Cellular Antioxidant Activity Assay Research Articles

Exploring the bioactive compounds from berry biowaste: Eugenia involucrata DC. as a study case

Aiming at valorising the Eugenia involucrata residue, the present work aimed in investigating the chemical profile and in vitro bioactivities of the residue (EIR) and its individual parts, i.e., seeds (EIS) and peels (EIP). Chromatographic analyses showed that EIP had the highest content of organic acids, tocopherols, and anthocyanins, whereas EIS was the richest in hydrolysable tannins. bis-HHDP-glucoside isomers and cyanidin-3-O-galactoside were the main detected phenolic compounds. By the cellular antioxidant activity (CAA) assay, the samples showed similar. antioxidant potential with 71%, 77%, and 80% inhibition for EIP, EIR, and EIS, respectively, while EIS demonstrated the best antioxidant activity by thiobarbituric acid reactive substances (TBARS, IC50 = 4.96 μg/mL) and oxidative inhibition assay (OxHLIA, IC50 = 40 μg/mL). The samples were able to inhibit the growth of seven bacteria and two fungi. Between the microorganisms tested, all samples showed a strong antibacterial activity (MIC = 0.156 mg/mL) against Yersinia enterocolitica and Pseudomonas aeruginosa (MIC = 0.156–0.625 mg/mL). Moreover, they also display a high antifungal activity against Arpergillus brasiliensis (MIC = 0.625 mg/mL). Furthermore, only EIR and EIS had anti-inflammatory effect via NO production inhibition (IC50 = 168–259 μg/mL, respectively) and anti-proliferative activity towards four tumour and one non-tumour cell lines. These findings suggest that the biowaste generated during the processing of E. involucrata fruit has potential as a source of bioactive compounds.

High-throughput synchronous erythrocyte cellular antioxidant activity and protection screening of phenolic-rich extracts: Protocol validation and applications

Oxidative/nitrosative damage takes part in chronic disease development, which generates an urgent need for intervention and better therapies to manage them. The scientific community has demanded easy-to-run, cheap, and reliable methods for cellular antioxidant activity assays. This work standardised and validated an erythrocyte cellular antioxidant activity and membrane protection/injury (HERYCA-P) protocol to study food-derive extracts. The method measures intracellular reactive oxygen species (ROS) generation, lipoperoxidation, and haemolysis induced by 2,2′-azobis(2-amidinopropane) dihydrochloride. Quercetin decreased ROS generation by 50.4% and haemolysis by 2.2%, while ascorbic acid inhibited lipid peroxidation by 40.1%. Total phenolic contents of teas were correlated with decreased ROS generation (r = −0.924), lipoperoxidation (r = −0.951), and haemolysis (r = −0.869). The erythrocyte ROS generation and lipoperoxidation were also associated with CUPRAC (r = −0.925; r = −0.951) and hydroxyl radical scavenging activity (r = −0.936; r = −0.949). The precision rates of antioxidant standards and tea samples were below 15%. HERYCA-P is feasible as a complementary antioxidant assay for food matrices.

Impact of In Vitro Digestion on the Digestibility, Amino Acid Release, and Antioxidant Activity of Amaranth (Amaranthus cruentus L.) and Cañihua (Chenopodium pallidicaule Aellen) Proteins in Caco-2 and HepG2 Cells.

This study evaluated the impact of in vitro gastrointestinal digestion on the digestibility, amino acid release, and antioxidant activity of proteins from amaranth (Amarantus cruentus L.) and cañihua (Chenopodium pallidicaule Aellen). Antioxidant activity was assessed using ORAC, ABTS, DPPH, and cellular antioxidant activity (CAA) assays in human intestinal Caco-2 and hepatic Hep-G2 cell lines. The results showed that amaranth had higher protein digestibility (79.19%) than cañihua (71.22%). In addition, intestinal digestion promoted the release of essential amino acids, such as leucine, lysine, and phenylalanine, in both protein concentrates. Concentrations of amaranth and cañihua proteins, ranging from 0.125 to 1.0 mg mL-1, were non-cytotoxic in both cell lines. At a concentration of 0.750 mg mL-1, simulated gastrointestinal digestion enhanced cellular antioxidant activity. Intestinal digest fractions containing peptides >5 kDa were the principal contributors to CAA in both cell lines. Notably, cañihua proteins exhibited high CAA, reaching values of 85.55% and 82.57% in Caco-2 and HepG2 cells, respectively, compared to amaranth proteins, which reached 84.68% in Caco-2 and 81.06% in HepG2 cells. In conclusion, both amaranth and cañihua proteins, after simulated gastrointestinal digestion, showcased high digestibility and released peptides and amino acids with potent antioxidant properties, underscoring their potential health benefits.

Phytochemical profiles, antioxidant activities, and synergistic antiproliferative effects of blueberry and apple peel extracts.

Blueberries and apples exhibit favorable bioactivity and health benefits due to their rich phytochemicals. Natural phytochemicals exist in complex forms, but there are few reports on whether have additive, synergistic or antagonistic effects between different phytochemicals. The aim of the present investigation was to elucidate the synergistic effects of blueberry extract (BE) and apple peel extract (APE) together in inhibiting the proliferation of HepG2 liver cancer cells. Meanwhile, phytochemical characterization of BE and APE were detected by HPLC, and total antioxidant activity was determined via cellular antioxidant activity (CAA) assay, oxygen radical absorption capacity (ORAC) assay and peroxy radical scavenging capacity (PSC) assay. The results showed that BE and APE were rich in phytochemicals and had potent antioxidant activities, which synergistically inhibited cell proliferation. In the bilateral combination, the dose reduction index (DRI) value increased 2-fold, and the combination index (CI) value at 95% inhibition was less than 1. Additionally, BE+APE supplementation could promote the expression levels of p53 and c-myc genes. In conclusion, the BE and APE had strong antioxidant activity and exhibited synergistic inhibition against HepG2 cells proliferation. This can provide a theoretical basis for the synergistic effect of different phytochemicals in health care. This article is protected by copyright. All rights reserved.

The Action Mechanisms, Anti-Cancer and Antibiotic-Modulation Potential of Vaccinium myrtillus L. Extract.

Herbal medicinal products containing Vaccinium myrtillus L. (bilberry) fruits and fruit extracts are widely available in the market. Although bilberry leaves and stems are considered as bio-waste, they contain much higher levels of phenolic compounds than fruits. The study aimed to investigate the antimicrobial and anticancer potential of aerial part extracts from Vaccinium myrtillus L. (V. myrtillus, VM) plants harvested at high altitudes in Armenian landscape and characterize the bioactive phytochemicals. For evaluation of antioxidant properties, chemical-based tests (total phenolic and flavonoid content, and antiradical activity in 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests) and cellular antioxidant activity (CAA) assay were applied. Genotoxicity and anticancer properties of the extract alone and in combination with fluorouracil were explored in human cancer and normal cell lines. Antibacterial properties of V. myrtillus extract alone and in combination with antibiotics, as well as their effect on proton-flux rate through cell membrane were explored on bacterial strains. The characterization of active phytochemicals was done using Liquid Chromatography-Quadrupole-Orbitrap High-Resolution Mass Spectrometry (LC-Q-Orbitrap HRMS). The V. myrtillus aerial part extract demonstrated promising antioxidant properties in all tests. The selective cytotoxic activity was documented against various cancer cell lines (human colon adenocarcinoma (HT29), human breast cancer (MCF-7) and human cervical carcinoma (HeLa)), while it did not inhibit the growth of tested human normal primary renal mixed epithelial cells (HREC) even at 10-fold higher concentrations. The extract did not have genotoxic properties in comet assay making it a potential source for the development of anticancer preparations. The investigated extract did not directly inhibit the growth of Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium) strains at up to 1 mg/mL concentration. However, V. myrtillus extract enhanced the kanamycin intake and increased its efficiency against E. coli strain. The phytochemical characterization of the extract showed the presence of different groups of phenolics. Based on obtained data, we suggest the aerial parts of the V. myrtillus plant as an alternative source of bioactive natural products for food supplements, nutraceuticals, functional foods and medicine.

Cellular antioxidant and viability efficacy of Harpephyllum caffrum peel and Syzygium guineense seed extracts

Crude phenolic extracts of indigenous sub-Saharan fruit byproducts have potent antioxidant activity. Purification of these extracts and evaluation of their antioxidant activity and viability using appropriate biological models, has, however, not yet been completed. The current study quantitatively evaluated the phenolic composition and in vitro antioxidant activity of crude and purified extracts (PE) of Harpephyllum caffrum peel (HCP) and Syzygium guineense seed (SGS), and cellular antioxidant and viability efficacy of the PE. Phenols were extracted by 60% ethanol (v/v) solvent system and purified using XAD7HP macroporous adsorption resin. The UPLC/Q-TOF-MS was used for the characterization and identification of phenols. Cellular antioxidant activity (CAA) assay was applied to assess the antioxidant efficacy and cytotoxicity of the PE. Analysis of variance of data was done using a generalized linear mixed model with fruit type, extract purity and their interaction as fixed effects. Purification increased (P ≤ 0.05) HCP total phenolic concentration (TPC) by over 200%, while SGS TPC decreased (P ≤ 0.05) by 20%. Regardless of fruit type, PE had higher (P ≤ 0.05) quantities of proanthocyanidins and anthocyanins than crude extracts. The HCP-PE had greater (P ≤ 0.05) in vitro antioxidant activity than the other extracts (P ≤ 0.05). Cellular antioxidant activity of HCP-PE was comparable to that of sodium metabisulphite (P > 0.05). Also, HCP-PE had lower (P ≤ 0.05) cytotoxic effects than SGS and sodium metabisulphite with increasing antioxidant concentration. It was concluded that HCP-PE is a potential source of safe natural antioxidants for nutraceutical applications.

"FORMATION OF HYDROGELS FROM ANTIOXIDATIVE SYNTHETIC HYDROXYCINNAMATE ESTER CONJUGATES BASED ON CORN BRAN ARABINOXYLAN "

Corn bran arabinoxylan (CAX) was extracted from corn bran and modified through conjugating to four hydroxycinnamic acids (HAs), namely ferulic acid (FA), p-coumaric acid (p-CA), caffeic acid (CA), and sinapic acid (SA). These HA arabinoxylan esters (HA-CAX-n) exhibited analogous n-degrees of substitution (DS), FA-CAX-0.31, p-CA-CAX-0.32, CA-CAX-0.32, and SA-CAX-0.31. The antioxidant capacity was evaluated by a chemical method – an oxygen radical antioxidant capacity (ORAC) assay – and cellular models – a cellular antioxidant activity (CAA) assay to healthy human skin fibroblasts (HSF). The results of the various analyses were comparable, demonstrating the order of antioxidant capability was: CA-CAX-0.32 > SA-CAX-0.31 > FA-CAX-0.31 > p-CA-CAX-0.32. HA-CAX-n gels with different crosslinking ability were obtained with the concentration of 0.5% (w/v) during oxidative gelation by laccase, SA-CAX-0.31 can form gels most easily, followed by FA-CAX-0.31, CA-CAX-0.32 and p-CA-CAX-0.32.

Comparative Analysis of Phytochemical Profiles and Antioxidant Activities between Sweet and Sour Wampee (Clausena lansium) Fruits.

As a local medicine and food, wampee fruit, with abundant bioactive compounds, is loved by local residents in Southern China. Titratable acid (TA), total sugar (TS), and total phenolic and flavonoid contents were detected, and phytochemical profiles and cellular antioxidant activities were analyzed by the HPLC and CAA (cellular antioxidant activity) assay in five sweet wampee varieties and five sour wampee varieties. Results showed that the average TS/TA ratio of sweet wampee varieties was 29 times higher than sour wampee varieties, while TA content was 19 times lower than sour wampee varieties. There were much lower levels of total phenolics, flavonoids, and antioxidant activities in sweet wampee varieties than those in sour wampee varieties. Eight phytochemicals were detected in sour wampee varieties, including syringin, rutin, benzoic acid, 2-methoxycinnamic acid, kaempferol, hesperetin, nobiletin, and tangeretin, while just four of them were detected in sweet wampee varieties. Syringin was the only one that was detected in all the sour wampee varieties and was not detected in all sweet wampee varieties. Correlation analysis showed significant positive correlations between TA with phenolics, flavonoids, and total and cellular (PBS wash) antioxidant activities, while there were significant negative correlations between TS/TA with phenolic and cellular (no PBS wash) antioxidant activities. This suggested that the content of titratable acid in wampee fruit might have some relationship with the contents of phenolics and flavonoids. Sour wampee varieties should be paid much attention by breeders for their high phytochemical contents and antioxidant activities for cultivating germplasms with high health care efficacy.

Assessment of the phytochemical profile and antioxidant activities of eight kiwi berry (Actinidia arguta (Siebold & Zuccarini) Miquel) varieties in China.

The kiwi berry (Actinidia arguta) is a new product on the market that expanding worldwide acceptance and consumption. This widespread interest has created an increasing demand to identify the nutritional and health benefits of kiwi berry. Many studies are being actively conducted to investigate the composition and health‐promoting effects of kiwi berry. In this study, the phytochemical content of free and bound fractions of eight kiwi berry varieties were systematically investigated in order to better understand the potential of this superfood crop. Nine phenolic monomers were identified and quantified by ultrahigh‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry and ultrahigh‐performance liquid chromatography‐PAD. Antioxidant activity was further determined via peroxyl radical scavenging capacity and cellular antioxidant activity assays. The free extracts had higher phytochemical contents and antioxidant activities than the corresponding bound extracts among the eight kiwi berry varieties. Bivariate Pearson's and multivariate correlation analyses showed that antioxidant activities were most related to the total phenolic, flavonoid, vitamin C, and phenolic acids contents. The results provide a theoretical basis for the selection of kiwi berry varieties and the utilization of functional foods.

Heat-induced gel properties and gastrointestinal digestive properties of egg white produced by hens fed with selenium-enriched yeast

The aim of this study was to investigate the effect of selenium enrichment on the gel properties and gastrointestinal digestive properties of egg white. Results of texture profile analysis, scanning electron microscopy and differential scanning calorimetry showed that the heat-induced gel of selenium-enriched egg white (EW-2) exhibited higher gel strength, smoother microstructure, and higher thermal denaturation temperature than ordinary egg white (EW-1), which might be due to the change of ovomucin and ovotransferrin content. The gastrointestinal digestive products of the EW-2 protein contained a higher proportion of small peptides and more free amino acids than those of EW-1. Results of oxygen radical absorbance capacity and cellular antioxidant activity assays indicated that digestive products of the EW-2 protein exhibited stronger antioxidant activity than those of the EW-1 protein. In summary, Se enrichment improved heat-induced gel properties of egg white, and promoted the gastrointestinal digestion of egg white protein.

The Antioxidant Effect of Small Extracellular Vesicles Derived from Aloe vera Peels for Wound Healing.

Extracellular vesicles (EVs) derived from plants have emerged as potential candidates for cosmetic and therapeutic applications. In this study, we isolated EVs from Aloe vera peels (A-EVs) and investigated the antioxidant and wound healing potential of A-EVs. A-EVs were isolated by ultracentrifugation and tangential flow filtration and were characterized using transmission electron microscopy, nanoparticle tracking analysis. The cytotoxicity and cellular uptake of A-EVs were investigated by WST-1 assay and flow cytometry. The antioxidant effect of A-EVs was evaluated by superoxide dismutase (SOD) activity assay and cellular antioxidant activity (CAA) assay. The wound healing potential was assessed by in vitro scratch assay using human keratinocytes (HaCaT) and fibroblasts (HDF). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and their associated genes was analyzed by quantitative RT-PCR. A-EVs displayed a round shape and had diameters from 50 to 200nm. A-EVs showed good cytocompatibility on human skin cells and were internalized into HaCaT cells via clathrin-, caveolae-mediated endocytosis, and membrane fusion. The SOD activity and CAA assays exhibited that A-EVs had antioxidant activity and reduced intracellular ROS levels in H2O2-treated HaCaT cells in a dose-dependent manner. A scratch assay showed that A-EVs enhanced the migration ability of HaCaT and HDF. Moreover, A-EVs significantly upregulated the mRNA expression of Nrf2, HO-1, CAT, and SOD genes in H2O2-treated HaCaT cells. Our findings reveal that A-EVs could activate the antioxidant defense mechanisms and wound healing process via the Nrf2 activation. Overall results suggest that the A-EVs are promising as a potential agent for skin regeneration.

Phenolics Profile and Antioxidant Activity Analysis of Kiwi Berry (Actinidia arguta) Flesh and Peel Extracts From Four Regions in China.

The kiwi berry (Actinidia arguta) has been widely studied because of its rich phenolic, flavonoid, and vitamin C contents. Numerous reports have demonstrated that fruit peels contain higher phenolic content and antioxidant activity than that of flesh. In this study, the phytochemical content and antioxidant activities of peel and flesh extracts of six kiwi berries were analyzed from four regions (namely, Dandong, Benxi, Taian, and Tonghua) in China. The antioxidant activity was determined using the peroxyl radical scavenging capacity (PSC) and cellular antioxidant activity (CAA) assays. The phenolic, flavonoid, and vitamin C contents of kiwi berry peel were 10.77, 13.09, and 10.38 times richer than that of kiwi berry flesh, respectively. In addition, the PSC and CAA values of kiwi berry peel were higher than those of kiwi berry flesh. The analysis of the separation and contents of phenolics were performed by the high-performance liquid chromatography (HPLC)-diode-array detectormass spectrometry/mass (DAD-MS/MS) system, and the results illustrated that protocatechuic acid, caffeic acid, chlorogenic acid, and quinic acid were the major phenolic compounds. In conclusion, this study indicated that kiwi berry peel contains a rich source of antioxidants. These data are of great significance for the full development and utilization of kiwi berries in these four regions of China to produce nutraceutical and functional foods.

Examining the Performance of Two Extraction Solvent Systems on Phenolic Constituents from U.S. Southeastern Blackberries

Two common extraction solvent systems, namely acidified aqueous methanol and acidified aqueous acetone, were used to extract blackberry phenolics, and the antioxidant properties of the recovered extracts were compared. The crude extracts were fractionated into low- and high-molecular-weight phenolics by Sephadex LH-20 column chromatography. The hydrophilic-oxygen radical absorbance capacity (H-ORACFL), ferric reducing antioxidant power (FRAP), and the cellular antioxidant activity (CAA) assays were employed as indices to assess antioxidant capacity of the extracts and their respective fractions. The methanolic solvent system displayed a greater efficiency at extracting anthocyanin and flavonol constituents from the blackberries, while the acetonic solvent system was better at extracting flavan-3-ols and tannins. Anthocyanins were the dominant phenolic class found in the blackberries with 138.7 ± 9.8 mg C3G eq./100 g f.w. when using methanol as the extractant and 114.6 ± 3.4 mg C3G eq./100 g f.w. when using acetone. In terms of overall antioxidant capacity of blackberry phenolics, the acetonic solvent system was superior. Though present only as a small percentage of the total phenolics in each crude extract, the flavan-3-ols (42.37 ± 2.44 and 51.44 ± 3.15 mg/100 g f.w. in MLF and ALF, respectively) and ellagitannins (5.15 ± 0.78 and 9.31 ± 0.63 mg/100 g f.w. in MHF and AHF, respectively) appear to account for the differences in the observed antioxidant activity between the two solvent systems.

Pacific Dulse Protein: A Promising Source of Bioactive Peptides

Abstract Objectives Pacific dulse (Devaleraea mollis) is a protein-rich seaweed in the Pacific Northwest and increasingly being cultivated as food resource. Our previous work showed that dietary supplementation of dulse exerted beneficial metabolic effects to the high-fat fed mouse model. This study aims to evaluate the potential of the seaweed protein from dulse as a precursor for bioactive peptide (BAP) generation. Methods The potential of Pacific dulse protein as BAP precursor was assessed using in vitro and in silico approaches. Hydrolysates were prepared from the hydrated freeze-dried protein isolates which were digested using commercial proteases. These were used for the in vitro screening for antioxidant and anti-inflammatory activities as well as the inhibition of Angiotensin-converting enzyme 1 (ACE-1), Renin, and Dipeptidyl peptidase IV (DPPIV). Peptides in the hydrolysates that exhibit the highest activities were identified using de novo sequencing. Identified peptides were shortlisted based on their potential to be bioactive using Peptide Ranker and specific bioactivity scores were obtained using in silico platforms such as iDPPIV-SCM and PreAIP. To determine whether the consumption of the seaweed protein may help give rise to some bioactive peptides, annotated protein sequences of the seaweed material were obtained from UniProtKB and were subjected to in silico digestion using pepsin, chymotrypsin, and trypsin through BIOPEP. Bioactivity indices of the generated peptide fragments were recorded as predictive values. Results In silico digestion showed that dulse proteins can give rise to ACE-1, DPP-IV, and renin inhibitors, as well as antioxidant peptides. Hydrolysates exerted inhibitory effects on human ACE-1, DPPIV, and renin in vitro. ABTS radical scavenging assay and cellular antioxidant activity assay in human hepatocytes indicated the strong antioxidant potential of dulse BAP. Peptides identified through de novo sequencing had high predictive scores for DDPIV inhibition and anti-inflammatory potential. Conclusions Pacific dulse proteins is a promising precursor for the generation of BAPs that may exert beneficial health activities and support metabolic health improvement. Funding Sources Oregon State University Agricultural Research Foundation.

Quercetin delivery characteristics of chitosan nanoparticles prepared with different molecular weight polyanion cross-linkers

The aim of the study was to investigate the effects of cross-linkers on quercetin (QUE) absorption characteristics of QUE-loaded chitosan nanoparticles (CS-NPs). CS-NPs (461.2–482.7 nm) were prepared by ionic gelation at pH 3.5 using tripolyphosphate (367.9 Da), dextran sulfate (>15 kDa), arabic gum (AG, >250 kDa), or hyaluronic acid (HA, >1000 kDa). Mucoadhesion and cell permeation of QUE were significantly increased by positive charged CS-NPs due to interactions with negatively charged mucosal layer. Moreover, CS-AG and CS-HA NPs prepared with relatively higher MW cross-linkers exhibited significantly higher adhesion and permeation than the others. These results were verified by a cellular antioxidant activity assay; CS-AG (137.5 unit) and CS-HA NPs (126.5 unit) showed significantly higher activities after internalization into Caco-2 cells. Therefore, encapsulation within CS-NPs prepared using high MW cross-linkers such as AG and HA is found to be potentially valuable techniques for improving the QUE absorption.

Therapeutic Potential of Tuna Backbone Peptide and Its Analogs: An In Vitro and In Silico Study.

Tuna backbone peptide (TBP) has been reported to exert potent inhibitory activity against lipid peroxidation in vitro. Since this bears relevant physiological implications, this study was undertaken to assess the impact of peptide modifications on its bioactivity and other therapeutic potential using in vitro and in silico approach. Some TBP analogs, despite lower purity than the parent peptide, exerted promising antioxidant activities in vitro demonstrated by ABTS radical scavenging assay and cellular antioxidant activity assay. In silico digestion of the peptides resulted in the generation of antioxidant, angiotensin-converting enzyme (ACE), and dipeptidyl peptidase-IV (DPPIV) inhibitory dipeptides. Using bioinformatics platforms, we found five stable TBP analogs that hold therapeutic potential with their predicted multifunctionality, stability, non-toxicity, and low bitterness intensity. This work shows how screening and prospecting for bioactive peptides can be improved with the use of in vitro and in silico approaches.

Seasonality Modulates the Cellular Antioxidant Activity and Antiproliferative Effect of Sonoran Desert Propolis.

The main chemical composition and pharmacological potential of propolis from arid and semi-arid regions of the Sonoran Desert have been previously reported. Caborca propolis (CP), from an arid zone of the Sonoran Desert, has shown a polyphenolic profile that suggests a mixed plant origin, presenting poplar-type markers, as well as a 6-methoxylated flavonoid, xanthomicrol, characteristic of Asteraceae plants. In addition, CP has shown significant antioxidant properties and antiproliferative activity on cancer cells. In this study, we analyzed the influence of collection time on the chemical constitution, antiproliferative activity and protective capacity of CP against reactive oxygen species (ROS), by using HPLC–UV–diode array detection (DAD) analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-Dimethyltetrazoliumbromide (MTT) and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assays, as well as cellular antioxidant activity (CAA) assay on murine B-cell lymphoma M12.C3.F6 cells. HPLC–UV–DAD analyses of seasonally collected CP (one-year period) revealed quantitative differences among the most abundant CP constituents: pinocembrin, galangin, chrysin and pinobanksin-3-O-acetate. Though all seasonal samples of CP induced an antiproliferative effect in M12.C3.F6 cells, CP from autumn showed the highest inhibitory activity (IC50: 5.9 ± 0.6 µg/mL). The DPPH assay pointed out that CP collected in autumn presented the highest antioxidant potential (IC50: 58.8 ± 6.7 µg/mL), followed by winter (65.7 ± 12.2 µg/mL) and spring (67.0 ± 7.5 µg/mL); meanwhile, the summer sample showed a lesser antioxidant capacity (IC50: 98.7 ± 2.5 µg/mL). The CAA assay demonstrated that CP induced a significant protective effect against ROS production elicited by H2O2 in M12.C3.F6 cells. Pretreatment of M12.C3.F6 cells with CP from spring and autumn (25 and 50 µg/mL for 1 h) showed the highest reduction in intracellular ROS induced by H2O2 (1 and 5 mM). These results indicate that the antiproliferative effect and cellular antioxidant activity of CP are modulated by quantitative fluctuations in its polyphenolic profile due to its collection time.

Effect of pH and Heat Treatment on the Antioxidant Activity of Egg White Protein-Derived Peptides after Simulated In-Vitro Gastrointestinal Digestion.

The study aimed to analyze pH and heat treatment’s effect in modulating the release of peptides with antioxidant activity after simulated gastrointestinal (GI) digestion of Egg white powder (EWP). EWP samples with neutral (EWPN) and alkaline (EWPA) pH were heat-treated at 20, 60, and 90 °C and analyzed for protein aggregation, solubility, and GI digestibility. Heat treatment decreased solubility and induced protein aggregation, which was higher for EWPN as compared to EWPA. The unfolding of EWPA proteins at 60 °C exhibited a higher GI digestibility and antioxidant activity via Oxygen Radical Absorbance Capacity (ORAC) assay as compared to EWPN. Interestingly, a reverse trend was observed in the cellular antioxidant assay, and the GI-digest of EWPN exhibited a higher antioxidant activity. The LC-MS/MS analysis are in concordance with cellular antioxidant activity assay and showed a higher intensity for peptides with potential antioxidant activity in the GI-digest of EWPN. The results indicate that heat treatment but not the pH is a critical factor in improving the protein digestibility and releasing peptides with antioxidant activity after GI digestion.

Callus Culture of Thai Basil Is an Effective Biological System for the Production of Antioxidants.

Thai basil is a renowned medicinal plant and a rich source of bioactive antioxidant compounds with several health benefits, with actions to prevent of cancer, diabetes and cardiovascular disease. Plant cell and tissue culture technologies can be routinely established as an important, sustainable and low-cost biomass source to produce high-value phytochemicals. The current study aimed at developing an effective protocol to produce Thai basil leaf-derived callus cultures with sustainable and high production of biomass and antioxidants as an alternative of leaves production. MS basal medium with various concentrations of plant growth regulators (PGRs) compatible with nutraceutical applications (i.e., gibberellic acid (GA3) and 6-benzylaminopurine (BAP) either alone or in combination with naphthalene acetic acid (NAA)) were evaluated. Among all tested PGRs, the combination BAP:NAA (5 mg/L:1 mg/L) yields the maximum biomass accumulation (fresh weight (FW): 190 g/L and dry weight (DW): 13.05 g/L) as well as enhanced phenolic (346.08 mg/L) production. HPLC quantification analysis indicated high productions of chicoric acid (35.77 mg/g DW) and rosmarinic acid (7.35 mg/g DW) under optimized callus culture conditions. Antioxidant potential was assessed using both in vitro cell free and in vivo cellular antioxidant assays. Maximum in vitro antioxidant activity DPPH (93.2% of radical scavenging activity) and ABTS (1322 µM Trolox equivalent antioxidant capacity) was also observed for the extracts from callus cultures grown in optimal conditions. In vivo cellular antioxidant activity assay confirmed the effective protection against oxidative stress of the corresponding extract by the maximum inhibition of ROS and RNS production. Compared to commercial leaves, callus extracts showed higher production of chicoric acid and rosmarinic acid associated with higher antioxidant capacity. In addition, this biological system also has a large capacity for continuous biomass production, thus demonstrating its high potential for possible nutraceutical applications.

Identification of Six Flavonoids as Novel Cellular Antioxidants and Their Structure-Activity Relationship.

This study is aimed at determining the relationship of flavonoid structures to their chemical and intracellular antioxidant activities. The antioxidant activities of 60 flavonoids were investigated by three different antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, oxygen radical absorption capacity (ORAC), and cellular antioxidant activity (CAA) assays. The result showed 6 flavonoids as good cellular antioxidants evaluated for the first time. The cellular antioxidant activities of compounds 7-methoxy-quercetin, 3-O-methylquercetin, 8-hydroxy-kaempferol, quercetin-3-O-α-arabinofuranose, kaempferol-7-O-glucopyranoside, and luteolin6-C-glucoside were linked with the upregulation of antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase). A structure-activity relationship suggested that 2,3-double bond, 4-keto groups, 3′,4′-catechol structure, and 3-hydroxyl in the flavonoid skeleton played important roles in the antioxidant behavior. Furthermore, the cell proliferative assay revealed a low cytotoxicity for 3-O-methylquercetin. The present results provide valuable information for the dietary application of flavonoids with different structures for high antioxidant.