Hepatoprotective effects of lycopene against carbon tetrachloride-induced acute liver injury in rats

Lycopene, the major carotenoid in tomatoes, is a known antioxidant that may lower oxidative stress biomarkers by a mechanism that is not fully elucidated. The intoxication of rats with carbon tetrachloride (CCl₄) resulted in significant histological hepatic degradation accompanied by a marked increase in reactive oxygen species (ROS) and in the number of apoptotic cells. The induced oxidative stress in turn results in a significant elevation of lipid peroxidation and H₂O₂ generation, together with a decrease in the concentration of reduced glutathione (GSH) and a significant reduction in activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S transferase (GST). CCl₄-intoxicated rats, pre-treated with lycopene, showed strongly reduced cell damage and ROS generation. The level of markers for hepatic integrity in lycopene pre-treated rats was close to the controls in the absence of CCl₄ treatment, indicating the protective effect of lycopene pre-treatment. In the same way, lycopene pre-treated rats significantly increased SOD, CAT, GPx, GST activities and GSH level. In addition, we measured an increased lipoxygenase (LOX) activity in CCl₄-intoxicated rats. This activity was reduced in lycopene pre-treated rats to values close to those observed in the controls, suggesting a potential pharmacological application of this dietary carotenoid.     

In vitro evaluation of the antioxidant activities in the differentially processed seeds from underutilized legume, Bauhinia vahlii Wight & Arn

Antioxidant potential and total phenolics content of 70% acetone extracts of the raw and processed seeds of Bauhinia vahlii were evaluated. The extract of raw seeds contained higher levels of total phenolics (30.8 g/100 g) and tannins (19.6 g/100 g) compared to dry heated and soaking followed by autoclaving seed extracts. Extracts were screened for antioxidant and free radical scavenging activities using various chemical and in vitro model systems. In all the models, except DPPH radical scavenging activity, the extract from raw seeds manifested the strongest antioxidant activity than that from processed seeds. In β-carotene/linoleic acid emulsion system and superoxide scavenging activity, the raw seed extract registered more activity when compared to the standards (butylated hydroxyanisole and α-tocopherol). Whereas, the extract from dry heated seed exhibited higher DPPH^· scavenging activity (IC₅₀ 70.77 μg/mL) than the raw seeds (IC₅₀ 74.4 μg/mL). This study has to some extent validated the antioxidant potential of the seeds of B. vahlii.     

Diphlorethohydroxycarmalol isolated from Pae (Ishige okamurae) protects high glucose-induced damage in RINm5F pancreatic β cells via its antioxidant effects

Pancreatic β cells are very sensitive to oxidative stress and this may play an important role in β cell death in diabetes. The protective effect of diphlorethohydroxycarmalol (DPHC), one of phlorotannin polyphenol compound purified from pae (Ishige okamurae) against high glucoseinduced oxidative stress was investigated using RINm5F pancreatic β cells. High glucose (30 mM) treatment induced RINm5F pancreatic β cells cell death, but DPHC, at concentration 10 or 50 μg/mL, significantly inhibited the high glucose-induced glucotoxicity and apoptosis. Furthermore, treatment with DPHC dose-dependently decreased thiobarbituric acid reactive substances (TBARS), intracellular reactive oxygen species (ROS) generation and nitric oxide level increased by high glucose. In addition, DPHC treatment increased activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in high glucose pretreated RINm5F pancreatic β cells. DPHC treatment improved the secretory responsiveness following stimulation with glucose. These findings indicate that DPHC might be used as potential nutraceutical agent which will protect the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.     

Hepatoprotective effect of the root extract of Decalepis hamiltonii against carbon tetrachloride-induced oxidative stress in rats

Decalepis hamiltonii, a climbing shrub, grows in the forests of peninsular India and is consumed for its health promoting properties. The hepatoprotective activity of the aqueous extract of the roots of D. hamiltonii with known antioxidant constituents was studied against carbon tetrachloride (CCl₄)-induced oxidative stress and liver injury in rats. Pretreatment of rats with aqueous extract of the roots of D. hamiltonii, single (50, 100 and 200 mg/kg b.w.) and multiple doses (50 and 100 mg/kg b.w. for 7 days) significantly prevented the CCl₄ (1 ml/kg b.w.) induced hepatic damage as indicated by the serum marker enzymes (AST, ALT, ALP, and LDH). Parallel to these changes, the root extract also prevented CCl₄-induced oxidative stress in the rat liver by inhibiting lipid peroxidation and protein carbonylation, and restoring the levels of antioxidant enzymes (SOD, CAT, GPx, GR, and GST) and glutathione. The biochemical changes were consistent with histopathological observations suggesting marked hepatoprotective effect of the root extract in a dose dependent manner. Protective effect of the aqueous extract of the roots of D. hamiltonii against CCl₄-induced acute hepatotoxicity could be attributed to the antioxidant constituents.     

Anthocyanin effectively scavenges free radicals and protects retinal cells from H<Subscript>2</Subscript>O<Subscript>2</Subscript>-triggered G2/M arrest

It has been previously shown that anthocyanins effectively neutralize free radicals and can act as an antioxidative and anti-aging agent and prevent dementia. In addition, anthocyanins promote expression of rhodopsin, which facilitates night vision impairment, blurred vision, eye fatigue due to physical and mental fatigue, and a loss in rhodopsin has been shown to result from various eye diseases. In this study, the free radical scavenging properties of anthocyanins were evaluated for the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, alkyl radical, and hydroxyl radical using electron spin resonance spectroscopy. The DPPH radical scavenging activity of anthocyanins increased in a dose-dependent manner, with a 50% inhibitory concentration (IC₅₀) value of 2.9 μg/mL. The alkyl radical scavenging activity of anthocyanin was also high, with a IC₅₀ value of 52.2 μg/mL. In addition, the hydroxyl radical scavenging activity of anthocyanins was concentration-dependent. The inhibitory effect of anthocyanins on lipid peroxidation was examined using the ferric thiocyanate and thiobarbituric acid assays. The inhibitory activity of anthocyanins was found to be comparable to that of Vitamin E. In addition, the ability of anthocyanins to reduce oxidative DNA damage was assessed in vitro by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. Also, we have found that anthocyanins’ inhibitory activity of the H₂O₂-induced G2/M phase arrest in ARPE-19 cells. Anthocyanins enhanced the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase in ARPE-19 cells. Taken together, the present results demonstrate that anthocyanins possess potent antioxidative activity.     

Antioxidant and tyrosinase inhibitory activities of different parts of guava (<Emphasis Type="Italic">Psidium guajava</Emphasis> L.)

The antioxidant and the tyrosinase inhibitory activities of 4 different solvents (acetone, ethanol, methanol, and water) for preparation of extracts from guava (branch, fruit, leaf, and seed) were evaluated by measuring total phenolic contents (TPC), DPPH radical scavenging activity, ABTS radical scavenging activity, reducing power (RP), and tyrosinase inhibitory activity. The extracts of branch and leaf showed relatively higher antioxidant properties than those of fruit and seed. The highest TPC (141.28 mg/g gallic acid equivalents), DPPH radical scavenging activity (IC₅₀=34.01 μg/mL), ABTS radical scavenging activity (IC₅₀=3.23 μg/mL), and RP (IC₅₀= 75.63 μg/mL) were found in acetone extract of leaf, while water extract of seed had the lowest antioxidant activity. The tyrosinase inhibitory activity of ethanol extract from guava leaf was 69.56%, which was the highest activity among the extracts. These results indicate that useful bioactive substances exist in the guava branch as well as leaf extracts.     

Effects of ethanol extracts from stalked sea squirt (Styela clava) on antioxidant potential,oxidative DNA damage and DNA repair

The aim of this study was to assess the total radical trapping antioxidant potential and antigenotoxic effects by comet assay of ethanol extracts of stalked sea squirt, Styela clava, (tunic, substrate, and whole). All extracts of stalked sea squirt effectively scavenged ABTS^{· +} in a dose dependent manner. Pretreatment with each extract of stalked sea squirt produced significant reductions in oxidative DNA damage at concentrations of 1–50 μg/mL, with whole extract of stalked sea squirt showing higher inhibition (16.1 μg/mL) of H₂O₂ induced DNA damage than substrate or tunic extracts based on ED₅₀ values. The addition of 50 μg/mL of stalked sea squirt extracts to human leukocytes after oxidative stimulus (200 μM H₂O₂) for 5 min positively influences the kinetics of DNA repair during 24 hr of incubation. These results indicate that the ethanol extracts of tunic, substrate, and whole stalked sea squirt have significant antioxidant activities that protect against oxidative DNA damage and improve DNA repair capacity.     

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.     

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.     

Protective activity of purple sweet potato extract-added soymilk fermented by Bacillus subtilis against oxidative stress

This study focused on the development of a Bacillus subtilis-fermented soymilk with strong antioxidative activity. In a previous study, we manufactured the protective effect of various kinds of Bacillus sp.-fermented soymilks (FSMs) against oxidative stress, and selected the FSM showing the strongest activity. To elevate the antioxidative activity and taste of FSM, 5% purple sweet potato extract (PSPE) was added to FSM based on the results of radical scavenging activities and sensory evaluations. The 5% PSPE added-FSM (PFSM) scavenged 2,2-diphenyl-1-picrylhydrazyl to 70.5% and ·OH to 97.4% at the concentration of 500 μg/mL. Furthermore, the protective effects of PFSM from nitric oxide, superoxide anion, and peroxynitrite produced by sodium nitroprusside, pyrogallol, and 2-morpholinosydnonimine were evaluated in LLC-PK₁ cells. The free radical generators led to losses of cell viability, while PFSM treatment significantly increased cell viability. The present study offers support for the development of a functional B. subtilis-fermented soymilk product using PSPE.     

Antioxidant activity of the differentially processed seeds of Jack bean (<Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L. DC)

Antioxidant activity of 70% acetone extracts of raw and processed seeds of Jack bean (Canavalia ensiformis L. DC) was evaluated by various in vitro antioxidant assays, including total antioxidant, free radical scavenging, reducing power, metal ion chelating, β-carotene/linoleic acid bleaching, and antihemolytic activities. The total phenolics and tannin contents were higher in the extract of seeds processed by autoclaving with 1% ash solution (3.2 and 1.6 g/100 g extract, respectively). In general, all the extracts of processed seeds exhibited higher activity in various antioxidant systems, when compared to raw seeds but significant differences were noticed between processing methods. The extract of seeds autoclaved with 1% sugar solution showed higher DPPH radical scavenging activity (IC₅₀ 10.6 mg/mL). Interestingly, the extract of dry heated seeds registered higher inhibition of hemolysis (76.1%) compared to standards butylated hydroxyanisole (BHA) (66.2%) and α-tocopherol (59.3%) at the concentration of 500 μg/mL.     

Protective effects of extract with phenolics from camellia (Camellia japonica) leaf against oxidative stress-induced neurotoxicity

Antioxidant and neuronal cell protective effects of aqueous extract from camellia (Camellia japonica) leaf (CJLE) were evaluated. The 1,1-phenyl-2-picrylhydrazyl (DPPH) radical scavenging and ferric reducing/antioxidant power (FRAP) assays of the CJLE were increased in a dose dependent manner. In neuronal cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), the aqueous extract showed protective effect against H₂O₂-induced neurotoxicity and lactate dehydrogenase (LDH) release into medium was also inhibited by CJLE (7.13–43.89%). The cell viability of CJLE was higher than vitamin C (200 μM) by neutral red uptake (NRU) assay at a concentration of 250–1,000 μg/mL. Phenolics of CJLE were 21.75 mg/g, and major phenolic compounds were quercetin (120.20mg/100 g) and kaempferol (88.13 mg/100 g). Therefore these data suggested that the CJLE including above phenolics may be useful in the natural antioxidant substance and reduce the risk of neurodegenerative disease such as Alzheimer’s disease.     

Radical scavenging activity and anti-obesity effects in 3T3-L1 preadipocyte differentiation of Ssuk (Artemisia princeps Pamp.) extract

Radical scavenging activity of ethanol extracts from ssuk (Artemisia princeps Pampan.) was determined by 2,2-diphenyl-1-pycryl-hydrazil (DPPH) and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) methods. Also, anti-obesity effects of ssuk extract were tested in vitro using 3T3-L1 preadipocyte cells. Total phenolic contents of ssuk extract were 42 μM tannic acid equivalent/mg and EC₅₀ of ssuk extract for scavenging DPPH and ABTS radicals were 2.02 and 1.03 mg/mL, respectively. Triacylglycerol content in 3T3-L1 preadipocyte cells treated with 25, 50, and 100 αg/mL ssuk extract decreased significantly by 63.3, 67.1, and 71.0%, respectively (p<0.05) compared to those in untreated cells. Also, ssuk extract induced the down-regulation of adipogenesis-related genes such as PPARγ, aP2, ACC, and GPDH. The ssuk extract possessed phenolic compounds with radical scavenging ability and had in vitro anti-obesity effects on 3T3-L1 preadipocyte cells. These results suggest that the extracts of ssuk may be used as obesity controlling food ingredients.     

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.     

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.     

The role of two putative nitroreductases,Frm2p and Hbn1p,in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H₂O₂ and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright © 2009 John Wiley & Sons, Ltd.     

Protective effects of loquat (Eriobotrya japonica) leaves against ethanol-induced toxicity in HepG2 cells transfected with CYP2E1

Hepatoprotective effects of loquat (Eriobotrya japonica) leaves were investigated in HepG2 cells overexpressing CYP2E1. When compared to cells treated with 200 mM ethanol alone, a concentration-dependent increase in cell viability was observed in the cells pretreated with 40 and 80 μg/mL of 5% ethanol extract (EJE) of loquat leaves (23 and 36%, respectively). Also, pretreatment with EJE lead to a decrease in intracellular reactive oxygen species formation and an increase in hepatic antioxidant activity. These results suggest that EJE attenuates oxidative stress by improving antioxidative potentials, which contribute to this herb’s protective profile against ethanol-induced toxicity in vitro.     

Antioxidant and antimicrobial activities of the methanol extracts from pummelo (Citrus grandis Osbeck) fruit albedo tissues

The present study was conducted to evaluate the antioxidant and antimicrobial properties of methanol (100 and 80% aqueous) extracts of pummelo fruits albedo (Citrus grandis Osbeck). The antioxidant and antibacterial activity for crude extracts and isolated compounds were evaluated using free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Paper disc diffusion method. A 100% Methanol (MeOH) extract was steeped in water at different pH levels and partitioned with ethyl acetate (EtOAc) to give basic, acidic, neutral, and phenolic fractions. The neutral extract was found to possess maximum antioxidant and antibacterial activity. Thereafter, neutral extract was carried out on a silica gel column and eluted with hexane:EtOAc:acetone and preparative TLC (PTLC) to give oil buntan compound, linoleic acid methyl ester, β-sitosterol, sigmasterol, limonin, nomilin and meranzin hydrate were isolated. While, 80% MeOH extract was fractionated also using a silica gel column and PTLC to give isomeranzin hydrate, p-coumaric acid and caffeic acid compound. The extract concentration providing 50% inhibition (IC₅₀) was as follows; oil buntan compound 95 μg/mL, caffeic acid 45 μg/mL, p-coumaric acid 105 μg/mL, limonin + nomilin (mixture) 135 μg/mL was lower than that of synthetic antioxidant butylated hydroxyanisole (BHA) 40 μg/mL. The inhibitory zone (mm) of bacteria tested was 2.9–4.1 mm caffeic acid and 11.6–15.1 mm p-coumaric acid.     

Antioxidant activities of the rice endosperm protein hydrolysate: identification of the active peptide

The defatted rice endosperm protein (REP) was digested using five different proteases (Alcalase, Chymotrypsin, Neutrase, Papain, and Flavorase) to produce the antioxidative peptide. The degree of hydrolysis of REP by Neutrase (20.00%) was slightly lower than that of Chymotrypsin, but higher than those of other enzymes. The Neutrase hydrolysate from rice endosperm protein (NHREP) took on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity similar to α-tocopherol. Also, the reaction condition of Neutrase hydrolysis was moderate (pH of 7.0 and temperature of 37 °C). Therefore, Neutrase was chosen to be the optimum enzyme for producing the antioxidative peptide from REP. In succession, the antioxidant activities of NHREP were further evaluated. The median effective concentration (EC₅₀) value of NHREP for hydroxyl radical scavenging activity was 2.0 mg/mL, while its superoxide radical scavenging activity did not surpassed 50% even at 6 mg/mL. The percentage inhibition of autooxidation in linoleic acid system by NHREP was 82.09%, similar to that of α-tocopherol (86.59%) on day 5 at the same concentration. NHREP displayed 89.15% chelating effect on ferrous ion at a concentration of 1000 μg/mL, and a high correlation was also observed between the reducing power and antioxidant activity of NHREP (r ² = 0.99678). Consequently, NHREP was purified and identified, and the sequence determination by MALDI-TOF/TOF MS/MS revealed that the active constituent contained eight amino acids in its sequence (Lys-His-Asn-Arg-Gly-Asp-Glu-Phe), with the molecular mass of 1002.5217 Da. After sequence interpretation and database searching, the MS/MS spectrum was matched to glutelin protein f (460–465).     

Characterization of growth and protein contents from microalgae Navicula incerta with the investigation of antioxidant activity of enzymatic hydrolysates

Microalgae are major primary producers of organic matters in aquatic environments through their photosynthetic activities. Benthic diatom Navicula incerta is the major component of phytoplankton and also relatively easy to cultivate, used as live food source in aquaculture. The growth characteristics of N. incerta were estimated under combinations of temperature, salinity, and light; and also its composition and antioxidant activities were determined. The maximum cell density of 87×10⁵cells/mL, was reached at 20°C, 250 μmol/m²·sec, 33‰ salinity, pH 8.3, 12:12 light:dark, and F/2 medium on 2 weeks of the culture period. The antioxidant enzymatic hydrolysates efficiently quenched different free radicals: 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) (pepsin IC₅₀=196.0 μg/mL), hydroxyl (α-chymotrypsin IC₅₀=102.0 μg/mL), and superoxide (neutrase IC₅₀=169.0 μg/mL). These results suggest that the enzymatic hydrolysate from N. incerta acts as a candidate against antioxidant and could be used as a potential functional food ingredient.