In Vitro and In Silico of Cholinesterases Inhibition and In Vitro and In Vivo Anti-Melanoma Activity Investigations of Extracts Obtained from Selected Berberis Species.

Berberis species have a long history of use in traditional Chinese medicine, Ayurvedic medicine, and Western herbal medicine. The aim of this study was the quantification of the main isoquinoline alkaloids in extracts obtained from various Berberis species by HPLC, in vitro and in silico determination of anti-cholinesterase activity, and in vitro and in vivo investigations of the cytotoxic activity of the investigated plant extracts and alkaloid standards. In particular, Berberis species whose activity had not been previously investigated were selected for the study. In the most investigated Berberis extracts, a high content of berberine and palmatine was determined. Alkaloid standards and most of the investigated plant extracts exhibit significant anti-cholinesterase activity. Molecular docking results confirmed that both alkaloids are more favourable for forming complexes with acetylcholinesterase compared to butyrylcholinesterase. The kinetic results obtained by HPLC-DAD indicated that berberine noncompetitively inhibited acetylcholinesterase, while butyrylcholinesterase was inhibited in a mixed mode. In turn, palmatine exhibited a mixed inhibition of acetylcholinesterase. The cytotoxic activity of berberine and palmatine standards and plant extracts were investigated against the human melanoma cell line (A375). The highest cytotoxicity was determined for extract obtained from Berberis pruinosa cortex. The cytotoxic properties of the extract were also determined in the in vivo investigations using the Danio rerio larvae xenograft model. The obtained results confirmed a significant effect of the Berberis pruinosa cortex extract on the number of cancer cells in a living organism. Our results showed that extracts obtained from Berberis species, especially the Berberis pruinosa cortex extract, can be recommended for further in vivo experiments in order to confirm the possibility of their application in the treatment of neurodegenerative diseases and human melanoma.

Isolation and Identification of Lactococcus lactis and Weissella cibaria Strains from Fermented Beetroot and an Investigation of Their Properties as Potential Starter Cultures and Probiotics.

The presence of certain microorganisms in dairy products or silage is highly desirable. Among them are probiotic strains of lactic acid bacteria (LAB), which show many beneficial features, including antimicrobial properties that support the development of beneficial microflora; in addition, owing to their biochemical activity, they influence the nutritional, dietary, and organoleptic properties of food products. Before being placed on the market, each strain requires separate testing to determine its probiotic properties and effectiveness. The aim of this study was to isolate LAB strains from a pickled beetroot sample that could be used in the dairy industry and with the potential to be considered as a probiotic in the future. Two strains identified using the MALDI technique were selected-Lactococcus lactis and Weissella cibaria. The optimal growth conditions of the strains were determined, and their proteolytic properties were assessed with the use of the o-PA reagent and spectrophotometry. The lipid profile was analyzed using the SALDI (surface-assisted laser desorption/ionization) technique and silver nanoparticles. High-performance liquid chromatography was used to assess the ability of the strains to synthesize beneficial metabolites, such as B vitamins (B2, B3, and B9) or lactic acid, and gas chromatography was used to analyze the substances responsible for organoleptic properties. Moreover, the ability to inhibit the growth of pathogenic strains was also tested in the selected strains. Both tested strains demonstrated the desired properties of starter cultures for future use in functional food production, showing that fermented plant products can serve as valuable potential probiotic sources.

Synthesis and physicochemical characterization of bovine lactoferrin supersaturated complex with iron (III) ions.

The aim of the study was to investigate the process of Fe3+ binding to bLTF. Moreover, the physicochemical characterization of the respective supersaturated complex was studied. The knowledge should be important for the description of processes that may take place in dairy products fortified with iron. Additionally, the synthesized complex can be utilized as a dietary supplement for the treatment of iron deficiency anemia (IDA). Finally, it was shown that formation of supersaturated iron-protein structures which include LTF often accompanies development of neurodegenerative diseases such as Alzheimer or Parkinson. Thus, the study can reveal some aspects of its pathogenesis process. The methodology of the investigation comprised the utilization of batch sorption study and applying Freundlich and Langmuir models. The complex also was characterized by numerous techniques: spectrometric (ICP-MS), spectroscopic (UV-Vis, ATR-FTIR), electron microscopy (TEM-EDX), SDS-PAGE. Based on obtained results the potential mechanisms of iron interaction with protein were described. Moreover, the molecular docking was applied to visualize possible metal binding sites. The respective complex contains ≈ 33.0 mg/g of iron which is nearly 50 Fe3+ per one protein molecule. The cytotoxicity of the obtained complex was evaluated by MTT reduction and LDH release assays on Caco-2 and nL929 cell lines.

Isoquinoline Alkaloid Contents in Macleaya cordata Extracts and Their Acetylcholinesterase and Butyrylcholinesterase Inhibition.

An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of Macleaya cordata collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman's reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-QqQ-ESI-MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the Macleaya cordata extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.

Lacticaseibacillus paracasei as a Modulator of Fatty Acid Compositions and Vitamin D3 in Cream.

Butter is an important source of essential fatty acids, lipid-soluble vitamins, and antioxidants in the diet. However, this study showed that the presence of the Lacticaseibacillus paracasei strain has a great influence on the fatty acid profile as well as provitamin D3 and vitamin D3 content in the cream-the raw material from which the butter is obtained. The addition of this lactic acid bacteria enriches the cream in 9-hexadecenoic acid, oleic acid, octadeca-9,12-dienoic acid, and conjugated linoleic acid, which exhibit antimutagenic and anticarcinogenic properties. Moreover, a higher level of monounsaturated fatty acids can extend the shelf life of butter in the future. In the present work, we observed that the presence of lactic acid bacteria contributed to an increase in the level of provitamin D after 6 h of incubation and an increase in the levels of vitamin D3 after 24 and 48 h. Fatty acid profiles and the content of vitamins were largely dependent on the presence of light and mixing, which are probably associated with the status of lipid peroxidation.

New sources of lactic acid bacteria with potential antibacterial properties.

In the face of the growing demand for functional food, the search for new sources of lactic acid bacteria (LAB) becomes a priority. In our research, we used multiplied culture conditions followed by identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry for seeking LAB strains in plant- and animal-derived sources. Furthermore, the selected LAB isolates were examined for their proteolytic activity as well as antimicrobial action against different bacterial pathogens. The applied method appeared to be useful tool for searching LAB strains within different types of the biological matrices. The best source of the LABs was from calf. Comparing properties of the two selected LABs, those isolated from calf demonstrated the greatest proteolytic and antibacterial properties suggesting that gastrointestinal microbiota are the most valuable LAB source. Nevertheless, second selected strain derived from pickled cucumber juice may be also treated as a promising source of potential probiotic strains.

The Study of Protein-Cyclitol Interactions.

Investigation of interactions between the target protein molecule and ligand allows for an understanding of the nature of the molecular recognition, functions, and biological activity of protein-ligand complexation. In the present work, non-specific interactions between a model protein (Bovine Serum Albumin) and four cyclitols were investigated. D-sorbitol and adonitol represent the group of linear-structure cyclitols, while shikimic acid and D-(-)-quinic acid have cyclic-structure molecules. Various analytical methods, including chromatographic analysis (HPLC-MS/MS), electrophoretic analysis (SDS-PAGE), spectroscopic analysis (spectrofluorimetry, Fourier transform infrared spectroscopy, and Raman spectroscopy), and isothermal titration calorimetry (ITC), were applied for the description of protein-cyclitol interactions. Additionally, computational calculations were performed to predict the possible binding places. Kinetic studies allowed us to clarify interaction mechanisms that may take place during BSA and cyclitol interaction. The results allow us, among other things, to evaluate the impact of the cyclitol's structure on the character of its interactions with the protein.

Impact of Ionic Strength of Carrier Liquid on Recovery in Flow Field-Flow Fractionation.

Asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) are techniques widely used in analytical, industrial and biological analyses. The main problem in all AF4 and HF5 analyses is sample loss due to analyte-membrane interactions. In this work the impact of liquid carrier composition on latex nanoparticles (NPs) separation in water and two different concentrations of NH4NO3 was studied. In AF4, a constant trend of decreasing the size of 60 and 121.9 nm particles induced by the ionic strength of the carrier liquid has been observed. In contrast, an agglomeration effect of the biggest 356 nm particles was observed when increasing ionic strength, which induced a significant drop of recovery to 35%. H5F provides better resolution and intensified peaks of NPs, but careful optimisation of system parameters is mandatory to obtain good separation.