Hypoglycemic active principles from the leaves of Bauhinia holophylla: Comprehensive phytochemical characterization and in vivo activity profile.

Bauhinia holophylla leaves, also known as "pata-de-vaca", are traditionally used in Brazil to treat diabetes. Although the hypoglycemic activity of this medicinal plant has already been described, the active compounds responsible for the hypoglycemic activity have not yet been identified. To rapidly obtain two fractions in large amounts compatible with further in vivo assay, the hydroalcoholic extract of B. holophylla leaves was fractionated by Vacuum Liquid Chromatography and then purified by medium pressure liquid chromatography combined with an in vivo Glucose Tolerance Test in diabetic mice. This approach resulted in the identification of eleven compounds (1-11), including an original non-cyanogenic cyanoglucoside derivative. The structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution mass spectrometry. One of the major compounds of the leaves, lithospermoside (3), exhibited strong hypoglycemic activity in diabetic mice at the doses of 10 and 20 mg/kg b.w. and prevents body weight loss. The proton nuclear magnetic resonance (1H NMR) quantification revealed that the hydroalcoholic leaves extract contained 1.7% of lithospermoside (3) and 3.1% of flavonoids. The NMR analysis also revealed the presence of a high amount of pinitol (4) (9.5%), a known compound possessing in vivo hypoglycemic activity. The hypoglycemic properties of the hydroalcoholic leaves extract and the traditional water infusion extracts of the leaves of B. holophylla seem thus to be the result of the activity of three unrelated classes of compounds. Such results support to some extent the traditional use of Bauhinia holophylla to treat diabetes.

Niazirin from Moringa oleifera Lam. attenuates high glucose-induced oxidative stress through PKCζ/Nox4 pathway.

BACKGROUND: Diabetic complications-coronary atherosclerosis is closely related to the increased reactive oxygen species (ROS) induced by hyperglycemia. ROS are reported to induce the abnormal proliferation of vascular smooth muscle cells (VSMCs) under high glucose conditions. Leaf and seed extracts from Moringa oleifera are found to exhibit antioxidant activity. However, few studies are evaluating the antioxidant activities of chemical compounds isolated from the M. oleifera especially in cardiovascular field. PURPOSE: The aim of this study is to explore the antioxidative effect during hyperglycemia of niazirin from M. oleifera. STUDY DESIGN: A cell model was applied. METHODS: After the taking the in vitro antioxidant experiment including ferric reducing antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Cell viability was carried out using high glucose-induced VSMCs model. ROS production was tested by 2',7'-dichlorofluorescein diacetate (DCF-DA) assay. The protein kinase C zeta (PKCζ) and NADPH oxidase 4 (Nox 4) expression in vitro and in vivo were measured by western blot analysis. RESULTS: Niazirin showed good free radical scavenging activity. Niazirin significantly attenuated the proliferation of high glucose-induced VSMCs. Furthermore, it could decrease the ROS and malondialdehyde (MDA) productions, while increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) as well as glutathione peroxidase (GPx) levels in high glucose-induced VSMCs and streptozotocin-induced mice. In addition, niazirin could eliminate the high glucose-induced PKCζ activation, indicated by Thr410 phosphorylation and inhibition of the Nox4 protein expression in vitro and in vivo. CONCLUSION: Niazirin from M. oleifera exhibited notably antioxidant activities and could be utilized as a potential natural antioxidant in preventing diabetic atherosclerosis.

The Effect of Grapefruit Juice on the Pharmacokinetics of Tadalafil in Rats.

We developed and validated a novel, sensitive, selective, and inexpensive high-performance liquid chromatography (HPLC) method for the determination of tadalafil in rats plasma and to investigate the effect of grapefruit juice on the pharmacokinetics of tadalafil in rats. The ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) chromatography column can be used to separate tadalafil and carbamazepine (internal standard, IS). A mixture of acetonitrile-0.2% trifluoroacetic acid-water (48 : 10 : 42, V/V/V) was used as the mobile phase with a flow rate of 1.0 mL/min. The column temperature was set at 35.0°C. The detection wavelength was set at 286 nm. The tadalafil was extracted by ethyl acetate from plasma at the alkaline condition. 12 healthy male Sprague-Dawley (SD) rats were randomly divided into two groups, Group A (experimental group, received grapefruit juice 5 mL/kg for 7 days) and Group B (control group, received normal saline for 7 days). All the rats were given a single dose of tadalafil (5 mg/kg) after the last administration. The main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the conditions of this experiment, the plasma concentrations of tadalafil in the range of 10-2000 ng/ml had a good linear relationship. The intra- and interday precision for tadalafil in plasma were less than 15%, and the relative recovery rate was good at low, medium, and high QC levels. The C max of tadalafil in the control group and the experimental group was (725.89 ± 161.59) ng/mL and (1271.60 ± 179.31) ng/mL, t 1/2 was (9.28 ± 2.07) h and (11.70 ± 1.47) h, AUC (0-t) was (7399.61 ± 696.85) ng·h/mL and (9586.52 ± 2048.81) ng·h/mL, and AUC(0-∞) was (7995.50 ± 707.23) ng·h/mL and (10639.43 ± 2235.94) ng·h/mL, respectively. Results show that the C max of tadalafil in group A was 75.17% higher than that in group B, the Vz/F was also reduced, and the t 1/2 was increased by 2.42 h. The developed HPLC-DAD method for the determination of tadalafil in rats plasma was accurate, reproducible, specific, and it was found to be suitable for the pharmacokinetics of tadalafil and food-drug interactions. Grapefruit juice can inhibit the metabolism of tadalafil and increase the exposure of tadalafil in rats.

Innate positive chemotaxis to paeonal from highly attractive Chinese medicinal herbs in the cigarette beetle, Lasioderma serricorne.

Lasioderma serricorne, also known as cigarette beetle, can exploit a wide variety of stored materials as foods, but it is particularly common on tobacco and herbs. This beetle is a dominant pest species of stored Chinese medicinal materials (CMMs) causing high economic damages, making effective control strategies urgently needed. Behavioural manipulation is an important component of Integrated Pest Management. To the best of our knowledge, plant-borne volatile organic compounds (VOCs) have never been explored to develop lures for managing L. serricorne. In this study, the behavioural responses of L. serricorne to VOCs from four selected CMMs (Euphorbia kansui, Aconitum carmichaelii, Eucommia ulmoides and Pinellia ternata) were studied and their components analysed. Then, the olfactory responses of L. serricorne to the most abundant VOC identified in the preferred CMM, i.e., paeonal, was tested. L. serricorne showed significant differences in its preferences for the VOCs from the four CMMs, i.e, E. kansui > A. carmichaelii > E. ulmoides > P. ternata. From the VOCs of E. kansui, A. carmichaelii, E. ulmoides, and P. ternata, 77, 74, 56, and 81 molecules, were identified, respectively. Paeonal (23.5%), junipene (17.2%), hexanal (17.1%), and benzeneacetonitrile (14.0%) were the most abundant, respectively. Since paeonal dominated the VOC spectrum of the most preferred CMM, this compound was selected for further studies. L. serricorne showed significant positive responses to paeonal tested at various doses, with the most attractive ones being 100 µg and 500 µg. Our findings shed light on the olfactory cues routing the food searching behaviour in the cigarette beetle, providing important information on how L. serricorne targets particular CMMs. The high attractiveness of paeonal at low doses tested here may be exploited further to develop novel monitoring and control tools (e.g., lure-and-kill strategies) against this important stored product pest.

Beneficial Effect of Jojoba Seed Extracts on Hyperglycemia-Induced Oxidative Stress in RINm5f Beta Cells.

Hyperglycemia occurs during diabetes and insulin resistance. It causes oxidative stress by increasing reactive oxygen species (ROS) levels, leading to cellular damage. Polyphenols play a central role in defense against oxidative stress. In our study, we investigated the antioxidant properties of simmondsin, a pure molecule present in jojoba seeds, and of the aqueous extract of jojoba seeds on fructose-induced oxidative stress in RINm5f beta cells. The exposure of RINm5f beta cells to fructose triggered the loss of cell viability (-48%, p < 0.001) and disruption of insulin secretion (p < 0.001) associated with of reactive oxygen species (ROS) production and a modulation of pro-oxidant and antioxidant signaling pathway. Cell pre-treatments with extracts considerably increased cell viability (+86% p < 0.001) for simmondsin and +74% (p < 0.001) for aqueous extract and insulin secretion. The extracts also markedly decreased ROS (-69% (p < 0.001) for simmondsin and -59% (p < 0.001) for aqueous extract) and caspase-3 activation and improved antioxidant defense, inhibiting p22phox and increasing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels (+70%, p < 0.001) for aqueous extract. Simmondsin had no impact on Nrf2 levels. The richness and diversity of molecules present in jojoba seed extract makes jojoba a powerful agent to prevent the destruction of RINm5f beta cells induced by hyperglycemia.

High-level expression and characterization of solvent-tolerant lipase.

In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future.

[JUN N-TERMINAL KINASES AND THEIR PHARMACOLOGICAL MODULATION OF ISCHE-MIC AND REPERFUSION INJURY OF THE BRAIN].

The article reviews the literature regarding the role of c-Jun-N-terminal kinases (JNK) and its inhibitors in brain damage in the settings of ischemia and reperfusion injury. The implication of JNK in signaling mechanisms involved in ischemia-reperfusion-induced cerebral injury are discussed. Described effects associated with JNK inhibition using synthetic and natural substances in experimental models of ischemic and reperfusion injury of the brain. Results of experimental studies demonstrated that JNK represent promising therapeutic targets for brain protection against ischemic stroke. However, multiple physiologic functions of various JNK family members do not allow for the systemic use of non-specific JNK inhibitors for therapeutic purposes. The authors conclude that the continuous search for selective inhibitors of JNK3 remains an important task.

Four New Cyclohexylideneacetonitrile Derivatives from the Hypocotyl of Mangrove (Bruguiera gymnorrhiza).

Four new cyclohexylideneacetonitrile derivatives 1-4, named menisdaurins B-E, as well as three known cyclohexylideneacetonitrile derivatives--menisdaurin (5), coclauril (6), and menisdaurilide (7)--were isolated from the hypocotyl of a mangrove (Bruguiera gymnorrhiza). The structures of the isolates were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-7 showed anti-Hepatitis B virus (HBV) activities, with EC50 values ranging from 5.1 ± 0.2 µg/mL to 87.7 ± 5.8 µg/mL.

Spongean alkaloids protect rat kidney cells against cisplatin-induced cytotoxicity.

Nephrotoxicity is the major dose-limiting adverse effect of cisplatin (CisPt) and results from CisPt-induced damage of tubular cells. Nephroprotective strategies are preferential to improve supportive care in cancer. We investigated a subset of purified substances originating from various plants or from marine sponges as to their potency to protect rat renal tubular cells (NRK-52E) against the cytotoxic and genotoxic effects of cisplatin. Cotreatment with a substance pool containing five purified substances originating from marine sponges increased the viability of NRK-52E cells following cisplatin treatment. Cytoprotection was accompanied by a reduced level of DNA damage as indicated by a lower amount of S139 phosphorylated histone H2AX (γH2AX) 24 h after treatment. Cytoprotection and genoprotection by the sponge substance pool did not comprise the anthracycline derivative doxorubicin. The spongean alkaloid aaptamine was identified as major bioactive compound that mediates cisplatin resistance. Aeroplysinin-1 was less cytoprotective than aaptamine. Notably, aaptamine preferentially conferred resistance to cisplatin, but not to oxaliplatin. Cytoprotection by aaptamine was also observed in rat glomerular endothelial cells, but not in RT-112 bladder cancer cells. Protection by aaptamine does not rest on a reduced formation of DNA damage caused by cisplatin treatment. Aaptamine and aeroplysinin-1 affected cisplatin-stimulated DDR as reflected on the level of S15-phosphorlyated p53 and S345-phosphorylated checkpoint kinase-1. Summarizing, the spongean alkaloid aaptamine alleviates cisplatin-induced damage in tubular and glomerular rat kidney cells. Therefore, we hypothesize that aaptamine might be useful to widen the therapeutic window of a cisplatin-based therapeutic regimen.

High dose of pyridoxine induces IGFBP-3 mRNA expression in MCF-7 cells and its induction is inhibited by the p53-specific inhibitor pifithrin-α.

It has been reported that supplementation with high-dose vitamin B(6) (B(6)) exerts antitumor effects in rodent models of cancer. However, the mechanism of these effects remains poorly understood. High-dose B(6) also suppresses cell proliferation and induces apoptosis of human breast adenocarcinoma MCF-7 cells. Based on preliminary experiments using DNA microarray analyses, we hypothesized that high-dose pyridoxine (PN) might induce IGF-binding protein-3 (IGFBP-3) expression in MCF-7 cells. In this study, we investigated IGFBP-3 induction by 3 or 10 mM PN using a quantitative real-time PCR method. We found that the induction reached a maximum of 24-fold with 10 mM PN for 72 h compared with non-treated cells. The induction of IGFBP-3 by PN was inhibited by a p53-specific inhibitor, pifithrin-α, in a dose-dependent manner, but was not affected by PD169316 (MAPK inhibitor), AS601245 (c-Jun N-terminal kinase inhibitor) or SL327 (MEK1/2 inhibitor). High-dose PN did not induce p53 mRNA expression. The IGFBP-3 induction by PN seemed to be related to p53 activation.

Method to screen substrates of apical sodium-dependent bile acid transporter.

Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.

Differential roles of p38-MAPK and JNKs in mediating early protection or apoptosis in the hyperthermic perfused amphibian heart.

In the present study the activation of p38 mitogen-activated protein kinase (p38-MAPK) and c-Jun N-terminal kinases (JNKs) by hyperthermia was investigated in the isolated perfused Rana ridibunda heart. Hyperthermia (42 degrees C) was found to profoundly stimulate p38-MAPK phosphorylation within 0.5 h, with maximal values being attained at 1 h [4.503(+/-0.577)-fold relative to control, P<0.01]. JNKs were also activated under these conditions in a sustained manner for at least 4 h [2.641(+/-0.217)-fold relative to control, P<0.01]. Regarding their substrates, heat shock protein 27 (Hsp27) was maximally phosphorylated at 1 h [2.261(+/-0.327)-fold relative to control, P<0.01] and c-Jun at a later phase [3 h: 5.367(+/-0.081)-fold relative to control, P<0.001]. Hyperthermia-induced p38-MAPK activation was found to be dependent on the Na+/H+ exchanger 1 (NHE1) and was also suppressed by catalase (Cat) and superoxide dismutase (SOD), implicating the generation of reactive oxygen species (ROS). ROS were also implicated in the activation of JNKs by hyperthermia, with the Na+/K+-ATPase acting as a mediator of this effect at an early stage and the NHE1 getting involved at a later time point. Finally, JNKs were found to be the principal mediators of the apoptosis induced under hyperthermic conditions, as their inhibition abolished poly(ADP-ribose) polymerase (PARP) cleavage after 4 h at 42 degrees C. Overall, to our knowledge, this study highlights for the first time the variable mediators implicated in the transduction of the hyperthermic signal in the isolated perfused heart of an ectotherm and deciphers a potential salutary effect of p38-MAPK as well as the fundamental role of JNKs in the induced apoptosis.

Suitability of commonly used excipients for electrophysiological in-vitro safety pharmacology assessment of effects on hERG potassium current and on rabbit Purkinje fiber action potential.

INTRODUCTION: Regulatory guidelines require investigation of the liability for delayed ventricular repolarization by new chemical entities within a broad concentration range in-vitro. However, investigation can be limited by poor drug aqueous solubility, and by solvent physicochemical attributes that disrupt cell membrane integrity. Although excipients or solubilizing agents may aid to achieve the necessary high concentrations, no comprehensive overview on the suitability of solvents for in-vitro electrophysiological safety studies exists. METHODS: Excipients were tested for potential interference with the hERG (human ether-a-go-go-related gene) K(+) current (whole-cell voltage-clamp, 23+/-2 degrees C), and the shape of rabbit Purkinje fiber action potentials (conventional glass microelectrode technique, 37+/-1 degrees C). RESULTS AND DISCUSSION: Water-soluble complexation builders/carriers had little effect on hERG K(+) current at up to 50 mg/ml (BSA, bovine serum albumin) and 11 mg/ml (HP-beta-CD, hydroxypropyl-beta-cyclodextrin; IC(20), concentration of 20% inhibition). Water-soluble organic (co)solvents inhibited hERG K(+) currents (IC(20), %/mM): 0.7/152, ethanol; 0.9/67, Transcutol; 1.2/154, DMSO (dimethylsulfoxide); 1.6/389, acetonitrile; 1.9/48, polyethylene glycol 400; 2.1/660, methanol. Part of their inhibitory effect is attributed to the osmolality of extracellular solutions, because hERG IC(20) and extrapolated osmolality at the hERG IC(20) strongly correlate. Water-soluble non-ionic solubilizers/surfactants are potent inhibitors of hERG K(+) current with IC(20) concentrations of 0.07% (Cremophor EL) or lower (Tween 20, Tween 80: approximately 0.001%). Part of this inhibitory effect is attributed to their interaction with lipid membranes, because hERG inhibition occurs close to critical micelle concentrations (Cremophor, approximately 0.009%; Tween 20, approximately 0.007%). Purkinje fiber action potentials are little affected by HP-beta-CD at up to 2 mg/ml, while DMSO tends to shorten the action potential duration at 1%. CONCLUSION: When conducting electrophysiological in-vitro assessments of drug effects, solubilizers/surfactants (Cremophor EL, Tween 20, Tween 80) should be avoided. Instead, water-soluble organic (co)solvents (methanol, acetonitrile, DMSO) or complexation builders/carriers (HP-beta-CD, BSA) appear to be more favorable.

Modulatory effects of luteolin on osteoblastic function and inflammatory mediators in osteoblastic MC3T3-E1 cells.

The effects of luteolin on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were investigated. Luteolin (1microM) caused a significant elevation of collagen content, alkaline phosphatase (ALP) activity, and osteocalcin secretion in the cells (P<0.05). The effect of luteolin in increasing collagen content and ALP activity was completely prevented by the presence of 10(-6)M cycloheximide and 10(-6)M tamoxifen, suggesting that luteolin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of luteolin on the 3-morpholinosydnonimine (SIN-1)-induced production of oxidative stress markers [nitric oxide (NO) and prostaglan E(2) (PGE(2))] and cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)] in osteoblasts. Luteolin (1 and 10microM) decreased the SIN-1-induced production of NO, PGE(2), TNF-alpha, and IL-6 in osteoblasts. These results suggest that inflammatory mediators can be regulated by luteolin stimulating osteoblastic function.

The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase.

BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization. RESULTS: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies. CONCLUSION: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.

Sample preparation improvement in polycyclic aromatic hydrocarbons determination in olive oils by gel permeation chromatography and liquid chromatography with fluorescence detection.

The determination of 15 polycyclic aromatic hydrocarbons (PAHs) in olive oil samples has been improved in order to obtain a fast methodology with a low limit of detection through the combination of liquid-liquid extraction with acetonitrile and preparative gel permeation chromatography (GPC) prior to the injection of purified extracts into a C18 column. Acetonitrile-water was used as the mobile phase with a gradient from 50 to 95%, w/w, acetonitrile in 30 min. The oven temperature was maintained at 15 degrees C, and fluorometric detection was made at a fixed excitation wavelength of 264 nm and variable, optimal emission wavelength for each analyte ranging from 352 nm for 11-H-benzo(b)fluorene to 500 nm for indeno(1,2,3-cd)pyrene. Recovery for all the compounds studied varied from 75 to 111%, and limit of detection values from 0.05 ng/g for benzo(k)fluoranthene to 0.48 ng/g for indeno(1,2,3-cd)pyrene, corresponding to 0.09 ng/g benzo(a)pyrene. Results were compared with those obtained by liquid-liquid extraction followed by a cleanup on silica and a direct GPC treatment of oil samples diluted in dichloromethane, 2 other methodologies that are appropriate for quantifying PAHs in olive oils. However, the proposed method improves the determination limits, reduces the time of analysis, and provides a highly stable baseline for sample chromatograms.

Screening mutant libraries of fungal laccases in the presence of organic solvents.

Reliable screening methods are being demanded by biocatalysts' engineers, especially when some features such as activity or stability are targets to improve under non-natural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase-expressed in Saccharomyces cerevisiae-highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/microL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu2+. Copper concentration was limited up to 10 microM CuSO4 where expression levels (approximately 14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents.

Selective extraction of derivates of p-hydroxy-benzoic acid from plant material by using a molecularly imprinted polymer.

Selective SPE of derivates of p-hydroxybenzoic acid (pHBA) from plant extract of Melissa officinalis is presented using a molecularly imprinted polymer (MIP) made with protocatechuic acid (PA) as template molecule. MIP was prepared with acrylamide as functional monomer, ethylene glycol dimethacrylate as crosslinking monomer and ACN as porogen. MIP was evaluated towards six phenolic acids: PA, gallic acid, pHBA, vanillic acid (VA), gentisic acid (GeA) and syringic acid (SyrA), and then steps of molecularly imprinted SPE (MISPE) procedure were optimized. The best specific binding capacity of MIP was obtained for PA in ACN (34.7 microg/g of MIP). Other tested acids were also bound on MIP if they were dissolved in this solvent. ACN was chosen as solvent for sample application. M. officinalis was extracted into methanol/water (4:1, v/v), the extract was then evaporated to dryness and dissolved in ACN before application on MIP. Water and ACN were used as washing solvents and elution of benzoic acids was performed by means of a mixture methanol/acetic acid (9:1, v/v). pHBA, GA, PA and VA were extracted with recoveries of 56.3-82.1% using this MISPE method. GeA was not determined in plant extract.

Simultaneous quantification of adrenergic amines and flavonoids in C. aurantium, various Citrus species, and dietary supplements by liquid chromatography.

An analytical method was developed for the simultaneous quantitative analysis of 6 amines and 20 flavonoids in fruits and extracts of 30 Citrus species, including C. aurantium, near-Citrus relatives, and dietary supplements by liquid chromatography with photodiode array detection. The separation was achieved with a Phenomenex Synergi Hydro reversed-phase column using gradient mobile phase of sodium acetate buffer (pH 5.5) and acetonitrile. Elution was run at a flow rate of 1.0 mL/min and UV at 254, 280, and 330 nm. Among the amines analyzed, synephrine, the main component, was present in the levels from 0.11 to 2.0 mg/g dry weight in 21 Citrus species and 0.07 to 18.62% in dietary supplements claiming to contain C. aurantium. The flavanones and flavones were analyzed in the same Citrus samples and were species-specific. The levels of flavones were very low compared with those of flavanones. The method facilitated the simultaneous quantification of 6 amines and 20 flavonoids in various Citrus species, the distinction between the different Citrus species, and the analysis of dietary supplements containing C. aurantium.

Simmondsin: effects on meal patterns and choice behavior in rats.

Simmondsin, a glycoside from jojoba meal, decreases food intake after oral administration. The present experiments are designed to clarify the mechanism of simmondsin's anorectic activity. The meal pattern analysis shows that simmondsin supplementation at different doses results in a dose-dependent food intake reduction, which is more pronounced after prior simmondsin experience. The effect of simmondsin on meal patterns (decreased meal size, meal duration and eating rate, increased latency to eat) is most severe at the highest concentration. Rats familiar with simmondsin more seriously postpone their first meal than with first contact, resulting in a decrease of the meal frequency and the day/night feeding ratio. Rats given the choice between a control diet and a simmondsin-supplemented (0.5%) diet, after half an hour, have a significant preference for the control diet. Simmondsin seems to have a specific flavor when mixed in the food since rats recognise the feeder containing simmondsin. The ability of simmondsin to induce conditioned taste aversion (CTA) was also investigated. Rats receiving simmondsin at concentrations of 0.15%, 0.25% or 0.5% during their conditioning develop significant taste aversions to the saccharin solutions. The performed experiments indicate that the simmondsin activity shows some analogy with the satiating molecule cholecystokinin (CCK) at first contact, but shows more analogy with the illness-inducing agent lithium chloride (LiCl) after prior experience with simmondsin. Rats familiar with simmondsin avoid simmondsin-supplemented food by directly monitoring its presence, and by learning to relate it to the postingestive consequences of consumption.