PEGylation of Chrysin Improves Its Water Solubility while Preserving the In Vitro Biological Activity.

Chrysin is a natural flavonoid that despite having numerous biological properties, its therapeutic value is limited due to its very low solubility in aqueous media. In this work, chrysin was conjugated with methoxypolyethylene glycols (mPEGs) of different molecular weights (350, 500, 750, and 2000 g/mol), affording PEGylated chrysins with high yields and excellent purities. In all cases, an increase in the water solubility of the conjugates was observed, which was highest when 500 g/mol of mPEG was used in the PEGylation reaction. Furthermore, in aqueous solution, PEGylated chrysins formed aggregates of ellipsoid shape. Electrochemical studies showed that the redox properties were conserved after PEGylation. While in vitro antibacterial and antifungal studies probed that the intrinsic activity was conserved, in vitro antitumor activities against HepG2 (liver carcinoma cells) and PC3 (prostate cancer cell) showed that PEGylated chrysins retained the cytotoxic activity and the ability of induction of apoptosis for the evaluated human cancer cells.

Cytotoxic, antioxidant, and cytoprotective properties of polyphenol-enriched extracts from pecan nutshells in MDA-MB-231 breast cancer cells.

Phenolic compounds present in plants have demonstrated several biological properties such as antioxidant, antitumor, cardioprotective, and antiproliferative. On the other hand, doxorubicin, a chemotherapeutic widely used to treat breast cancer, usually exhibits chronic cardiotoxicity associated with oxidative stress. Therefore, we aimed to study the effects of phenolic compound-enriched extract (PCEE) with doxorubicin in breast cancer. To achieve this, after an SPE-C18 -column purification process of crude extracts obtained from pecan nutshells (Carya illinoinensis), the resulting PCEE was used to evaluate the cytotoxicity and antioxidant properties against the human breast cancer cell line MDA-MB-231 and the normal-hamster ovary cell line CHO-K1. PCEE was selectively cytotoxic against both cell lines, with an IC50 value (≈26.34 mg/L) for MDA-MB-231 lower than that obtained for CHO-K1 (≈55.63 mg/L). As a cytotoxic mechanism, PCEE inhibited cell growth by G2/M cell cycle arrest in MDA-MB-231 cells. Simultaneously, the study of the antioxidant activity showed that PCEE had a cytoprotective effect, evidenced by reduced ROS production in cells with oxidative stress caused by doxorubicin. The results highlight PCEE as a potential antitumor agent, thus revaluing it as an agro-industrial residue.

In vitro gastrointestinal digestion and cytotoxic effect of ovalbumin-conjugated linoleic acid nanocomplexes.

The aim of this work was to examine the behavior of conjugated linoleic acid (CLA) delivery systems based on ovalbumin (OVA) and their derived nanoparticles (OVAn1 and OVAn2), under static in vitro gastrointestinal digestion model. In addition, potential cytotoxic effect of these inclusion complexes on a human colon cancer cell line (HT-29) was evaluated. OVA was resistant to gastric and intestinal digestion, while OVA nanoparticles were very susceptible to digestive enzymes hydrolysis. Particle size distribution (PDS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for OVA evidenced the presence of a protein fragment of similar size after simulated digestive process. Conversely, for nanoparticles, partial and total hydrolysis in gastric and intestinal phases, respectively, was evidenced. After in vitro gastrointestinal digestion, released CLA (RCLA) was assayed. In case of OVA, as CLA carrier, RCLA was 37%, while for OVA nanoparticles, lower RCLA values (~10-20%) were obtained. From cytotoxic assays, it was observed that CLA molecule was responsible for cell death, whereas OVA or their derived nanoparticles were not cytotoxic on HT-29 cells. On the other hand, flow cytometry analysis revealed that main death mechanism for CLA, and their inclusion complexes was apoptosis. OVA-CLA and OVAn1-CLA inclusion complexes displayed the highest potential cytotoxic activity and apoptotic index. Information derived from this work could be relevant for the design of CLA delivery systems as promising nanosupplements for production of new functional and excipient foods for both prevention and control of colon cancer.

BMP2, 4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development.

Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1.

Efficiency of a recombinant MSA-2c-based ELISA to establish the persistence of antibodies in cattle vaccinated with Babesia bovis.

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.