Chemical characterization and chemo-protective activity of cranberry phenolic powders in a model cell culture. Response of the antioxidant defenses and regulation of signaling pathways

Oxidative stress and reactive oxygen species (ROS)-mediated cell damage are implicated in various chronic pathologies. Emerging studies show that polyphenols may act by increasing endogenous antioxidant defense potential. Cranberry has one of the highest polyphenol content among commonly consumed fruits. In this study, the hepato-protective activity of a cranberry juice (CJ) and cranberry extract (CE) powders against oxidative stress was screened using HepG2 cells, looking at ROS production, intracellular non-enzymatic and enzymatic antioxidant defenses by reduced glutathione concentration (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) activity and lipid peroxidation biomarker malondialdehyde (MDA). Involvement of major protein kinase signaling pathways was also evaluated. Both powders in basal conditions did not affect cell viability but decreased ROS production and increased GPx activity, conditions that may place the cells in favorable conditions against oxidative stress. Powder pre-treatment of HepG2 cells for 20 h significantly reduced cell damage induced by 400 μM tert-butylhydroperoxide (t-BOOH) for 2 h. Both powders (5–50 μg/ml) reduced t-BOOH-induced increase of MDA by 20% (CJ) and 25% (CE), and significantly reduced over-activated GPx and GR. CE, with a significantly higher amount of polyphenols than CJ, prevented a reduction in GSH and significantly reduced ROS production. CJ reversed the t-BOOH-induced increase in phospho-c-Jun N-terminal kinase. This study demonstrates that cranberry polyphenols may help protect liver cells against oxidative insult by modulating GSH concentration, ROS and MDA generation, antioxidant enzyme activity and cell signaling pathways.     

Hydroxytyrosyl acetate contributes to the protective effects against oxidative stress of virgin olive oil

The effects of virgin olive oil phenols, hydroxytyrosyl acetate (HTy-Ac) and hydroxytyrosol (HTy), on cell integrity and steady-state values of cellular redox status were assessed in HepG2 cells, as well as their potential protective effects against oxidative stress induced by tert-butyl hydroperoxide (t-BOOH). Direct treatment for 20 h with 0.5-10 μM HTy or HTy-Ac reduced ROS generation and increased glutathione peroxidase activity at the higher concentrations. Furthermore, after t-BOOH exposure, pretreatment with HTy-Ac and HTy for 2 or 20 h counteracted cell viability damage from 1 μM, counterbalanced reduced glutathione levels from 0.5 μM, protected against lipid peroxidation from 0.5 μM, decreased ROS generation from 1 μM as well as antioxidant enzyme activities from 1 μM. All these changes were statistically significant.HTy-Ac presents antioxidative stress protective effects at physiological concentrations similar to or even slightly higher than that of HTy, thus contributing to the protective role of virgin olive oil.     

Antiproliferative and cytoprotective activities of a phenolic-rich juice in HepG2 cells

Antioxidant phytochemicals, especially the phenolics found in fruits and vegetables, have been proposed as the major bioactive compounds providing the health benefits associated with diets rich in plant-foods. In the present study, we aimed to evaluate the modulatory effect of a functional juice rich in phenolic compounds on cell proliferation and on the protection from oxidative stress caused by tert-butylhydroperoxide (t-BOOH) and hydrogen peroxide (H₂O₂) in HepG2 cells. Cell growth and cell viability were evaluated by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation was measured as the thiobarbituric acid reacting substances (TBARS) assay. Glutathione peroxidase (GPx) and glutathione-s-transferase (GST) activities were assayed by colorimetric assays. In order to elucidate the action mechanisms of juice phenolics, the protective effects of the juice were compared to those of the radical scavenger N,N′-diphenyl-1,4-phenylene-diamine (DPPD) and the iron chelator o-phenanthroline (o-phe). Cell proliferation was inhibited in a dose-dependent manner by juice extracts. Sub-toxic concentrations of phenolics (2–30 μg/mL of medium) increased cell survival and a decreased lipid peroxidation after exposure to both H₂O₂ and t-BOOH. Both juice and o-phe decreased the t-BOOH-induced GPx activation, but not that of GST. DPPD suppressed the t-BOOH-induced GST activation but not that of GPx. Our results support the efficacy of natural phenolics from grapes and berries in offering protection against oxidative stress. The possible role of an iron chelating mechanism, potentially of biological relevance, is proposed as being partially responsible for the health benefits provided by phenolic-rich processed foods.     

Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells

The objective of this study was to investigate the intracellular reactive oxygen species (ROS) scavenging activities and antioxidant enzyme regulating capacities of corn gluten peptide fractions (CPFs) in HepG2 cells. A cellular antioxidant activity (CAA) assay was used to assess their antioxidant activities and revealed that both CPF1 (molecular weight < 1 kDa) and CPF2 (molecular weight between 1 and 3 kDa) exhibited high cellular antioxidant activities with EC₅₀ values of 2.85 ± 0.19 mg/mL and 5.05 ± 0.32 mg/mL, respectively. Both CPFs also exhibited cytoprotective effects and intracellular ROS scavenging activities in HepG2 cells subjected to oxidative stress by oxidation with H₂O₂. In addition, at concentrations of 2.50 mg/mL, the CPFs increased the activity levels of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), as well as the total glutathione (GSH) levels in oxidized HepG2 cells (from 86.54% to 114.14% (CPF1) or 109.72% (CPF2) for SOD activity; from 71.91% to 107.64% (CPF1) or 106.50% (CPF2) for CAT activity; from 70.52% to 103.01% (CPF1) or 104.10% (CPF2) for GR activity; and from 81.39% to 114.00% (CPF1) or 108.82% (CPF2) for total GSH levels). These results suggested that both CPF1 and CPF2 exhibited positive effects on the activities of the intracellular antioxidant enzymes SOD, CAT and GR, as well as on the total GSH levels in HepG2 cells under conditions of oxidative stress. Furthermore, size exclusion gel chromatography and MALDI-TOF/TOF mass spectrometry revealed that the molecular weights of the antioxidant peptides in CPF1 were between 500 Da to 900 Da, and a novel antioxidant peptide consisting of GLLLPH (Gly-Leu-Leu-Leu-Pro-His) was identified in CPF1.     

Antioxidant potential of mate tea (Ilex paraguariensis) in type 2 diabetic mellitus and pre-diabetic individuals

The effect of long-term mate tea (Ilex paraguariensis) consumption on the oxidative stress biomarkers of type 2 diabetic mellitus and pre-diabetic individuals was investigated. A 60-day intervention pilot study where 11 T2DM and 11 pre-diabetic volunteers ingested 1 L/day of mate tea was carried out. Ferric reducing antioxidant power (FRAP), erythrocyte reduced glutathione (GSH), serum lipid hydroperoxides (LOOH) using ferrous oxidation-xylenol orange (FOX2), advanced glycation end-products (AGEs), and glycaemic and lipid profiles were assessed at baseline and after 20, 40, and 60 days of intervention. Mate tea consumption promoted a significant increase of GSH concentration and a decrease of LOOH levels in T2DM and pre-diabetic subjects. In addition, GSH concentration was inversely correlated with LOOH in T2DM and pre-diabetic individuals and with AGEs in T2DM subjects. No correlations between glycaemic and lipid profiles with oxidative stress biomarkers were found. Thus, ingestion of mate tea attenuated oxidative stress in T2DM and pre-diabetic subjects, which may prevent diabetes complications.     

Comparison of protective effects of three varieties of sugarcane leaves on oxidative stress in Clone 9 cells

The protective effects of water extracts of sugarcane (Saccharum officinarum L.) leaves among three varieties, including 28NG256, wild type, and ROC10, on t-BHP-induced oxidative stress in Clone 9 cells were systematically compared. Among these three varieties, 28NG256 showed the highest protective effect against 0.2 mM t-BHP-induced oxidative stress in Clone 9 cells. In addition, 28NG256 displayed higher inhibitory effects on ROS generation than wild type and ROC10. Moreover, 28NG256 showed higher positive regulated GSH levels and antioxidant enzymes as well as higher protective potential against cell death by inhibiting caspase-3 activity and mitochondrial membrane depolarization. Chlorogenic and caffeic acids present in 28NG256 decreased significantly the generation of ROS, which may partly be responsible for the effect of Clone 9 cell growth. Thus, 28NG256, among the three varieties studied, showed the most protective effects against t-BHP-induced oxidative stress in Clone 9 cells.     

Protective effects of phenolic constituents from Cytisus multiflorus,Lamium album L. and Thymus citriodorus on liver cells

The present study investigated the antioxidant and cytoprotective effects of purified ethanolic extracts of Cytisus multiflorus, Lamium album L. and Thymus citriodorus plants. These extracts showed high antioxidant activity in DPPH and reducing power assays. Using a model of chemical stress induced by potassium dichromate (DK) in human hepatoblastoma HepG2 cells, 50 μg/mL of C. multiflorus, L. album and T. citriodorus extracts decreased the rate of reactive oxygen species (ROS) production by 35%, 26% and 20%, respectively, when exposed to 25 μM of DK. This effect was also observed for the treatment of cells with individual polyphenolic compounds determined in the extracts, or with mixtures prepared with individual polyphenolic compounds simulating the phenolic composition of the extracts. Additionally, the purified ethanolic extracts and the prepared polyphenolic mixtures showed a cytoprotective effect against DK-induced toxicity. The overall results emphasize the contribution of polyphenols in antioxidant and cytoprotective properties of the studied plants.     

Hepatoprotection using sweet orange peel and its bioactive compound,hesperidin, for CCl4-induced liver injury in vivo

The hepatoprotective effect of water extracts of sweet orange (Citrus sinensis) peel (WESP) and its biological compound, hesperidin (HD), on oxidative stress in vivo, were investigated. HD was the major compounds among the ten compounds identified using HPLC-DAD and HPLC-MS/MS analysis. Oral administration of WESP to rats at 10 and 100 mg/kg bw for 28 consecutive days before a single dose of CCl₄ (2 ml/kg bw) demonstrates a significant protective effect by lowering the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and by improving the histological architecture of the rat liver. WESP attenuated oxidative stress by increasing the content of hepatic glutathione (GSH), and by a dramatic increase in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). WESP induced a significant CYP2E1 activity, which suggests that WESP may be a substrate of CYP2E1. WESP at a dose of 1.0 mg/kg bw and HD at 0.1 mg/kg bw did not sustain the protective effect against oxidative stress, in vivo. This study demonstrated that citrus peel protects rat liver from CCl₄-induced injury by attenuating hepatic oxidative stress, which suggests that WESP can be used as a therapeutic antihepatotoxic agent for the treatment of hepatic injury.     

Chemo-protective activity and characterization of phenolic extracts from Corema album

There is currently substantial interest in the cyto-protective effects of natural compounds against oxidative stress and in studying of the defense mechanisms involved. Corema album fruit is an edible berry consumed along the Atlantic littoral of the Iberian Peninsula. The aim of this study was to characterize the phenolic composition and evaluate the chemo-protective effects against oxidative stress of three phenolic extracts from this fruit on liver cells.Characterization of phenolic compounds, achieved by liquid chromatography and diode-array, mass spectrometry and electrospray ionization-time of flight-mass spectrometry detection, showed a main fraction of hydroxycinnamic acids. Liver HepG2 cells were treated with 1–40 μg/mL of the extracts and exposed to oxidative stress chemically induced. Cell viability, reactive oxygen species (ROS), reduced glutathione (GSH), antioxidant enzymes and biomarkers of oxidative damage were evaluated.Treatment of HepG2 cells with the extracts partially prevented ROS increase, GSH depletion, antioxidant enzymes over-activity and oxidative damage to proteins and lipids induced by stress. The results support the traditional use of C. album as a medicinal plant and suggest that inclusion of its berries in the diet would contribute to the protection afforded by fruits, vegetables and plant-derived beverages against oxidative stress related diseases.     

Effect of apparent density of sliced food particles on the efficiency of pulsed electric field treatment

This work discusses the effects of pulsed electric field (PEF) (electric field strength, E, 100–1000 V; pulse duration, t_p, 100 μs) on disintegration of potato slices treated between parallel plate electrodes by using a laboratory compression chamber equipped with a PEF-treatment system. The apparent density of slices (bed of particles), ρ, was varied within 0.313 g/cm³ and 1 g/cm³. The electrical conductivity σ(ρ) of the packing of slices versus volume fraction of particles ϕ was approximated by the percolation law σ ∝ (φ − φ_c)^t, where ϕ_c ≈ 0.290, t = t_i ≈ 0.46 and t = t_d ≈ 1.39 for the intact and completely damaged tissues, respectively. The impact of electric field strength and apparent density of slices on PEF-induced damage kinetics was studied. The more accelerated kinetics of damage was observed for more dense packing of slices. The approximated relation between the applied, E, and effective, E_e, electric field strengths accounting for the σ(ρ) dependence was derived.Industrial relevanceThe practical applications of PEF treatment (e.g., pressing, drying, extraction, etc.) demand operations with slices of food particles. In this study, research towards the electroporation efficiency of PEF applied to the porous packing of sliced food particles is provided.     

High molecular weight persimmon tannin is a potent antioxidant both ex vivo and in vivo

The antioxidant activities of high molecular weight persimmon condensed tannin (HMWPT) were evaluated in an ex vivo tissue system and in vivo. Addition of HMWPT to mouse liver homogenate protected the samples against auto-peroxidation or H₂O₂- or Fe²⁺/ascorbic acid-induced lipid peroxidation. The IC₅₀ values for HMWPT were 4.32 ± 0.20 μg/mL (auto-oxidation), 1.36 ± 0.40 μg/mL (H₂O₂-induced peroxidation) or 0.20 ± 0.09 mg/mL (Fe²⁺/ascorbic acid-induced peroxidation). Mice were oxidatively stressed with bromobenzene to test the antioxidant activity of HMWPT in vivo. An oral dose of HMWPT at 200 or 400 mg/kg HMWPT significantly (P < 0.01) prevented the bromobenzene-induced decrease in serum and liver superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and decreased liver malondialdehyde levels in bromobenzene-treated mice (P < 0.01). The results suggest that dietary HMWPT may provide protection from oxidative damage both ex vivo and in vivo.     

Bioactivity of ellagic acid-, lutein- or sesamol-enriched meat patties assessed using an in vitro digestion and Caco-2 cell model system

Interest exists in the manufacture of functional meat products whereby synthetic antioxidants are replaced with naturally-sourced compounds. Therefore the aim of this study was to determine the bioactivity of pork and beef patties containing ellagic acid (600 μg/g), lutein (200 μg/g) or sesamol (500 μg/g). Cooked pork and beef patties were subjected to an in vitro digestion procedure and the resultant micelles were added to human intestinal Caco-2 cells. Supplementation with micelles from lutein-enriched pork patties protected (P < 0.05) against H₂O₂-induced cell injury whereas the presence of control beef, lutein-enriched beef or ellagic acid-enriched beef, at levels ?20% (v/v), enhanced (P < 0.05) oxidant-induced cytotoxicity. None of the pork patties significantly modulated cellular glutathione content. Micelles from all the enriched pork patties significantly protected against H₂O₂-induced DNA damage. In conclusion, the cytoprotective and genoprotective effects of ellagic acid, lutein, and sesamol, when incorporated into meat systems, depend greatly on the food matrix.     

Cyanidin-3-glucoside,nutritionally important constituents and in vitro antioxidant activities of Santalum album L. berries

Anthocyanins are ubiquitous plant pigments. They are hydrophilic, and have high tinctorial value hence, used as natural food colorant. The main aim of this study was characterization of Santalum album L. berries for its pigment content, nutritionally important phytoconstituents and functional attributes. Major pigment identified was cyanidin-3-glucoside (0.21% FW) as confirmed by spectral characteristics including LC–MS. Nutrients such as total carbohydrates (8.5), proteins (5.8), free amino acids (0.8 g), oil (1.5 g), ascorbic acid (1.5 μg), tocopherols (0.3 mg) and niacin (5.2 mg) per 100 g berries were determined along with total soluble solids (13.4°Brix) and phenols (3.1 mg GAE/g). Reducing power and DPPH· scavenging assays showed highest activity in methanol extract, which also efficiently protected bleaching of β-carotene (EC₅₀ 285 μg/mL) in β-carotene–linoleate model. Pigment rich crude extract was not toxic to HepG2 cells during 24 h exposure, whereas cyanidin-3-glucoside was cytotoxic (IC₅₀ 0.1 μg/mL).     

The in vivo antioxidant and antifibrotic properties of green tea (Camellia sinensis,Theaceae)

The in vivo antioxidant and antifibrotic properties of green tea (Camellia sinensis, Theaceae) were investigated with a study of carbon tetrachloride (CCl₄)-induced oxidative stress and hepatic fibrosis in male ICR mice. Oral administration of green tea extract at doses of 125, 625 and 1250 mg/kg for 8 weeks significantly reduced (p < 0.05) the levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyls in the liver by at least 28% compared with that was induced by CCl₄ (1 mL/kg) in mice. Moreover, green tea extract administration significantly increased (p < 0.05) the activities of catalase, glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) in the liver. Our study found that oral administration of green tea extract prevented CCl₄-induced hepatic fibrosis, as evidenced by a decreased hydroxyproline level in the liver and a reduced incidence of hepatic fibrosis by histological observations. These results indicate that green tea exhibits potent protective effects against CCl₄-induced oxidative stress and hepatic fibrosis in mice by inhibiting oxidative damage and increasing antioxidant enzyme activities.     

Antioxidative and anti-inflammatory effects of polyphenol-rich litchi (Litchi chinensis Sonn.)-flower-water-extract on livers of high-fat-diet fed hamsters

Gentisic acid and epicatechin are two major compounds in phenolic acids and flavonoids of litchi-flower-water extracts (LFWEs), respectively. Increased (p < 0.05) serum lipids and liver size/lipid, damage/inflammatory indices, TBARS value, CRP level, MMP-9 activity, and decreased (p < 0.05) liver GSH and TEAC levels, and SOD, CAT and GSH-Px activities were observed in high-fat-diet fed hamsters compared to normal-fat-dietary hamsters. Those biochemical values of high-fat-diet fed hamsters were significant improved (p < 0.05) by drinking LFWEs. In addition, these improvements on liver damage induced by a high-fat diet were also evidenced in the histopathological examination of livers where less microvesicular steatosis and no necrotic/inflammatory cells were observed in high-fat-diet fed hamster drinking LFWEs. Therefore, protective effects of LFWEs on liver damage of high-fat-diet fed hamsters can be accounted for antioxidative properties and anti-inflammatory effects of LFWEs.     

Isolation and identification of an antiproliferative substance from fructose–tyrosine Maillard reaction products

We isolated a substance from fructose–tyrosine Maillard reaction products (MRPs) and investigated its antiproliferative effect on six human cancer cell lines. The ethyl acetate fraction of fructose–tyrosine MRPs showed a strong antiproliferative effect; this fraction was isolated and purified using silica gel column chromatography, semipreparative RP-HPLC, and recycling HPLC. The structure of the purified compound was determined using spectroscopic methods. The isolated compound was identified as 2,4-bis(p-hydroxyphenyl)-2-butenal (C₁₆H₁₄O₃, HPB242). HPB242 inhibited cell growth in a dose-dependent manner (10–80 μg/ml) on the six human cancer cell lines. The IC₅₀ values of HPB242 on the six human cancer cell lines were 17.34 μg/ml (MCF-7), 29.21 μg/ml (HCT-116), 34.57 μg/ml (H-460), 34.87 μg/ml (HepG2), 48.77 μg/ml (PC-3), and 55.83 μg/ml (MKN-45).     

Effects of a citrus based juice on biomarkers of oxidative stress in metabolic syndrome patients

Bioactive substances found in numerous foods can be successfully and safely used to modify various cellular functions and affect the oxidative stress. The aim of this study was to analyze the effect of a citrus-based juice (juice citrus (95%) with 5% of aronia extract (Aronia melanocarpa)) on biomarkers of oxidative stress in patients with metabolic syndrome compared with healthy individuals. The study comprised 20 healthy subjects and 33 patients with metabolic syndrome. Eighteen patients consumed daily 300 mL of a citrus-based juice during 6 months and 15 patients consumed 300 mL of a placebo beverage. The control group consumed a citrus-based juice (CJ). Before, and at sixth months after consuming of a citrus-based juice the following parameters were determined: 15-isoprostane F₂, 8-hydroxydeoxyguanosine (8-OHdG), reduced and oxidized glutathione (GSH/GSSH), carbonyl groups and oxidized LDL (ox-LDL). After consuming CJ during 6 months the values of 8-OHdG, carbonyl groups and LDL-ox decreased in both analyzed groups and the values of GSH/GSSH increased. Significant differences were observed in both groups. Thus consumption of citrus-based juice improved the biomarkers of oxidative stress in metabolic syndrome patients.     

In vitro antioxidant activity of a peptide isolated from Nile tilapia (Oreochromis niloticus) scale gelatin in free radical-mediated oxidative systems

In the present study, a peptide possessing antioxidant properties was isolated from Nile tilapia (Oreochromis niloticus) scale gelatin. Gelatin protein was hydrolyzed using alcalase, pronase E, trypsin and pepsin. Antioxidant efficacy of respective hydrolysates were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical anion scavenging activities. Moreover, protective effect on DNA damage caused by hydroxyl radicals generated was determined. Further, the level of reactive oxygen species (ROS) was determined using a fluorescence probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW 264.7 cells. Among hydrolysates, alcalase-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, it was further analyzed and the sequence of an active peptide present in it was identified as Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da). This peptide showed no cytotoxic effect on mouse macrophages (RAW 264.7) and human lung fibroblasts (MRC-5). In addition, it scavenged hydroxyl, DPPH and superoxide radicals at the IC₅₀ values of 7.56, 8.82 and 17.83 μM, respectively. These results suggest that the peptide derived from Nile tilapia (O. niloticus) scale gelatin acts as a candidate against oxidative stress and could be used as a potential functional food ingredient.     

Alleviation effect of heat-treated and in vitro gastrointestinal digested soymilks on AAPH-induced oxidative stress in human erythrocytes: Digested soymilks alleviate oxidative stress

To investigate the potential protective effect of raw and heat-treated soymilks after gastrointestinal digestion against chemical oxidative stress induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) on human erythrocytes, soymilk was subjected to heat treatment and in vitro gastrointestinal digestion. The inhibition rate of hemolysis, generation of reactive oxygen species (ROS), concentration of malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSH) and the enzyme activity of total superoxide dismutase (SOD) and cellular glutathione peroxidase (GPx) were evaluated as the biomarkers of oxidative status. Hemolysis of erythrocytes induced by AAPH was significantly inhibited by pretreatment with the digested raw soymilk (DRS) and digested heat-treated soymilk (DHS). Moreover, heat treatment prior to gastrointestinal digestion improved the inhibition effect of soymilk on erythrocytes hemolysis. The soymilk treated at 95 °C showed the highest inhibition rate, followed by 121 °C and 143 °C, revealed that the increase of temperature caused the decrease of hemolysis inhibition rate of DHS. Preincubation with the digested soymilks reduced the accumulation of MDA in erythrocytes, indicating the inhibition effect of the digested soymilks on lipid peroxidation. Results revealed that DRS and DHS alleviated the hemolysis of erythrocytes and lipid peroxidation resulted from oxidative stress by suppressing the accumulation of ROS, reducing the increase of SOD activity and decrease of non-enzymatic antioxidant GSH and enzymatic antioxidant GPx activity. Compared with raw soymilk, heat treatment improved the protective effect of the digested soymilk on erythrocytes against oxidative stress via enhancing the free radicals scavenging activity instead of improving the inhibition effect on the generation of free radicals.