In Vitro and Sensory Evaluation of Capsaicin-Loaded Nanoformulations

Capsaicin has known health beneficial and therapeutic properties. It is also able to enhance the permeability of drugs across epithelial tissues. Unfortunately, due to its pungency the oral administration of capsaicin is limited. To this end, we assessed the effect of nanoencapsulation of capsaicin, under the hypothesis that this would reduce its pungency. Core-shell nanocapsules with an oily core and stabilized with phospholipids were used. This system was used with or without chitosan coating. In this work, we investigated the in vitro release behavior of capsaicin-loaded formulations in different physiological media (including simulated saliva fluid). We also evaluated the influence of encapsulation of capsaicin on the cell viability of buccal cells (TR146). To study the changes in pungency after encapsulation we carried out a sensory analysis with a trained panel of 24 students. The in vitro release study showed that the systems discharged capsaicin slowly in a monotonic manner and that the chitosan coating had an effect on the release profile. The cytotoxic response of TR146 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, was reduced following its encapsulation. The sensory study revealed that a chitosan coating results in a lower threshold of perception of the formulation. The nanoencapsulation of capsaicin resulted in attenuation of the sensation of pungency significantly. However, the presence of a chitosan shell around the nanoformulations did not mask the pungency, when compared with uncoated systems.     

Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade

Hepatitis B virus (HBV) pre-S2 mutant can induce hepatocellular carcinoma (HCC) via the induction of endoplasmic reticulum stress to activate mammalian target of rapamycin (MTOR) signaling. The association of metabolic syndrome with HBV-related HCC raises the possibility that pre-S2 mutant-induced MTOR activation may drive the development of metabolic disorders to promote tumorigenesis in chronic HBV infection. To address this issue, glucose metabolism and gene expression profiles were analyzed in transgenic mice livers harboring pre-S2 mutant and in an in vitro culture system. The pre-S2 mutant transgenic HCCs showed glycogen depletion. The pre-S2 mutant initiated an MTOR-dependent glycolytic pathway, involving the eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), Yin Yang 1 (YY1), and myelocytomatosis oncogene (MYC) to activate the solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1), contributing to aberrant glucose uptake and lactate production at the advanced stage of pre-S2 mutant transgenic tumorigenesis. Such a glycolysis-associated MTOR signal cascade was validated in human HBV-related HCC tissues and shown to mediate the inhibitory effect of a model of combined resveratrol and silymarin product on tumor growth. Our results provide the mechanism of pre-S2 mutant-induced MTOR activation in the metabolic switch in HBV tumorigenesis. Chemoprevention can be designed along this line to prevent HCC development in high-risk HBV carriers.     

Computational study on the structural and optoelectronic properties of a carbazole-benzothiadiazole based conjugated oligomer with various alkyl side-chain lengths

The effect of the alkyl side-chain length on the structural and optoelectronic properties of poly[N-9′-heptadecanyl-27-carbazole-alt-55-(4′,7′-di-2-thienyl-2′,1′,3′-benzothiadiazole)] (PCDTBT) conjugated oligomers have been studied by density functional theory (DFT) and time-dependent density functional theory (TD-DFT). The study was carried out by varying the length of alkyl side-chain attached to the nitrogen atom of the carbazole unit of the PCDTBT oligomers. The structural properties of the optimised oligomers were then studied by determining the bond-length alternation and dihedral angles (Φ) for various side-chain lengths. Total energy calculations for the determination of HOMO energy (E_HOMO), LUMO energy (E_LUMO), and fundamental energy gap (E_Gap) were performed using DFT at the B3LYP/6-31G(d), while the first singlet excitation energies (E_Opt) were calculated by TD-DFT also at the same level of theory. It was observed that there are no significant structural changes occurring as the alkyl chain lengths are varied. For the electronic properties, very small differences (i.e. ~0.01 eV) were observed for E_Gap and E_Opt while the exciton binding energies (E_B) were virtually the same. The results suggest that using shorter alkyl side-chains do not significantly affect the structural and optoelectronic properties of the carbazole-benzothiadiazole based polymer. The observations can aid future computational design studies of analogous systems by reducing large structures thus decreasing computational costs.     

Interaction between cystatin-derived peptides and papain

The interaction between papain and synthetic peptides which tentatively mimic cystatin surfaces was investigated both enzymatically and structurally. Measurements of dissociation equilibrium constants for the interaction of papain with these peptides modified by successive deletions or substitutions demonstrated that the QVVAG segment, which is highly conserved throughout members of the cystatin superfamily, is essential for the interaction. The glycylcontaining (N-terminal) fragments and PW-containing (C-terminal) fragments were found to be of lesser importance, since each could be deleted without significantly modifying the interaction. These fragments improved the stability of the interacting QVVAG region, which appeared to be substrate-like in all peptides tested, as it was cleaved at the A-G bond upon peptide-papain interaction. Replacement of the A residue at the scissile bond of the QVVAG by a blocked cysteinyl residue reduced the rate of cleavage of the susceptible bond and therefore shifted the resulting peptide from a substrate to an inhibitor. Derivatization of this substituted peptide at its N- and C-terminal ends by fluoresceinyl groups resulted in a dramatic decrease in theK _i to 0.5 µM. This improvement in the inhibitory properties of the substituted and derivatized peptides was correlated with structural changes as analyzed by molecular dynamic calculations. The results were compared to those proposed for the mechanism of inhibition by natural inhibitors of the cystatin superfamily.     

Development of a Efficient and Sensitive Dispersive Liquid–Liquid Microextraction Technique for Extraction and Preconcentration of 10 β2-Agonists in Animal Urine

Dispersive liquid–liquid microextraction (DLLME) coupled with ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was developed for the extraction and determination of 10 β₂-agonists in animal urine. Some experimental parameters, such as the type and volume of the extraction solvent, the concentration of the dispersant, the salt concentration, the pH value of the sample solution, the extraction time and the speed of centrifugation, were investigated and optimized. Under the optimized conditions, a good enrichment factors (4.8 to 32.3) were obtained for the extraction. The enrichment factor show that the concentration rate of DLLME is significantly higher than other pretreatment methods, and the detection sensitivity has been greatly improved. The calibration curves were linear, the correlation coefficient ranged from 0.9928 to 0.9999 for the concentration range of 0.05 to 50 ngmL^-1 and 0.1 to 50 ngmL^-1, and the relative standard deviations (RSDs, n = 15, intra and inter-day precision) at a concentration of 5 ngmL^-1 were in the range of 1.8 to 14.6%. The limits of detection (LODs) for the 10 β₂-agonists, based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.01 to 0.03 ngmL^-1. The proposed method was used to identify β₂-agonists in three types of animal urine (swine, cattle, sheep), and the relative recoveries from each matrix were in the range of 89.2 to 106.8%, 90.0 to 109.8% and 89.2 to 107.2%, respectively.