Cellular delivery of cationic lipid nanoparticle-based SMAD3 antisense oligonucleotides for the inhibition of collagen production in keloid fibroblasts.

SMAD3 is a key player in the TGFß signaling pathway as a primary inducer of fibrosis. The inhibition of SMAD3 production is one strategy to alleviate fibrosis in keloid fibroblasts. In the present study, antisense oligonucleotides (ASOs) against SMAD3 were designed to specifically block the expression of SMAD3. The cationic lipid nanoparticles (cLNs) were formulated to enhance an intracellular activity of SMAD3 ASOs in keloid fibroblasts. This formulation was prepared using melt-homogenization method, composed of 3-[N-(N',N'-dimethylaminoethane)-carbamol] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE), Tween20, and trimyristin as a lipid core (1:1:1:1.3, w/w). The size and zeta potential of cLNs and cLN/ASO complexes were measured using light scattering. AFM was used to confirm the morphology and the size distribution of cLNs and cLN/ASO complexes. The prepared cLNs had a nano-scale sized spherical shape with highly positive charge, which were physically stable without aggregation during the storage. The cLN/SMAD3 ASO complexes were successfully generated and internalized onto keloid fibroblasts without toxicity. After the treatment with cLN/ASO complexes, SMAD3 was inhibited and collagen type I was also significantly suppressed in keloid fibroblasts. These results suggest that SMAD3 ASOs complexed with cLNs have a therapeutic potential to suppress collagen deposition in fibrotic diseases. Therefore, this strategy might be developed to lead to anti-fibrotic therapies.

Crystal structure of tuguaconitine, a diterpene alkaloid from Aconitum sibiricum.

The three-dimensional structure of a natural product, tuguaconitine (C23H35NO7) from Aconitumrn sibiricum was determined by single crystal X-ray diffraction analysis. The compound was recrystallized from a methanol solution into orthorhombic crystal system, with a = 18.093(4), b = 12.075(11), c = 9.824(4) A, Dm = 1.35, Dx= 1.354 g/cm3, space group P2(1)2(1)2(1), and Z=4. The structure was solved by direct method and refined by least-squares procedure to the final R value of 0.046 for 1495 observed reflections with I > or = 2sigma(I). Aconitines are diterpene alkaloid composed of six rings. In the molecule, two 6-membered rings have stable chair conformation, but other two 6-membered rings have unstable boat conformation because of their bonding strain and hydrogen bonds. Based on the three-dimensional structure of tuguaconitine, we suggest another 2D-diagram for the compound which is far better in imagination of the spatial shape of the molecule than the ordinary one.

Development of HPLC method for the determination of levosulpiride in human plasma.

A rapid and simple high performance liquid chromatography (HPLC) method was developed and validated for determination of levosulpiride in human plasma. After extraction with ethylacetate/methylene chloride (5:1, v/v), analysis of levosulpiride in plasma samples was carried out using a reverse phase C18 column with fluorescence detector (maximum excitation at 300 nm and maximum emission at 365 nm) for separation and quantification. A mixture of methanol-20 mM phosphate buffer (pH 3.5, 16:84, v/v) was used as a mobile phase. The method was specific and sensitive with a limit of quantification of 5 ng/ml. This HPLC method was validated by examining the precision and accuracy for inter- and intra-day analysis in the concentration range of 5-150 ng/ml. The relative standard deviation (R.S.D.) in inter- and intra-day validation were 8.16-19.75 and 3.90-11.69%, respectively. In stability tests, levosulpiride in human plasma was stable during the storage and assay procedure. The method was applied to the bioequivalence study of two levosulpiride tablet formulations (25 mg) after a single oral administration.

Hepatoprotective pyrrole derivatives of Lycium chinense fruits.

As a part of our search for hepatoprotective compounds from Lycium chinense fruits, three new pyrrole derivatives (1-3) were isolated. These compounds and a related synthetic methylated compound (4) were evaluated for their biological activity and structure-activity relationship, and compounds 1 and 2 showed hepatoprotective effects comparable to silybin at the concentration of 0.1 microM (64.4 and 65.8%, respectively).

High-performance liquid chromatographic analysis of chrysin derivatives on a Nova-Pak C18 column.

A high-performance liquid chromatographic method has been developed for the separation and quantification of chrysin and synthetic chrysin derivatives (12 chrysin alkyl and 7 chrysin acyl derivatives). The chromatography was performed using a Nova-Pak C18 column. A RP-HPLC was performed by using a binary mixture (MeOH-10 mM H3PO4) as a mobile phase, and the column temperature was maintained at room temperature. A flow rate was 1.0 ml/min, and the effluent was monitored at a wavelenth of 280 nm. The retention times for chrysin acyl and alkyl derivatives were within 10 minutes and 20 minutes, respectively. The absolute recovery of samples were all over 96%. The detection limits were 0.1-18 ng at S/N = 3 ratio.

Crystal structure of byakangelicin (C17H18O7).

The crystal structure of byakangelicin, one of furanocoumarin aldose reductase inhibitors, was determined by X-ray diffraction method. The crystal is triclinic, with a = 8.114(1), b = 10.194(1), c = 11.428(1)A, a = 111.50(1), beta= 95.57(1), gamma = 112.52(1) degrees , Dx = 1.41, Dm = 1.39 g/cm3, space group P1 and Z = 2. The intensity data were collected by omega-2theta scan method with CuK(a) radiations. The structure was solved by direct method and refined by full matrix least-squares procedure to the final R-value of 0.056. There are two molecules with different conformations in an asymmetric unit. The molecules are kept by two intermolecular O-HO type hydrogen bonds and van der Waal's forces in the crystal. The absolute configuration of the molecules was estimated to S-form by the 'Eta refinement' procedure.

Phytochemical constituents from the herba of Artemisia apiacea.

Four compounds of a coumarin, a steroid and two flavonoids were isolated from the herba of Artemisia apiacea by open column chromatography. Their structures were elucidated as artemicapin C, apigenin, daucosterol and cacticin by chemical and spectroscopic analysis. This is the first report of the isolation of these compounds from this plant.

Rheological evaluation of thermosensitive and mucoadhesive vaginal gels in physiological conditions.

The timely gelation and retention of in situ-gelling vaginal formulations would be fundamental to improve the efficacy of drugs. In this study, various rheological properties of clotrimazole gels were evaluated for predicting their performance in vagina. Two kinds of thermosensitive and mucoadhesive formulations were composed of poloxamer 407 (P407, 15%), polycarbophil (0.2%), and different amounts of P188 (15 vs. 20%). Both formulations were Newtonian at 20 degrees C but non-Newtonian at 37 degrees C. Although both liquid formulations gelled below the vaginal temperature, they differed in gelation time and viscoelastic properties in the presence of vaginal fluid simulant. At body temperature, the formulation with 20% of P188 gelled within 35 s but it took two times longer for the other one gelled. Upon dilution with simulated vaginal fluid, the formulation with 20% of P188 retained the rheology of a gel, but the other one lost the viscoelastic properties typical for a gel. Moreover, after dilution with simulated vaginal fluid, the elastic modulus was orders of magnitude higher in the formulations with 20% of P188 relative to the other one. These results indicate that the rheological evaluation at the physiologic conditions needs to be preceded to develop more effective in situ-gelling vaginal formulations.

The crystal structure of KR-21042, an analgesic capsaicinoid.

The crystal structure of KR-21042, N-(3-Phenylpropyl)-4-hydroxy-3-methoxyphenylacetamide, was determined by single crystal X-ray diffraction analysis. The compound was recrystallized from a mixture of ethylacetate and n-hexane in monoclinic, space group P21/c, with a = 16.622(1), b = 6.215(1), c = 15.802(1) A, P= 104.97(1), and Z = 4. The calculated density is 1.261 g/cm3. The structure was solved by the direct method and refined by full matrix least-squares procedure to the final R value of 0.068 for 2332 observed reflections.