Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

Extraction, Enrichment, and Quantification of Main Antioxidant Aglycones of Flavonoids and Tannins from Melastoma Dodecandrum Lour.: Guided by UPLC-ESI-MS/MS

The objective of this work was to determine the main antioxidant aglycones of flavonoids and tannins in antioxidant extracts from Melastoma dodecandrum Lour. (MD), based on its extraction and purification process optimization and component identification by UPLC-ESI-MS/MS. Firstly, the ultrasound-assisted extraction (UAE) process for antioxidants was established by using single factor tests and response surface optimization. Evaluating by DPPH radical scavenging assay, the antioxidant capacity of MD could reach 2742.27 ± 93.86 (μmol Trolox/g) under the optimized conditions including methanol concentration (61%, v/v), extraction time (45 min), liquid-solid ratio (0.46 ml/mg), and extraction temperature (25°C). Then, the antioxidants in the solution were enriched by using macroporous resins. Because of the highest adsorption capacity and desorption ratio, HPD 500 was selected out of 14 macroporous resins for further study based on the static adsorption and desorption tests. The adsorption mechanism of the HPD 500 resin presented that pseudo-first-order kinetics model and Freundlich isotherm model could adequately explain the adsorption process. After the antioxidants were dynamically saturated by HPD 500, the column was eluted with different concentrations of ethanol (0, 25, 50, 75, and 100%, v/v). Among the above five ethanol fractions, 50% ethanol fraction showed the strongest antioxidant activity and more than 95% antioxidants adsorbed in HPD500 could be eluted by 75% ethanol. Guided by analysis results of UPLC-ESI-MS/MS, main antioxidant aglycones of flavonoids and tannins in each ethanol elution fraction were quantitatively detected. Finally, according to the correlation analysis between the antioxidant capacity of each ethanol elution fraction and its content of 10 main flavonoids and phenolic acids, the antioxidant activities of MD could mainly contribute to tannins containing phenolic units such as ellagic acid and/or gallic acid. The results of this work would provide useful information for the production of antioxidants from MD. Thus, the analysis methods could also be a quality control tool for material or products related with MD.

Temperature-induced alanine oxidation in a psychrotrophic Pseudomonas

A psychrotrophic pseudomonad isolated from iced fish oxidized alanine at temperatures close to 0 °C and grew over the range 0 °C–35 °C. The rate of oxidation of alanine, measured manometrically, by cells grown at 2 °C was lower than that of cells grown at 22 °C. However, the consumption of oxygen after heat treatment at 35 °C for 35 min was reduced considerably by 2 °C grown cells. Alanine oxidase activity was tested in an extract from cells grown at 2 °C and 22 °C with alanine as the sole carbon, nitrogen, and energy source. Cells grown at 2 °C produced an alanine oxidase with a temperature optimum of 35 °C and pH optimum of 8, which lost about 80% activity by heat treatment at 40 °C for 30 min. There was no change in activity after dialysis at pH 7, 8, or 9. Extracts from cells grown at 22 °C contained an alanine oxidase system with an optimum temperature of 45 °C, a pH optimum above 8, and only about 30% reduction of activity after heat treatment. This enzyme activity was concentrated in the 0.5 M elution fraction from a Sephadex column, and dialysis reduced the activity at pH 7 and 8. Mesophilic enzyme synthesis apparently started around a growth temperature of 10 °C.The crude alanine oxidase systems of Pseudomonas aeruginosa derived from cells grown at 13 °C and 37 °C had a common optimum temperature of 45 °C. These data suggest that one mechanism of psychrophilic growth by psychrotrophic bacteria may be the induction of enzymes with low optimum temperatures in response to low temperature conditions.