Capsaicin enhances astaxanthin action in liposomes

We previously demonstrated that co-encapsulation of the potent antioxidant astaxanthin (Asx) and tocotrienols into liposomes results in synergistically higher antioxidative activity than the calculated additive activity of each individual antioxidant-containing liposome, due to intermolecular interactions between terminal ring moieties of the two antioxidants and the polyene chain and the triene moiety. We reported that intermolecular interactions depend on the stereochemistry of Asx, and change the electronic state of the Asx polyene moiety. Based on these findings, we hypothesized that antioxidants that interact with Asx at the terminal ring and polyene moieties may enhance the antioxidative activity. Herein, we selected two candidate antioxidants, capsaicin (Cap) and resveratrol, based on their structures, in which the compounds exhibit similar characteristics to tocotrienols. We evaluated the antioxidative capacities of liposomes co-encapsulating Asx and the selected candidates. Based on hydroxyl radical scavenging activity, Cap was found to synergistically enhance the antioxidative activity of Asx at an optimal Asx/Cap ratio. Intermolecular interactions between Asx and Cap are necessary for the synergistic effect, and the Asx stereoisomer 3R,3’R-form (Asx-R) was predicted to most potently interact. Liposomes co-encapsulating Asx-R and Cap exhibited clear synergistic antioxidative activity at an optimal ratio, whereas liposomes co-encapsulating the other Asx stereoisomer and Cap did not demonstrate such activity. Computational chemistry analysis showed that changes in the electronic state of the polyene moiety of Asx-R are crucial for the synergistic activity. These results suggest that antioxidants that can change the electronic state of Asx via intermolecular interactions may enhance the function of Asx

GIPC-phospholipase D in plants

Previously, we detected an unknown sphingophospholipid in cabbage leaves and identified it as phytoceramide-1-phosphate (PC1P). We also found an enzyme activity that produces PC1P by glycosylinositol phosphoceramide (GIPC)-specific hydrolysis in cabbage leaves. To characterize the GIPC-specific phospholipase D (GIPC-PLD) activity, we investigated distributions of GIPC-PLD activity in 25 tissues of 10 plants. In most plants, the GIPC-PLD activity was the highest in roots. Young leaves of cabbage and Welsh onion had higher activities than corresponding aged outer leaves. The GIPC-PLD activities in leaves, stems and roots of mung bean were higher in the sprouting stage than in more mature stages. We also examined distribution of substrate GIPC and product PC1P, and found that GIPC was ubiquitously distributed at 50-280 nmol/g (wet wt) in tissues of plants, whereas PC1P was detectable (3-60 nmol/g wet wt.) only in tissues showing considerable GIPC-PLD activity. These results suggest a possibility that GIPC-PLD activity is involved in plant growth

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices

Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables

Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20mg/100g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2mg/100g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules

Efficacy and Safety of Switching Prostaglandin Analog Monotherapy to Tafluprost/Timolol Fixed-Combination Therapy

Purpose. To assess the efficacy and safety of switching from prostaglandin analog (PGA) monotherapy to tafluprost/timolol fixed-combination (Taf/Tim) therapy. Subjects and Methods. Patients with primary open-angle glaucoma, normal-tension glaucoma, or ocular hypertension who had received PGA monotherapy for at least 3 months were enrolled. Patients were examined at 1, 2, and 3 months after changing therapies. Subsequently, the patients were returned to PGA monotherapy. The examined parameters included intraocular pressure (IOP) and adverse events. A questionnaire survey was conducted after the switch to Taf/Tim therapy. Results. Forty patients with a mean age of 66.5 ± 10.3 years were enrolled; 39 of these patients completed the study protocol. Switching to Taf/Tim significantly reduced the IOP from 18.2 ± 2.6 mmHg at baseline to 14.8 ± 2.5 mmHg at 1 month, 15.2 ± 2.8 mmHg at 2 months, and 14.9 ± 2.5 mmHg at 3 months (P<0.001). Switching back to the original PGA monotherapy returned the IOP values to baseline levels. Taf/Tim reduced the pulse rate insignificantly. No significant differences were observed in blood pressure, conjunctival hyperemia, or corneal adverse events. A questionnaire showed that the introduction of Taf/Tim did not significantly influence symptoms. Conclusions. Compared with PGA monotherapy, Taf/Tim fixed-combination therapy significantly reduced IOP without severe adverse events