Characterization and in vitro biological activities of phenolic compounds from pea (Pisum sativum L.)

Porodica mahunarki (Fabaceae) obiluje ekonomski i poljoprivredno značajnim vrstama među kojima se po zastupljenosti gajenja u Srbiji izdvaja grašak (Pisum sativum L.) sa udelom od 4,5% u ukupnoj povrtarskoj proizvodnji. Semenjače graška, posebno genotipova sa izrazito pigmentisanim tamnim semenima predstavljaju značajan izvor fenolnih jedinjenja čiji su sastav i biološke aktivnosti nedovoljno ispitani, te stoga ni potencijal ovog materijala nije iskorišćen na odgovarajući način. Semenjače graška čine i do 10% suve mase semena i uglavnom predstavljaju otpadni materijal koji nastaje u procesu industrijske izolacije proteina semena koji se koriste u humanoj i animalnoj ishrani. U ovom radu je ispitivan sadržaj fenolnih jedinjenja prisutnih u semenjačama deset evropskih genotipova graška kao i njihove biološke aktivnosti. Pored toga je ispitivan i uticaj različitih rastvarača, termičke obrade i in vitro digestije semena graška na prinos fenolnih jedinjenja i antioksidativne aktivnosti dobijenih ekstrakata. Dobijeni rezultati su pokazali da termički tretman i in vitro digestija dovode do statistički značajnog povećanja sadržaja fenolnih jedinjenja, kako u vodenim ekstraktima semena graška tako i u ekstraktima pripremljenim pomoću organskog rastvarača. Utvrđeno je da peptidi koji nastaju u toku in vitro digestije proteina semena graška mogu značajno interferirati sa testovima za određivanje antioksidativne aktivnosti, pogotovo u vodenim rastvorima, pošto i sami poseduju detektabilne antioksidativne aktivnosti. Pokazano je da kombinacija rastvarača koja sadrži 70% acetona, 29% destilovane vode i 1% sirćetne kiseline najpodesnije sredstvo za ekstrakciju ukupnih fenola iz semenjače graška, koje daje najveći prinos kako kod genotipova sa bledom, tako i kod genotipova sa izrazito tamno pigmentisanom semenjačom. Sadržaj ukupnih fenola u ekstraktima dobijenim primenom pomenutog rastvarača se kod pojedinih genotipova (Assas, MBK 168 и Aslaug) kretao u opsegu 41,8-46,6 mg GAE/ g s.m., što su najviše do sada zabeležene vrednosti kod graška. Isti genotipovi su ujedno sadržali najviše taninskih materija i antocijanina i pokazivali su najizraženije sposobnosti uklanjanja DPPH radikala i redukcije Fe3+ jona. Analizom ekstrakata semenjača pomoću UHPLC–LTQ OrbiTrap MS tehnike identifikovano je 25 najzastupljenijih fenolnih jedinjenja i tom prilokom je detektovano 9 jedinjenja čije prisustvo do sad nije bilo zabeleženo u semanjačama graška...The family of legumes (Fabaceae) is rich in economicaly and agriculturally important species among which the most abundant in Serbia stands peas (Pisum sativum L.) with a share of 4.5% of the total vegetable production. Pea seed coats, especially in genotypes with highly pigmented dark seeds are an important source of phenolic compounds, whose composition and biological activity have not been tested in detail, thus this valuable material is still underutilized. Pea seed coat makes up to 10% of the seed dry weight and largely represents waste material generated in the process of industrial isolation of seed proteins, which are used in human and animal nutrition. In this study the content of phenolic compounds in seeds of ten European pea genotypes, as well as their biological activities has been examined. In addition, the effect of different solvents, heat treatment and in vitro digestion of pea seeds on yield of phenolic compounds and antioxidant activity in extracts was also studied. The obtained results showed that the thermal treatment and in vitro digestion led to a statistically significant increase of the total phenolic content, both in aqueous extracts of pea seeds and in the extracts prepared by using an organic solvent. It was shown that peptides which were formed during the in vitro digestion of the pea seed proteins can significantly interfere with the assays for determination of antioxidant activity, especially in aqueous solutions, since they possess a detectable antioxidant activity. It has been shown that the combination of solvents containing 70% acetone, 29% distilled water and 1% acetic acid was the most suitable means for the extraction of total phenols from pea seed coats, which gave the highest yield in both bright colored and dark colored seed genotypes. Total phenolic content of extracts obtained by mentioned solvent in certain genotypes (Assas, MBK 168 and Aslaug) ranged from 41.8 to 46.6 mg GAE / g s.m., which are the highest recorded values in pea. The same genotypes also possessed the highest tannin and anthocyanin content and showed the most pronounced ability for DPPH radical scavenging as well as for the reduction of Fe 3+ ions. The analysis of seed coat extracts, using the LTQ Orbitrap UHPLC-MS technique, identified 25 most abundant phenolic compounds, from which 9 compounds have not been recorded in pea seed coats of previously studied genotypes..

A simple and efficient DNA isolation method for Ornithogalum L. species (Hyacinthaceae, Asparagales)

We report an efficient, simple and cost-effective protocol for the isolation of genomic DNA from Ornithogalum species. Our modification of the standard CTAB protocol includes two polyphenol adsorbents (insoluble PVPP and activated charcoal), high NaCl concentrations (4 M) for removing polysaccharides, and addition of phenol to remove proteins and other contaminants. DNA yield obtained with our protocol was 223 and 312 μg DNA g-1 of dry leaf tissue. The absorbance ratio 260/280 nm was 1.879 (O. refractum) and 1.753 (O. sibthorpii), and the absorbance ratio 260/230 nm was 1.779 (O. refractum) and 1.545 (O. sibthorpii), revealing lack of contamination. PCR amplifications of one nuclear marker (26S rDNA) indicated that this DNA isolation protocol may be used for Ornithogalum plants containing many interfering compounds for further analyses in population genetics and phylogeographic studies

Synthesis and antioxidant activity of 1,3,4-oxadiazoles and their diacylhydrazine precursors derived from phenolic acids

Eight 1,3,4-oxadiazole derivatives containing phenolic acid moieties (7a-h) and eight of their diacylhydrazine precursors (6a-h) were synthesized, characterized using spectroscopic methods and examined by scavenging of stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals. The most potent phenolic 1,3,4-oxadiazoles showed better DPPH scavenging activity in comparison with their corresponding diacylhydrazine precursors as a result of participation of both aromatic rings and a 1,3,4-oxadiazole moiety in resonance stabilization of the formed phenoxyl radical. Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxadiazoles (7d, 7e, 7g and 7h) with the best DPPH scavenging activity, were chosen for further evaluation of their antioxidant potential through various assays. The investigated compounds exerted pronounced ABTS radical scavenging capacity, moderate to good H2O2 scavenging properties and strong ferric ion reducing capacity. Further in vitro evaluation of the antioxidant properties of the most active compounds demonstrated their protective effects in normal lung fibroblasts MRC-5 against hydrogen peroxide induced oxidative stress. Diacylhydrazine 6h increased two times the activity of glutathione peroxidase in treated cells in comparison with a control sample and did not affect the superoxide dismutase activity.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2980

Lactococcin B Is Inactivated by Intrinsic Proteinase PrtP Digestion in Lactococcus lactis subsp. Lactis BGMN1-501

In our previous study we demonstrated that proteinase PrtP is able to impair bacteriocin LcnB activity, despite being produced by the same organism and encoded by the same plasmid. However, precise mechanism of this action, i.e., the exact cleavage site within LcnB bacteriocin, as well as its effect on antimicrobial activity of the resulting peptide remained vague. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that this unusual hydrolysis indeed occurs in vivo, between the sixth and seventh amino acid on the N terminus of LcnB. To address whether the cleaved form of LcnB retains any level of activity, both recombinant and chemically synthesized variant of truncated LcnB were engineered and produced, but demonstrated no antimicrobial activity. When LcnB was recombinantly overexpressed and subjected to PrtP digestion, the change in its antimicrobial activity was monitored and the degradation products analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin. In addition, it was demonstrated that, once truncated, LcnB is not able to bind its receptor and is susceptible to additional hydrolysis. This is the first report on proteolytic inactivation of bacteriocins inside the same bacterial host.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3299

Antioxidant activity of pea protein hydrolysates produced by batch fermentation with lactic acid bacteria

Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO) activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW lt 10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity) and the recently developed direct current (DC) polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins

Identification of Seed Coat Phenolic Compounds from Differently Colored Pea Varieties and Characterization of Their Antioxidant Activity

The phenolic composition of seed coats in four differently colored pea varieties (Pisum sativum L.) was investigated using UHPLC-LTQ OrbiTrap MS. The obtained findings revealed that the seed coats of the examined pea genotypes possess a unique phenolic composition compared to previously studied European cultivars. In total, 41 phenolic compounds have been identified. The seed coats of the studied cultivars contained certain amounts of rosmarinic acid, rutin, galangin, morin, naringin, hesperetin and pinocembrin as well as ten flavonol glycosides that had not been reported previously. Additionally, the total phenolic content, antioxidant activity and metal chelating capacity of extracts was determined using Folin-Ciocalteu's method, 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric ion-reducing capacity and ferrous ion-chelating capacity assay, respectively. Dark colored genotypes MBK 168 and MBK 173 possessed the highest total phenolic contents as well the strongest antioxidant activities. On the other hand, bright colored genotypes MBK 88 and MBK 90 exhibited the strongest metal-chelating capacities. The examined pea seed coats may be considered as important potential contributors to human health due to the presence of bioactive phenolic constituents. In addition, our results could be used as a guideline for breeding new pea cultivars with high antioxidant activities applicable in the formulation of functional food products

Identification of seed coat phenolic compounds from differently colored pea varieties and characterization of their antioxidant activity

The phenolic composition of seed coats in four differently colored pea varieties (Pisum sativum L.) was investigated using UHPLC-LTQ OrbiTrap MS. The obtained findings revealed that the seed coats of the examined pea genotypes possess a unique phenolic composition compared to previously studied European cultivars. In total, 41 phenolic compounds have been identified. The seed coats of the studied cultivars contained certain amounts of rosmarinic acid, rutin, galangin, morin, naringin, hesperetin and pinocembrin as well as ten flavonol glycosides that had not been reported previously. Additionally, the total phenolic content, antioxidant activity and metal chelating capacity of extracts was determined using Folin-Ciocalteu's method, 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric ion-reducing capacity and ferrous ion-chelating capacity assay, respectively. Dark colored genotypes MBK 168 and MBK 173 possessed the highest total phenolic contents as well the strongest antioxidant activities. On the other hand, bright colored genotypes MBK 88 and MBK 90 exhibited the strongest metal-chelating capacities. The examined pea seed coats may be considered as important potential contributors to human health due to the presence of bioactive phenolic constituents. In addition, our results could be used as a guideline for breeding new pea cultivars with high antioxidant activities applicable in the formulation of functional food products

Supplementary material for the article: Stanisavljević, N. S.; Ilić, M. D.; Matić, I. Z.; Jovanović, Ž. S.; Čupić, T.; Dabić, D. Č.; Natić, M. M.; Tešić, Ž. L. Identification of Phenolic Compounds from Seed Coats of Differently Colored European Varieties of Pea (Pisum Sativum L.) and Characterization of Their Antioxidant and In Vitro Anticancer Activities. Nutrition and Cancer 2016, 68 (6), 988–1000. https://doi.org/10.1080/01635581.2016.1190019.

Supplementary material: [https://doi.org/10.1080/01635581.2016.1190019]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2291

Supplementary data for article: Mihailovic, N.; Marković, V.; Matić, I. Z.; Stanisavljević, N. S.; Jovanovic, Z. S.; Trifunović, S. S.; Joksović, L. Synthesis and Antioxidant Activity of 1,3,4-Oxadiazoles and Their Diacylhydrazine Precursors Derived from Phenolic Acids. RSC Advances 2017, 7 (14), 8550–8560. https://doi.org/10.1039/c6ra28787e

Supplementary material for: [https://doi.org/10.1039/c6ra28787e]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2398

In vitro digestion of meat- and cereal-based food matrix enriched with grape extracts: How are polyphenol composition, bioaccessibility and antioxidant activity affected?

The aim of this study was to evaluate the effect of enriching a complex food matrix (FM) with grape extracts on polyphenol content, composition, bioaccessibility and antioxidant activity during digestion. The grape extracts and FM were separately tested under the same conditions as controls. The FM by itself contains a significant amount of phenolic acids and flavonols, influencing the final recovery of polyphenols from grape extracts. The FM significantly increased the total recovery of polyphenols after digestion of grape seed extracts compared to those digested without the FM; however, a low recovery of proantocyanidins and total flavonoids was observed. Digestive fluids and FM compounds significantly increased the total polyphenol content of grape digests and significantly contributed to their ABTS(center dot+) scavenging activity and ferrous-ion-chelating capacity. The present study suggested that enrichment of meat-and cereal-based products with grape polyphenol extracts could be a good strategy to formulate a healthier diet