Chemopreventive Potential of Ethanolic Extracts of Luobuma Leaves (Apocynum venetum L.) in Androgen Insensitive Prostate Cancer.

Luobuma (Apocynum venetum L. (AVL)) is a popular beverage in Asia and has been reportedly to be associated with the bioactivities such as cardiotonic, diuretic, antioxidative, and antihypertensive. However, its biofunction as chemoprevention activity is seldom addressed. Herein, we aimed to characterize the anti-androgen-insensitive-prostate-cancer (anti-AIPC) bioactive compounds of Luobuma, and to investigate the associated molecular mechanisms. Activity-guided-fractionation (antioxidative activity and cell survivability) of Luobuma ethanolic extracts was performed to isolate and characterize the major bioactive compounds using Ultra Performance Liquid Chromatography (UPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR). Plant sterols (lupeol, stigamasterol and ß-sitosterol) and polyphenolics (isorhamnetin, kaempferol, and quercetin) were identified. Lupeol, a triterpene found in the fraction (F8) eluted by 10% ethyl acetate/90% hexane and accounted for 19.3% (w/w) of F8, inhibited the proliferation of PC3 cells. Both lupeol and F8 induced G2/M arrest, inhibition of ß-catenin signaling, regulation of apoptotic signal molecules (cytochrome c, Bcl-2, P53, and caspase 3 and 8), and suppression DNA repair enzyme expression (Uracil-DNA glycosylase (UNG)). To our knowledge, our study is the first report that lupeol inhibited the expression of UNG to elicit the cytotoxicity against androgen-insensitive-prostate-cancer cells. Collectively, Luobuma, which contains several antitumor bioactive compounds, holds the potential to be a dietary chemopreventive agent for prostate cancer.

Hyaluronic acid-nimesulide conjugates as anticancer drugs against CD44-overexpressing HT-29 colorectal cancer in vitro and in vivo.

Carrier-mediated drug delivery systems are promising therapeutics for targeted delivery and improved efficacy and safety of potent cytotoxic drugs. Nimesulide is a multifactorial cyclooxygenase 2 nonsteroidal anti-inflammatory drug with analgesic, antipyretic and potent anticancer properties; however, the low solubility of nimesulide limits its applications. Drugs conjugated with hyaluronic acid (HA) are innovative carrier-mediated drug delivery systems characterized by CD44-mediated endocytosis of HA and intracellular drug release. In this study, hydrophobic nimesulide was conjugated to HA of two different molecular weights (360 kDa as HA with high molecular weight [HAH] and 43kDa as HA with low molecular weight [HAL]) to improve its tumor-targeting ability and hydrophilicity. Our results showed that hydrogenated nimesulide (N-[4-amino-2-phenoxyphenyl]methanesulfonamide) was successfully conjugated with both HA types by carbodiimide coupling and the degree of substitution of nimesulide was 1%, which was characterized by 1H nuclear magnetic resonance 400 MHz and total correlation spectroscopy. Both Alexa Fluor® 647 labeled HAH and HAL could selectively accumulate in CD44-overexpressing HT-29 colorectal tumor area in vivo, as observed by in vivo imaging system. In the in vitro cytotoxic test, HA-nimesulide conjugate displayed >46% cell killing ability at a nimesulide concentration of 400 µM in HT-29 cells, whereas exiguous cytotoxic effects were observed on HCT-15 cells, indicating that HA-nimesulide causes cell death in CD44-overexpressing HT-29 cells. Regarding in vivo antitumor study, both HAL-nimesulide and HAH-nimesulide caused rapid tumor shrinkage within 3 days and successfully inhibited tumor growth, which reached 82.3% and 76.4% at day 24 through apoptotic mechanism in HT-29 xenografted mice, without noticeable morphologic differences in the liver or kidney, respectively. These results indicated that HA-nimesulide with improved selectivity through HA/CD44 receptor interactions has the potential to enhance the therapeutic efficacy and safety of nimesulide for cancer treatment.

Ameliorative Effect of Curcumin-Encapsulated Hyaluronic Acid-PLA Nanoparticles on Thioacetamide-Induced Murine Hepatic Fibrosis.

In this study, we developed curcumin-encapsulated hyaluronic acid-polylactide nanoparticles (CEHPNPs) to be used for liver fibrosis amelioration. CD44, the hyaluronic acid (HA) receptor, is upregulated on the surface of cancer cells and on activated hepatic stellate cells (aHSCs) rather than normal cells. CEHPNPs could bind to CD44 and be internalized effectively through endocytosis to release curcumin, a poor water-soluble liver protective agent. Thus, CEHPNPs were potentially not only improving drug efficiency, but also targeting aHSCs. HA and polylactide (PLA) were crosslinked by adipic acid dihydrazide (ADH). The synthesis of HA-PLA was monitored by Fourier-transform infrared (FTIR) and Nuclear Magnetic Resonance (NMR). The average particle size was approximately 60-70 nm as determined by dynamic light scattering (DLS) and scanning electron microscope (SEM). Zeta potential was around -30 mV, which suggested a good stability of the particles. This drug delivery system induced significant aHSC cell death without affecting quiescent HSCs, hepatic epithelial, and parenchymal cells. This system reduced drug dosage without sacrificing therapeutic efficacy. The cytotoxicity IC50 (inhibitory concentration at 50%) value of CEHPNPs was approximately 1/30 to that of the free drug treated group in vitro. Additionally, the therapeutic effects of CEHPNPs were as effective as the group treated with the same curcumin dose intensity in vivo. CEHPNPs significantly reduced serum aspartate transaminase/alanine transaminase (ALT/AST) significantly, and attenuated tissue collagen production and cell proliferation as revealed by liver biopsy. Conclusively, the advantages of superior biosafety and satisfactory therapeutic effect mean that CEHPNPs hold great potential for treating hepatic fibrosis.

Comparison of Antioxidant Capability after Isopropanol Salting-Out Pretreatment and n-Butanol Partition Extraction, and Identification and Evaluation of Antioxidants of Sedum formosanum N.E.Br.

Crude extracts of Sedum formosanum N.E.Br. obtained from n-butanol partition (BP) and isopropanol salting-out pretreatment (ISP) were analyzed using antioxidation assays. The results indicated that the extract from ISP contained more potent antioxidants and thus exhibited more antioxidant activity in all the assays. The superoxide radical-scavenging activity and inhibition of nitric oxide radicals achieved after ISP were 3.65 and 2.18 times higher than those achieved through BP, respectively. Eight bioactive natural products were isolated and identified according to an analysis of antioxidation activity in different fractions of the ISP crude extract, namely three cyanophoric glycosides 1-3, three flavonoids 4-6 and two phenolic compounds (7 and a new compound 8). Among them, compounds 5 and 6 exhibit the highest antioxidation capability, and the ISP is suitable for obtaining compounds 5 and 6 using HPLC chromatograms. Therefore, ISP is an excellent extraction technology that can be used to extract antioxidant compounds in the nutraceutical and pharmaceutical industries.

Enzymatic synthesis of rose aromatic ester (2-phenylethyl acetate) by lipase.

BACKGROUND: 2-Phenylethyl acetate (2-PEAc) is a highly valued natural volatile ester with a rose-like odour that is widely used to add scent or flavour to cosmetics, soaps, foods and drinks. In this study, 2-PEAc was synthesised enzymatically by transesterification of vinyl acetate with 2-phenethyl alcohol catalysed by immobilised lipase (Novozym(®) 435) from Candida antarctic RESULTS: Response surface methodology and a three-level/three-factor Box-Behnken design were used to evaluate the effects of time, temperature and enzyme amount on the molar conversion % of 2-PEAc. The results showed that temperature was the most important variable. Based on the ridge max analysis results, optimum enzymatic synthesis conditions were predicted as a reaction time of 79 min, a temperature of 57.8 °C and an enzyme amount of 122.5 mg. The predicted and experimental yields were 86.4 and 85.4% respectively. CONCLUSION: Three immobilised lipases were screened and 15 reaction conditions were tested in order to find the combination for maximum yield. The optimisation of 2-PEAc synthesis catalysed by Novozym(®) 435 was successfully developed. The kinetic study of this transesterification reaction showed that it followed an ordered ping-pong bi-bi mechanism without any inhibition by reactants.

Quantum dot enhancement of peptide detection by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a rapid and sensitive tool for characterizing a wide variety of biomolecules. However, invisible "sweet spots" that form during heterogeneous cocrystallization minimize the analytical throughput and affect the reproducibility of MALDI analysis. In this study, visible "sweet spots" were generated on a metallic sample plate by quantum dots (QDs)-assisted MALDI analysis. To the best of our knowledge, this is the first report to demonstrate that "sweet spots" can be observed directly without using a microscope. The proper proportion of matrix to QDs that results in optimal crystallization was also examined. The signals of standard peptides and phosphopeptides obtained by QD-assisted MALDI analysis were 5- and 3-fold higher, respectively, than those obtained by conventional MALDI analysis. For peptide mixtures, the QD-assisted MALDI analysis not only resulted in more intense peptide signals but also resulted in a greater number of peaks, thereby allowing for better detection of individual peptides in peptide mixtures. Moreover, we demonstrated that application of QDs to a radiate microstructure chip followed by MALDI analysis enhanced the detection of peptide signals. Overall, we show that this method is a simple, sensitive, and high-throughput technique for peptide detection.

Folic acid-anchored PEGgylated phospholipid bioconjugate and its application in a liposomal immunodiagnostic assay for folic acid.

A folic acid-anchored, poly(ethylene glycol)-linked (PEGgylated) phospholipid and an immunoaffinity chromatographic column were prepared and employed to develop a liposomal immunodiagnostic assay for the direct determination of folic acid (FA) in this study. Distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-folic acid (DSPE-PEG2000-FA) was synthesized through carbodiimide-mediated coupling of FA and DSPE-PEG2000-amine and characterized using thin layer chromatography, 1H nuclear magnetic resonance spectroscopy, and electrospray ionization-mass spectrometry. Liposomal biolabels were constructed using the synthesized DSPE-PEG2000-FA in conjunction with other phospholipids. A stationary phase having affinity for FA was prepared by covalently linking purified anti-FA monoclonal antibodies onto N-hydroxysuccinimide-activated Sepharose beads, which were subsequently packed into a 1.9 cm diameter polypropylene column. The calibration curve for FA had a linear range from 10(-8) to 10(-4) M. The limit of detection was 6.8 ng (equivalent to 500 microL of 3.1 x 10(-8) M FA). The elution buffer (35% methanol in Tris buffered saline containing 0.1% Tween 20) also served as the regeneration buffer, which allowed the same column to be used for up to 50 times without any observable loss of reactivity. The immunoaffinity chromatographic column was reusable and capable of concentrating analytes from sample solution; in conjunction with folic acid-sensitized liposomal biolabels, however, they hold great potential as sensitive immunoaffinity assays for the determination for FA. To confirm the feasibility of using this system in the analysis of real samples, the folic acid contents of three over-the-counter vitamin supplements were tested. The recoveries of folic acid of 90-112% for these three samples were obtained, suggesting contents that were consistent with the information obtained from their nutritional facts panels.

Gene families encoding isoforms of two major sesame seed storage proteins, 11S globulin and 2S albumin.

Sesame (Sesamum indicum L.) seed has been recognized as a nutritional protein source owing to its richness in methionine. Storage proteins have been implicated in allergenic responses to sesame consumption. Two abundant storage proteins, 11S globulin and 2S albumin, constitute 60-70 and 15-25% of total sesame proteins, respectively. Two gene families separately encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3328 expressed sequence tag (EST) sequences from maturing sesame seeds. Full-length cDNA sequences derived from these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The amino acid compositions of these deduced storage proteins revealed that the richness in methionine is attributed mainly to two 2S albumin isoforms and partly to one 11S globulin isoform. The presence of four 11S globulin and three 2S albumin isoforms resolved in SDS-PAGE was confirmed by MALDI-MS analyses. The abundance of these isoforms was in accord with the occurrence frequency of their EST sequences in the database. A comprehensive understanding of these storage proteins at the molecular level may also facilitate the identification of allergens in crude sesame products that have caused severe allergic reactions increasingly reported in the past decade.