HPLC Retention Times Research Articles

Anti-Hyperuricemic, Anti-Inflammatory and Analgesic Effects of Siegesbeckia orientalis L. Resulting from the Fraction with High Phenolic Content

BackgroundThe medicinal plant Siegesbeckia orientalis L. has been commonly used for the treatment of acute arthritis, rheumatism, and gout in Vietnam. However, pharmacological research of this plant associated with gout has not been reported. Anti-hyperuricemic and anti-inflammatory effects were evaluated and observed for the crude ethanol extract (CEE) of S. orientalis. Retention of these biological properties was found in a n-butanol-soluble fraction (BuOH fr.) of the extract, and therefore further biological and chemical investigations were undertaken on the BuOH fr. to support the medical relevance of this plant.MethodsThe aerial part of S. orientalis was obtained in the mountainous region of Vietnam. The crude ethanol extract (CEE) and its BuOH fr. were prepared from the plant materials. Anti-hyperuricemic activities of the CEE and BuOH fr. were tested in vivo using the model oxonate-induced hyperuricemia rats through determination of serum uric acid levels and inhibitory effects on xanthine oxidase (XO) in the rat liver. Anti-inflammatory activities of the BuOH fr. were also evaluated in vivo using carrageenan-induced paw edema and urate-induced synovitis in rats. Active components of the BuOH fr. were characterized by comparison of HPLC retention time (tR) and spectroscopic data (UV, 1H–NMR) with those of reference compounds.ResultsThe CEE of S. orientalis displayed anti-hyperuricemic activity, and the BuOH fr. was found to be the most active portion of the extract. Further in vivo studies on this fraction showed 31.4% decrease of serum uric acid levels, 32.7% inhibition of xanthine oxidase (XO), 30.4% reduction of paw edema volume, symptomatic relief in urate-induced synovitis and significant analgesic effect at the dose of 120 mg/kg, as compared to the corresponding values of the control groups. Chemical analysis of the BuOH fr. revealed high phenolic content, identified as caffeic acid analogues and flavonones.ConclusionsThis study suggested that anti-hyperuricemic and anti-inflammatory mechanism of S. orientalis is related to XO inhibitory effect of the phenolic components. Our findings support the use of this plant as the treatment of gout and other inflammatory diseases.

Fermented blueberry juice extract and its specific fractions have an anti-adipogenic effect in 3\xa0T3-L1 cells

BackgroundObesity and Type 2 diabetes have reached epidemic status worldwide. Wild lowbush blueberry (Vaccinium angustifolium Aiton) is a plant of the North American Aboriginal traditional pharmacopeia with antidiabetic potential, especially when it is fermented with Serratia vaccinii.MethodsA phytochemical fractionation scheme was used to identify potential bioactive compounds as confirmed by HPLC retention times and UV–Vis spectra. 3 T3-L1 cells were differentiated for 7 days with either Normal Blueberry Extract (NBE), Fermented Blueberry Extract (FBE/F1), seven fractions and four pure compounds. Triglyceride content was measured. Examination of selected intracellular signalling components (p-Akt, p-AMPK) and transcriptional factors (SREBP-1c and PPARγ) was carried out by Western blot analysis.ResultsThe inhibitory effect of FBE/F1 on adipocyte triglyceride accumulation was attributed to total phenolic (F2) and chlorogenic acid enriched (F3-2) fractions that both inhibited by 75%. Pure compounds catechol (CAT) and chlorogenic acid (CA) also inhibited adipogenesis by 70%. Treatment with NBE, F1, F3-2, CAT and CA decreased p-AKT, whereas p-AMPK tended to increase with F1. The expression of SREBP1-c was not significantly modulated. In contrast, PPARγ decreased in all experimental groups that inhibited adipogenesis.ConclusionsThese results demonstrate that fermented blueberry extract contains compounds with anti-adipogenic activity, which can serve to standardize nutraceutical preparations from fermented blueberry juice and to develop novel compounds with anti-obesity properties.

Purification and electronic characterisation of 18 isomers of the OPV acceptor material bis-[60]PCBM.

The as-produced isomer mixture of the organic photovoltaic device acceptor material bis-[60]PCBM has been purified into its constituents by peak-recycling HPLC, and those individual isomers were characterised by UV-Vis absorption spectroscopy and cyclic voltammetry. A total of 18 isomers were purified from the mixture to a standard exceeding 99.5% with respect to other isomers. The HOMOs, LUMOs, and HOMO-LUMO gaps of the purified isomers vary from -5.673 to -5.444 eV, -3.901 to -3.729 eV, and 1.664 to 1.883 eV, respectively. We also find a correlation between HPLC retention time and the relative positions of the addends; in that generally the closer the addends are to each other the longer the retention time of the isomer, and vice versa.

Biotransformation of bromhexine by Cunninghamella elegans, C. echinulata and C. blakesleeana.

Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography–mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard – clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4 min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7 min when RLM were incubated with a CYP3A4 enzyme inhibitor – clarithromycin.

Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo.

Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (∼30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous "block" in the middle of the segment or "scrambled" throughout the sequence) and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a "threshold hydrophilicity" is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomalous translocation/insertion.

Sulfoureido Lipopeptides from the Marine Sponge Discodermia kiiensis.

New N-sulfoureidylated lipopeptides, sulfolipodiscamides A-C (1-3), were isolated by gel filtration chromatography of the n-butanol fraction of the marine sponge Discodermia kiiensis. By extensive NMR analyses and high-resolution mass spectrometry, the structures of 1-3 were elucidated as having an unprecedented N-sulfoureidyl group on the d-citrulline residue, a distinct feature that was not found in the structurally related lipodiscamides A-C (4-6), derived from the ether fraction of the same sponge. Furthermore, the absolute configurations of 1-3 were confirmed by comparisons of the HPLC retention times of the hydrolytic products and the corresponding authentic lipodiscamides. Interestingly, sulfolipodiscamide A displayed a 2.3-fold increase in cytotoxicity against murine leukemia (P388) cells, compared to the unconjugated parent compound.

Fully automated determination of the sterol composition and total content in edible oils and fats by online liquid chromatography–gas chromatography–flame ionization detection

Sterol analysis of edible oils and fats is important in authenticity control. The gas chromatographic determination of the sterol distribution and total content is described by ISO norm 12228. Extraction, purification, and detection of the sterols are time-consuming and error-prone. Collaborative trials prove this regularly. Purification by thin-layer chromatography (TLC) and robust GC determination of all mentioned sterols is not straightforward. Therefore, a fully automated LC-GC-FID method was developed to facilitate the determination of sterols. The only manual step left was to weigh the sample into an autosampler vial. Saponification and extraction were performed by an autosampler while purification, separation, and detection were accomplished by online coupled normal-phase LC-GC-FID. Interlacing of sample preparation and analysis allowed an average sample throughput of one sample per hour. The obtained quantitative results were fully comparable with the ISO method with one apparent exception. In the case of sunflower oils, an additional unknown sterol was detected generally missed by ISO 12228. The reason was found in the omission of sterol silylation before subjection to GC-FID. The derivatization reaction changed the retention time and hid this compound behind a major sterol. The compound could be identified as 14-methyl fecosterol. Its structure was elucidated by GC–MS and ensured by HPLC and GC retention times. Finally, validation of the designed method confirmed its suitability for routine environments.

Cercosporin From Pseudocercosporella capsellae and its Critical Role in White Leaf Spot Development.

Pseudocercosporella capsellae, the causative agent of white leaf spot disease in Brassicaceae, can produce a purple-pink pigment on artificial media resembling, but not previously confirmed as, the toxin cercosporin. Chemical extraction with ethyl acetate from growing hyphae followed by quantitative (thin-layer chromatography [TLC] and high-performance liquid chromatography [HPLC]) and qualitative methods showed an identical absorption spectrum, with similar retardation factor (Rf) values on TLC papers and an identical peak with the same retention time in HPLC as for a standard for cercosporin. We believe this is the first report to confirm that the purple-pink pigment produced by P. capsellae is cercosporin. Confocal microscopy detected green autofluorescence of cercosporin-producing hyphae, confirming the presence of cercosporin inside hyphae. The highly virulent UWA Wlra-7 isolate of P. capsellae produced the greatest quantity of cercosporin (10.69 mg g-1). The phytotoxicity and role of cercosporin in disease initiation across each of three Brassicaceae host species (Brassica juncea, B. napus, and Raphanus raphanistrum) was also studied. Culture filtrates containing cercosporin were phytotoxic to all three host plant species, producing large, white lesions on highly sensitive B. juncea, only water-soaked areas on least sensitive R. raphanistrum, and intermediate lesions on B. napus. It is noteworthy that sensitivity to cercosporin of these three host species was analogous to their susceptibility to the pathogen, viz., B. juncea the most susceptible, R. raphanistrum the least susceptible, and B. napus intermediate. The presence of cercosporin in the inoculum significantly increased disease severity on the highly cercosporin-sensitive B. juncea. We believe that this is the first study to demonstrate that P. capsellae produces cercosporin in liquid culture rather than agar media. Finally, this study highlights an important role of cercosporin as a pathogenicity factor in white leaf spot disease on Brassicaceae as evidenced by the ability of the cercosporin-rich culture filtrate to reproduce white leaf spot lesions on host plants and by the enhanced virulence of P. capsellae in the presence of cercosporin.

DATATOC: a novel conjugate for kit-type 68Ga labelling of TOC at ambient temperature

BackgroundThe widespread acceptance and application of 68Ga-PET depends on our ability to develop radiopharmaceuticals that can be prepared in a convenient and suitable manner. A kit-type labelling protocol provides such characteristics and requires chelators that can be radiolabelled under exceptionally mild conditions. Recently the DATA chelators have been introduced that fulfil these requirements. In continuing their development, the synthesis and radiolabelling of the first DATA bifunctional chelator (BFC) and peptide conjugate are described.ResultsA BFC derived from the DATA ligand (2,2’-(6-((carboxymethyl)amino)-1,4-diazepane-1,4-diyl)diacetic acid) has been synthesised in five steps from simple building blocks, with an overall yield of 8 %. DATAM5-3tBu (5-[1,4-Bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-methyl-amino)-[1, 4]diazepan-6-yl]-pentanoic acid) has been coupled to [DPhe1][Tyr3]-octreotide (TOC) and the resulting peptide conjugate (DATATOC) radiolabelled with purified 68Ga derived via four different 68Ge/68Ga generator post-processing (PP) methods. The stability and lipophilicity of the radiotracer have been assessed and a kit-type formulation for radiolabelling evaluated. 68Ga-DATATOC has been prepared with a > 95 % radiochemical yield (RCY) within 1 (fractionated and acetone-PP) and 10 min (ethanol- and NaCl-PP) at 23 °C (pH 4.2–4.9, 13 nmol). The radiolabelled peptide is stable in the presence of human serum. Lipophilicity of 68Ga-DATATOC was calculated as logP = −3.2 ± 0.3, with a HPLC retention time (tR = 10.4 min) similar to 68Ga-DOTATOC (logP = −2.9 ± 0.4, tR = 10.3 min). Kit-type labelling from a lyophilised solid using acetone-PP based labelling achieves > 95 % RCY in 10 min at 23 °C.ConclusionsThe favourable labelling properties of the DATA chelators have been retained for DATATOC. High radiochemical purity can be achieved at 23 °C in less than 1 min and from a kit formulation. The speed, reliability, ease, flexibility and simplicity with which 68Ga-DATATOC can be prepared makes it a very attractive alternative to current standards.

Can the pathophysiology of autism be explained by the nature of the discovered urine peptides and dietary antigens?

PurposeA: 1. To develop the urine analysis for exorphins for routine use in blood and cerebrospinal fluid (CSF).2. Disorders where patient related validation must be carried out: schizophrenia, depression (uni- and bipolar) and autism.MethodA: HPLC-MS/MS (fragmentation mass spectrometry) technology.With both a specific HPLC retention time and MS/MS (fragmentation) this method is close to an absolute technique for peptide recognition.B: ELISA against specific proteins (gliadin, gluten and casein and transglutaminase 6) (Table 1 og 2).BackgroundA: schizophrenia: increased opioid peptide levels have been found in Schizophrenia using HPLC, immune assay and behavioral tests. [1–6] as part of a general peptide increase in urine. Since peptides are signaling compounds inhibition of peptidases during transport and work up of samples is critical to prevent break down, which is as expected fast at room temperature.Strongly supporting is view is the data on postpartum psychoses (a very symptom rich psychosis) where also amino acid sequence of human casomorphin found increased, has been done [7–8]. The opioids can explain most of the symptoms of the psychotic schizophrenic state [6]. It is of paramount importance then to measure these peptides in carefully diagnosed patients on and without medication, in urine, blood and spinal fluid.As can be seen in Table 1, it is important to measure IgA and IgG antibodies against the precursor proteins for the exorphins, which are found increased by several groups, and also have direct effects on the nervous system [9].B. In depression increase levels of peptides has been found [18,28,29] and also opioid levels measured as opium receptor binding peptides [28]. In schizoaffective psychosis MS/MS exact detection of exorphins have been published [6]. Also in this syndrome it is critical to be able to measure the exorphins in blood and CSF, especially since the peptidases involved in break down of exorphins are decreased in depressions [30,31]. Inflammatory interleukins are also increased in depressions both uni- and bipolar [32] indicative of inflammatory processes probably in the gut. Inflammatory interleukins increase the permeability of epithelial membranes [33].C. Autism. Considerable work has been done using HPLC with UV detection and co-chromatography [12,34–40]. However, with HPLC–MS/MS we can ensure that we are measuring only the exorphins and not chromatographically similar peaks that hide inside the main peak [41–43]. We therefore need to validate the new method in autism for both urine, blood and CSF (CSF collected only when spinal tap has to be done in any case).Inhibition of break down in urine, blood and cerebrospinal fluid (CSF)After extensive testing we have been left with three inhibitors. Citric acid 0.2 M; acetic acid 0.2 M and aprotenine [44,45].These body fluids will be provided by Prof Dr E. Severance and Prof Dr R. Yolken (Johns Hopkins Univ.) and Prof Dr. Cunningham (Uppsala Univ. Sweden). Lab 1 provides monovettes with citric acid as peptidase inhibitor for urine collection. Blood will be collected in EDTA – aprotenin vacuum test tubes (Vacutainer) as will be CSF.HPLC and MS/MS detection.The amount of urine analyzed on the HPLC after work up = 250 nanomles creatinine. To pick out generally active peptides in any one disorder, five and five autistic children or schizophrenic derived and depressive derived urines are mixed, creatinine re-determined and rerun. Peaks that are common to all patients increase or remain the same, while individual peaks of material on the HPLC runs are diluted out.The complete procedure is published in detail [48]. If we use reporter ions we do not have to match all the peaks as shown in attached figures. On Fig. 1, synthetic bovine β-casomorphine 1-4 (Y-P-F-P) is compared to biologically isolated compound from a batch of five autistic children. On Fig. 2, the faster routine analysis using reporter ions is shown for bovine β-casomorhne 1-4. Top trace is synthetic casomorphin 1-4 and bottom trace is biologically isolated compound. The complete analysis for a series of opioids is published [48].Program is then in sequence:– A: further validation of method for urine in the different disorders;– B: validation of method for blood in the same disorders;– C: validation of method for CSF (spinal fluid) in schizophrenics and depressive patients.NB.To avoid overlooking new compounds a complete HPLC run with UV 215 nm (peptide bonds); 280 nm (aromatic groups) and 325 nm (Indolyl-acryloid) shall be run for urines. If sufficient serum is available and spinal fluid these will also be run on HPLC in addition to MS/MS detection.Antibody assays will be done at Johns Hopkins using ELISA, Transglutaminase 6 antibodies at Lab 1 also using ELISA assay.Figures and references not available in the abstract.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Synthesis, Characterization, and In Vitro Evaluation of New (99m)Tc/Re(V)-Cyclized Octreotide Analogues: An Experimental and Computational Approach.

Radiolabeled proteolytic degradation-resistant somatostatin analogues have been of long-standing interest as cancer imaging and radiotherapy agents for targeting somatostatin receptor-positive tumors. Our interest in developing (186)Re- and (188)Re-based therapeutic radiopharmaceuticals led to investigation of a new Re(V)-cyclized octreotide analogue, Re(V)-cyclized, thiolated-DPhe(1)-Cys(2)-Tyr(3)-DTrp(4)-Lys(5)-Thr(6)-Cys(7)-Thr(OH)(8) (Re-SDPhe-TATE) using both experimental and quantum chemical methods. The metal is directly coordinated to SDPhe-TATE through cyclization of the peptide around the [ReO](3+) core. Upon complexation, four isomers were observed; the isolated/semi-isolated isomers exhibited different somatostatin receptor (sstr) binding affinities, 0.13 to 1.5 μM, in rat pancreatic tumor cells. Two-dimensional NMR experiments and electronic structure calculations were employed to elucidate the structural differences among the different isomers. According to NMR studies, the metal is coordinated to three thiolates and the backbone amide of Cys(2) in isomers 1 and 4, whereas the metal is coordinated to three thiolates and the backbone amide of Tyr(3) in isomer 2. Quantum chemical methods clarified the stereochemistry of Re-SDPhe-TATE and the possible peptide arrangements around the [ReO](3+) core. The re-cyclization reaction was translated to the (99m)Tc radiotracer level with four isomers observed on complexation with comparable HPLC retention times as the Re-SDPhe-TATE isomers. About 85% total (99m)Tc labeling yield was achieved by ligand exchange from (99m)Tc-glucoheptonate at 60 °C for an hour. About 100% and 51% of (99m)Tc(V)-cyclized SDPhe-TATE remained intact in phosphate buffered saline and 1 mM cysteine solution under physiological conditions at 6 h, respectively.

Analysis of 3-Methyl-4-nitrophenol, A Major Metabolite of Fenitrothion, In Mice Urine Using HPLC

Introduction: 3-methyl-4-nitrophenol is a major metabolite of fenitrothion and is considered as a potential risk to public health owning to its genotoxicity and carcinogenicity. Objective: To develop and validate an HPLC method for the quantification of 3-methyl-4-nitrophenol in urine. Methods: A sample of mice urine was collected following the administration of fenitrothion solution through oral gavage and subsequently, 3-methyl-4-nitrophenol was extracted using a simple liquid-liquid extraction method. The newly developed method uses acetonitrile: water 60:40 mobile phase at 270 nm with a flow rate of 1 mL/min HPLC condition. An optimization to a previously used extraction method was made by using ethyl acetate instead of acetonitrile to avoid extraction of water-soluble components of urine other than 3-methyl- 4-nitrophenol. The developed method was validated using the ICH guideline for the validation of analytical procedures. Accordingly, the selectivity, linearity, limit of detection and quantification, accuracy, precision, extraction efficacy, robustness and stability of the method was determined. Results: All of the validation parameters showed that the method is valid for the determination of 3-methyl-4-nitrophenol in urine sample. Using this method it was possible to detect low concentration of 3-methyl-4-nitrophenol in urine samples down to 0.87 μg/mL. Conclusion: The sample was analyzed with this validated method and the respective concentration of the 3-methyl-4-nitrophenol was found to be 31.11 μg/mL. Accordingly, 10.37% of the total fenitrothion administered to mice was found metabolized into 3-methyl-4-nitrophenol. The HPLC retention time for 3-methyl-4-nitrophenol was 2.81 minutes. Key words: Fenitrothion, 3-methyl-4-nitrophenol, HPLC, Metabolites, Method development, Validation.

Optimization of elicitation condition with Jasmonic Acid, characterization and antimicrobial activity of Psoralen from direct regenerated plants of Psoralea corylifolia L

Abstract In present study, estimation and characterization of Psoralen were done for Jasmonic Acid treated in vitro plants of Psoralea corylifolia L. The elicitor Jasmonic Acid at different concentrations (50,100,150, 200 and 250 µM/L) was used along with 6% sucrose and plant growth regulators to enhance psoralen content in leaves and roots of P. corylifolia . Shoot tip explant cultured on MSB 5 medium containing 2.0 mg/L BAP showed higher induction and proliferation of multiple shoots with 82.2% of response and 16.3 shoots/explant. In vitro rooting was best achieved with 82.6% of response and 18.6 roots/shoot at 0.8 mg/L IAA. JA at 150 µM/L concentration was found to enhance Psoralen content in leaves and roots of in vitro regenerated plants of P. corylifolia . The presence and structure of psoralen in methanolic extract of leaves and roots were confirmed through HPLC (retention time 21.007 in root, 20.659 in leaf sample), UV (213 nm), FT-IR, and NMR (C 11 H 6 O 3 ). The amount of Psoralen in JA treated leaf and root sample were found to be higher 1.17 mg/mL and 3.93 mg/mL respectively when compared to naturally growing P. corylifolia (0.56 mg/mL). The methanolic extract of root sample showed effective antimicrobial activity against tested bacterial and fungal pathogens. The present work provides the possibility of increasing production of psoralen in in vitro plants of P. corylifolia using Jasmonic Acid as an elicitor.

Cyclic alpha-conotoxin peptidomimetic chimeras as potent GLP-1R agonists

Type 2 diabetes mellitus (T2DM) results from compromised pancreatic β-cell function, reduced insulin production, and lowered insulin sensitivity in target organs resulting in hyperglycemia. The GLP-1 hormone has two biologically active forms, GLP-1-(7–37) and GLP-1-(7–36)amide, which are equipotent at the glucagon-like peptide-1 receptor (GLP-1R). These peptides are central both to normal glucose metabolism and dysregulation in T2DM. Several structurally modified GLP-1 analogues are now approved drugs, and a number of other analogues are in clinical trials. None of these compounds is orally bioavailable and all require parenteral delivery. Recently, a number of smaller peptidomimetics containing 11–12 natural and unnatural amino acids have been identified that have similar insulin regulating profiles as GLP-1. The α-conotoxins are a class of disulfide rich peptide venoms isolated from cone snails, and are known for their highly constrained structures and resistance to enzymatic degradation. In this study, we examined whether 11-residue peptidomimetics incorporated into α-conotoxin scaffolds, forming monocyclic or bicyclic compounds constrained by disulfide bonds and/or backbone cyclization, could activate the GLP-1 receptor (GLP-1R). Several compounds showed potent (nanomolar) agonist activity at GLP-1R, as evaluated via cAMP signaling. In addition, HPLC retention times and in silico calculations suggested that mono- and bicyclic compounds had more favorable n-octanol/water partition coefficients according to the virtual partition coefficient model (vLogP), while maintaining a smaller radius of gyration compared to corresponding uncyclized peptidomimetics. Our findings suggest that cyclic peptidomimetics provide a potential avenue for future design of potent, compact ligands targeting GLP-1R and possessing improved physicochemical properties.

Identification of new DNA adducts of phenylnitrenium.

Phenylnitrenium ion (PhNH(+)) may bind to nucleophiles through nitrogen as well as through C2 or C4 carbons. However, only adducts of the former type have been hitherto reported after its reaction with purine nucleosides. In this study, reactions of N-acetoxyaniline (PhNHOAc), a precursor to PhNH(+), with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), and with DNA in vitro at physiological conditions are described. The reaction of PhNHOAc with dA followed by a hydrolytic deribosylation afforded 8-phenylaminoadenine (C8-PhNHA) together with a smaller amount of N(6)-(4-aminophenyl)adenine (N(6)-4APA). A similar reaction with dG afforded 8-phenylaminoguanine (C8-PhNHG) together with traces of 7-(4-aminophenyl)guanine (N7-4APG). The same adducts were found also in the DNA treated with PhNHOAc, and all of them were identified by comparison of their HPLC retention times and MS(2) spectra with a set of synthesized authentic adenine adducts at C2, C8, N7, and N(6) positions and guanine adducts at C8, N7, and N(2) positions. The newly identified minor adduct, N7-4APG, represents the first proof of arylnitrenium adduction at the N7 position of dG, which is the prominent site of attack by most C-electrophiles.

Monitoring sialylation levels of Fc-fusion protein using size-exclusion chromatography as a process analytical technology tool.

To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples. A statistical significant correlation between the sialic acid content and size-exclusion chromatography (SEC)-HPLC retention time of an Fc-fusion protein was observed when analyzing the titer of the samples. Using linear fitting analysis, the data fit the model well with R (2) = 0.985. Based on the SDS-PAGE and oligosaccharide analysis, we speculate that the amounts of the glycans could expand the structure of the Fc-fusion protein. This was manifested by the SEC-HPLC method in which proteins were separated based on its molecular size. In order to development a robust PAT method, an internal standard was used to improve the precision of the method by reducing systematic errors. We found the change of SEC retention time (delta t) and sialic acid content were highly correlated (R (2) = 0.992). This method was further validated by a 1500 l production process. SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.

Lovastatin-producing endophytic fungus isolated from a medicinal plant Solanum xanthocarpum

Lovastatin is a potent drug for lowering blood cholesterol. An endophytic fungus Phomopsis vexans was isolated from the healthy leaf tissues of Solanum xanthocarpum, a medicinal plant, and screened for lovastatin production. The fungus was identified by their characteristic cultural morphology and molecular analysis. The strain had a component with the same TLC Rf value and HPLC retention time as authentic lovastatin. The presence of lovastatin was further confirmed by FT-IR, UV, 1H, 13C NMR and LC–MS analyses. The amount of lovastatin produced by this endophytic fungus was quantified to be 550 mg/L, and thus the fungus can serve as a potential material to improve the production of lovastatin.

Anthocyanins in the Bracts of Curcuma Species and Relationship of the Species Based on Anthocyanin Composition

Five anthocyanins, delphinidin 3-O-rutinoside, cyanidin 3-O-rutinoside, petunidin 3-O-rutinoside, malvidin 3-O-glucoside and malvidin 3-O-rutinoside, were identified. Three anthocyanins, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside and pelargonidin 3-O-rutinoside, were putatively identified based on C18 HPLC retention time, absorption spectrum, including λmax, and comparisons with those of corresponding standard anthocyanins, as the compounds responsible for the pink to purple-red pigmentation of the bracts of Curcuma alismatifolia and five related species. Cluster analysis based on four major anthocyanins formed two clusters. One consisted of only one species, C. alismatifolia, and the other consisted of five. Each cluster further formed sub-clusters depending on either species or habitats.

Prediction of HPLC retention times of tebipenem pivoxyl and its degradation products in solid state by applying adaptive artificial neural network with recursive features elimination

A sensitive and fast HPLC method using ultraviolet diode-array detector (DAD)/electrospray ionization tandem mass spectrometry (Q-TOF-MS/MS) was developed for the determination of tebipenem pivoxyl and in the presence of degradation products formed during thermolysis. The chromatographic separations were performed on stationary phases produced in core–shell technology with particle diameter of 5.0µm. The mobile phases consisted of formic acid (0.1%) and acetonitrile at different ratios. The flow rate was 0.8mL/min while the wavelength was set at 331nm. The stability characteristics of tebipenem pivoxyl were studied by performing stress tests in the solid state in dry air (RH=0%) and at an increased relative air humidity (RH=90%). The validation parameters such as selectivity, accuracy, precision and sensitivity were found to be satisfying. The satisfied selectivity and precision of determination were obtained for the separation of tebipenem pivoxyl from its degradation products using a stationary phase with 5.0µm particles. The evaluation of the chemical structure of the 9 degradation products of tebipenem pivoxyl was conducted following separation based on the stationary phase with a 5.0µm particle size by applying a Q-TOF-MS/MS detector. The main degradation products of tebipenem pivoxyl were identified: a product resulting from the condensation of the substituents of 1-(4,5-dihydro-1,3-thiazol-2-yl)-3-azetidinyl]sulfanyl and acid and ester forms of tebipenem with an open β-lactam ring in dry air at an increased temperature (RH=0%, T=393K) as well as acid and ester forms of tebipenem with an open β-lactam ring at an increased relative air humidity and an elevated temperature (RH=90%, T=333K). Retention times of tebipenem pivoxyl and its degradation products were used as training data set for predictive model of quantitative structure–retention relationship. An artificial neural network with adaptation protocol and extensive feature selection process was created. Input parameters for model were calculated from molecular geometries optimized with application of Density Functional Theory. The model was prepared and optimized especially for small data sets such as degradation products of specific compound. Validation of the model with statistical test against requirements for QSAR showed its ability for prediction of retention times within given data set. Mean error of 24.75% (0.8min) was achieved with utilization of topological, geometrical and electronic descriptors.

New Assay Method for Allene Oxide Cyclase, an Important Enzyme in Jasmonic Acid Biosynthesis

Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method. Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo(10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC. Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan. Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were Original Research Article Keimei and Yoichiro; BBJ, 9(2): 1-7, 2015; Article no.BBJ.20089 2 purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system. Results: We found in the presence of 50 nmol of purified AOS together with 20 M 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system. Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.