Статті в журналах з теми "Y-Cyclodextrine"

The effect of the reaction conditions (substrate concentration, enzyme dosage, and pH) on cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium was investigated by applying mathematical modeling methods. Adequate models were developed and they were used for determination of the optimal conditions for maximal formation of beta-cyclodextrins at minimal concentrations of a- and gamma-cydclodextrins. The main factor affecting the ratio of the products was pH of the reaction mixture. At pH 9 the enzyme formed mainly beta- and y-cyclodextrins and the ratio a:beta:gamma was 2.6:83.5:13.9; at pH 5 the ratio changed to 8.6:84.6:6.8. Mathematical models were used for determination of the conditions for maximal conversion of the substrate into cyclodextrins. 45.88% conversion of starch was achieved at 5% substrate concentration, 3.5 U/g enzyme dosage, and pH 7.4.

Currently, the percentage of infections with invasive (parasitic) diseases is quite large; therefore, the treatment of helminthiases is an urgent problem in veterinary medicine. Parasitic worms inflict significant damage on animal husbandry, leading to the death of animals, shortage of meat, dairy products, and wool. The most common active ingredient in antihelmintics is praziquantel, which is well known as an effective broad-spectrum anthelmintic. At the same time, praziquantel has low solubility in water and a pronounced bitter taste, which represents a significant obstacle in developing liquid forms of drugs that are convenient for administration to animals. One way to solve these problems is the complexation of medicinal substances with various (natural and synthetic) compounds. In this regard, this paper aims to study the complexation of praziquantel with α-, β-, and -cyclodextrins in aqueous-alcoholic solutions. The studies were carried out by the method of ultraviolet spectroscopy. It was found that the addition of cyclodextrins to aqueous-alcoholic solutions of praziquantel leads to spectral changes indicating the presence of intermolecular interactions and complexation. The isomolar series method showed that in dilute solutions, praziquantel forms complex compounds with cyclodextrins 1:1, that is, one molecule of praziquantel falls on one molecule of α-, β- or y-cyclodextrin. The stability constants of the resulting complexes were calculated using the molar ratio method. It is shown that in the range of 296-316 K, the composition of complex compounds remains unchanged (1:1), and their stability decreases with increasing temperature. The study of the temperature dependences of the stability constants made it possible to determine the standard values of changes in the Gibbs energy, enthalpy, and complexation entropy.

The reaction of 1-bromo-2-X-2-(Y-phenyl) ethane derivatives (1: X = Y = H; 2: X = Ph, Y = H; 3: X = H, Y = 4-Ac; 4: X = H, Y = 3-NO2; 5: X = H, Y = 4-NO2; 6: X = H, Y = 3-Me; 7: X = H, Y = 4-Me) in basic solution was studied, and in most cases, only the elimination product is formed. Only (2-bromo-1-phenylethyl)benzene, 2, yielded significant substitution product, and this yield decreased with the concentration of HO-. Addition of cyclodextrin (β-CD) diminished (about half for 0.02 M cyclodextrin concentration) the reaction rate of all substrates but 4 and 5. In the latter two cases, the rate rises. The observed rate-constant value at 0.5 M NaOH is 6.78 × 10-4 s-1 (at 40°C) and 1.80 × 10-3 s-1 (at 25°C) for 4 and 5, respectively. Under the same reaction conditions but with 0.01 M β-CD, the corresponding rates were 7.70 × 10-4 s-1 and 5.20 × 10-3 s-1. The elimination yield for 2 increased from 64 to 98% when the β-CD changed from zero to 0.02 M at 0.5 M NaHO. Also, there was an increase in the relative elimination products of 20-40% for compounds 6 and 7. The Hammet ρ values were 1.3 and 2.3 for the reaction in pure solvent and in the presence of β-cyclodextrin, indicating an increase in the negative character of the transition state for the reactions in the latter conditions. The results are interpreted in terms of the formation of an inclusion complex whose structure depends on the substrate.Key words: cyclodextrin, elimination reactions, inhibition, catalysis.

The complexation of cationic pyronine B (PB+) and pyronine Y (PY+) by β-cyclodextrin (βCD) and two linked βCD dimers, N,N′-bis((2AS,3AS)-3A-deoxy-β-cyclodextrin-3A-yl)succinamide, 33βCD2suc, and N,N′-bis(6A-deoxy-β-cyclodextrin-6A-yl)succinamide, 66βCD2suc, in aqueous solution has been studied by UV-vis, fluorescence, and 1H NMR spectroscopy. The complexation constants for the 1:1 complexes: βCD.PB+, 33βCD2suc.PB+, 66βCD2suc.PB+, and the analogous PY+ complexes are reported as are the dimerization constants for PB+ and PY+. The modes of complexation, dimerization, and fluorescence quenching are discussed.

Introduction: Cyclodextrins (CD), are known to form inclusion complexes with a variety of guest molecules both in solution and in the solid state. This can lead to the alteration of properties of guest molecules. Unfortunately, the complexation efficiency of CD is rather low, and can be enhanced by formation of ternary complexes using aminoacids (AA). Sulfadiazine (SDZ) is an antibiotic with extremely low water solubility which limits its therapeutic applications and bioavailability. Objetives: The aim of this work was to increase the aqueous solubility of SDZ by preparing ternary complexes of this drug with β-cyclodextrin (βCD) and an AA as a third auxiliary substance. Materials y Methods: Complex formation was studied by phase solubility analysis (PSA), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TG) and scanning electron microscopy (SEM). Results: The apparent stability constants (KC) of the multicomponent complexes were calculated from the solubility diagrams. By the analysis of the NMR spectra, it could be said that the shifts of some protons evidenced the important role of the AA in the formation of multicomponent complexes. Among the AA, Arginine (ARG) proved to have better solubilizing properties for SDZ, reaching an improvement up to 70 times. The use of DSC, TG and SEM suggested the formation of new solid phases between SDZ:βCD:AA. Conclusions: As a result of this research, it was determined that ternary products were more effective in improving drug solubility than the corresponding SDZ:βCD binary system.

Eosin B (EoB) and eosin Y (EoY), two xanthene dye derivatives with photosensitizing ability were prepared in high purity through an improved synthetic route. The dyes were grafted to a 6-monoamino-β-cyclodextrin scaffold under mild reaction conditions through a stable amide linkage using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The molecular conjugates, well soluble in aqueous medium, were extensively characterized by 1D and 2D NMR spectroscopy and mass spectrometry. Preliminary spectroscopic investigations showed that the β-cyclodextrin–EoY conjugate retains both the fluorescence properties and the capability to photogenerate singlet oxygen of the unbound chromophore. In contrast, the corresponding β-cyclodextrin–EoB conjugate did not show either relevant emission or photosensitizing activity probably due to aggregation in aqueous medium, which precludes any response to light excitation.

A β-cyclodextrin-core star copolymer with Y-shaped ABC miktoarms was designed which exhibits unimolecular micelles in aqueous solution. It is a good platform for unimolecular container encapsulating hydrophobic molecules with release of the payload exhibiting pH-sensitivity.

Studies of cyclodextrin chemistry using quantum chemical methods are mainly adopted to investigate the formation of the inclusion complex causing changes in the physicochemical properties of the cyclodextrin guest. In this paper, we conducted a computational modeling study of the inclusion complexes of β-cyclodextrin (β-CD) with m-Methyl Red (m-MR) by using parametric method 6 (PM6), the semi empirical molecular orbital calculations and the natural bond orbital method (NBO). The inclusion process is carried out by maintaining the coordinates of the β-CD fixed and by displacing the guest molecule. The different relative positions between m-MR and β-CD are measured with respect to the distance between the reference atom (N) in the guest molecule and the origin of the coordinates from the equatorial plane of β-CD. The m-MR/β-CD (B) inclusion complex has a lower negative value of ΔG compared to another m-MR/β-CD (A) complex, highlighting the spontaneous behavior of the inclusion process. In addition, during the process of inclusion, the complexation energy is negative, which allows us to affirm that the complexation of m-MR in the β-CD is thermodynamically favorable. Among two directions A and B, the minimum energy generated from the PM6 was obtained in the orientation B and the guest molecule is partially encapsulated in the cavity of β-CD. In the NBO analysis, the stabilization energy is also usually used to characterize the hydrogen bond interaction between a lone pair (LP(Y)) of an atom Y and an anti-bonding orbital (BD٭(X-H)).

CANCERA winning combinationAndrogen receptor antagonists compromise the T cell response against prostate cancer, leading to early tumor relapse.Y. Pu, M. Xu, Y. Liang, K. Yang, Y. Guo, X. Yang, Y.-X. Fu, Androgen receptor antagonists compromise T cell response against prostate cancer leading to early tumor relapse. Sci. Transl. Med.8, 333ra47 (2016). [Abstract]NEUROTOXICITYInsights into neuronal cell deathA method to culture human cortical neurons that yielded a balanced network of excitatory and inhibitory neurons revealed that these cells die in a PARP-dependent manner after neurotoxic insult.J.-C. Xu, J. Fan, X. Wang, S. M. Eacker, T.-I. Kam, L. Chen, X. Yin, J. Zhu, Z. Chi, H. Jiang, R. Chen, T. M. Dawson, V. L. Dawson, Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity. Sci. Transl. Med.8, 333ra48 (2016). [Abstract]ATHEROSCLEROSISDissolving away cholesterolThe cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin facilitates atheroprotective mechanisms through oxysterol-mediated reprogramming of macrophages.S. Zimmer, A. Grebe, S. S. Bakke, N. Bode, B. Halvorsen, T. Ulas, M. Skjelland, D. De Nardo, L. I. Labzin, A. Kerksiek, C. Hempel, M. T. Heneka, V. Hawxhurst, M. L. Fitzgerald, J. Trebicka, I. Björkhem, J.-Å. Gustafsson, M. Westerterp, A. R. Tall, S. D. Wright, T. Espevik, J. L. Schultze, G. Nickenig, D. Lütjohann, E. Latz, Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming. Sci. Transl. Med. 8, 333ra50 (2016). [Abstract]

Hyperlipidemia is a risk factor for abnormal cerebrovascular events. Rafts are cholesterol-enriched membrane microdomains that influence signal transduction. We previously showed that Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle (VSM) induced by sphingosylphosphorylcholine (SPC) has a pivotal role in cerebral vasospasm. The goals of the study were to show SPC-Rho-kinase-mediated VSM contraction in vivo and to link this effect to cholesterol and rafts. The SPC-induced VSM contraction measured using a cranial window model was reversed by Y-27632, a Rho-kinase inhibitor, in rats fed a control diet. The extent of SPC-induced contraction correlated with serum total cholesterol. Total cholesterol levels in the internal carotid artery (ICA) were significantly higher in rats fed a cholesterol diet compared with a control diet or a β-cyclodextrin diet, which depletes VSM cholesterol. Western blotting and real-time PCR revealed increases in flotillin-1, a raft marker, and flotillin-1 mRNA in the ICA in rats fed a cholesterol diet, but not in rats fed the β-cyclodextrin diet. Depletion of cholesterol decreased rafts in VSM cells, and prevention of an increase in cholesterol by β-cyclodextrin inhibited SPC-induced contraction in a cranial window model. These results indicate that cholesterol potentiates SPC-Rho-kinase-mediated contractions of importance in cerebral vasospasm and are compatible with a role for rafts in this process.

Purpose. Study the complexation of dexamethasone in combinations of y-cyclodextrin (yCD) and 2-hydroxypropyl-y-cyclodextrin (HPyCD) with emphasis on solid characterization and development of aqueous dexamethasone eye drop suspension for drug delivery through sclera. Methods. Dexamethasone/cyclodextrin (dexamethasone/CD) solid complex systems were prepared and characterized by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and by in vitro drug dissolution testing. Sample eye drop suspensions were prepared applying solubilizer/suspender consisting of yCD/HPyCD mixtures, poloxamer 407 (P407) and polyvinylpyrrolidone. The eye drop suspension was characterized by its physicochemical properties. Results. The solid characterization techniques applied suggested that solid complexes were being formed. The results indicated that dexamethasone formed non-inclusion or micelle-like aggregates with HPyCD and the yCD/HPyCD mixture. The dissolution and dexamethasone release from the solid dexamethasone/yCD/HPyCD complexes was much faster than from the solid dexamethasone/yCD and dexamethasone/HPyCD complexes. The diameter of the solid particles in the dexamethasone eye drop suspension formulations were in all cases less than 10 μm with a mean diameter from 2.5 to 5.8 μm. The particle size decreased with increasing amount of P407. Permeation studies through semi-permeable membrane and porcine sclera showed that increasing the amount HPyCD could enhance drug transport through the membrane barriers and this was related to enhanced drug solubility. The permeation rates were, however, decreased compared to formulation containing yCD alone due to larger hydrodynamic diameter of dexamethasone/yCD/HPyCD complex aggregates. All formulations were both chemically stable for at least 8 months at 25oC and 40oC. Conclusions. Combination of yCD and HPyCD, i.e., formation of dexamethasone/yCD/HPyCD complexes, resulted in synergistic effect. That is the mixture had greater solubilizing effect than the individual CD, resulted in enhanced dissolution and drug delivery through membranes. Furthermore, it is possible to control the drug release rate by adjusting the yCD:HPyCD ratio in the solid dexamethasone/yCD/HPyCD complexes.

Memantine (memary) oligosaccharides (gluco-oligosaccharides) were synthesized by enzymatic glycosylation, using cyclodextrin glucanotransferase as a biocatalyst. Although memantine hardly crosses the blood–brain barrier (BBB) in the mouse brain, memantine oligosaccharides crossed the BBB of mouse brain and were incorporated into the mouse brain tissue. Our investigations indicated that memantine modified with oligosaccharides might have gained a BBB-crossing ability. Furthermore, during the Y-maze test using senescence-accelerated mouse prone 8, our study revealed that the time spent in the novel Y-maze arm by the memantine-oligosaccharides-treated mouse was longer than that spent in the novel arm by the memantine-treated-mouse. Therefore, this study established that since memantine oligosaccharides could penetrate the BBB of mouse brain and be incorporated into the mouse’s brain tissue, they could also enhance spatial learning.

This study synthesized resveratrol oligosaccharides (gluco oligosaccharides) by two-step enzymatic glycosylation, using glucosyltransferase from Phytolacca americana and cyclodextrin glucanotransferase. Although resveratrol hardly crosses the blood–brain barrier (BBB) in the mouse brain, the synthesized resveratrol oligosaccharides incorporated into the mouse brain tissue crossed the BBB. Our study indicated that resveratrol modified with oligosaccharides might have gained a BBB-crossing ability. Furthermore, during the Y-maze test using senescence-accelerated mouse prone 8, our investigations revealed that the time spent in the novel Y-maze arm by the resveratrol-oligosaccharides-treated mice was longer than that spent in the novel arm by the resveratrol-treated-mice. Therefore, this study established that since resveratrol oligosaccharides could penetrate the BBB of mouse brain samples and be incorporated into the mouse’s brain tissue, they could also enhance spatial learning.

This study investigates whether Andrographolide-loaded Lactose β-Cyclodextrin (ALN-βCD) nanoparticles enhance cognitive function, particularly spatial learning and memory. The successful conjugation of lactose to β-cyclodextrin was confirmed via 1H NMR spectroscopy, facilitating neuronal cell entry. The solvent evaporation method was used to create the nanoparticles, which were characterised for particle size, PDI, zeta potential, and drug release. The nanoparticles exhibited a size of 247.9 ± 3.2 nm, a PDI of 0.5 ± 0.02, and a zeta potential of 26.8 ± 2.5 mV. FTIR and TEM analyses, along with in vitro drug release and BBB permeability studies, confirmed their stability and efficacy. Behavioural tests, including the Elevated Plus Maze, Y-Maze, Object Recognition, and Locomotor Activity tests, demonstrated significant improvements in memory, motor coordination, and exploration time in the nanoparticle-treated groups. The group treated with ALN-βCD at a dose of 100 mg/kg/p.o. showed superior cognitive performance compared to the group receiving free andrographolides (AG). Biochemical assays indicated a significant reduction in acetylcholinesterase activity and lipid peroxidation, suggesting increased acetylcholine levels and reduced oxidative stress. Histopathological examination showed improved neuronal function without toxicity. The results showed significant improvements (p < 0.001) in memory and cognitive abilities in experimental animals, highlighting the potential of ALN-βCD nanoparticles as a non-invasive treatment for memory loss. These promising findings warrant further exploration through clinical trials.

Montelukast sodium (MLK) is a worldwide antiasthmatic drug. Commercial formulations still have some issues with solubility and instability to light and humidity. To overcome them, the present work reports inclusion compounds of MLK and γ-cyclodextrin (γ-CD). As a molecular capsule, CDs have the ability to protect the inclusion guest from degradation, enhance its solubility and alter the pharmacokinetic parameters. MLKγ-CD inclusion compounds were prepared by mechanochemistry. Without using any solvent, γ-CD was pre-milled and then co-milled with an equimolar quantity of MLK in a ball mill at 600 cycles·min−1. After 120 min of milling, the formation of MLK·γ-CD inclusion compounds was confirmed by powder X-ray diffraction and scanning electron microscopy. Additional studies, performed under pharmacopeia guidelines, showed that the prepared MLKγ-CD inclusion compounds can indeed increase the dissolution of MLK when in ultrapure water or simulated intestinal fluid (without pancreatin). This way, the MLKγ-CD inclusion compounds that are presented in this work are a promising solution for improving the therapeutic effectiveness of MLK.

Abstract We evaluated the performance of two homogeneous assays for quantifying HDL cholesterol (HDL-C) and compared them with the phosphotungstic acid (PTA)/MgCl2 assay. Both homogeneous HDL-C assays were precise, having a within-run CV of &lt;1.20% and a between-run CV of &lt;4.07%. The HDL-C values (y) measured by the two homogeneous methods correlated well with those by the PTA/MgCl2 method (x): y = 1.00x + 64.98 mg/L, r = 0.987, Sy|x = 27.99 mg/L (n = 152) for the polyethylene glycol-modified enzymes/α-cyclodextrin sulfate (PEGME) assay (Kyowa), and y = 0.84x + 106.51 mg/L, r = 0.984, Sy|x = 26.10 mg/L (n = 152) for the polyanion–polymer/detergent (PPD) assay (Daiichi). The specificity of the PEGME method seemed better than that of the PPD method, as the PPD method was markedly interfered with by supplemental LDL-C. Addition of 20 g/L triglycerides produced a negative error of ∼18% in both homogeneous assays. Bilirubin and hemoglobin had little influence on the PEGME method; hemoglobin had little effect on the PPD method. Bilirubin, however, markedly decreased the readings by the PPD method. We found the PEGME assay superior to the PPD assay for routine HDL-C testing, because the PPD assay is relatively inaccurate and not specific.

The aim of this research is to use capillary electrophoresis to establish/determine the binding constants for ofloxacin and ornidazole enantiomers with the negatively charged chiral selector sulfated-β-cyclodextrin (S-β-CD). Using electrophoretic mobility values of ofloxacin and ornidazole enantiomers at various concentrations of S-β-CD used in the context of background electrolyte (BGE), binding constants were calculated using three separate linearization plots, namely double-reciprocal, X-reciprocal and Y-reciprocal. The R-ofloxacin enantiomer-S-β-CD complex had the highest inclusion affinity of the ofloxacin and ornidazole enantiomers, which matched with previously reported estimation. Every enantiomer-S-β-CD complex’s binding constants, as well as thermodynamic binding parameters, were calculated at different temperatures. The host-guest binding constants using double reciprocal fit showed greater linearity (R2 > 0.99) at all temperature ranges measured (15-30 ºC) as compared to the other two fit approaches. The thermodynamic complexation parameters were found to be dependent on the temperature of the enantiomers, as seen by the linear van’t Hoff (15-30 ºC) plot.

Bioproductos de la industria de la yuca: sustratos alternativos para la producción de ciclodextrina glucosiltransferasa por alcalófilo Bacillus trypoxylicola SM-02 En el presente trabajo estudiamos el uso licor de maíz fermentado (LMF), harina de cáscara de yuca (HCY) y aguas residuales de yuca para la producción de ciclodextina glicosiltransferasa (CGTase) por un nuevo aislado alcalófilo de Bacillus trypoxylicola SM-02 en fermentación sumergida. Los experimentos se realizaron por Diseño Central Compuesto Rotativo 22 totalizando 11 ensayos. La mayor actividad enzimática de 352.53 U/mL se obtuvo con 1.5 g de HCY y 0.6 g de LMF. La temperatura y el pH óptimos fueron 55 ºC y pH 8.0, respectivamente. CGTase mostró una actividad relativa superior al 50% durante 120 min. a la temperatura óptima. Solo el CaCl2 mostró actividad positiva para CGTasa. Los resultados apuntaron a un buen potencial de B. trypoxylicola SM-02 para la producción de CGTasa usando substratos residuales. In the present work was studied the use of cassava peel flour (CPF), corn steep liquor (CSL), and cassava wastewater as substrates to produce cyclodextrin glycosyltransferase (CGTase) from a new alkalophilic isolate of Bacillus trypoxylicola SM-02 by submerged fermentation. The experiments were performed as a Central Composite Design 22, totalizing 11 assays. An enzymatic activity of 352.53 U/mL was obtained using 1.5 g of CPF and 0.6 g of CSL. The optimum temperature and pH of CGTase was 55 °C and 8.0, respectively. The CGTase depicted a relative activity of more than 50% for 120 min at the optimum temperature. The only salt that positively influenced the CGTase activity was CaCl2. The results are indicative of a potential role of B. trypoxylicola SM-02 in the production of CGTase using residual substrates.

ABSTRACTThe current treatment of Chagas disease (CD), based on nifurtimox and benznidazole (Bz), is unsatisfactory. In this context, we performed the phenotypicin vitroscreening of novel mono- and diamidines and drug interaction assays with selected compounds. Ten novel amidines were tested for their activities against bloodstream trypomastigote (BT) and amastigote forms ofTrypanosoma cruzi(Y and Tulahuen strains) and their toxicities for mammalian host cells (L929 cells and cardiac cells). Seven of 10 molecules were more active than Bz against BT, with the most active compound being the diamidine DB2267 (50% effective concentration [EC50] = 0.23 μM; selectivity index = 417), which was 28-fold more active and about 3 times more selective than the standard drug. Five of the six monoamidines were also more active than Bz. The combination of DB2267 and DB2236 in fixed-ratio proportions showed an additive effect (sum of fractional inhibitory concentrations < 4) on BT. Interestingly, when intracellular forms were exposed to DB2267, its activity was dependent on the parasite strain, being effective (EC50= 0.87 ± 0.05 μM) against a discrete typing unit (DTU) II strain (strain Y) but not against a representative DTU VI strain (strain Tulahuen) even when different vehicles (β-cyclodextrin and dimethyl sulfoxide) were used. The intrinsic fluorescence of several diamidines allowed their uptake to be studied. Testing of the uptake of DB2236 (inactive) and DB2267 (active) by amastigotes of the Y strain showed that the two compounds were localized intracellularly in different compartments: DB2236 in the cytoplasm and DB2267 in the nucleus. Our present data encourage further studies regarding the activities of amidines and provide information which will help with the identification of novel agents for the treatment of CD.

Self-healing is a measure of material's ability to repair damage. Physical and chemical processes have been used to obtain self-healing polymers for various applications. Different approaches are involved for these systems, such as shape-memory effects, covalent-bond reform, heterogeneous systems, diffusion and flow, and the dynamics of supramolecular chemistry. Different approaches are used to achieve self-healing polymers, in particular, the role of the healing system in chemistry is highlighted, which enables thermal transients, damage repairing and reconnection. Moreover, self-healing systems and their energy storage applications are currently getting great importance. Inspired by the dynamic network structure of animal dermis, in which collagen fibril (rigid and strong) and elastin fibril (soft and elastic) crosslink through supramolecular interactions to form a sturdy and flexible material we hypothesized that the combination of a rigid conductive polymer with a soft hydrophilic polymer through proper supramolecular interactions would yield a strong and robust polymer binder. According to the literature, the external stimulus such as heat, light, pH and redox initiate the healing process. This process is known as non-autonomous self-healing when some additional external stimulus is needed. Considering the battery chemistry, it is the most functional method to choose self-healing structures and batteries with the help of hydrogen bonds in order to make the self-healing process occurs autonomously. In the literature, polymerized β-cyclodextrin (β-CD) has been used as an advanced binder for Si-nanoparticle anodes, and cracks in the electrode can be avoided with its reversible properties through supramolecular cyclodextrin or hydrogen bonding compounds[1]. Dynamic bonds such as hydrogen bonds between composites positively affect the durability of the Si anode performance. For example, the Si anode was coated by the researchers with hydrogen bond-oriented polymer binder layers based on polyimide polymer such as Upy, resulting in electrochemical performance ten times longer than conventional silicon anodes[2]. Horizon projects such as BAT4EVER and HIDDEN under Battery 2030+ initiative have been investigate the integration and optimization of self-healing polymers in Li-ion battery [3]. In the scope of the BAT4EVER project, supramolecular coupling of polyaniline and polyvinyl alcohol via dynamic boronate bond yields polyaniline-polyvinyl alcohol hydrogel with outstanding tensile strength and electrochemical performance. The self-healing chemistry in the Si anode prevents unbalanced volumetric changes and cracks on the electrode surface. Thus, electrochemical performance losses are decreased. The self-healing functionalized components of lithium-ion batteries, which focus on improving and optimizing properties such as high energy density, high voltage, long life, and cycle stability, are of great importance for next-generation batteries. References Jeong, Y.K., Kwon, T.W., Lee, I., Kim, T.S., Coskun, A., Choi, J.W.: Hyperbranched β-cyclodextrin polymer as an effective multidimensional binder for silicon anodes in lithium rechargeable batteries. Nano Lett. 14, 864–870 (2014). https://doi.org/10.1021/NL404237J/SUPPL_FILE/NL404237J_SI_001.PDF Yang, J., Zhang, L., Zhang, T., Wang, X., Gao, Y., Fang, Q.: Self-healing strategy for Si nanoparticles towards practical application as anode materials for Li-ion batteries. Electrochem commun. 87, 22–26 (2018). https://doi.org/10.1016/J.ELECOM.2017.12.023 https://battery2030.eu/ This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 957225 (BAT4EVER).

Abstract Stimulation of human acute monocytic cells with sublytic complement leads to an increase of intracellular calcium resulting in the release of two forms of microvesicles, one derived from the surface membrane (between 0.1-1 μm), which we term plasma membrane-derived vesicles (PMVs) and exosomes (50-100nm) released by exocytosis. Much work has been documented on the release of PMVs, using various stimuli, and the roles they play in disease, but comparatively little is available on inhibition of PMV release. Although upon stimulation cells release exosomes in addition to PMVs, little has been reported on ways of differentiating between these two microvesicle types. Our aim was therefore to identify inhibitors of PMV release and to effectively differentiate between PMVs and exosomes. Methyl-β -cyclodextrin, an inhibitor of lipid rafts, calpeptin, which inhibits calpain and Rho-kinase inhibitor, Y-27632 were found to inhibit effectively the release of PMVs. Exosomes separated from PMVs by sucrose gradient were confirmed by electron microscopy and compared to PMVs were found to express less phosphatidylserine, but more CD63. Of the various cytokines carried by PMVs and exosomes, macrophage migration inhibition factor was found at a four-fold higher level in PMVs. Source of Research Support: Royal Society, London.

For four decades, the functionalization of electrodes by biomaterials based on electrogenerated polymers, carbon nanotubes and / or nano-objects, was widely used in the field of analytical chemistry and energy conversion. Recently, we redesigned enzymatic bioelectrodes in order to produce original objects. An innovative concept of reagentless biosensors based on enzymatic reduction of oxygen was developed by trapping and releasing the substrate of the enzyme (catechol) from the structure of the biosensor. A polyurethane support modified by two perforated microcapsules filled with catechol was easily transformed into a bioelectrode by the successive deposition of multiwalled carbon nanotubes and a mixture of laponite clay and tyrosinase enzyme [1]. On the other hand, the development of glyconanoparticles resulting from the self-assembly of block copolymers composed of polystyrene and cyclodextrin as an inclusion site will be reported. These glyconanoparticles, which are stable in suspension in aqueous media, have an outer layer composed of cyclodextrin [2]. The latter allows a post-functionalization of the nanoparticle by hydrophobic molecules through host-guest interactions. It appears that it is possible to modulate the site density of βCD at the surface of the shell of the hybrid glyconanoparticles while maintaining its inclusion properties. Moreover, the anchoring of glyconanoparticles to the surface of electrodes has been carried out by host-guest interactions with electrogenerated polymers. The efficient immobilization of the nanoparticles allows the anchoring of multilayers of biotinylated glucose oxidase. Finally, a new generation of bioelectrodes for oxygen reduction and electrical energy production will be described. This approach is based on the design of flat electrodes made of permeable and conductive materials defining a very thin microcavity with a large surface area containing, in a microvolume, enzymes in powder form. The concept has been demonstrated with bilirubin oxidase (BOD) for the electroenzymatic reduction of oxygen in water. Besides the influence of the amount of enzyme trapped in the microcavity on the performance of the bioelectrode, the functionalization of the electrodes by adsorption of redox mediators or compounds allowing an orientation of the BOD favorable to electron transfer was also explored. After 5 months of storage in an aqueous buffer, the bioelectrode exhibits 20% of its initial electro-enzymatic activity. Y. Nedellec, C. Gondran, K. Gorgy, S. MC Murtry, O. El Mazria, P. Agostini, S. Cosnier. Biosens. Bioelectron., 180, 113137-113141(2021). M. Carrière, P. H. M. Buzzetti, K. Gorgy, M. Mumtaz, C. Travelet, R. Borsali, S. Cosnier. Nanomaterials 11 (2021) 1162. doi.org/10.3390/nano11051162.

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H1) and 5-hydroxytryptamine (serotonin) receptors (5-HT2A) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-β-cyclodextrin (MβCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (∼80%), 5-HT (100%), α-methyl-5-HT (100%), and KCl (∼30%). Cholesterol-loaded MβCD (CL-MβCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (∼85%) and 5-HT (∼59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MβCD and partially reestablished by CL-MβCD. The increase in intracellular Ca2+ that was induced by both agonists was reduced by MβCD. The presence of caveolin-1 (Cav-1), H1, 5-HT2A, and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H1 receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT2A receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H1 and 5-HT2A receptors through their Rho/ROCK signaling.

The antioxidant capacity of polyphenols have been widely used in food and pharmaceutical industries. Quercetin (Quer) is a polyphenolic flavonoid that shows several biological effects such as antioxidant, antitumor, antibacterial and antiproliferative effects both in-vitro and in-vivo. However, the solubility of quercetin in water is poor. Thus, it is essential to improve solubility of quercetin in pharmaceuticals by making its complexation with other compounds. In this study, the synthesis of the 2-hydroxypropyl-β-cyclodextrin complex with quercetin (Quer-HPβCD) in the form of nanoparticles in water-ethanol solvents has been carried out. The results showed that the obtained yield of (Quer-HPβCD) complexation in binary solvent was greater than that in pure water. The highest Y value was 80% in a binary solvent with 20% v/v of ethanol. The composition, morphology, structural and thermodynamic properties of the nanoparticles Quer-HPβCD have been determined. This study demonstrated that using mixed water- ethanol solvent and lyophilization technique was able to produce quercetin nanoparticles with significantly smaller particle size. The nanoparticles have a spherical shape with an average size of about 40-60 nm. The results of the phase solubility diagram showed that in water the solubility of quercetin increased and linearly depended on the concentration of host’s molecule while Quer and HP-βCD obtained a 1:1 stoichiometric complex. The stability constant of (Quer-HPβCD) complex was found to be logK = 2.56. The Gibbs energy change of the complexation reaction was found to be -14.60 kJ/mol.

ABSTRACT Bluetongue virus (BTV) is a nonenveloped double-stranded RNA virus belonging to the family Reoviridae. The two outer capsid proteins, VP2 and VP5, are responsible for virus entry. However, little is known about the roles of these two proteins, particularly VP5, in virus trafficking and assembly. In this study, we used density gradient fractionation and methyl beta cyclodextrin, a cholesterol-sequestering drug, to demonstrate not only that VP5 copurifies with lipid raft domains in both transfected and infected cells, but also that raft domain integrity is required for BTV assembly. Previously, we showed that BTV nonstructural protein 3 (NS3) interacts with VP2 and also with cellular exocytosis and ESCRT pathway proteins, indicating its involvement in virus egress (A. R. Beaton, J. Rodriguez, Y. K. Reddy, and P. Roy, Proc. Natl. Acad. Sci. USA 99:13154-13159, 2002; C. Wirblich, B. Bhattacharya, and P. Roy J. Virol. 80:460-473, 2006). Here, we show by pull-down and confocal analysis that NS3 also interacts with VP5. Further, a conserved membrane-docking domain similar to the motif in synaptotagmin, a protein belonging to the SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) family was identified in the VP5 sequence. By site-directed mutagenesis, followed by flotation and confocal analyses, we demonstrated that raft association of VP5 depends on this domain. Together, these results indicate that VP5 possesses an autonomous signal for its membrane targeting and that the interaction of VP5 with membrane-associated NS3 might play an important role in virus assembly.

Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl−/HCO3−(OH−) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl−/HCO3−(OH−) exchange activity (∼2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl−/HCO3−(OH−) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl−/HCO3−(OH−) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (∼45%) by NPY compared with control. MβCD significantly decreased Cl−/HCO3−(OH−) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive 36Cl− uptake (∼50%). Our results demonstrate that NPY modulates Cl−/HCO3−(OH−) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl−/HCO3−(OH−) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.

Various dietary supplements have been shown to extend the life span of Drosophila melanogaster, including several that promote autophagy, such as rapamycin and spermidine. The goal of the study presented here was to test numerous additional potential anti-aging supplements, primarily inhibitors of the target of rapamycin (TOR) and/or phosphatidylinositol 3-kinase (PI3K). Using a single, comparatively long-lived y w test strain, screening was performed in male flies supplemented either throughout adulthood or, in a few cases, beginning in middle or late adult life, with concentrations spanning 4–6 orders of magnitude in most cases. Supplementation with PP242 and deferiprone, an iron chelator, beginning in late adult life had no positive effect on life span. Lifelong supplementation with Ku-0063794, LY294002, PX-866-17OH, Torin2 and WYE-28 had no effect at any dose. Rapamycin, spermidine and wortmannin all had significant life-shortening effects at the highest doses tested. AZD8055, PI-103 hydrochloride and WYE-132 yielded slight beneficial effects at 1–2 doses, but only 100 nM AZD8055 was confirmed to have a minor (1.3%) effect in a replicate experiment, which was encompassed by other control groups within the same study. These compounds had no effect on fly fecundity (egg laying) or fertility (development of progeny to adulthood), but equivalent high doses of rapamycin abolished fertility. The solvent DMSO had no significant effect on life span at the concentrations used to solubilize most compounds in the fly medium, but it drastically curtailed both survival and fertility at higher concentrations. 2-Hydroxypropyl-β-cyclodextrin also failed to extend the life span when provided throughout adulthood or beginning in mid-adult life. Collectively, the results suggest that inhibition of the TOR/PI3K pathway and autophagy through dietary intervention is not a straightforward anti-aging strategy in Drosophila and that further extension of life is difficult in comparatively long-lived flies.

Abstract Hydroxyl radicals were generated radiolytically in N2O -and N2O / O2(4: 1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atom s. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radio­ lysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity in­ duced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the con ­ tributions of the different com ponents cannot be adequately resolved. At pH7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t1/2 = 0.6 ms; at pH 10, t1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductom etric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scat­ tering technique is best operated in the low -dose range. In the presence of oxygen a pro­ nounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 × 10-7 mol J-1 and at pH 10.4, 4.8 × 10-7 mol J-7. The radiolytic yields of CO2, H2O2, organic hydroperoxides, O2·- and oxygen consum ption have been determined in y-irradiated N2O/ 0 2(4: 1)-saturated solutions of both hyaluronic acid and β-cyclodextrin.

At present, the class of rhodanine com-pounds containing 4-oxo-2-thioxo-1,3-thiazolidine residues is very promising in the development of medical and veterinary drugs. The purpose of the work is to develop a chemical compound 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one (XC-3-2-FE-2-TO-1,3T-4-OH) based on clathrate with β- cyclodextrin (CD) - a supramolecular com-pound (CMC) with improved biopharmaceu-tical properties (solubility and bioavailabil-ity). The lattice clathrates KhS-3-2-FE-2-TO-1,3T-4-OHa with CD were synthesized at the ratios of their masses 1: 5 and 1:10. CMC is a crystalline powder of white with a yellow tint with a particle size of less than 100 nm. For the first time, a method was developed for the quantitative determination of CHS-3-2-FE-2-TO-1,3T-4-OHa in the blood plasma of dogs by high performance liquid chromatography (HPLC). In the range of 0.2-4.0 μg / ml, the calibration curve is linear for ChS-3-2-FE-2-TO-1,3T-4-OH, while the regression equation is described by y = 3.977 + 30.343 × X, s the coefficient of accuracy of the approximation equal to R2 = 0.999. The lower limit of quantitative detec-tion is 10 ng / ml. The average maximum concentrations (Cmax) of ChC-3-2-FE-2-TO-1,3T-4-OHa in the blood plasma of dogs with it, as well as in the form of a clathrate single oral administration, respectively, were 0.66 ± 0, 25 and 3.03 ± 0.82 μg / ml (P≤0.05). The average values of the parame-ter AUC (0 → t) with the introduction of ChS-3-2-FE-2-TO-1,3T-4-On and its clath-rate with CD, respectively, were 4.07 ± 2.15 μg × h / ml and 24.58 ± 5.66 μg × h / ml (P≤0.05). According to a pharmacokinetic study, the best bioavailability (DB) is pos-sessed by the C-3-2-FE-2-TO-1,3T-4-OHa clathrate with CD in a ratio of 1: 5, the ob-tained DB value is 6.4 times higher than the same the value of the original compound. It was concluded that, in terms of biopharma-ceutical properties, the most promising agent for medical and veterinary use is the XC-3-2-FE-2-TO-1,3T-4-OHa clathrate with CD with a mass ratio of 1: 5 and an average par-ticle size of 40.5 nm

Abstract Mutations in FLT3 are detected in approximately 30% of AML and are associated with poor overall survival. Although first (PKC412, sorafenib and CEP701) and second generation (AC220) FLT3 tyrosine kinase inhibitors (TKI) induce remissions, resistance-causing gatekeeper (F691I/L), activation loop (AL) mutations (D835V/Y/F) or compound FLT3-ITD/F691I AL mutations are known to impair the in vitro and in vivo activity of the FLT3-TKIs. The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., MYC and BCL-2. BRD4 antagonists JQ1 and I-BET151 disrupt the binding of the bromodomain of BRD4 to acetylated lysines on histone proteins, thereby inhibiting expressions of c-MYC and BCL-2 and inducing apoptosis of AML cells. Based on this, we evaluated the in vitro and in vivo activity of JQ1 and FLT3 antagonists AC220 and ponatinib against cultured mouse lymphoid (Ba/F3/FLT3-ITD), as well as human cultured (MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs) expressing FLT3-ITD. JQ1, but not its inactive enantiomer R-JQ1, potently induced apoptosis of not only Ba/F3/FLT3-ITD but also of Ba/F3/FLT3-ITD expressing the highly FLT3 TKI-resistant mutations F691L and D835V (IC50 values for JQ1 were 697, 1588 and 909 nM, in the three cell lines, respectively). This was associated with attenuation of c-MYC, but the induction of BIM levels. Both JQ1 and I-BET151 dose-dependently induced apoptosis of MOLM13 and MV4-11 cells, as well as of primary AML BPCs expressing FLT3-ITD. Concomitantly, JQ1 treatment attenuated c-MYC, BCL2 and CDK6, while inducing p21, p27, BIM and cleaved PARP levels. JQ1 and I-BET151 did not induce apoptosis of CD34+ normal bone marrow progenitor cells. Following engraftment of NOD/SCID mice with MOLM13 cell xenograft, treatment with JQ1 (50 mg/kg, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, daily x 5 days per week x 3 weeks), versus treatment with vehicle control, significantly improved survival of the mice (p< 0.05), without causing any toxicity. This was associated with the in vivo attenuation of c-MYC and BCL-2 levels in the harvested AML cells from the mice. Co-treatment with JQ1 or I-BET151 and FLT3 antagonist AC220 or ponatinib synergistically induced apoptosis of MOLM13 and MV4-11 cells. This was associated with greater reduction in the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. Knockdown of BRD4 by treatment with specific shRNA phenocopied the effects of JQ1 and sensitized MOLM13 cells to ponatinib and AC220. As compared to each agent alone, treatment with JQ1 and ponatinib or AC220 also induced more apoptosis of primary AML BPCs expressing FLT3-ITD, associated again with greater reduction of the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. We also determined the effects of JQ1 against MOLM13/TKIR cells that were generated under the continuous selection pressure of FLT3 TKI, and exhibited > 10 fold resistance to ponatinib but > 50 fold resistance to AC220. Importantly, as compared to the parental MOLM13, the MOLM13/TKIR cells were markedly more sensitive to JQ1-induced apoptosis (p< 0.001). Additionally, co-treatment with JQ1 and ponatinib but not AC220 synergistically induced apoptosis of MOLM13/TKIR cells. Supporting our previous findings (Blood. 2005;105:1768) that FLT3-ITD is a heat shock protein (hsp) 90 client-oncoprotein, the non-geldanamycin hsp90 inhibitor AUY922 was equally effective in inducing apoptosis of MOLM13 versus MOLM13/TKIR cells. Collectively, these findings demonstrate that BRD4 antagonist exhibits potent activity against cultured and primary AML cells expressing FLT-3-ITD, as well as against cellular models of FLT3 with gate-keeper and activation loop mutations. These findings also highlight the novel and synergistic activity of the combination of BRD4 antagonist and AC220 or ponatinib against AML BPCs expressing FLT3-ITD, and support the rationale for testing ponatinib and BRD4 antagonist against TKI-refractory AML expressing FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 82 Hematopoietic stem/progenitor cells (HSPCs) reside in a bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for their proliferation and survival. In co-cultures of osteoblasts with primary human normal CD34+ cells, CD34+CD38- cells, or the KG1a progenitor cell line, we previously showed, using live cell imaging approaches, that HSPCs made prolonged contact with the osteoblast surface via a specialized membrane domain enriched in prominin 1 (CD133), the very late antigen-4 (VLA-4), the phosphatidylethanolamine (PE) analogue rhodamine-PE, and the tetraspanins CD63 and CD81. At the contact site, portions of the specialized domain of the CD34+ cells containing these molecules were taken up by osteoblasts and internalized into signaling endosomes within the osteoblasts. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow (Gillette J. et al, Nat. Cell Biol. 11(3): 303–311, 2009). We have now evaluated the functional significance of these specialized membrane domains for in vivo homing of normal HSPCs to the bone marrow microenvironment. G-CSF mobilized human peripheral blood CD34+ cells from two normal donors were treated for 30 minutes with the cholesterol sequestration agent methyl-β-cyclodextrin (MβCD). This treatment resulted in disruption of the CD34+ cell membrane domains but had no effect on cell viability, proliferation or colony forming capacity in vitro. However, in two independent experiments, we observed a three-fold decrease in homing of MβCD-treated CD34+ cells to the bone marrow of NOD/SCID IL2rψcnull mice 16 hours after transplantation as compared to mock-treated CD34+ cells (p=0.0002). In contrast to homing studies, long-term human cell engraftment determined by CD45 cell surface expression 2 months after transplantation in two independent experiments was not significantly different in mice transplanted with MβCD-treated CD34+ cells compared to mock-treated CD34+ cells (p=0.13). Rapid repolarization of the membrane domain after transplantation may have resulted in engraftment of MβCD-treated CD34+ cells at levels similar to those observed with mock-treated CD34+ cells. Given the known homing/engraftment defect of actively cycling HSPC, we compared membrane domains on quiescent and cycling CD34+ cells from two normal donors. At baseline, 55–68% of CD34+ cells were in the G0 phase of the cell cycle as measured by Hoechst/Pyronin Y staining and specialized membrane domains were detected on a similar percentage of CD34+ cells for both donors. After culture for 4 days in the presence of stimulatory cytokines (SCF, TPO and Flt3), less than 10% of the CD34+ cells remained in G0 and, similarly, less than 10% of the cells analyzed by microscopy had a specialized membrane domain. After 4 days, cells were continued in culture for 2 days under non-stimulatory conditions (SCF alone). Under these conditions, for one donor, the percentage of cells in G0 increased from 9% to 18% and the percentage of cells with membrane domains increased similarly from 6% to 11%. For the other donor, the percentage of cells in G0 and with membrane domains both remained unchanged after 2 days in non-stimulatory culture. Surprisingly, the polarized membrane domains detected on normal CD34+ cells were not found on peripheral blood blast cells from patients diagnosed with relapsed AML (n=2 patients) or CML (n=1 patient). Accordingly, in a preliminary experiment, no difference in homing was observed 16 hours after transplantation of MβCD- or mock-treated peripheral blood cells from an AML patient in NOD/SCID IL2rψcnull mice. Additional homing and engraftment studies using cells from leukemic patients are ongoing. In combination, these findings indicate that specialized membrane domains are found on normal but not leukemic HSPCs and that these domains are required for homing to the bone marrow microenvironment. Disruption of these domains in actively cycling progenitor cells may provide an explanation for the previously demonstrated homing/engraftment defect of cycling cells compared to quiescent cells in the G0 phase of the cell cycle. Disclosures: No relevant conflicts of interest to declare.

Plants for medicinal purposes are considered as the main source of health care for the majority of the world population. In order to promote medicinal plants in Algeria, the present work aimed to assess the physico-chemical characterization, hemolytic, and antioxidant activities of Ammodaucus leucotrichus essential oil of an endemic plant from south-west of Algeria. The plant was harvested from the region of Bechar (south-west of Algeria). The oil was obtained by hydrodistillation method using Clevenger apparatus. The antioxidant activity of the oil was carried out using DPPH scavenging assay, and its effect on erythrocyte cells was evaluated by measuring the hemolytic degree. The obtained results showed that this oil presented a low antioxidant activity compared to positive control (ascorbic acid). The hemolytic activity was very low since the oil diluted to 1/10, while it was low in the pure state. This fact proves the safety of A. leucotrichus oil, which can explain its use without risk by the indigenous population. REFERENCES: Abu Zarga, M.H., Al-Jaber, H.I., Baba Amer, Z.Y., Sakhrib, L., Al-Qudah, M.A., Alhumaidi, J.Y.G., Abaza, I.F., & Afifi, FU. (2013). Chemical composition, antimicrobialand antitumor activities of essential oil of Ammodaucus leucotrichus growing in Algeria.Journal of Biologically Active Products from Nature, 3, 224–231.http://doi.org/10.1080/22311866.2013.833469.Adorjan, B., & Buchbauer, G. (2010). Biological properties of essential oils: An updatedreview. Flavour and Fragrance Journal, 25, 407–426. http://doi.org/10.1002/ffj.2024.AFNOR. (1992). Association Française de Normalisation «Recueil des normes françaises surles huiles essentielles». II édition, Paris.Amorati, R., Foti, M.C., & Valgimigli, L. (2013). Antioxidant activity of essential oils.Journal of Agricultural and Food Chemistry, 61, 10835–10847.http://doi.org/10.1021/jf403496k.Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M. (2008). Biological effects of essentialoils- a review. Food and Chemical Toxicology, 46, 446–475.http://doi.org/10.1016/j.fct.2007.09106.Barao Paixao, V.L., & de Carvalho, J.F. (2021). Essential oil therapy in rheumatic diseases:A systematic review. Complementary Therapies in Clinical Practice, 43, 101391.http://doi.org/10.1016/j.ctcp.2021.101391Benhouhou, S. (2005). A Guide to Medicinal plants in North Africa: database on Medicinalplants, IUCN center for Mediterranean cooperation. Mâlaga.Bouaziz, M., Yangui, T., Sayadi, S., & Dhouib, A. (2009). Disinfectant properties of essentialoils from Salvia officinalis L. cultivated in Tunisia. Food and Chemical Toxicology, 47,2755–2760. http://doi.org/10.1016/j.fct.2009.08.005.Burt, S. (2004). Essential oils: their antibacterial properties applications in foods-a review.International Journal of Food Microbiology, 94, 223–253.http://doi.org/10.1016/j.ijfoodmicro.2004.03.022.Conforti, F., Sosa, S., Marrelli, M., Menichini, F., Statti, G.A., Uzunov, D., Tubaro, A., &Menichini, F. (2009). The protective ability of Mediterranean dietary plants against theoxidative damage: The role of radical oxygen species in inflammation and thepolyphenol, flavonoid and sterol contents. Food Chemistry, 112, 587–594.http://doi.org/10.1016/j.foodchem.2008.06.013.Dalar, A., Türker, M., & Konczak, I. (2012). Antioxidant capacity and phenolic constituentsof Malva neglecta Wallr. and Plantago lanceolata L. from Eastern Anatolia region ofTurkey. Journal of Herbal Medicine, 2, 42–51.http://doi.org/10.1016/j.hermed.2012.03.001.Dawidowicz, A.L., Szewczyk, J., & Dybowski, M.P. (2016). Modified application of HSSPME for quality evaluation of essential oil plant materials. Talanta, 146, 195–202.http://doi.org/10.1016/j.talanta.2015.08.043.El Haci, I.A., Bekhechi. C., Atik-Bekkara, F., Mazari, W., Gherib, M., Bighelli, A.,Casanova, J., & Fellix, T. (2014). Antimicrobial activity of Ammodaucus leucotrichusfruit oil from Algerian Sahara. Natural Product Communications, 9, 711–712.http://doi.org/10.1177/1934578X1400900533 Essid, R., Rahali, F.Z., Msaada, K., Sghair, I., Hammami, M., Bouratbine, A., Aoun, K., &Limam, F. (2015). Antileishmanial and cytotoxic potential of essential oils frommedicinal plants in Northern Tunisia. Industrial Crops and Products, 77, 795–802.http://doi.org/10.1016/j.indcrop.2015.09.049.Fillatre, Y., Rondeau, D., Daguin, A., & Communal, P.Y. (2016). A work flow for multiclassdetermination of 256 pesticides in essential oils by liquid chromatography tandem massspectrometry using evaporation and dilution approaches: Application to lavandin, lemonand cypress essential oils. Talanta, 149, 178–186.http://doi.org/10.1016/j.talanta.2015.11.052.Gainza, Y.A., Domingues, L.F., Perez, O.P., Rabelo, M.D., Lopez, E.R., & Chagas, A.C.S.(2015). Anthelmintic activity in vitro of Citrus sinensis and Melaleuca quinquenerviaessential oil from Cuba on Haemonchus contortus. Industrial Crops and Products, 76,647–652. http://doi.org/10.1016/j.indcrop.2015.07.056.Gomes, M.R.F., Schuh, R.S., Jacques, A.L.B., Dorneles, G.G., Montanha, J., Roehe, P.M.,Bordignon, S., Dallegrave, E., Leal, M.B., & Limberger, R.P. (2013). Biologicalassessment (antiviral and antioxidant) and acute toxicity of essential oils from Drimysangustifolia and D. brasiliensis. Revista Brasileira de Farmacognosia, 23, 284–290.http://doi.org/10.1590/S0102-695X2012005000142.Halla, N., Helenoa, S.A., Costa, P., Fernandes, I.P., Calhelha, R.C., Boucherit, K., Rodrigues,A.E., Ferreira, I.C.F.R., & Barreiro, M.F. (2018). Chemical profile and bioactiveproperties of the essential oil isolated from Ammodaucus leucotrichus fruits growing inSahara and its evaluation as a cosmeceutical ingredient. Industrial Crops and Products,119, 249–254. http://doi.org/10.1016/j.indcrop.2018.04.043.Hammiche, V., & Maiza, K. (2006). Traditional medicine in central Sahara: pharmacopoeiaof Tassili N’ajjer. Journal of Ethnopharmacology, 105, 358–367.http://doi.org/10.1016/j.jep.2005.11.028.Hossain, M.H., Shah, M.D., Sang, S.V., & Sakari, M. (2012). Chemical composition andantibacterial properties of the essential oils and crude extracts of Merremia borneensis.Journal of King Saud University –Science, 24, 243–249.http://doi.org/10.1016/j.jksus.2011.03.006.Hussein, G., Miyashiro, H., Nakamura, N., Hattori, M., Kakiuchi, N., & Shimotohno, K.(2000). Inhibitory effects of Sudanese medical plant extracts on hepatitis C virus (HCV)protease. Phytotherapy Research, 14, 510–516. http://doi.org/10.1002/1099-1573(200011)14:7<510::aid-ptr646>3.0.co;2-b.Idm’hand, E., Msanda, F., & Cherifi, K. (2020). Medicinal uses, phytochemistry andpharmacology of Ammodaucus leucotrichus. Clinical Phytoscience, 6,6.http://doi.org/10.1186/s40816-020-0154-7.Khaldi, A., Meddah, B., Moussaoui, A., & Sonnet, P. (2017). Anti-mycotoxin effect andantifungal properties of essential oil from Ammodaucus leucotrichus Coss. & Dur. onAspergillus flavus and Aspergillus ochraceus. Journal of Essential Oil and BearingPlants, 20, 36–44. http://doi.org/10.1080/0972060X.2017.1282840.Lee, J.Y., Cho, P.Y., Kim, T.Y., Kang, S.Y., Song, K.Y., & Hong, S.J. (2002). Hemolyticactivity and developmental expression of pore-forming peptide, clonorin. Biochemicaland Biophysical Research Communications, 296, 1238–1244.http://doi.org/10.1016/s0006-291x(02)02062-4.Manssouri, M., Znini, M., & Majidi, L. (2020). Studies on the antioxidant activity of essentialoil and various extracts of Ammodaucus leucotrichus Coss. & Dur. fruits from Morocco.Journal of Taibah University for Science, 14, 124–130.http://doi.org/10.1080/16583655.2019.1710394. Pavlović, I., Petrović, S., Radenković, M., Milenković, M., Couladis, M., Branković, S.,Pavlović, M.D., & Niketic, D.M. (2012). Composition, antimicrobial, antiradical andspasmolytic activity of Ferula heuffelii Griseb. ex Heuffel (Apiaceae) essential oil. FoodChemistry, 130, 310–315. http://doi.org/10.1016/j.foodchem.2011.07.043.Pillai, S., Mahmud, R., Lee, W.C., & Perumal, S. (2012). Anti-parasitic Activity of Myristicafragrans Houtt. essential oil against Toxoplasma Gondii parasite. APCBEE Procedia, 2,92–96. http://doi.org/10.1016/j.apcbee.2012.06.017.Quezel, P., & Santa, S. (1963). Nouvelle flore de l’Algérie et des régions désertiquesméridionales, CNRS, Paris.Riaz, M., Rasool, N., Bukhari, I.H., Shahid, M., Zubair, M., Rizwan, K., & Rashid, U.(2012). In vitro antimicrobial, antioxidant, cytotoxicity and GC/MS analysis of Mazusgoodenifolius. Molecules, 17, 14275–14287. http://doi.org/10.3390/molecules171214275Sadaoui, N., Bec, N., Barragnan-Montero, V., Kadri, N., Cuisinier, F., Larroque, C., Arab,K., & Khettal, B. (2018). The essential oil of Algerian Ammodaucus leucotrichus Coss.& Dur. and its effect on the cholinesterase and monoamine oxidase activities.Fitoterapia, 130, 1–5. http://doi.org/10.1016/j.fitote.2018.07.015Saleh, M.A., Clark, S., Woodard, B., & Deolu-Sobogun, S.A. (2010). Antioxidant and freeradical scavenging activities of essential oils. Ethnicity and Disease, 20 Suppl 1, 78–82.Salem, M.Z.M., Zidan, Y.E., Mansour, M.A.M., El Hadidi, N.M.N., & Abo Elgat, W.A.A.(2016). Antifungal activities of two essential oils used in the treatment of threecommercial woods deteriorated by five common mold fungi. InternationalBiodeterioration and Biodegradation, 106, 88–96.http://doi.org/10.1016/j.ibiod.2015.10.010.Sebaaly, C., Charcosset, C., Stainmesse, S., Fessi, H., & Greige-Gergesa, H. (2016). Cloveessential oil-in-cyclodextrin-in-liposomes in the aqueous and lyophilized states: fromlaboratory to large scale using a membrane contactor. Carbohydrate Polymers, 138, 75–85. http://doi.org/10.1016/j.carbpol.2015.11.053.Sharma, P., Sharma, J.D. (2001). In vitro hemolysis of human erythrocytes by plant extractswith antiplasmodial activity. Journal of Ethnopharmacology, 74, 239–243.http://doi.org/10.1016/S0378-8741(00)00370-6.Tampieri, M.P., Galuppi, R., Macchioni, F., Carelle, M.S., Falcioni, L., Cioni, P.L., &Morelli, I. (2005). The inhibition of Candida albicans by selected essential oils and theirmajor components. Mycopathologia, 159, 339–345. http://doi.org/10.1007/s11046-003-4790-5.Turek, C., & Stintzing, F.C. (2012). Impact of different storage conditions on the quality ofselected essential oils. Food Research International, 46, 341–353.http://doi.org/10.1016/j.foodres.2011.12.028.Velasco-Negueruela, A., Pérez-Alonso, M.J., Pérez de Paz, P.L., Palá-Paúl, J., & Sanz, J.(2006). Analysis by gas chromatography-mass spectrometry of the volatiles from thefruits of Ammodaucus leucotrichus subsp. leucotrichus and subsp. nanocarpus grown inNorth Africa and the Canary Islands, respectively. Journal of Chromatography A, 1108,273–275. http://doi.org/10.1016/j.chroma.2006.01.031.

V. Rama Subbaraoa* aNatural Products Laboratory, Organic Chemistry Division-I, Indian Institute of Chemical Technology, Habsiguda, Hyderabad 500007, India. Study and optimization of Diels-Alder reaction of piperine in aqueous ionic solutions using Gn.HCl as a catalyst. The semi-synthesis of these products using intermolecular [4+2] cycloaddition reaction has been described. Obtained products were characterized using IR, HNMR, CNMR and Mass Spectroscopy. Introduction An outsized number of phenomena concern to and are conducted in liquid phase involving ionic species (Millions of years ago, Mother Nature discovered the secrets of water molecule) in different biological and other natural processes. Salt present in the oceans, a striking example from Nature, is a multi component salt solution reflecting the distant marine origin of life on earth together with the composition of physiological fluids. In general the ionic solutions play roles in several industrial and geological processes in addition to their deep impact on the biological molecules. This enormous power of ionic solutions is based on the interactions of ion with solvent. In this work, we present some interesting results with comprehensive implications on the application of ion-solvent (i-s) interactions on organic reactions. Ion-Solvent interactions Cohesion among molecules in the liquid phase results from intermolecular forces. These forces include hydrogen-bonding, dipole-dipole, multi polar, dispersion interactions and also interactions emerging from the repulsion between two molecules. The cohesion due to intermolecular forces gives rise to a 'pressure' which is experienced by the solvent molecules. A liquid undergoing a small, isothermal volume expansion does work against the cohesive forces which causes a change in the internal energy, U. The function (∂U/∂V)T, is called as internal pressure (Pi) of a liquid and is supported by the equation of state. Internal pressure increases upon the addition of some solutes like NaCl, KCI, etc. and decreases by salts like of guanidinium salts. Diels-Alder Reaction in aqueous medium For long time water was not a popular solvent for the Diels-Alder reaction. Before 1980 its use had been reported only incidentally. Diels and Alder themselves performed the reaction between furan and maleic acid in an aqueous medium in 1931,27 an experiment which was repeated by Woodward and Baer in 1948. 28 They noticed a change in endo-exo selectivity when comparing the reaction in water with ether. The extreme influence of water can exert on the Diels-Alder reaction was rediscovered by Breslow in 1980, much by coincidence 29,30 while studying the effect of β-cyclodextrin on the rate of a Diels-Alder reaction in water, accidentally. Schem 1. Alternatively, Grieco et al., have repeatedly invoked the internal pressure of water as an explanation of the rate enhancement of Diels-Alder reactions in these solvents. 31 They probably inspired by the well known large effects of the external pressure on rates of cycloadditions. However the internal pressure of water is very low and offers no valid explanation for its effects on the Diels-Alder reaction. The internal pressure is defined as the energy required bringing about an infinitesimal change in the volume of the solvent at constant temperature. Due to the open and relatively flexible hydrogen-bond network of water, a small change in volume of these solvents does not require much energy. A related, but much more applicable solvent parameter is the cohesive energy density. This quantity is a measure of energy required for evaporation of the solvent per unit volume. The reactions in water were less accelerated by pressure than those in organic solvents, which is in line with notion that pressure diminishes hydrophobic interactions. The effect of water on the selectivity of Diels-Alder reactions Three years after the Breslow report on the large effects of water on the rate of the Diels-Alder reaction, he also demonstrated that the endo-exo selectivity of this reaction benefits markedly from employing aqueous media. Based on the influence of salting-in and salting-out agents, Breslow pinpoints hydrophobic effects as the most important contributor to the enhanced endo-exo selectivity. Hydrophobic effects are assured to stabilize the more compact endo transition state more than the extended exo transition state. In Breslow option the polarity of water significantly enhances the endo-exo selectivity. In conclusion, the special influence of water on the endo-exo selectivity seems to be a result of the fact that this solvent combines in it three characteristics that all favors formation of the endo/exo adduct. 1. water is strong hydrogen bond donor 2. water is polar and water induces hydrophobic interactions. Study of salting-out and salting-in reagents towards the Diels-Alder reaction of piperine (1): The special effects of water as solvent for valuable Diels-Alder reaction (Scheme 1) of piperine (1), greatly altered by the addition of ionic solutes (Table 1) such as LiCl, LiBr, LiClO4,- NaCl, NaBr, KF, KCl, KBr, MgCl2, CaCl2, guanidinium chloride, guanidinium carbonate, guanidinium nitrate. Aqueous salts solutions accelerated cycloaddition reactions (Scheme 1) of piperine (1) to give resultant cycloadducts 2, 3 and 4 among them 2 is major ortho-exo cyclohexene type dimeric amide alkaloid and also known as chabamide, which is previously isolated from this plant, isomer 3 is also known adduct and previously isolated from Piper nigrum. 21 Cycloadduct 4 was synthesized from piperine by Diels-Alder reaction by Wei. et al. its physical and spectroscopic data were identical with reported data22 (1H-NMR & Mass spectra). Table 1: Study of different salts towards the Diels-Alder reaction of piperine (1). aOverall yield of adducts after HPLC, un-reacted piperine was recovered in all reactions. Reaction showed good overall yield and more exo selectivity. This reaction showed completely regioselectivity (yield of 2+3>4) due to maximum involvement of α-double bond rather than γ-double bond of 1 during Diels-Alder reaction. Table 2: Comparision of salting-out and salting-in reagents towards the Diels-Alder reaction of piperine (1). Study of Salting-out reagents Increased rate in Diels-Alder reaction (over all yield up to 79 %) of piperine (1) has been attributed to the hydrophobic effect. Owing to the difference in polarity between water and the reactants, water molecules tend to associate amongst themselves, excluding the organic reagents and forcing them to associate together forming small drops surrounded by water. A further method of increasing the rate of Diels-Alder reaction in water is so called ‘salting-out’ effect. Among the salting-out reagents used (Table 1) in this methodology CaCl2 is the best reagent and gave 79 % over all yield. If anion size increases, reaction yield decreases, where as cation size increases, reaction yield increases. Here a salt such as calcium chloride is added to the aqueous solution. In this case water molecules attracted to the polar ions, increasing the internal pressure and reducing the volume. This has the effect of further excluding the organic reagents. For reactions such as Diels-Alder, which have negative activation volumes, the rates are enhanced by this increase in internal pressure in much the same way as expected for an increase in external pressure. This salting-out reagent showed good exo selectivity, due to formation of cycloadduct 2 (ortho-exo) is major up to 69 % (cycloadduct ratio) compare to cycloadducts 3 (21 %, meta-exo) and 4 (10%, meta-exo) are poor in yield. Schem 2. Plausible mechanism of Diels-Alder reaction catalyzed by Gn.HCl. Study of Salting-in reagents Among the tested salting-in reagents used in this methodology (Table 1) guanidinium chloride (Gn.HCL) is the best reagent and gave 81 % overall yield, where as LiClO4 end up with only 15 % overall yield. Gn.HCL reagent exhibited well selectivity towards the Diels-Alder reaction of piperine in given conditions (scheme 1). Formation of cycloadduct 2 in 80 %, 3 in 15 % and 4 in 5 % ratio is clearly indicates this methodology received good attention towards the exo selectivity in Diels-Alder reaction of piperine. Overall yield is also high with salting-in reagents when compare to salting-out reagents. Procedure for aqueous ionic salts catalyzed Diels–Alder reactions of piperine (1): To a stirred mixture of piperine (1) (50.0 mg, 0.175 mmol), 6M aqueous guanidinium. Hydrochloride (2 mL) in a round bottom flask fitted with condenser and refluxed for 70 h in an oil bath. After completion of the reaction, monitored by TLC (dipped in 5% solution of phosphomolybdic acid in methanol and heating), the reaction mixture was cooled to room temperature and diluted with water (3 mL). Then extracted with EtOAc (2x5 mL), the combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue obtained was then purified by reversed-phase (RP) HPLC (column: Phenomenex Luna C18, 250 x 10 mm, 10µ), solvent system: 80% acetonitrile in water, flow rate: 1.5 mL/min, to give pure compounds of adducts (2) 0.065 g, (3) 0.012 g and (4) 0.004 g. Cycloaddition reaction between piperine (1a) and pellitorine (1b): Our aim of this cycloaddition reaction is to explain to study different cycloadducts and selectivity of diene among piperine and pellitorine (Scheme 4). This biomimetic synthesis will explain the probability of diene, which participated in Diels-Alder reaction between piperine (1a) and pellitorine (1b) both were isolated from same plant (P. chaba). Nigramide N, which is formed biosynthetically via cycloaddition reaction between piperine and pellitorine, this adduct previously isolated from roots of P. nigrum 21 by Wei. et. al. Lewis acid catalyzed cycloaddition reactions of piperine (1a) and pellitorine (1b) under organic and aqueous solvent conditions to give resultant cycloadducts 2c, 3c, 4c, 2a and 3b. Cycloadduct 2c and 3c is new cycloadducts and their structures were illustrated by 1D and 2D spectral data. Structure elucidation of compound 2c: Compound 2c was obtained as pale yellow liquid. The molecular formula of 2c was established as C31H44N2O4 by HRESIMS (Fig-18), which provided a molecular ion peak at m/z 509.3381 [M++H], in conjunction with its 13C NMR spectrum (Fig-12). The IR spectrum displayed absorption bands diagnostic of carbonyl (1640 cm-1) (Fig-10). The 300 MHz 1H NMR spectrum (in CDCl3) indicated the presence of two signals at δ 5.86 (dd, J = 15.6, 10.1 Hz) and 6.27 (d, J = 15.6 Hz), which were assigned to trans-olefinic protons by the coupling constant of 15.6 Hz. It also displayed aromatic protons due to two 1, 3, 4-trisubstituted aromatic rings at δ 6.82 (1H, br s), 6.76 (1H, dd, J = 7.8, 1.4 Hz), 6.75 (1H, d, J = 7.8 Hz) (Fig-11), (Table 4). In addition to the above-mentioned moieties, combined inspection of 1H NMR and 1H–1H COSY revealed the presence of cyclohexene ring, one isobutylamide and one pyrrolidine ring. The 13C NMR spectrum (Fig-12) displayed the presence of 31 carbon atoms and were further confirmed by DEPT experiments (Fig-13) into categories of 11 methylenes, 12 methines and 5 quaternary carbons including two carbonyls (δ 173.01 and 172.50). On the basis of these characteristic features, database and literature search led the skeleton of compound 2c as a dimeric alkaloidal framework. A comprehensive analysis of the 2D NMR data of compound 2c facilitated the proton and carbon assignments. 1H–1H COSY spectrum (Fig-16) suggested the sequential correlations of δ 3.51 (dq, J = 5.0, 2.6 Hz)/5.62 (dt, J = 9.8, 2.6 Hz)/6.10 (ddd, J = 9.8, 1.5 Hz)/2.20 (m)/2.72 (ddd, J = 11.1, 10.1, 5.2 Hz)/3.35 (dd, J = 11.1, 9.8 Hz) assignable to H-2-H-3-H-4-H-5-H-3"-H-2" of the cyclohexene ring. Concerning the connections of the n-amyl and 3, 4-methylenedioxy styryl groups, HMBC spectrum (Fig-15) showed correlations of H-4, H-6, H-7/C-5; H-5", H-4"/C-3", which implies that these units were bonded to the cyclohexene ring at C-5 and C-3". Further, HMBC correlations of two methylene protons at δ 5.95 with 147.91 (C-8"), 146.87 (C-9"), confirmed the location of methylenedioxy group at C-8", and C-9". Remaining units, isobutylamine and pyrrolidine (rings) were connected through carbonyl groups at C-2 and C-2", which was confirmed by HMBC correlations of H-2 and H-1' to C-1 (δ 173.01) and H-2" and H-1''' to C-1" (δ 172.50). The assignment of the relative configuration of compound 2c, and confirmation of overall structure were achieved by the interpretation of the NOESY spectral data and by analysis of 1H NMR coupling constants. The large vicinal coupling constants of H-2"/H-2 (11.1 Hz) and H-2"/H-3" (11.1 Hz) indicated anti-relations of H-2"/H-2 and H-2"/H-3" and the axial orientations for these protons. In the NOESY spectrum (Fig-17), the occurrence of the correlations between H-2/H-3" and the absence of NOE effects between H-2/H-2" and H-2"/H-3" supported the above result. This data indicated β-orientation for H-2" and α-orientation for H-2 and H-3". The α-orientation of H-5 was suggested by the coupling constant of H-5/H-3" (5.2 Hz) and the absence of the NOESY correlations between H-3" and H-2". On the basis of these spectral data, the structure of compound 2c was unambiguously established and trivially named as chabamide M. Compound 3a: IR (KBr) nmax: 2923, 2855,1628, 1489, 1242, 1128, 1035 cm-1 d ppm 0.69 & 1.25 (2H, m, H-2'"), 1.15 & 1.23 (2H, m, H-4'"), 1.31 & 1.40 (2H, m, H-3'"), 1.52 (2H, m, H-2'), 1.56 (2H, m, 4'), 1.61 (2H, m, H-3'), 2.94 (1H, td, J = 10.1, 10.1, 5.5 Hz, H-3"), 3.02 & 3.60 (2H, m, H-5'"), 3.09 & 3.32 (2H, m, H-1'"), 3.51 (2H, m, H-1'), 3.61 (1H, m, H-2), 3.61 (2H, m, H-5'), 3.78 (1H, dq, J = 10.0, 2.3 Hz, H-5), 4.07 (1H, t, J = 10.1, H-2"), 5.72 (1H, ddd, J = 9.8, 5.0, 2.7 Hz, H-3), 5.88 (2H, s, H-12), 5.89 (1H, dt, 10.3, 1.8 Hz, H-4), 5.90 (1H, J =15.8, 9.8 Hz, H-4"), 5.92 (1H, s, H-12"), 6.37 (1H, d, J = 15.8 Hz, H-5"), 6.68 (1H, brs, H-7), 6.69 (1H, d, J = 8.0 Hz, H-10"), 6.70 (1H, dd, J = 8.0, 1.4 Hz, H-11), 6.69 (1H, d, J = 8.0 Hz, H-10), 6.74 (1H, dd, J = 8.0, 1.6 Hz, H-11"), 6.79 (1H, brs, H-7"). ESIMS (m/z): 571 [M+ +H] Table 4: 1H & 13C NMR data of cycloadduct 2c in CDCl3 (300 MHz, δ in ppm, mult, J in Hz) Compound 4a: IR (KBr) nmax: 2926, 2857,1627, 1484, 1440, 1240, 1034 cm-1 1H NMR (300 MHz, CDCl3): d ppm 0.81 & 1.35 (2H, m, H-2'), 1.29 & 1.47 (2H, m, H-4'), 1.35 (2H, m, H-2"'), 1.36 & 1.51 (1H, m, H-3'), 1.47 (2H, m, H-4"'), 1.51 (2H, m, H-3"'), 2.92 (2H, m, H-1"'), 2.99 (1H, ddd, J = 12.5, 9.7, 5.5 Hz, H-4"), 3.22 (2H, m, H-1'), 3.29 & 3.71 (2H, m, H-5'), 3.38 (1H, m, H-4"'), 3.44 (1H, dd, J = 12.1, 10.1 Hz, H-5"), 3.59 (1H, t, J = 5.3 Hz, H-5), 3.70 (1H, dq, J = 12.1, 2.1, H-2), 5.65 (1H, dd, J = 15.6, 9.5 Hz, H-3"), 5.70 (1H, dt, J = 9.9, 1.6, H-3), 5.81 (1H, d, J = 15.6 Hz, H-2"), 5.84 (1H, s, H-12"), 5.90-5.92 (2H, brs, H-12), 5.96 (1H, ddd, J = 9.2, 5.8, 2.6 Hz, H-4), 6.55 (1H, dd, J =7.9, 1.5 Hz, H-11"), 6.61 (1H, d, J = 8.2 Hz, H-10"), 6.62 (1H, d, J = 1.4 Hz, H-7"), 6.79 (1H, d, J = 7.9 Hz, H-10), 6.92 (1H, dd, J = 8.0, 1.5 Hz, H-11), 7.01 (1H, d, J = 1.5 Hz, H-7). ESIMS (m/z): 571 [M+ +H] Acknowledgements The authors are thankful to Director IICT for his constant encouragement and CSIR New Delhi for providing the fellowship References Braun, M. Synth. Highlights 1991, 232 Robinson, R. Chem. Soc. 1917, 762. Stork, G.; Burgstahler, A. W. Am. Chem. Soc. 1955, 38, 1890. Johnson, W. S.; Gravestock, M. B.; McCarry, B. E. Am. Chem. Soc. 1971, 93, 4332. Chapman, O. L.; Engel, M. R.; Springer, J. P.; Clardy, J. C. Am. Chem. Soc. 1971, 93, 6696. Bandaranayake, W. M.; Banfield, J. E.; Black, D. St. C. Chem. Soc., Chem Commun. 1980, 902. Nicolaou, K. C.; Zipkin, R. E.; Petasis, N. A. Am. Chem. Soc. 1982, 104, 5558. O.; Alder, K. Ann. 1928, 460, 98. Woodward, R. B.; Hoffmann, R. Chem. 1969, 81, 797. Fakui, K. Chem. Res. 1971, 4, 57. Houk, K. N. Chem.. Res. 1975, 8, 361. Houk, K. N.; Li, Y.; Evanseck, D. Angew Chem., Ed. Engl. 1992, 31, 682. Alder, K.; Stein, G. Chem. 1937, 50, 510. Fotiadu, F.; Michel, F.; Buono, G. Tetraheron Lett. 1990, 34, 4863. Gleiter, R.; Bohm, M. C. Pure Appl. Chem. 1983, 55, 237. Woodward, R. B.; Katz, T. J. Terahedron 1958, 5, 70. Kakushima, M. J. Chem. 1979, 57, 2564. Houk, K. N. Tetrahedron Lett. 1970, 30, 2621. Houk, K. N.; Luskus, L. J. Am. Chem. Soc. 1971, 93, 4606. Otto, S.; Bertoncin, F.; Engberts, J.B. F. N. Am. Chem. Soc., 1996, 118, 7702–7707. Wei, K.; Li, W.; Koike, K.; Chen, Y-J.; Nikaido, T. Org. Chem. 2005, 70, 1164. Wei, K.; Li, W.; Koike, K.; Chen, Y-J.; Nikaido, T. Lett. 2005, 7, 2833–2835. Rukachaisirikul, T.; Prabpai, S.; Champung, P.; Suksamrarn, A. Planta Med. 2002, 68, 850-853. Nagao, Y.; Seno, K.; Kawabata, K.; Miyasaka, T.; Takao, S.; Fujita, Tetrahedron Lett. 1980, 21, 841. Otto, S.; Boccaletti, G.; Engberts, J. B. F. N. Am. Chem. Soc. 1998, 120, 4238–4239. Otto, S.; Bertoncin, F.; Engberts, J. B. F. N. Am. Chem. Soc. 1996, 118, 7702–7707. O.; Alder, K. Ann. 1931, 490, 243. Woodward, R. B.; Baer, H. Am. Chem. Soc. 1948, 70, 1161. Breslow, R.; Rideout, D. C. Am. Chem. Soc. 1980, 102, 7816. Breslow, R.; Guo, T. Am. Chem. Soc. 1988, 110, 5613. Grieco, P.A.; Nunes, J. J.; Gaul, M. D. Am. Chem. Soc. 1990, 112, 4595.

This study aims to ameliorate the water solubility of flurbiprofen by using β-cyclodextrin (β-CD). The drug/ligand 1:1 (M/M) stoichiometry was determined based on the effect of β-CD on the solubility of flurbiprofen. Several methods of preparing flurbiprofen/β-CD inclusion complex were investigated and a solvent method using hot water to dissolve the starting materials was selected. The selected method showed a lot of advantages such as high complexing ability, good product yield, simple and eco-friendly process. The obtained product was characterized using various analytical techniques such as high-performance liquid chromatography, differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. The product had a predominantly amorphous form with clathrate particles of about 2-7 µm in size, irregular edges and rough surfaces. The study results show that in the complexing process, flurbiprofen replaced water molecules located in the conical cavity of β-CD. The complex contained 19.91% flurbiprofen by mass with water solubility at 37°C was 1,100 µg/ml. The results also show that the complexing with β-CD significantly improved the water solubility of flurbiprofen by both speed and level. Keywords Flurbiprofen, β-cyclodextrin, inclusion complex, water solubility, preparation, characterization. References [1] K. Maroof, F. Zafar, H. Ali, S. Naveed, Flurbiprofen: a potent pain reliever, J. Bioequiv. Availab. 7(1) (2015) 056-058. https://doi.org/10.4172/jbb.1000214.[2] J.J. Thebault, G. Lagrue, C.E. Blatrix, L. Cheynier, R. Cluzan, Clinical pharmacology of flurbiprofen: a novel inhibitor of platelet aggregation, Curr. Med. Res. Opin. 5(1) (1977) 130-134. https://doi.org/10.1185/03007997709108990.[3] H. Geerts, Drug evaluation: (R)-flurbiprofen - an enantiomer of flurbiprofen for the treatment of Alzheimer's disease, Idrugs. 10(2) (2007) 121-133.[4] P.L. McGeer, M. Schulzer, E.G. McGeer, Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies, Neurology. 47(2) (1996) 425-432. https://doi.org/10.1212/WNL.47.2.425.[5] S. Meister, I. Zlatev, J. Stab, D. Docter, S. Baches, et al., Nanoparticulate flurbiprofen reduces amyloid-β 42 generation in an in vitro blood-brain barrier model, Alzheimers Res. Ther. 5(6) (2013) 51-63. https://doi.org/10.1186/alzrt225.[6] K.P. Townsend, D. Praticò, Novel therapeutic opportunities for Alzheimer’s disease: focus on nonsteroidal anti-inflammatory drugs, FASEB J. 19(12) (2005) 1592-1601. https://doi.org/10.1096/fj.04-3620rev.[7] C.K. Kim, Y.S. Yoon, J.Y. Kong, Preparation and evaluation of flurbiprofen dry elixir as a novel dosage form using a spray-drying technique, Int. J. Pharm. 120 (1995) 21-31. https://doi.org/10.1016/0378-5173(94)00375-F.[8] M.J. Habib, M.T. Phan, G. Owusu-Ababio, Dissolution profiles of flurbiprofen in phospholipid solid dispersions, Drug Dev. Ind. Pharm. 24 (1998) 1077-1082. https://doi.org/10.3109/03639049809089952.[9] D.H. Oh, Y.J. Park, J.H. Kang, C.S. Yong, H.G. Choi, Physicochemical characterization and in vivo evaluation of flurbiprofen-loaded solid dispersion without crystalline change, Drug. Deliv. 18 (2010) 46-53. https://doi.org/10.3109/10717544.2010.509365.[10] G.D. Gupta, S. Jain, N.K. Jain, Formulation of an aqueous injection of flurbiprofen, Pharmazie. 52 (1997) 709-712. https://doi.org/10.1080/10826079708005547.[11] K.W. Ambade, S.L. Jadhav, M.N. Gambhire, S.D. Kurmi, V.J. Kadam, K.R. Jadhav, Formulation and evaluation of flurbiprofen microemulsion, Curr. Drug Deliv. 5 (2008) 32–41[12] H.H. Baek, S.Y. Kwon, S.J. Rho, W.S. Lee, H.J. Yang, J.M. Hah, H.G. Choi, Y.R. Kim, C.S. Yong, Enhanced solubility and bioavailability of flurbiprofen by cycloamylose, Arch. Pharm. Res. 34 (2011) 391-397. https://doi.org/10.1007/s12272-011-0306-x.[13] A. Muraoka, T. Tokumura, Y. Machida, Evaluation of the bioavailability of flurbiprofen and its β-cyclodextrin inclusion complex in four different doses upon oral administration to rats, Eur. J. Pharm. Biopharm. 58(3) (2004) 667-671. https://doi.org/10.1016/j.ejpb.2004.03.030.[14] T. Tokumura, A. Muraoka, Y. Machida, Improvement of oral bioavailability of flurbiprofen from flurbiprofen/beta-cyclodextrin inclusion complex by action of cinnarizine, Eur. J. Pharm. Biopharm. 73 (2009) 202-204. https://doi.org/10.1016/j.ejpb.2009.04.018.[15] D. Li, M. Han, P. Balakrishnan, Y. Yan, D. Oh, et al., Enhanced oral bioavailability of flurbiprofen by combined use of micelle solution and inclusion compound, Arch. Pharm. Res. 33(1) (2010) 95-101. https://doi.org/10.1007/s12272-010-2231-9.[16] H. Arima, K. Motoyama, T. Irie, Recent findings on safety profiles of cyclodextrins, cyclodextrin conjugates, and polypseudorotaxanes, in: E. Bilensoy (Ed.), Cyclodextrins in Pharmaceutics, Cosmetics, and Biomedicine: Current and Future Industrial Applications, John Wiley & Sons Inc., Hoboken, 2011, pp. 91-122. https://doi.org/10.1002/9780470926819.ch5.[17] T.J. Grattan, Inclusion complexes of beta-cyclodextrin with flurbiprofen, ketoprofen and naproxen, International patent WO1995007104A1, March 16, 1995.[18] M. Cirri, C. Rangoni, F. Maestrelli, G. Corti, P. Mura, Development of fast-dissolving tablets of flurbiprofen-cyclodextrin complexes, Drug Dev. Ind. Pharm. 31(7) (2005) 697-707. https://doi.org/10.1080/03639040500253694.[19] M. Tirunagari, N. Mehveen, M.F. Qureshi, J.P. Sultana, V. Tirunagari (2012), Solubility Enhancement of Flurbiprofen using Different Solubilization Techniques, Int. J. Pharm. Pharm. Sci. 4(4) (2012) 97-100.[20] P. Saokham, C. Muankaew, P. Jansook, T. Loftsson, Solubility of cyclodextrins and drug/cyclodextrin complexes, Molecules. 23(5) (2018) 1161-1175. https://doi.org/10.3390/molecules23051161.[21] S. Pereva, T. Sarafska, S. Bogdanova, T. Spassov, Efficiency of “cyclodextrin-ibuprofen” inclusion complex formation, J. Drug Deliv. Sci. Tec. 35 (2016) 34-39. https://doi.org/10.1016/j.jddst.2016.04.006.[22] T.T.B. Nguyen, N.A. Dang, M.A. Tran, T.H. Nguyen, Validation of a high-performance liquid chromatographic method with diod array detection for the quantification of flurbiprofen in 100 mg film-coated tablet, VNU Journal of Science: Medical and Pharmaceutical Sciences. 33(2) (2017) 41-49. https://doi.org/10.25073/2588-1132/vnumps.4085.[23] T. Higuchi, K.A. Connors, Phase-solubility techniques, Adv. Anal. Chem. Instrum. 4 (1965) 117-122.

Abstract. Allopurinol is an antigout drug therapy, commonly used in the treatment of chronic gout or hyperuricaemia associated with treatment of diuretic conditions. In the present study, new formulations based on Allopurinol, have been prepared with the microencapsulation by solvent evaporation process. Microspheres were prepared using pure Allopurinol and polymeric matrices (ethylcellulose EC, poly (ε-caprolactone) PCL, β-cyclodextrin CD and hydroxypropylmethylcellulose HPMC) at different compositions and stirring speeds to investigate the effect of these parameters on loading efficiency and drug release kinetics. The formulations produced were characterized by various methods : Fourier transforms infrared spectroscopy (FTIR), X-ray powder diffractometry, optical microscopy, surface morphology by scanning electron microscopy (SEM) and drug loading, as well as in vitro release studies in the simulated stomach tract. Depending on the stirring speed and the composition of the microparticles, the active ingredient loading is in a range from 10.46 ± 1.45 to 46.40 ± 0.5%. The microspheres are spherical and the mean Sauter diameter (d32) of the microparticles obtained is smaller and is in the range of 47.71 to 151.01 µm. Different release profiles were obtained and show that the release rate is strongly influenced by the characteristics of the microparticles ; namely, the stirring rates and the composition of the microparticles. The release mechanism was identified by modelling using Higuchi and Korsmeyer-Peppas models. Resumen. Alopurinol es una droga terapéutica para tratar la gota, y se utiliza en el tratamiento de gota crónica o hiperuricemia asociada con el tratamiento de condiciones diuréticas. En este estudio, nuevas formulaciones basadas en Alopurinol se prepararon mediante microencapsulación por el proceso de evaporación de disolvente. Microesferas se prepararon usando Alopurinol puro y diferentes matrices poliméricas (etil-celulosa EC, poli(-caprolactona) PCL, β-cyclodextrina CD e hidroxipropil-metil-celulose HPMC) en diferentes composiciones y velocidades de agitación, para investigar el efecto de estos parámetros en la eficiencia de carga y en la cinética de liberación del fármaco. Las formulaciones obtenidas fueron caracterizadas por diferentes técnicas : Espectroscopía infrarroja de transformadas de Fourier (FTIR), difractometría de rayos X de polvos, microscopía óptica, morfología de superficies mediante microscopía electrónica de barrido electrónico, y la eficiencia de carga del fármaco, así como estudios de liberación in vitro en tracto estomacal simulado. Dependiendo de la velocidad de agitación y la composición de las micropartículas, la carga del ingrediente activo se encuentra en el rango de 10.46 ± 1.45 a 46.40 ± 0.5%. Las microesferas son esféricas y el diámetro medio de Sauter (d32) de las micropartículas obtenidas es menor, y se encuentra en el rango de 47.71 a 151.01 µm. Se obtuvieron diferentes perfiles de liberación y se observa que la velocidad de liberación está influenciada principalmente por las características propias de la producción de las micropartículas ; en particualr, las velocidades de agitación y las composición de las micropartículas. El mecanismo de liberación se ajusta mejor a los modelos matemáticos de Higuchi and Korsmeyer-Peppas.

Eplerenone, a BCS class II drug with low bioavailability and t1/2 of 3-6 hrs is primarily used to treat Congestive Heart Failure (CHF) and hypertension. So, to develop the biological performance of Eplerenone, solid dispersion along with oral disintegrates was prepared by employing HP ?-Cyclodextrin and ?-Cyclodextrin. Eplerenone solid dispersions were repared with various carriers in varying ratios of carrier and drug respectively (0.5:1, 1:1 and 1.5:1). Results of prepared Eplerenone solid dispersions through solvent evaporation technique were demonstrated which comprise melti g point determination, solubility, entrapment efficiency, drug content uniformness and in-vitro breakup studies. Characterization of solid state was done by FT-IR. From comparison of all the formulation characteristics, formulation (F3) containing Eplerenone + Hp ?-cyclodextrin (1:1.5) showed better results y solvent evaporation technique. As maximum drug was released from F3 at the end of 60 min, this formulation was decided as the best. From the optimized formulation, Fast dissolving tablets were formulated employing various disintegrates in varying concentrations. The pre and post compression parameters were calculated and the results were pecified. All the results were within the acceptable range. An in-vitro drug discharge study of the formulated drug was done victimisation pH 6.8 buffers. F6 formulation containing crospovidone (13.5mg) exhibited 97.36 % drug release within 20mins. The optimized formulation follows zero-order release kinetics.

Bacterial growth in fruits and vegetables causes a large percentage of the loss and waste of these foods worldwide. For this reason, the objective of this study was to develop antibacterial gels based on sodium alginate, β-cyclodextrin, and allyl isothiocyanate inclusion complexes (β-CD:AITC) for the potential packaging of fresh fruits and vegetables. The β-CD:AITC complexes were prepared by the co-precipitation method with a 1:1 molar ratio, and was further verified by FESEM microscopy. On the other hand, the antibacterial gels were prepared using the external gelation method, and the effect of the order of incorporation of the components, the conditions of the components, and the resting conditions of the dispersions on their visual appearance was evaluated. Furthermore, the antibacterial activity of the gels against Escherichia coli and Listeria innocua was evaluated using a headspace system. FESEM micrographs showed a crystalline block-like morphology in the inclusion complexes. The order of incorporation (1) glucone-delta-lactone (2) complexes (3) alginate allowed for obtaining more homogeneous gels with a smooth surface. The presence of glucono-delta-lactone, the concentration of 0.05 M CaCl2, a cross-linking time of 20 hr, and the cooling of the dispersion allowed more uniform gels to be obtained. Finally, better antibacterial activity against E. coli was obtained with the gels loaded with 10% β-CD:AITC complexes. According to these results, the developed materials could be used as antimicrobial packaging materials for fresh fruits and vegetables. Keywords: β-cyclodextrin, allyl isothiocyanate, alginate, food packaging, antimicrobial. Resumen El crecimiento bacteriano en frutas y verduras provoca un gran porcentaje de pérdida y desperdicio de estos alimentos a nivel mundial. Por esta razón, el objetivo de este estudio fue desarrollar geles antibacterianos a base de alginato de sodio y complejos de inclusión de β-ciclodextrina e isotiocianato de alilo (β-CD:AITC) para el potencial envasado de frutas y verduras frescas. Los complejos β-CD:AITC se prepararon mediante el método de co-precipitación con una relación molar 1:1, y su obtención se verificó mediante microscopía FESEM. Por otro lado, los geles antibacterianos se prepararon mediante el método de gelificación externa, y se evaluó el efecto del orden de incorporación de los componentes, las condiciones de los componentes y de reposo de las dispersiones en su apariencia visual. Además, se evaluó la actividad antibacteriana de los geles frente a Escherichia coli y Listeria innocua mediante un sistema de espacio de cabeza. Las micrografías FESEM mostraron una morfología del tipo bloque cristalino en los complejos de inclusión. El orden de incorporación (1)Glucono-deltalactona-( 2)Complejos-(3)Alginato permitió obtener geles más homogéneos y con una superficie lisa. La presencia de la Glucono-delta-lactona, la concentración de 0,05 M de CaCl2, un tiempo de entrecruzamiento de 20 horas y la refrigeración de la dispersión permitió obtener geles más uniformes. Finalmente, se obtuvo una mejor actividad antibacteriana frente a E. coli con los geles cargados con un 10% de complejos β-CD:AITC. De acuerdo con estos resultados, los materiales desarrollados podrían ser utilizados como materiales de envase antimicrobiano para frutas y verduras frescas. Palabras Clave: β-ciclodextrina, isotiocianato de alilo, alginato, envases de alimentos, antimicrobiano.