The Response Surface Optimization of Supercritical CO2 Modified with Ethanol Extraction of p-Anisic Acid from Acacia mearnsii Flowers and Mathematical Modeling of the Mass Transfer.

A widely disseminated native species from Australia, Acacia mearnsii, which is mainly cultivated in Brazil and South Africa, represents a rich source of natural tannins used in the tanning process. Many flowers of the Acacia species are used as sources of compounds of interest for the cosmetic industry, such as phenolic compounds. In this study, supercritical fluid extraction was used to obtain non-volatile compounds from A. mearnsii flowers for the first time. The extract showed antimicrobial activity and the presence of p-anisic acid, a substance with industrial and pharmaceutical applications. The fractionation of the extract was performed using a chromatographic column and the fraction containing p-anisic acid presented better minimum inhibitory concentration (MIC) results than the crude extract. Thus, the extraction process was optimized to maximize the p-anisic acid extraction. The response surface methodology and the Box-Behnken design was used to evaluate the pressure, temperature, the cosolvent, and the influence of the particle size on the extraction process. After the optimization process, the p-anisic acid yield was 2.51% w/w and the extraction curve was plotted as a function of time. The simulation of the extraction process was performed using the three models available in the literature.

4-Ethoxybenzoic acid inhibits Staphylococcus aureus biofilm formation and potentiates biofilm sensitivity to vancomycin.

The adverse health effects of Staphylococcus aureus biofilm infections coupled with an increased global prevalence of antibiotic resistance highlight the need for novel anti-pathogenic, anti-biofilm compounds. The authors recently determined that ethyl-4-ethoxybenzoic acid (EEB) had anti-pathogenic, anti-biofilm activity. Based on this finding, a structure-activity analysis was undertaken to identify more effective compounds. Microtitre crystal violet assays followed by plate counts were conducted to measure the dose-dependent anti-biofilm and antimicrobial activities of 13 phenolic compounds related to EEB. By displaying these characteristics on a two-component plot, 4-ethoxybenzoic acid (4EB) and methyl gallate were identified as two anti-pathogenic, anti-biofilm compounds of interest. To characterize their mechanisms of activity, their effects on cell hydrophobicity, hemolysis activity, membrane integrity, extracellular polymeric substance production and vancomycin sensitivity were examined. Both 4EB and methyl gallate inhibited up to 87% of biofilm formation with minimal impact on the viability of stationary-phase cells or bacterial growth. Combination treatments of 4EB and vancomycin decreased the viability of biofilm-dwelling cells by up to 85% compared with vancomycin alone, indicating a synergistic effect. Methyl gallate did not potentiate vancomycin. 4EB decreased the percentage of hydrophobic cells in culture from 78% to 49%, indicating that 4EB may prevent biofilm formation by altering cell membrane hydrophobicity. These findings suggest that 4EB has potential as an anti-pathogenic, anti-biofilm agent for the prevention of S. aureus biofilms, or as a treatment for established biofilms when combined with antibiotics.

The Novel Lipid-Lowering Drug D-47 Ameliorates Hepatic Steatosis and Promotes Brown/Beige-Like Change of White Adipose Tissue in db/db Mice.

D-47 is a newly developed solid dispersion of the arginine salt of (S)-(+)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidin-4-yl]methoxybenzoic acid (S-2E), which inhibits sterol and fatty acid synthesis. D-47 was recently shown to lower the serum level and hepatic content of both triglyceride and cholesterol in a rabbit model of familial hypercholesterolemia. We here investigated the effects of D-47 on dyslipidemia and hepatic steatosis in comparison with those of bezafibrate in the db/db mouse model of obesity. Treatment of db/db mice with D-47 or bezafibrate for 14 days lowered the serum triglyceride concentration without affecting that of cholesterol. D-47, but not bezafibrate, almost completely eliminated lipid droplets in hepatocytes and markedly lowered the triglyceride content of the liver in these mice. The two agents induced similar changes in the hepatic expression of genes including those related to ß-oxidation or fatty acid synthesis. D-47 however significantly reduced the mass of white adipose tissue and up-regulated the expression of genes related to energy expenditure, mitochondrial function, fatty acid oxidation or lipolysis in this tissue, indicating that D-47 induced the brown/beige adipocyte-like change in white adipose tissue, whereas bezafibrate had no such effects. Treatment of 3T3-L1 adipocytes with D-47 provoked the expression of genes related to mitochondrial function, fatty acid oxidation or lipolysis. Our data have thus shown that D-47 ameliorated hypertriglyceridemia and hepatic steatosis in an animal model of obesity, and they suggest that this latter effect might be mediated through the change of adipose tissue characteristics.

Two New Anisic Acid Derivatives from Endophytic Fungus Rhizopycnis vagum Nitaf22 and Their Antibacterial Activity.

Rhizopycnis acids A (1) and B (2), two new anisic acid derivatives, were obtained from the ethyl acetate extract of the fermentation cultures of Rhizopycnis vagum, an endophytic fungus isolated from the healthy tissues of Nicotiana tabacum. The structures of the two compounds were determined through a series of 1D and 2D NMR and HRMS spectral analyses. Both compounds were the first anisic acid derivatives containing methylbutanoic/methylbutenoic acid group found in fungi. 1 and 2 displayed antibacterial activity against six tested bacteria with IC50 values in the range 16.1~81.3 µg/mL.

Ameliorating effects of D-47, a newly developed compound, on lipid metabolism in an animal model of familial hypercholesterolemia (WHHLMI rabbits).

Improvements induced in lipid metabolism in the liver by D-47, a newly developed compound, were examined herein. WHHLMI rabbits, an animal model of hypercholesterolemia and coronary atherosclerosis, was fed D-47-supplemented chow for 5 weeks at a dose of 30mg/kg. Lipid concentration were assayed using enzymatic methods. Plasma lipoproteins were fractionated with an ultracentrifuge. mRNA expression was analyzed with real-time PCR. Lipidome analyses of lipoproteins were performed using supercritical fluid chromatography mass spectrometry. In the D-47-treated group, serum lipid levels decreased by 23% for total cholesterol and by 40% for triglycerides. These reductions were mainly attributed to decreases in the VLDL fraction. Compared with the control, in the D-47 group, lipid contents in the liver were decreased by 22% in cholesterol and by 69% in triglycerides, and fat accumulation was decreased by 57% in pericardial fat and by 17% in mesenteric fat. In lipidome analyses of VLDL fraction, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylethanolamine plasmalogen, sphingomyelin, and ceramide were decreased by the D-47 treatment. mRNA expression in the liver was 51% lower for FAS and 24% lower for MTP, but 5.9- and 5.1-fold higher for CYP7A1 and CPT-1, respectively, in the D-47 group than in the control. mRNA expression was 72%, 64%, and 36% higher for LPL, CTP-1, and PPARγ, respectively, in mesenteric fat in the D-47 group. D-47 is a potent lipid-lowering compound that uses a different mechanism of action from that of statins. It has potential as a compound in the treatment of steatohepatitis and metabolic syndrome.

Antifungal and Cytotoxic ß-Resorcylic Acid Lactones from a Paecilomyces Species.

Eight new ß-resorcylic acid lactones (RALs), including the hypothemycin-type compounds paecilomycins N-P (1-3) and the radicicol-type metabolites dechloropochonin I (4), monocillins VI (5) and VII (6), 4'-hydroxymonocillin IV (7), and 4'-methoxymonocillin IV (8), along with nine known RALs (9-17), were isolated from the cultures of Paecilomyces sp. SC0924. Compounds 1 and 2 feature a novel 6/11/5 ring system, and 3 is the first 5'-keto RAL. The structures of 1-8 were elucidated on the basis of extensive spectroscopic analysis, X-ray diffraction analysis, and theoretical calculations of ECD spectra. Compounds 3, 5, and 6 exhibit cytotoxicity against MCF-7, A549, and HeLa cells, and compounds 5 and 7 display antifungal activity against Peronophythora litchii.

A novel lipoprotein (a) lowering drug, D-47, decreases neointima thickening after vascular injury.

Although Lp(a) have been thought to be a cardiovascular risk factor, it is unclear whether lowering Lp(a) levels reduces the risk of cardiovascular diseases. No pharmacological agents which selectively reduce serum Lp(a) levels, and Lp(a) is present in primate but absent in common laboratory animals such as mice and pigs. In the present study we used transgenic mice of human Lp(a) and tested effect a novel Lp(a) lowering drug D-47 on neointima formation after vascular injury. D-47 successfully decreased plasma levels of Lp(a) and possibly inhibited neointima formation in Lp(a) transgenic mice. The results indicate that we can modulate plasma Lp(a) levels by pharmacologic agents and inhibit atherogenic properties of Lp(a) by reducing plasma levels of Lp(a). J. Med. Invest. 64: 64-67, February, 2017.

Designing of Biodegradable and Biocompatible Release and Delivery Systems of Selected Antioxidants Used in Cosmetology.

Conjugates of antioxidants p-anisic (p-AA) and vanillic (VA) acids with nontoxic, biocompatible, and biodegradedable oligo-(R,S)-(3-hydoxybutyrate) carrier were synthesized, and their structural and biological characterization was performed. The molecular structure of the bioconjugates, in which antioxidants are covalently bonded with oligo(3-hydroxybutyrate) (OHB) chains, has been proven by mass spectrometry supported by NMR. The bioconjugate hydrolytic degradation studies allowed gaining thorough insight into the hydrolysis process and confirmed the release of p-AA and VA. In vitro studies demonstrated that all of the conjugates studied were well tolerated by KB and HaCaT cell lines, as they had no marked cytotoxicity, while conjugates with a relatively short OHB carrier are optimal to support keratinocyte function. The preliminary study of the biological activity confirmed the protective effect of VA-OHB conjugates against H2O2-induced lipid peroxidation in human keratinocytes (HaCaT). It was also demonstrated that the selected bioconjugates can penetrate all layers of the skin, which shows their functionality and opens up their potential application in cosmetology.

Microwave assisted synthesis and biomedical potency of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives.

A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7-19 from appropriate steroidal precursors 1-6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more successful regarding reaction time and product yields. It was more environmentally friendly too, compared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on hydroxysteroid dehydrogenase enzymes (Δ(5)-3ßHSD, 17ßHSD2 and 17ßHSD3). All of the tested compounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging new compounds were effective, but less than reference compounds. 2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition potency. Compounds 9, 10 and 11 were the best inhibitors of 17ßHSD2 enzyme. X-ray structure analysis and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds 10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their molecules conformations are stable even after releasing of the influence of crystalline field and that the influence of crystal packing on molecular conformation is not predominant.

Synthesis and organic surface modification of luminescent, lanthanide-doped core/shell nanomaterials (LnF3@SiO2@NH2@organic acid) for potential bioapplications: spectroscopic, structural, and in vitro cytotoxicity evaluation.

A facile coprecipitation reaction between Ce(3+), Gd(3+), Tb(3+), and F(-) ions, in the presence of glycerine as a capping agent, led to the formation of ultrafine, nanocrystalline CeF3:Tb(3+) 5%, Gd(3+) 5% (LnF3). The as-prepared fluoride nanoparticles were successfully coated with an amine modified silica shell. Subsequently, the obtained LnF3@SiO2@NH2 nanostructures were conjugated with 4-ethoxybenzoic acid in order to prove the possibility of organic modification and obtain a new functional nanomaterial. All of the nanophosphors synthesized exhibited intense green luminescence under UV light irradiation. Based on TEM (transmission electron microscopy) measurements, the diameters of the cores (≈12 nm) and core/shell particles (≈50 nm) were determined. To evaluate the cytotoxic activity of the nanomaterials obtained, their effect on human erythrocytes was investigated. LnF3 nanoparticles were bound to the erythrocyte membrane, without inducing any cytotoxic effects. After coating with silica, the nanoparticles revealed significant cytotoxicity. However, further functionalization of the nanomaterial with -NH2 groups as well as conjugation with 4-ethoxybenzoic acid entailed a decrease in cytotoxicity of the core/shell nanoparticles.

Intermolecular interaction of voriconazole analogues with model membrane by DSC and NMR, and their antifungal activity using NMR based metabolic profiling.

The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer's structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time (1)H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.

Inhibition of toxic actions of phospholipase A2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds.

BACKGROUND & OBJECTIVES: Phospholipase A2 (PLA2 ) is one of the major constituents of krait venom associated with several pathophysiological actions like myotoxicity, cardiotoxicity, neurotoxicity, etc. As there was no specific antiserum available against Bungarus fasciatus venom, this study was done with synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum to neutralize the PLA2 induced toxicities in experimental models. METHODS: B. fasciatus venom phospholipase A2 fraction 38 (BF-38) was isolated by ion exchange chromatography, molecular weight was determined by mass spectrometry and its N terminal amino acid sequence was identified. Monospecific rabbit antiserum was raised against the PLA2 in presence of Freund complete adjuvant. The neutralization of PLA2 induced toxicities was done in in vitro and in in vivo models using synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum. RESULTS: A toxic PLA2 (BF-38) was purified from the B. fasciatus venom by CM-cellulose and HPLC, of 13.17 kDa and a minor band of 7.3 kDa using ESI-MS. The 13.17 kDa PLA2 sequence was NLYQFKNMIQC. The 7.3 kDa toxin sequence was RKCLTKYSQDNES and was found to be <10 per cent w/w. Anti PLA2 rabbit antiserum produced faint precipitant band in immunogel diffusion and showed low titre value. The commercial polyvalent snake venom antiserum, anti PLA2 rabbit antiserum and the synthetic herbal compounds neutralized the PLA2 induced toxicities at different intensities. INTERPRETATION & CONCLUSIONS: Our results suggested that synthetic herbal compound (BA) along with antiserum might provide effective protection against PLA2 induced toxicities of B. fasciatus venom.