HPLC purification of antioxidant and antibacterial peptides from a lichen "Parmotrema perlatum (Huds.) M. Choisy": Identification by LC-MS/MS peptide mass fingerprinting.

Parmotrema perlatum, a lichen belonging to the family Parmeliaceae, is well known for its culinary benefits and aroma used as a condiment in Indian homes is also known as the "black stone flower" or "kalpasi" in India. This research intends to analyze the antioxidant power of the crude extracts using four pH-based buffers solubilized proteins/peptides and RP-HPLC fractions of P. perlatum obtained by purification. The proteins that were extracted from the four different buffers were examined using LC-MS/MS-based peptide mass fingerprinting. When compared to the other buffers, the 0.1 M of Tris-HCl buffer pH 8.0 solubilized proteins/peptides had the strongest antioxidant capacity. The sequential purification of the peptide was carried out by using a 3-kDa cut-off membrane filter and semipreparative RP-HPLC. Additionally, the purified fractions of the peptide's antioxidant activity were assessed, and effects were compared with those of the crude and 3 kDa cut--off membrane filtrates. The peptide fractions were sequenced by LC-MS/MS, which reveals that fraction 2 from RP-HPLC with the sequence LSWFMVVAP has shown the highest antioxidant potential in comparison with other fractions which can serve as the potential natural antioxidant drug. Further, fraction 2 also showed antibacterial activity against the selected microorganisms.

Metabolite profiling from different parts of tender fruit endosperm of Borassus flabellifer L. (ice apple).

Borassus flabellifer L., commonly known as Asian palmyra, is native to South and Southeast Asia. The endosperms of B. flabellifer (known as nungu in Dravidian culture) are widely consumed during the summer season. It is rich in various nutrients and helps in reducing weight, treating skin and digestive issues, lowering body temperature, and managing migraines and diabetes. This study focuses on identifying the small molecules and proteins from the two varieties of B. flabellifer tender fruit endosperms collected from districts around Chennai, Tamil Nadu, India. The collected free nuclear endosperm was subjected to direct extraction and the mesocarp and cellular endosperms were lyophilized and homogenized. Metabolites were extracted by hexane, methanol, and chloroform and investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The compounds identified were from the classes of carboxylic acids, flavonoids, amino acids, alkaloids, fatty acids, oligopeptides, vitamins, and glycosides. High-performance liquid chromatography (HPLC) technique was employed to estimate the quantity of amino acids, wherein the total amino acid in the green variety was found to be higher than in the black variety. Proteins were identified after simulating with a gastrointestinal enzyme using liquid chromatography tandem mass spectrometry (LC-MS/MS)-based peptide mass fingerprinting. The different mineral oxides present in the tender fruit endosperm were identified using X-ray diffraction studies, which confirmed the presence of mineral oxides, such as Br1.25ClO2.75Pb3.88, calcium zirconium tantalum oxide, and barium fluoroniobate. This study validates the presence of bioactive metabolites in green and black varieties of B. flabellifer tender fruit endosperm with a range of activities, such as anti-inflammatory, antibacterial, anticancer, and anti-diabetic properties.

Effect of incubation period on the glycosylated protein content in germinated and ungerminated seeds of mung bean (Vigna radiata (L.) Wilczek).

The effects of different incubation periods on the contents of amino acids, proteins, glycosylated proteins and metabolites in germinated and ungerminated mung bean seeds were investigated in this study. The study employs soaking of mung bean seeds in water under laboratory conditions at 28 °C for 3, 6, and 9 h, followed by germination for 12, 24, 36, and 48 h. Seeds collected from different period of imbibition and germination were subjected to total protein extraction for phytochemical analysis. Germination of the seeds was found to be most successful after 6 h of soaking (rather than 9 h of incubation). Hence, seeds imbibed for 6 h were further investigated for germination at 28 °C for 12, 24, 36, and 48 h. Total protein was extracted from both imbibed and germinated seeds, followed by trypsin digestion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based peptide mass fingerprinting revealed 38 proteins in 6 h water-imbibed seeds and 50 proteins in 24 h germinated seeds. Among these, 16 were identified as glycosylated proteins and the maximum number of glycosylated proteins were detected in 6 h water-imbibed seeds and 24 h germinated seeds. Moreover, High Performance Liquid Chromatography (HPLC) was used to quantify amino acids from the extracted proteins. A total of 15 amino acids were detected, of which eight were essential and the remaining were non-essential; amino acid concentrations increased following 3, 6, and 9 h of imbibition when compared to the control. It was concluded from the study that seeds with 6 h of imbibition and 24 h of germination can be used as potential nutritional source of different amino acids, proteins, glycosylated proteins, and other bioactive metabolites in human diet.

Modulation of quorum sensing-controlled virulence factors in Chromobacterium violaceum by selective amino acids.

Bacterial pathogenesis regulation requires N-acyl homoserine lactone (AHL)-based quorum sensing (QS). The main objective of this study was to assess the anti-QS and anti-biofilm potential of five different amino acids, namely serine, aspartic acid, lysine, leucine and tryptophan. The selected amino acids were assessed for their ability to inhibit QS activity via such mechanisms as exopolysaccharide (EPS) production, biofilm formation, pigment production in Chromobacterium violaceum and swarming motility. At 0.684 mM concentration, lysine inhibited the biofilm formation by 16% at 24 h, chitinolytic activity by 88.3% at 24 h and EPS production by 12.5% at 24 h. It also exhibited inhibition of swarming motility in C. violaceum. Confocal laser scanning microscopy revealed a decrease in the average thickness of the biofilms when treated with lysine. Modulation in the expression of cvi I and cvi A was observed when treated with all the amino acids, with the greatest reduction observed with lysine. Our results conclude that the amino acid lysine showed anti-QS and significant anti-biofilm activities; it could be further exploited as a main constituent in the synthesis of peptide/protein, and testing the same for treatment of bacterial infections, eventually reducing the utilization of conventional antibiotics.

Simple mass spectrometric method for the estimation of boron and aluminum in water at the parts per billion level.

The coordinating nature of the hydroxy carboxylic acids, such as tartaric and citric acids, has been utilized for the in-situ formation of anions representing the trivalent elements boron and aluminum and two dianions of the hydroxy acid selected under negative electrospray ionization mass spectral conditions. The abundance of these ions could be used for the quantification of boron and aluminum in water at concentrations ranging from 4.0 ppb to 535.0 ppb. For a period of six months, the validity of this method was tested with citric acid as the coordinating agent. Thus, the developed method offers a simple means for the quantification of boron and aluminum in water by negative electrospray ionization mass spectrometry with a single quadrupole mass spectrometer.