Physicochemical and Functional Modifications of Hemp Protein Concentrate by the Application of Ultrasonication and pH Shifting Treatments.

According to the Food and Agriculture Organization (FAO), protein demand is expected to increase globally by around 40% by 2030 as a response to the world's population growth. Due to their clean label, vegan or vegetarian based applications, nutritional value, and cost-efficient properties, plant-based proteins have been widely studied. However, most of the alternatives currently found in the market have some challenges because of their poor solubility, emulsifying, gelling, and foaming attributes. Hemp seed protein has gained increasing attention due to its unique amino acids and fatty acids profiles. In this study, commercial HPC mixtures were adjusted to pH 2, 4, 6, 8, 10, and 12 followed by ultrasonication (US) for 5 min (5 s on: 5 s off) and incubated for an hour before neutralizing to pH 7. Following the treatments, the samples were analyzed for their hydrodynamic diameter, conductivity, zeta potential, polydispersity index, surface hydrophobicity, solubility, electrophoresis (SDS-PAGE), free sulfhydryl group, and optical characteristics. The samples treated with ultrasound at pH 8 and 10 significantly (p < 0.05) enhanced the solubility of the hemp seed protein by 12.12% and 19.05%, respectively. Similarly, the samples treated with ultrasonication and pH shifting at pH 6, 8, and 10 also significantly increased the amount of free sulfhydryl content (p < 0.05) to 41.6, 58.72, and 46.54 mmol/g from 32.8 mmol/g, respectively. This study shows that the application of ultrasonication and pH shifting is a promising alternative method to modify the functional properties of HPC and widen their applications in the food, cosmetics, and pharmaceutical industries.

Chemical and in vitro toxicity analysis of a supercritical fluid extract of Kava kava (Piper methysticum).

ETHNOPHARMACOLOGICAL RELEVANCE: Kava and kava extracts have shown great potential as a way to minimize anxiety-associated symptoms and to help alleviate pain. Hepatoxicity has been associated with the consumption of kava products. The chemical compounds, kavalactones (KL) and flavokavains (FK) have been implicated in kava's psychotropic and possible hepatotoxic properties. AIM OF THE STUDY: To investigate the kavalactone and flavokavain content and in vitro toxicity of KAVOA™, a supercritical carbon dioxide extraction (SFE) of kava. MATERIALS AND METHODS: Kavalactone and flavokavain content of SFE kava and noble kava root were determined following extraction in acetone, cell culture media, and water using ultra high-performance liquid chromatography (UHPLC). Using water extractions of the kava products, the cell viability and toxicity on the human hepatocellular carcinoma cell line (HepG2) were determined using luminescent and fluorescent assays, respectively. The half maximal inhibitory concentration (IC50) of the SFE kava and noble kava root, extracted in cell culture media, were determined utilizing a luminescent cell viability assay. RESULTS: Quantification of the KAVOA™, a SFE extract of kava and kava root showed similar profiles of kavalactone and flavokavain content. Water extracted SFE and root kava did not show a negative impact on cell viability and toxicity when compared to the vehicle control treated cells. IC50 values were determined for the SFE kava and kava root extracted in cell culture media in respect to cell viability, 78.63 and 47.65 µg/mL, respectively. CONCLUSIONS: KAVOA™, a supercritical carbon dioxide extract of kava displays a similar kavalactone profile to a noble variety of kava. In relation to total kavalactone content, KAVOA™ also has a lower content of the cytotoxic compound FKB. Aqueous extractions of KAVOA™ and noble kava root had no significant negative impact on cell viability and toxicity on HepG2 cells when compared to vehicle controlled treated cells. Results indicate KAVOA™ demonstrates a similar in vitro safety profile to that of noble kava root when experiments are normalized to kavalactone content.

Genetic dependence of central corneal thickness among inbred strains of mice.

PURPOSE: Central corneal thickness (CCT) exhibits broad variability. For unknown reasons, CCT also associates with diseases not typically considered corneal, particularly glaucoma. The purpose of this study was to test the strain dependence of CCT variability among inbred mice and identify cellular and molecular factors associated with differing CCT. METHODS: Methodology for measuring murine CCT with ultrasound pachymetry was developed and used to measure CCT among 17 strains of mice. Corneas from three strains with nonoverlapping differences in CCT (C57BLKS/J, C57BL/6J, and SJL/J) were compared by histology, transmission electron microscopy, and expression profiling with gene microarrays. RESULTS: CCT in mice was highly strain dependent. CCT exhibited continuous variation from 89.2 microm in C57BLKS/J to 123.8 microm in SJL/J. Stromal thickness was the major determinant of the varying murine CCT, with epithelial thickness also contributing. Corneal expression levels of many genes differed between strains with differing CCT, but most of these changes did not correlate with the changes observed in previously studied corneal diseases nor did they correlate with genes encoding major structural proteins of the cornea. CONCLUSIONS: Murine CCT has been measured with a variety of different techniques, but only among a limited number of different strains. Here, pachymetry was established as an additional tool and used to conduct a broad survey of different strains of inbred mice. These results demonstrated that murine CCT was highly influenced by genetic background and established a baseline for future genetic approaches to further elucidate mechanisms regulating CCT and its disease associations.

Lyst mutation in mice recapitulates iris defects of human exfoliation syndrome.

PURPOSE: Human eyes with exfoliation syndrome (XFS) exhibit a distinctive pattern of iris transillumination defects that are recapitulated in Lyst mutant mice carrying the beige allele. The purpose of this study was to determine the anatomic basis for Lyst-mediated transillumination defects, test whether Lyst mutant mice develop other features of XFS, and describe the molecular basis of the beige mutation. METHODS: Lyst mutant mice and strain-matched controls were compared by clinical, histologic, immunohistochemical, and molecular genetic analyses. RESULTS: Slit-lamp examination showed that Lyst mutant mice uniformly exhibit XFS-like transillumination defects. Histologic analysis showed that these defects correlate with a sawtooth morphology of the iris pigment epithelium. Lyst mutant mice also produce an exfoliative-like material and exhibit pronounced pigment dispersion. Despite these insults, Lyst mutation does not cause increased intraocular pressure or optic nerve damage in the C57BL/6J genetic background. Sequence analysis identified that the beige mutation is predicted to delete a single isoleucine from the WD40 domain of the LYST protein, suggesting that this mutation is likely to disrupt a protein-protein interaction. CONCLUSIONS: Lyst mutant eyes exhibit multiple features of XFS. Recent human genetic association studies have identified changes occurring in the LOXL1 gene as an important risk factor for XFS but also indicated that other factors contributing to risk likely exist. These results demonstrated that mutation of the Lyst gene can produce ocular features of human XFS and suggested that LYST or LYST-interacting genes may contribute to XFS.

Scanning transmission X-ray microscopic analysis of purified melanosomes of the mouse iris.

Melanosomes are specialized intracellular membrane bound organelles that produce and store melanin pigment. The composition of melanin and distribution of melanosomes determine the color of many mammalian tissues, including the hair, skin, and iris. However, the presence of melanosomes within a tissue carries potentially detrimental risks related to the cytotoxic indole-quinone intermediates produced during melanin synthesis. In order to study melanosomal molecules, including melanin and melanin-related intermediates, we have refined methods allowing spectromicroscopic analysis of purified melanosomes using scanning transmission X-ray microscopy. Here, we present for the first time absorption data for melanosomes at the carbon absorption edge ranging from 284 to 290 eV. High-resolution images of melanosomes at discrete energies demonstrate that fully melanized mature melanosomes are internally non-homogeneous, suggesting the presence of an organized internal sub-structure. Spectra of purified melanosomes are complex, partially described by a predominating absorption band at 288.4 eV with additional contributions from several minor bands. Differences in these spectra were detectable between samples from two strains of inbred mice known to harbor genetically determined melanosomal differences, DBA/2J and C57BL/6J, and are likely to represent signatures arising from biologically relevant and tractable phenomena.