Incubation In Buffer Solution Research Articles

Whey protein based electrosprayed nanospheres for encapsulation and controlled release of bioactive compounds from Tinospora cordifolia extract

The synergistic effects of bioactive compounds of Tinospora cordifolia have insulin mimicking and hypoglycemic activity, however, low bioavailability and poor stability limits its potential. In the present study, an appropriate delivery system was developed for the controlled release of its anti-diabetic activity. The bioactive compounds such as palmatine, berberine and palmatoside had better binding energy as observed in docking studies compared to that of the commercial active compounds. However, as these biocompounds from Tinospora cordifolia are associated with low stability and poor bioavailability, these compounds were encapsulated in a core-shell matrix of whey protein isolate. The bioactive compounds had highest antidiabetic activity in chloroform extract with an IC50 concentration of 11.34 mg/ml. An increase in 28.12% activity was observed in nanoemulsion form with an average size of 82.68 ± 4.37 nm. The bioactive compounds were further encapsulated by electrospray technique for increased stability. The particles had an encapsulation efficiency of 91.2 ± 3.27% with an average particle size of 187 ± 2.71 nm. The kinetic study revealed the complete release of bioactive compounds after 24 h of incubation in buffer solution. This formulation can be further explored as novel nutraceutical delivery system with minimal side effects as compared to their synthetic counterparts. Considering the potential application of this developed technology, further upscaling as well as in-vivo experimentation on small as well as large animals should be performed.

Immobilization and Activity of Pepsin in Silicone Elastomers

Pepsin [EC, 3.4.23.1] from Porcine stomach mucosa was immobilized in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). Two network precursor chain molar masses were used in this investigation: in pepsin–silicone (A), M n ∼26,000 g mol−1 and in pepsin–silicone (B) M n ∼750 g mol−1. Tetraethyl orthosilicate (TEOS) was used as the cross-linking agent and dibutyltin dilaurate was used as the catalyst. The activity and stability of free pepsin and pepsin immobilized in PDMS were studied with respect to pH, temperature, cross-link density, solvents and storage time using a hemoglobin assay. A notable finding is that free pepsin has zero activity in neutral buffer solution (pH 7) after incubation for 5 h, while pepsin immobilized in the silicone elastomers was found to retain more than 70% of its maximum normalized activity. There was no marked improvement in the thermal stability of the PDMS immobilized pepsin when compared to free pepsin and all the three systems showed no activity at and above 70 °C. From the Lineweaver–Burk kinetic analyses, the apparent K m (g L−1 hemoglobin) for free pepsin was 4.5, for pepsin–silicone (A) was 5.1, and for pepsin–silicone (B) was 3.9, the V max (U/mg of pepsin) for free pepsin was 14,000, for pepsin–silicone (A) was 11,710, and for pepsin–silicone (B) was 8,510, respectively after incubation in buffer solution at pH 2 and 37 °C. The activity of the free and the PDMS immobilized pepsin in six different organic solvents was also studied. The pepsin retained high activity in non-polar solvents such as n-hexane, isooctane and toluene, but the enzyme performed poorly in methanol, ethanol and tetrahydrofuran. The degree of swelling of the pepsin immobilized silicone elastomers in these solvents had no impact on the activity of the pepsin. When stored at room temperature for time periods up to 6 months, pepsin immobilized in silicone elastomers was observed to retain its full activity. The results reported herein demonstrate that cross-linked PDMS is a promising support material for the immobilization of hydrolytic enzymes such as pepsin.

Amperometric Glucose Sensor Using Thermostable Co-Factor Binding Glucose Dehydrogenase

A thermostable mediator-type enzyme glucose sensor was constructed. The electrode was fabricated using chemically cross-linked thermostable co-factor binding glucose dehydrogenase (GDH) from thermophilic bacteria in carbon paste matrix. The electrode responded directly proportional to D-glucose concentration from 0.01 mM to 3 mM in stirred buffer containing 1 mM 1-methoxyphenazinemethosulfate as a mediator with the steady-state mode. The storage stability was examined by incubating the enzyme electrode at 50oC during the measurement. The cross-linked GDH immobilized electrode showed good storage stability. Ninety percent of its initial response was retained after incubation in buffer solution for 9 days at 50oC. The flow injection analysis (FIA) glucose sensing system was also constructed by immobilizing the cross-linked GDH and ferrocene as a mediator in the carbon paste matrix. The FIA system was able to measure 600 samples for 100 h.

Adsorption of proteins on electro-conductive polymer films

Shapable electro-conductive (SEC) polymer films (polyanion-doped polypyrrole films) show several interesting properties for bioelectrochemical applications. The SEC film can be used as an inert, stable and hydrophobic electrode in aqueous solution over a wide potential range. In this study, the physical and the potential-assisted adsorption of various proteins on the SEC film is described. Because of the hydrophobic surface characteristic proteins easily adsorb and retain on the film surface by strong hydrophobic interactions. The amount of the adsorbed protein varies from 2.2 to 4.8 μg cm −2 depending on the protein when the film is incubated for 22 h in the protein solution. The adsorption is effectively accelerated and enhanced by applying a positive potential in the range from 0.4 V to 1.0 V (vs. Ag/AgCl). The potential-assisted adsorption process is completed by 10–15 min and the amount of the adsorbed protein is nearly doubled as compared to the adsorption without potential. The adsorbed protein is chemically very stable in comparison with the protein in solution. More than 85% of the initial adsorbed proteins retains on the surface after three weeks of incubation in buffer solution. The initial adsorption rate is studied by quartz crystal micro-balance measurements on a thin polymer film coated quartz crystal. In addition, the SEC film surface is etched with air plasma which leads to a four-fold increase of the adsorption of proteins.