Crosslinking Efficacy and Cytotoxicity of Genipin and Its Activated Form Prepared by Warming It in a Phosphate Buffer: A Comparative Study

The aim of the present study was to compare the acute and cumulative cytotoxicity of intact (n-GE) and warmed genipin (w-GE), while investigating the differences in crosslinking capabilities of these two genipins by rheological and mechanical tests. The n-GE solution was prepared by dissolving genipin powder in a sodium phosphate buffer solution. The w-GE solution was prepared by warming the n-GE solution at 37 °C for 24 h. The mechanical tests for chitosan (CH)/genipin gels showed the crosslinking rate of w-GE was much greater than that of n-GE up until 6 h after preparation, whereas the degree of crosslinking of CH/n-GE gels became higher at 12 h. The ISO 10993-5 standard method, which is established specifically for evaluating cumulative cytotoxicity, determined equivalent IC50 for w-GE (0.173 mM) and n-GE (0.166 mM). On the other hand, custom-made cytotoxicity tests using a WST-8 assay after 1 h of cultivation showed that the acute cytotoxicity of w-GE was significantly higher than that of n-GE at concentrations between 0.1–5 mM. The acute cytotoxicity of w-GE should be taken into consideration in its practical uses, despite the fact that the much faster crosslinking of w-GE is useful as an effective cross linker for in-situ forming gels.

Extracting Sweetening and Bioactive Compounds From Stevia Rebaudiana Using Cellulase Enzyme

This study was conducted to find out the sweetener and bioactive compounds in the Stevia plant’s enzyme extract and its antioxidant and antibacterial effect. The enzyme extract was used in a ratio of 1:15 (w: v) with the use of sodium phosphate buffer solution (pH=4) in an equal mixing ratio with the enzyme. Extraction was carried out at 55°C with a time of 25 minutes to extract and determine steviol glycosides using HPLC. Stevioside and Rebaudioside-A (5.101 and 3.027 mg/g) were obtained, respectively. The study showed that Stevia contains high amounts of phenols and flavonoids (83.052 and 71,765) mg/ml, respectively. The enzyme extract gave an antioxidant activity of 71.367% compared to BHT which gave an activity of 77.267%. It gave the highest inhibitory activity against Bacillus subtilis, which was 14 mm, followed by Staphylococcus aureus and E. coli. The bioactive compounds were diagnosed by GC-MS and contained important bioactive compounds such as Hydroxydehydrostevic acid (steviol), 1,2-Benzenediol, 4-Ethylcatechol, 9-Octadecenamide, (Z)-, Isosteviol methyl ester, gamma-Sitosterol.

Investigation of guanidine hydrochloride induced unfolding of apolipoprotein A-IMilano

A guanidine hydrochloride (GuHCl) induced unfolding study of apolipoprotein A-IMilano(apo A-IM) has been performed. The unfolding was followed by circular dichroism (CD) measurements at 222 nm and by isothermal titration (ITC) calorimetry at 25°C. In the ITC experiments enthalpies of transfer were determined for apo A-IMfrom aqueous sodium phosphate buffer solution into solutions of different concentrations of GuHCl. The CD data and the ITC data give complementary and consistent results of the complex unfolding process. Analytical ultracentrifugation experiments were made on apo A-IMin sodium phosphate buffer and in 0.6 M GuHCl, respectively. Analyses of the obtained sedimentation velocity data show that apo A-IMis highly aggregated in sodium phosphate buffer. The aggregates are almost completely dissociated in 0.6 M GuHCl. Aggregation of the protein in sodium phosphate buffer solution induces an increase in α-helical content. The loss of α-helical secondary structural element of the protein upon dissociation of the aggregates destabilises the protein resulting in a low GuHCl concentration of unfolding, [GuHCl]m=1.1 M. The unfolded protein has a significant α-helical content at the unfolded state. From ITC- and CD data we suggest that increased binding of GuHCl to the unfolded protein results in a disruption of the residual secondary structure.