% and the relative error was 0.8-13.2%. The method was validated and successfully applied to the pharmacokinetic study of KR-62980 in rat.

Manzamenones L-N, new dimeric fatty-acid derivatives from an Okinawan marine sponge Plakortis sp.

Three new polyketides, manzamenones L-N (1-3), have been isolated from an Okinawan marine sponge of the genus Plakortis. The structures of 1-3 were elucidated on the basis of spectroscopic data. Manzamenones L-N (1-3) were new dimeric fatty-acid derivatives consisting of a tetrahydroindenone with three carboxy groups and two hexadecanyl chains. Manzamenones M (2) and N (3) showed antimicrobial activity against several bacteria and fungi.

Three phases hollow fiber LPME combined with HPLC-UV for extraction, preconcentration and determination of valerenic acid in Valeriana officinalis.

In the present work, the applicability of hollow fiber-based liquid phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of valerenic acid prior to its determination by reversed-phase HPLC/UV. The target drug was extracted from 5.0 mL of aqueous solution with pH 3.5 into an organic extracting solvent (dihexyl ether) impregnated in the pores of a hollow fiber and finally back extracted into 10 µ L of aqueous solution with pH 9.5 located inside the lumen of the hollow fiber. In order to obtain high extraction efficiency, the parameters affecting the HF-LPME, including pH of the donor and acceptor phases, type of organic phase, ionic strength, the volume ratio of donor to acceptor phase, stirring rate and extraction time were studied and optimized. Under the optimized conditions, enrichment factor up to 446 was achieved and the relative standard deviation (RSD) of the method was 4.36% (n = 9). The linear range was 7.5-850 µg L⁻¹ with correlation coefficient (r²=0.999), detection limits was 2.5 µg L⁻¹ and the LOQ was 7.5 µg L⁻¹. The proposed method was evaluated by extraction and determination of valerenic acid in some Iranian wild species of Valerianaceae.

Antifungal activity of plumericin and isoplumericin.

This study evaluated the in vitro antifungal activity of the chloroform extract of Plumeria bicolor and its phytoconstituents plumericin and isoplumericin against Candida species and Cryptococcus neoformans by measuring the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). Plumericin's consistently high activity against Candida albicans, C. krusei, C. glabrata, C. tropicalis and Cryptococcus neoformans was more potent than isoplumericin and the standard antifungal drug nystatin suggesting its potential as a drug candidate for candidiasis and cryptococcosis.

Two new resveratrol tetramers isolated from Cayratia japonica (Thunb.) Gagn. with strong inhibitory activity on fatty acid synthase and antioxidant activity.

Two new resveratrol tetramers, cajyphenol A (1) and cajyphenol B (2), together with two known resveratrol dimers, quadrangularin A (3) and pallidol (4), and resveratrol (5) were isolated from the stems of Cayratia japonica (Thunb.) Gagn. Their structures were established by means of NMR (¹H,¹H-COSY, ¹H, ¹³C-HSQC, HMBC, and NOESY) and ESI-MS analyses. Compounds 1-5 showed fast-binding inhibitory activities on fatty acid synthase with the IC50 values of 1.63±0.02, 1.49±0.03, 7.50±0.01, 11.1±0.01, and 10.2±0.01 µM, respectively. Compounds 1-5 also exhibited antioxidant activities in the ORAC assay.

[Studies on chemical constituents of Valeriana officinalis].

From Valeriana officinalis L., 4 compounds were isolated and identified by various spectral analysis and chemical conversion, as valerenic acid, beta-sitosterol, ursolic acid, 4, 4', 8, 8'-tetrahydroxy-3, 3'-dimethoxyl-dibenzyl-ditetrahydrofuran and caryophyllene acide,valerane, naphthalene, linoleic acid, ethyl ester, myrtenyl acetate were identified by GC-MS. Ursolic acid and 4, 4', 8, 8'-tetrahydroxy-3, 3'-dimethoxyl-dibenzyl-ditetrahydrofuran were discovered in this plant for the first time.