Preparation and Characterization of Zein/Sodium Caseinate/Xanthan Gum Complex for Encapsulation of Piperine and its In Vitro Release Study

To encapsulate piperine (Pip), as a poor water-soluble bioactive compound, zein-sodium caseinate-xanthan gum (Z-SG-XG) nanocomplex was prepared as a colloidal delivery system. The effect of different parameters involved in complexation process, including concentration of proteins, polysaccharide, and Pip on the encapsulation efficiency of Pip, particle size and stability of the nanocomplexes was investigated. Powders obtained by freeze-drying of the colloidal solution had relatively uniform particles compared to those obtained from conventional drying system and showed well redispersibility in water. At the optimal condition, a stable and homogeneous nanocomplex with a mean particle size of 145.9 ± 2.7 nm, PDI of 0.27 ± 0.01, and ζ-potential of −39.7 ± 1.3 mV was obtained. The antioxidant activity of Pip was significantly improved by encapsulation into the Z-SC-XG nanocomplex. Also, the in vitro release of Pip from the synthesized nanocomplexes in phosphate-buffer saline (PBS) solution and simulated gastrointestinal fluids (SGIF) was investigated and the release kinetic was studied as well. The Pip/Z-SG-XG nanocomplex showed a slower release in SGIF compared to the free Pip and nanoparticles without XG and SC, while its antioxidant activity was remarkable. Results suggested a possible utilization of Z-SC-XG nanocomplex for improving the water solubility, bioavailability and storage stability of Pip.

     

Statistical medium optimization and biodegradative capacity of <Emphasis Type=Italic>Ralstonia eutropha</Emphasis> toward <Emphasis Type=Italic>p</Emphasis>-nitrophenol

The effect of p-nitrophenol (PNP) concentration with or without glucose and yeast extract on the growth and biodegradative capacity of Ralstonia eutropha was examined. The chemical constituents of the culture medium were modeled using a response surface methodology. The experiments were performed according to the central composite design arrangement considering PNP, glucose and yeast extract as the selected variables whose influences on the degradation was evaluated (shaking in reciprocal mode, temperature of 30°C, pH 7 and test time of about 9 h). Quadratic polynomial regression equations were used to quantitatively explain variations between and within the models (responses: the biodegradation capacity and the biomass formation). The coefficient of determination was high (R _adjusted² = 0.9783), indicating the constructed polynomial model for PNP biodegradative capacity explains the variation between the regressors fairly well. A PNP removal efficiency of 74.5% occurred within 9 h (15 mg/L as the initial concentration of PNP with use of yeast extract at 0.5 g/L).     

Modulating furin activity with designed mini-PDX peptides: synthesis and in vitro kinetic evaluation

A peptide was designed from reactive site loop structure of alpha1 Antitrypsin Portland known as alpha1 PDX as a novel mini-PDX inhibitor of furin. The sequence was derived from (367-394) that contains the crucial furin cleavage motif RIPR382. A P3 mutant replacing Ile380 by Leu was prepared as a first model peptide. A Cys residue was inserted at each terminal of the peptide for purpose of cyclisation which was accomplished by air or iodine-induced oxidation. This mini-PDX peptide both cyclic and acyclic form inhibited in vitro furin activity (IC50 in nM) when measured against either substrates Boc-RVRRdown double arrow MCA or QVEGF-C [Abz-QVHSIIRRdown double arrow SLP-Y(NO2)-A-CONH2, Abz=2-amino benzoic acid and Y(NO2)=3-nitro tyrosine], latter being derived from vascular endothelial growth factor-C (VEGF-C) processing site. The geometrically constrained structure mimicking PDX reactive loop is crucial for enzyme inhibition. Our study further revealed that both mini-PDX peptides inactivate furin in a slow tight binding manner, with disulfide-bridged cyclic form being slightly more potent. Unlike PDX, these peptides inhibit furin via a different mechanistic pathway. The study provides an alternate strategy for development of efficient peptide-based inhibitors of Proprotein Convertases including furin.     

Optimization of Influencing Parameters on Phenanthrene Removal Efficiency in Soil Washing Process by Using Response Surface Methodology

Response surface methodology (RSM) under Box–Behnken design (BBD) was applied to evaluate the effect of the influencing parameters including surfactant concentration, liquid/soil ratio, Humic Acid concentration, and washing time on phenanthrene removal efficiency in soil washing process by using the nonionic surfactant Tween 80 and find an optimal operational conditions to achieve the highest removal efficiency. A polynomial quadratic model was used to correlate phenanthrene removal efficiency and four independent variables (R² = 0.9719). Based on the obtained results the most influential parameter on phenanthrene removal efficiency was surfactant concentration with an impact value of 69.519%. Liquid/soil ratio was also another factor that significantly influenced on removal efficiency with an impact value of 25.014%. The interaction between surfactant concentration and liquid/soil ratio was also shown to have a positive significant effect on removal efficiency (p_value = 0.0027). However, the other independent variables Humic Acid concentration and time were not significant in the ranges selected in this study. Based on the optimization results maximum removal efficiency of 70.692 ± 3.647% was achieved under the conditions of surfactant concentration 5000 mg L^?1, liquid/soil ratio 30 v/w, HA concentration 9.88 mg L^?1, and washing time 2 h, which was in good agreement with predicted value (66.643%).     

A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma

Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH₄NO₃, KH₂PO₄, KNO₃ and CaCl₂. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.     

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.     

Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.