Oxidative stress inbibits distant metastasis by human melanoma cells

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Effect of the Lycium barbarum polysaccharides

Journal of Ethnopharmacology111(2007)504–511Effect of the Lycium barbarum polysaccharideson age-related oxidative stress in aged miceX.M.Li a,∗,Y.L.Ma b,X.J.Liu ca School of Food Science and Technology of the XingJiang Agriculture University,Urumqili City,XinJiang830000,PR Chinab School of Traditional Chinese Medicine of the NanZhou University,NanZhou City,GanSu720000,PR Chinac Department of Chinese Herb Medicine of the XingJiang University,Urumqili City,XinJiang830000,PR ChinaReceived7October2006;received in revised form2December2006;accepted14December2006Available online28December2006AbstractOxidative damage of biomolecules increases with age and is postulated to be a major causal factor of various physiological function disorders. Consequently,the concept of anti-age by antioxidants has been developed.Lycium barbarum fruits have been used as a traditional Chinese herbal medicine and the data obtained in in vitro models have clearly established the antioxidant potency of the polysaccharides isolated from the fruits.In the present study,the age-dependent changes in the antioxidant enzyme activity,immune function and lipid peroxidation product were investigated and effect of Lycium barbarum polysaccharides on age-induced oxidative stress in different organs of aged mice was checked.Lycium barbarum polysaccharides(200,350and500mg/kg b.w.in physiological saline)were orally administrated to aged mice over a period of30days.Aged mice receiving vitamin C served as positive control.Enzymatic and non-enzymatic antioxidants,lipid peroxides in serum and tested organs,and immune function were measured.Result showed that increased endogenous lipid peroxidation,and decreased antioxidant activities,as assessed by superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)and total antioxidant capacity(TAOC),and immune function were observed in aged mice and restored to normal levels in the polysaccharides-treated groups.Antioxidant activities of Lycium barbarum polysaccharides can be compable with normal antioxidant,vitamin C.Moreover,addition of vitamin C to the polysaccharides further increased the in vivo antioxidant activity of the latter.It is concluded that the Lycium barbarum polysaccharides can be used in compensating the decline in TAOC,immune function and the activities of antioxidant enzymes and thereby reduces the risks of lipid peroxidation accelerated by age-induced free radical.©2007Elsevier Ireland Ltd.All rights reserved.Keywords:Oxidative stress;Vitamin C;Lycium barbarum polysaccharides;Antioxidant;Lipid peroxidation;Anti-aging;Superoxide anions1.IntroductionA major characteristic of an aging organism is its progres-sive functional decline,including a loss of adaptive responses to stresses,with the passage of time(Ian and Grotewiel,2006). One currently major cause of aging is the concept of oxidative stress as a root of aging(Golden and Melov,2001).Oxida-tive stress is described generally as a condition under which increased production of free radicals,reactive species(including singlet oxygen and reactive lipid peroxidation products,such as reactive aldehydes and peroxides),and oxidant-related reactions occur that result in damage.∗Corresponding author.Tel.:+869915667541.E-mail address:xj.goodli@(X.M.Li).Current studies suggest that development of anti-aging drugs from Chinese medicinal herbs may be one of the possible inter-ventions(Chang,2001;Bastianetto and Quirion,2002;Lei et al.,2003).Oriental herbal medicine has been widely investi-gated for drug development because it has fewer side effects (Wong et al.,1994).Lycium barbarum belongs to the plant fam-ily Solanaceae.Red-colored fruits of Lycium barbarum have been used as a traditional Chinese herbal medicine for thousands of years(Gao et al.,2000).The earliest known Chinese medici-nal monograph documented medicinal use of Lycium barbarum around2300years ago.Lycium barbarum fruits have a large variety of biological activities and pharmacological functions and play an important role in preventing and treating various chronic diseases,such as diabetes,hyperlipidemia,cancer,hep-atitis,hypo-immunity function,thrombosis,and male infertility (Gao et al.,2000;Li,2001).It is well recognized that freeX.M.Li et al./Journal of Ethnopharmacology111(2007)504–511505radical scavengers or antioxidants plays a important role in slow-ing down biological aging(Andr`e s et al.,2006;Linnane and Eastwood,2006).The evidence suggests that Lycium barbarum is effective to be an anti-aging agent as well as nourishment of eyes,livers and kidneys.The anti-aging property of Lycium barbarum is found in the polysaccharides isolated from the red-colored fruits and has been investigated in different models(Qi et al.,2001;Peng et al.,2001;Wang et al.,2002;Gan et al., 2003,2004;Zhang et al.,2005).For example,extracts of Lycium barbarum have anti-decrepit effect in brain and heart tissues in mice by increasing the activity of superoxide dismutase(SOD) (Xu and Fang,2000).The extracts can still prolongs the life span of Drosophila(Xu,2003).Polysaccharides isolated from Lycium barbarum fruits exhibit anti-aging function in fruitﬂies and mice(Wang et al.,2002).Although numerous studies have been published on humans and animals examining the health aspects of Lycium barbarum polysaccharides,to our knowl-edge,there have been scarce studies to investigate its beneﬁcial effects on health from the aspect of its antioxidant activity in vivo.Therefore,in the present study,we investigated age-dependent changes in the activity of antioxidant enzymes and the immune function in the mice studied and assess the regulatory effects of polysaccharides isolated from Lycium barbarum fruits on oxidative stress in aged mice to improve the understanding of the health beneﬁts of these polysaccharides.2.Methods and materials2.1.Preparation of polysaccharidesFruits of Lycium barbarum,family solanacae,originated from china were purchased from JingHe county herb market (Xinjiang,China),and identiﬁed by Professor D.S.Chen,School of Traditional Chinese Medicine,Xinjiang University.V oucher specimens(HYT-PM040008)were preserved in XinJiang Nat-ural Product Research Institute.Polysaccharides from Lycium barbarum was prepared by the method of Luo et al.(2004).The dried fruit samples(100g) were ground toﬁne powder and put in1.5l of boiling water and decocted for2h by a traditional method for Chinese medici-nal herbs.The decoction was left to cool at room temperature,ﬁltered and then freeze-dried to obtain crude polysaccharides. The dried crude polysaccharides were reﬂuxed three times to remove lipids with150ml of chloroform:methanol solvent (2:1)(v/v).Afterﬁltering the residues were air-dried.The result product was extracted three times in300ml of hot water (90◦C)and thenﬁltered.The combinedﬁltrate was precipi-tated using150ml of95%ethanol,100%ethanol and acetone, respectively.Afterﬁltering and centrifuging,the precipitate was collected and vacuum-dried,giving desired polysaccharides (13g).The content of the polysaccharides was measured by phenol-sulfuric method(Masuko et al.,2005).Result showed that 2.2.Determination of in vivo antioxidant activity of the polysaccharides2.2.1.Animals grouping and treatingSixty20-month-old,body weight28–40g,aged Kunming mice and ten3-month-old,body weight19–26g,young Kun-ming mice were provided by Laboratory Animal breeding Center attached to our institute.The animals were kept under controlled conditions(temperature:23±2◦C;humidity: 55±5%;14h light–10h dark cycle)using an isolator caging system(Niki Shoji,Co.,Tokyo)and allowed free access to standard laboratory pellet diet and water throughout the exper-imental period.All experimental animals were overseen and approved by the Animal Care and Use Committee of XinJiang Medical University before and during experiments.Aged Kunming mice were randomly divided into six groups (10for each):Group II(the aged control),Group III,Group IV,Group V,Group VI and Group VII.Young Kunming mice (Group I)served as normal control.The polysaccharides and vitamin C were administered orally to test animals using vehicle solution(physiological saline)by using a gastric gavage. Group I:Normal control mice were maintained on standard laboratory pellet diet and water ad libitum,withoutadministering medicine for30consecutive days. Group II:Aged control mice were maintained on standard laboratory pellet diet and water ad libitum,withoutadministering medicine for30consecutive days. Group III:Aged mice received polysaccharides(200mg/kgb.w.)in appropriate volumes of physiological salineby using gastric gavage and allowed free accessto standard laboratory pellet diet and water for30consecutive days.Group IV:Aged mice received polysaccharides(350mg/kgb.w.)in appropriate volumes of physiological salineby using gastric gavage and allowed free accessto standard laboratory pellet diet and water for30consecutive days.Group V:Aged mice received polysaccharides(500mg/kgb.w.)in appropriate volumes of physiological salineby using gastric gavage and allowed free accessto standard laboratory pellet diet and water for30consecutive days.Group VI:Aged mice received medicine(polysaccharides plus vitamin C;1/1)(500mg/kg b.w.)in appropri-ate volumes of physiological saline by using gastricgavage and allowed free access to standard labora-tory pellet diet and water for30consecutive days. Group VII:Aged mice received vitamin C(500mg/kg b.w.) in appropriate volumes of physiological saline byusing gastric gavage and allowed free access tostandard laboratory pellet diet and water for30consecutive days.506X.M.Li et al./Journal of Ethnopharmacology111(2007)504–511were harvested,kept at−20◦C until analyzed.Blood sam-ples were centrifuged at4000rpm for3min at4◦C and the serum was separated.The serum MDA level was measured. The organs(including liver,heart,brain,kidney and lung)were removed,weighed and homogenized immediately with DY89-II homogenizer(NingBo Scientz Biotechnology Co.,Ltd.)ﬁtted with teﬂon plunger,in ice chilled10%KCl solution(10ml/g of tissue).The suspension was centrifuged at671×g at4◦C for10min and clear supernatant was used for the following estimations of activity of SOD,CAT,GSH-Px,TAOC,and levels of MDA by spectrophotometric methods.Spleen and thymus were removed and kept frozen at−80◦C until mea-surement.2.2.2.Analytical methodsSuperoxide dismutase activity was measured according to the method described by Misra and Fridovich(1972)based on the inhibition of auto-oxidation of epinephrine by SOD at480nm in a LKB Ultraspec-2spectrophotometer.Tissue homogenate (0.5ml)was diluted to1.5ml with distilled water,and250␮l of chilled ethanol and100␮l of chilled chloroform were added.The mixture was shaken and centrifuged.The supernatant was used for the assay of enzyme activity.1.5ml of the supernatant was added to1.5ml of0.1mol/l carbonate–bicarbonate buffer,pH 10.2,containing0.3mmol/l EDTA.The contents were mixed, and the reaction was initiated by adding200␮l of epinephrine (pH3.0,3mmol/l)to the buffered reaction mixture.The change in optical density per minute was measured at480nm.The enzyme activity was expressed as unit per milligram of pro-tein,where1U is deﬁned as the enzyme concentration required to inhibit50%of epinephrine auto-oxidation in1min under the assay conditions.The assay of Beers and Sizer(1952)was used to measure CAT.Substrate solution for CAT was59mM H2O2in50mM potassium phosphate buffer at pH7.0.Assays were initiated by the addition of0.1ml of supernatant to1.9ml of deionized water and1ml of substrate solution.The disappearance of H2O2 was measured as the decrease in absorbance at240nm.Catalase activity was expressed as U/mg protein(1U is the amount of enzyme that utilizes1␮mol of hydrogen peroxide/min).The GS-Px activity of the supernatant was determined spectrophotometrically at423nm.The reaction mixture was composed of GSH,distilled water,and the supernatant.The reac-tion was stopped by adding trichloroacetic acid.The content of residual GSH was then measured using5-5 -dithiobis-(2-nitrobenzoic acid)(DTNB).One unit of GS-Px activity was deﬁned as1␮mol/l GSH consumption per minute(Liu and Ng, 2000).Lipid peroxidation(LPO)was measured by high performance liquid chromatography(Shimadzu LC10A,Shimadzu,Kyoto, Japan)as described previously(Nielsen et al.,1997).The pho-todiode array detector(Shimadzu SPD-M10A VP)and a C18 column(4.6␮m×25.5␮m Shimpack HRC-ODS)were used for assay.After pretreatment with thiobarbituric acid,the sam-ple was injected and an isocratic elution was carried out with at a wavelength of532nm.Peak authenticity was conﬁrmed by use of pure1,1,3,3-tetraethoxipropane standards.The malondi-aldehyde(MDA)content of the samples was expressed as nmol MDA formed/mg protein.Total antioxidant capacity(TAOC)was measured on an Olympus AU-600analyzer using the TAOC kit(Medikon SA, Gerakas,Greece)as described previously(Kampa et al.,2002). Brieﬂy,antioxidants in the sample inhibit the bleaching of crocin from2,2-azobis-(2-amidinopropane)dihydrochloride(ABAP) to a degree that is proportional to their concentration.The assay was performed at37◦C in the following steps:2␮l of sample, calibrator or control were mixed with250␮l of crocin reagent (R1)and incubated for160s.Subsequently,250␮l of ABAP (R2)were added and the decrease in absorbance at450nm was measured26s later.Values of TAOC were expressed as U/mg protein.Lipofuscin(LPF)contents were determined by the method of Vernet et al.(1988)and Hill and Womersley(1991).For lipofus-cin measurement,0.5ml of tissue homogenate was suspended in3ml of isopropanol and2ml of chloroform.This was allowed to stand for30min and centrifuged at1800×g in a refrigerated centrifuge.Theﬂuorescence was measured using a spectroﬂu-orimeter with extinction at360nm and emission at440nm. Lipofuscin concentrations were expressed as␮g/g tissue.The phagocytic activity of neutrophils in whole blood was conducted as per the method described by Panasiuk et al.(2005). Brieﬂy,standard strain of Staphylococcus aureus was procured from the Division of Standardization(IVRI).Eighteen hours culture was opsonised with pooled mice serum in an incuba-tor for1h.An equal volume(500␮l)of PMNs and500␮l opsonised bacterial suspension was incubated at37◦C for half an hour,maintaining PMNs and bacteria at a1:5ratio.Thereafter, it was stained with500␮l of Acridine orange stain(0.015%, Sigma,St.Louis,MO,USA),vortexed and centrifuged at4◦C, 13000rpm to obtain cell pellet.Finally,500␮l crystal violet was added and centrifuged as above.The pellet was resus-pended in cold sterile PBS(500␮l)and wet mount seen under ultraviolet source with excitationﬁlter of530nm.Phagocytic activity,expressed by the percentage of phagocytosed neutrophil in100cells and phagocytic index,determined on the unit of Staphylococci ingested by single PMNs,was counted in100 cells.The spleen and thymus of the mice were also removed and weighed to obtain the index of the spleen and thymus.The thy-mus and spleen indices were assayed according to the method of Zhang et al.(2003)and calculated according to the follow-ing formula:Thymus or spleen index=weight of thymus or spleen/body weight×100.2.3.Statistical analysesAll data in table are expressed as mean±S.D.(n=10) and differences between groups were assessed by analysis of variance(ANOV A)and Student’s t-test.Differences were con-sidered to be statistically signiﬁcant if P<0.05.All statisticalX.M.Li et al./Journal of Ethnopharmacology111(2007)504–5115073.Results3.1.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in lungs in aged mice As shown in Table1,there was signiﬁcant difference in SOD activities,MDA level,TAOC observed in lung between the aged control and young mice control(P<0.05)but not in GSH-Px, and CAT activity.Declined antioxidant enzymes activity(SOD, CAT activity,TAOC)or increased lipid peroxidation product (MDA)in aged tissues(groups III–VII)were signiﬁcantly ele-vated or reduced with administration of polysaccharides and vitamin C in a dose-dependent manner.The antioxidant activity of polysaccharides is stronger than that of vitamin C at identical dose of500mg/kg b.w.3.2.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in livers in aged mice As was shown in Table1,signiﬁcantly decreased antioxidant enzymes activity(SOD,GSH-Px,CAT,TAOC)and increased MDA level were observed in livers in aged control mice compared with normal control(P<0.01).Administration of polysaccharides and vitamin C dose-dependently increased the activity of antioxidant enzymes,reduced the level of MDA in livers in aged mice(groups III–VII).Moreover,the inhibition by polysaccharide administration of age-induced oxidation is stronger than that of vitamin C at identical dose of500mg/kgb.w.3.3.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in hearts in aged miceA marked increase in MDA production and decrease of antioxidant enzymes activity(SOD,GSH-Px,CAT,TAOC), were observed in hearts of aged control mice(Table1)when compared with normal control(P<0.01).Polysaccharides and vitamin C treatment signiﬁcantly inhibited the formation of MDA in mice hearts and raised antioxidant enzymes activity in a dose-dependent manner(groups III–VII).Likewise,polysac-charides exhibited stronger antioxidation effects than vitamin C at identical dose of500mg/kg b.w.Table1Effect of polysaccharides on activities of SOD(NU/mg protein),CAT(U/mg protein),GSH-Px(U/mg protein),TAOC(U/mg protein)and levels of MDA(nmol/mg protein or/ml serum)in tested organs in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VII LungSOD11.41±1.1310.18±0.77c9.98±0.58b12.98±0.60b13.99±1.21b15.52±0.78b11.59±1.64a CAT 4.82±0.57 4.52±0.47 4.46±0.46 4.68±0.64 5.05±0.56a 5.47±0.61b 4.74±0.39 GSH-Px 3.11±0.61 2.77±0.39 3.98±0.47b 4.43±0.33b 5.92±0.53b 6.21±0.65b 3.91±0.43b TAOC 1.13±0.090.96±0.06c 1.04±0.14 1.32±0.14b 1.49±0.13b 1.71±0.09b 1.30±0.13b MDA 2.34±0.49 2.85±0.34c 2.37±0.23b 2.20±0.27b 2.03±0.24b 1.87±0.21b 2.51±0.17a LiverSOD8.70±0.677.58±0.66d8.45±0.38b9.45±1.15b15.88±1.06b18.17±0.94b14.02±0.97b CAT 2.14±0.20 1.65±0.19d 1.80±0.11a 1.92±0.15b 2.15±0.17b 2.86±0.40b 1.86±0.16a GSH-Px10.91±0.928.78±0.81d10.41±0.81b12.69±1.18b14.24±1.07b17.62±1.53b10.97±0.84b TAOC 2.01±0.190.84±0.11d 1.16±0.19b 1.36±0.13b 1.87±0.28b 2.44±0.41b 1.48±0.16b MDA13.46±0.9615.64±0.64d14.53±0.66b12.57±1.11b10.56±0.75b8.75±0.41b9.83±0.73b HeartSOD16.44±0.8314.53±0.94d15.12±0.7216.58±0.55b22.58±0.91b28.43±0.69b16.91±0.68b CAT 2.25±0.32 1.55±0.26d 1.58±0.21 1.65±0.21 1.86±0.19b 1.91±0.20b 1.65±0.27 GSH-Px9.91±1.228.84±0.53d9.54±0.23b10.38±0.25b10.97±0.51b11.93±0.48b10.52±0.93b TAOC0.98±0.170.71±0.09d0.85±0.11b0.97±0.08b 1.31±0.13b 1.77±0.08b 1.06±0.12b MDA 3.97±0.29 5.13±0.30d 4.67±0.17b 3.96±0.21b 3.57±0.24b 2.95±0.12b 4.69±0.12b BrainSOD19.11±0.9717.8±0.51d18.54±1.0520.58±1.32b23.89±1.35b27.82±1.12b20.63±0.86b CAT 4.41±0.37 4.12±0.24 4.23±0.45 4.95±0.11b 5.21±0.27b 5.76±0.31b 4.31±0.09b GSH-Px 4.30±0.50 2.95±0.43d 3.35±0.32 4.41±0.52b 6.03±0.75b8.16±0.48b 5.73±0.33b TAOC0.92±0.230.70±0.08d0.76±0.17 1.84±0.37b 2.33±0.11b 2.89±0.19b 2.04±0.09b MDA9.74±1.3811.28±1.23c10.71±0.888.27±1.51b 6.56±0.85b 4.27±0.16b7.95±0.63b SerumMDA17.34±2.1232.49±2.97d28.78±2.04b23.83±1.77b19.56±1.83b16.63±1.55b24.82±2.79b The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.a P<0.05,compared with aged control group(II).508X.M.Li et al./Journal of Ethnopharmacology111(2007)504–511Table2Effect of polysaccharides on thymus and spleen index in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VII Thymus index0.385±0.0340.263±0.027c0.273±0.0250.336±0.040b0.457±0.043b0.572±0.063b0.431±0.034b Spleen index0.533±0.0520.452±0.044c0.486±0.0820.512±0.068a0.550±0.080b0.591±0.076b0.512±0.042b The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.Thymus or spleen index=weight of thymus or spleen/body weight×100.a P<0.05,compared with aged control group(II).b P<0.01,compared with aged control group(II).c P<0.01,compared with normal group(I).3.4.Effect of the Lycium barbarum polysaccharides on antioxidant enzymes activity in brains in aged mice Data on age-induced changes in brains are summarised in Table1;there was only a slight change in CAT activity (P>0.05),but signiﬁcant decreases in SOD,GSH-Px activity, and TAOC,and an increase in MDA level with age compared with control young animals(P<0.05,P<0.01).Administration of polysaccharides and vitamin C dose-dependently elevated these antioxidant enzymes activity and reduced MDA level in brains(groups III–VII).Antioxidant activity of the polysaccha-rides in vivo was better than vitamin C at500mg/kg b.w.3.5.Effect of the Lycium barbarum polysaccharides on thymus and spleen index in aged miceAs was shown in Table2,signiﬁcantly decreased thymus and spleen weight were observed with age(P<0.01)in compari-son with normal control.Administration of polysaccharides and vitamin C are seen to have remarkable effects on increasing the two indices in immune organ in aged mice in a dose-dependent manner(groups III–VII).The reversal of age-induced decreased thymus and spleen weight by polysaccharides administration is stronger than that by vitamin C at identical dose of500mg/kgb.w.3.6.Effect of the Lycium barbarum polysaccharides on macrophage function in aged miceLikewise,in the present study we observed that the tested indices(phagocytic index and phagocytic activity)markedly decreased with age(Table3)(P<0.01)in comparison with nor-mal control.Supplementation of polysaccharides and vitamin C both signiﬁcantly raised the two indices in a dose-independent manner in aged mice(groups III–VII).In terms of the effect on phagocytic indices,polysaccharides administration was basically consistent with vitamin C of identical given dose but stronger than the latter on stimulating phagocytic activity.3.7.Effect of the Lycium barbarum polysaccharides onLPF level in tested organs in aged miceTable4represents the effect of polysaccharides and vitamin C on the levels of LPF in different tested organs in control and experimental animals.The LPF level in aged mice is markedly higher than that in young mice(P<0.01).A signiﬁcant reduc-tion(P<0.01)was found in the levels of LPF in all experiment animals groups(groups V–VII),when compared with aged con-trol.Moreover,the level was dose-independently decreased in polysaccharides-treated animals(groups III–VI).It was found in the present study that polysaccharides administration was still more effective in reducing LPF level than vitamin C at the identical dose.3.8.Effect of the Lycium barbarum polysaccharides on serum MDA level in aged miceEffect of the Lycium barbarum polysaccharides on serum MDA level in aged mice was shown in Table1.Signiﬁcant differ-ences in serum MDA level were detected between normal control and aged control groups(P<0.05).Data from polysaccharides treatment were pooled and compared to the aged control.Result indicated that polysaccharides treatment signiﬁcantly decreased serum MDA level(P<0.05)compared with aged control.Table3Effect of polysaccharides on macrophage function in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VII Phagocytic index(k) 3.32±0.44 1.93±0.25d 2.17±0.22 2.65±0.43b 2.87±0.58b 3.12±0.45b 3.18±0.33b Phagocytic activity(α)87.31±5.6572.06±2.98c73.83±4.3279.97±7.1181.42±7.38a86.43±8.39a76.51±7.39 The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.Phagocytic activity,expressed by the percentage of phagocytosed neutrophil in100cells and phagocytic index,determined on the unit of Staphylococci ingested by single PMNs,was counted in100cells.a P<0.05,compared with aged control group(II).X.M.Li et al./Journal of Ethnopharmacology111(2007)504–511509 Table4Effect of polysaccharides on LPF level(␮g/g tissue)in tested organs in aged miceParameters Group I Group II Group III Group IV Group V Group VI Group VIIBrain0.224±0.0270.440±0.041b0.434±0.0360.358±0.051a0.312±0.031a0.285±0.027a0.398±0.022a Heart0.115±0.0090.187±0.014b0.180±0.0240.174±0.011a0.160±0.012a0.133±0.013a0.184±0.023 Liver0.245±0.0190.444±0.036b0.360±0.019a0.351±0.014a0.330±0.011a0.284±0.027a0.348±0.019a Kidney0.206±0.0110.382±0.023b0.371±0.0250.344±0.019a0.305±0.019a0.284±0.013a0.369±0.024The data were presented as means±S.D.(n=10)and evaluated by one-way ANOV A followed by the Student’s t-test to detect inter-group differences.Differences were considered to be statistically signiﬁcant if P<0.05.a P<0.01,compared with aged control group(II).b P<0.01,compared with normal group(I).Moreover,the compound of polysaccharides plus vitamin C exhibited stronger antioxidant activity than either of the two antioxidants.4.DiscussionAging is a progressive deterioration of physiological function that impairs the ability of an organism to maintain homeostasis and consequently increases the organism’s susceptibility to dis-ease and death(Nohl and Hegner,1978).Nearly all organisms manifest functional declines as a result of aging.It is widely accepted that disorganizing free radical reactions linked to oxy-gen metabolism or“oxidative stress”(Chance et al.,1979;Sies, 1986;Gutteridge,1987)play an important role not only in normal aging(Harman,1956;Lesser,2006)but also in many age-related degenerative processes(Harman,1981).Oxidation of lipids produces lipid peroxides that can reduce membraneﬂuidity,inactivate membrane-bound proteins and decompose into cytotoxic aldehydes such as malondialdehyde or hydroxynonenal(Richter,1987).Accumulation of hydrox-ynonenal increases with age in several Drosophila tissues (Zhang and Xu,2006)and the level of malondialdehyde and hydroxynonenal-conjugated collagen protein increases with age in rat tissue(Noberasco et al.,1991).We have also observed an increase in the levels of MDA,a marker of lipid peroxidation in the test organs of aged mice.Hence,lipid oxidation is closely associated to aging.On the contrary,LBP treatment demon-strated decreased level of lipid peroxides and this could be in part due to reduced formation of lipid peroxides from age-dependent free radicals.A vast number of evidence implicates that aging is associated with a decrease in antioxidant status and that age-dependent increases in lipid peroxidation are a consequence of dimin-ished antioxidant protection(Schuessel et al.,2006;Alvarado et al.,2006),being in agreement with our current study.The major antioxidant enzymes,including SOD,GPX and CAT, are regarded as theﬁrst line of the antioxidant defense sys-tem against reactive oxygen species generated in vivo during oxidative stress.SOD dismutates superoxide radicals to form hydrogen peroxide,which in turn is decomposed to water and oxygen by GSH-Px and CAT,thereby preventing the formation of hydroxyl radicals(Yao et al.,2005).Therefore,these enzymes act cooperatively at different sites in the metabolic pathway of in various organs.For example,some previous reports(Y¨u ksel and Asma,2006;Miquel et al.,2006)have shown that some of the antioxidant enzymes in important organs such as liver, heart,kidney,and brain,were decreased with aging,whereas, other investigators have indicated no alteration or increased activities in the antioxidant enzymes(Rolo and Palmeira,2006; Masztalerz et al.,2006).The differences among those data might be,in part,due to differences in the animal’s sex,strain,and age used,assay method,the enzyme property examined,and/or experimental conditions used.In our study along with increased lipid peroxidation,age-induced oxidative injury was found to reduce the total antioxidant capacity(TAOC),which reﬂects the non-enzymatic antioxidant defense system,as well as antiox-idant enzyme levels(SOD,CAT,GSH-Px)in test organs of aged mice and this observation concurs with earlierﬁndings (Kogan et al.,2005).Due to depletion in antioxidant levels, the free radicals are not neutralized and aged organs show enhanced susceptibility to lipid peroxidation.The observation that LBP treatment signiﬁcantly restores the marker enzymes activity of aged mice compared to aged control suggests the reversal by these drugs administration of age-induced oxida-tion.The enhanced activity of SOD,CAT and GSH-Px and increased TAOC in the aging animals can be very effective in scavenging the various types of oxygen free radicals and their products.So the inhibitory effect of the Lycium barbarum polysaccharides on lipid peroxidation might be,at least in part, attributed to its inﬂuence on the antioxidant enzymes and non-enzymatic ind et al.(1990)have reported that,in general,the age-related changes in the activities of SOD,GSH-Px,and CAT were paralleled by a similar change in the relative level of the mRNA expressions coding for these enzymes in brain,hepatocytes,and kidney.As for lung,Gomi and Matsuo (2002)have shown that aging decreased the mRNA expres-sions of SOD and GSH-Px but did not change CAT.Their study also revealed the discrepancy between the activity and mRNA expression of either SOD or GSH-Px.Thus,theseﬁndings, including the currentﬁndings,may suggest that the activities of antioxidant enzymes in aged tissues could be controlled by translational process and/or post-translational process,but not by transcriptional process.Future investigation,however,will be required to determine additional mechanisms.It is possible that the effect of the Lycium barbarum polysaccharides on SOD, CAT and GSH-Px was associated with its effect on translational。

Antioxidative and ACE inhibitory activities of protein hydrolysates from the muscle of brownstripe r

Process Biochemistry 46(2011)318–327Contents lists available at ScienceDirectProcessBiochemistryj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p r o c b ioAntioxidative and ACE inhibitory activities of protein hydrolysates from themuscle of brownstripe red snapper prepared using pyloric caeca and commercial proteasesSutheera Khantaphant a ,Soottawat Benjakul a ,∗,Hideki Kishimura ba Department of Food Technology,Faculty of Agro-Industry,Prince of Songkla University,Hat Yai,Songkhla,90112,ThailandbLaboratory of Marine Products and Food Science,Research Faculty of Fisheries Science,Hokkaido University,Hakodate 041-8611,Japana r t i c l e i n f o Article history:Received 4June 2010Received in revised form 10September 2010Accepted 10September 2010Keywords:Protein hydrolysate Antioxidative activityGastrointestinal model system Oxidation model system Two-step hydrolysisa b s t r a c tProtein hydrolysates from the muscle of brownstripe red snapper (Lutjanus vitta )prepared using Alcalase or Flavourzyme as the ﬁrst step with 40%degree of hydrolysis (DH),followed by hydrolysis with pyloric caeca protease (PCP)as the second step for 2(HAP)and 1h (HFP),respectively,were prepared and determined for their antioxidative and angiotensin I-converting enzyme (ACE)inhibitory activities.HAP had the higher DPPH and ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP),while HFP showed the higher ferrous chelating activity and ACE inhibitory activity (p <0.05).Both HAP and HFP were able to retard lipid oxidations in lecithin–liposome and ␤-carotene–linoleic acid oxidation model systems in dose-dependent manner.HAP and HFP contained 87.36and 86.55%protein (wet basis),respectively with glutamic acid/glutamine as the major amino acids,followed by aspartic acid/asparagine,lysine,alanine and leucine,respectively.HFP showed a slightly greater efﬁciency in prevention of lipid oxidation in all systems tested.Antioxidative activities,except DPPH radical scavenging activity,of both HAP and HFP after being subjected to gastrointestinal tract model system (GIMs)increased,suggesting the enhancement of antioxidative activities of both hydrolysates after ingestion.©2010Elsevier Ltd.All rights reserved.1.IntroductionLipid oxidation is one of the major deteriorative processes in many types of foods,leading to the changes in food quality and nutritional value.Additionally,potentially toxic reaction products can be produced.Lipid oxidations have been known to be the major causes of many serious human diseases,such as cardiovascular dis-ease,cancer,and neurological disorders as well as the aging process [1,2].To prevent oxidative deterioration of foods and to provide protection against serious diseases,such as cancer and atheroscle-rosis [3,4],it is important to inhibit the oxidation of lipids and the formation of free radicals occurring in the foodstuff and living body.To tackle the problem,antioxidants,both natural and synthetic ones,have been used widely.Nevertheless,synthetic antioxidants have been suspected of being responsible for toxicity in the long term and their use in foodstuffs is restricted or prohibited in some countries [5–7].Therefore,there is a growing interest on natu-ral antioxidants,especially peptides derived from hydrolyzed food proteins.Protein hydrolysates from several ﬁsh species includ-ing round scad (Decapterus maruadsi )[8],yellow stripe trevally∗Corresponding author.Tel.:+6674286334;fax:+6674558866.E-mail address:soottawat.b@psu.ac.th (S.Benjakul).(Selaroides leptolepis )[9],Paciﬁc hake (Merluccius productus )[10],tilapia (Oreochromis niloticus )[11],silver carp (Hypophthalmichthys molitrix )[12]and smooth hound (Mustelus mustelus )[13]have been reported to possess antioxidative activities.Hypertension has been considered as the most common seri-ous chronic health problem [14].Since angiotensin I-converting enzyme (ACE)(EC 3.4.15.1)is physiologically important in raising blood pressure,the inhibition of ACE activity can lead to an over-all antihypertensive effect.The synthetic ACE inhibitors are now widely used as pro-drugs but these synthetic ACE inhibitors can cause many signiﬁcant undesirable side effects [14,15].Therefore,the natural safe compounds are desirable for prevention of hyper-tension instead of the synthetic counterpart.Among those,food protein derived peptides are promising natural products exhibiting ACE inhibitory activities.Protein hydrolysates with antihyperten-sive activity have also produced from sardinelle (Sardina pilchardus )by-products [16],tuna cooking juice [17],salmon protein [18]and tilapia [19].In Thailand,brownstripe red snapper (Lutjanus vitta )is one of the raw materials for surimi production [20].Besides being pro-duced into surimi,this species and its viscera,especially pyloric caeca,can be used as raw material for production of protein hydrolysate and as the source of proteases,respectively.Produc-tion of ﬁsh protein hydrolysates with bioactivity can pave the way1359-5113/$–see front matter ©2010Elsevier Ltd.All rights reserved.doi:10.1016/j.procbio.2010.09.005S.Khantaphant et al./Process Biochemistry46(2011)318–327319for full utilization of these species.Many factors affect the bioac-tivity of protein hydrolysates,e.g.type of proteases[9],steps of hydrolysis[21],etc.This work aims to study antioxidative and ACE inhibitory activ-ities of protein hydrolysate from brownstripe red snapper muscle prepared using its pyloric caeca protease in combination with commercial proteases via2-step hydrolysis and to investigate the bioactivities of selected hydrolysate after digestion in gastrointesti-nal tract model system.2.Materials and methods2.1.Enzymes and chemicalsAlcalase2.4L(E.C.3.4.21.62)(2.4AU/g)and Flavourzyme500L(E.C.3.4.21.77) (500LAPU/g)were provided by Novozyme(Bagsvaerd,Denmark).2,2 -Azinobis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),1,1-diphenyl-2-picrylhydrazyl (DPPH),2,4,6-trinitrobenzenesulfonic acid(TNBS),1,1,3,3-tetramethoxypropane, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4 ,4 -disulfonic acid(ferrozine),2,4,6-tripyridyl-triazine(TPTZ),l-␣-phosphatidylcholine(lecithin)and linoleic acid were purchased from Sigma(St.Louis,MO,USA).Thiobarbituric acid(TBA),potassium persulfate,␤-carotene and Tween40were obtained from Fluka(Buchs,Switzer-land).Tris(hydroxymethyl)aminomethane(Tris–HCl)was procured from Merck (Darmstadt,Germany)and sodium sulﬁte was obtained from Riedel-deHaën(Seelze, Germany).All chemicals were of analytical grade.2.2.Preparation of pretreatedﬁsh minceBrownstripe red snapper,stored in ice and off-loaded approximately24–36h after capture,were purchased from a dock in Songkhla province,Thailand.Fish were transported in ice with theﬁsh/ice ratio of1:2(w/w)to the Department of Food Technology,Prince of Songkla University within1h.Upon arrival,wholeﬁsh were washed and onlyﬂesh was separated manually.Flesh was minced to uniformity using Moulinex AY46blender(Group SEB,Lyon,France)and phospholipid mem-brane was removed by further homogenizing with nine volumes of cold8mmol L−1 CaCl2solution containing5mmol L−1citric acid[22]using an IKA Labortechnik homogenizer(Selangor,Malaysia)at a speed of11000rpm for2min.After contin-uous stirring for60min at4◦C,the sample was centrifuged at4000×g for15min at4◦C using a Beckman Coulter centrifuge Model Avant J-E(Beckman Coulter,Inc., Fullerton,CA,USA).Thereafter,the pellet was washed by homogenizing withﬁve volumes of cold distilled water using a homogenizer at a speed of11000rpm for 2min,followed by stirring at4◦C for15min prior to centrifuging at9600×g for 10min at4◦C.The washing process was repeated twice.Pretreated mince obtained was kept in polyethylene bags and placed in ice until use,but not longer than2h.2.3.Preparation of proteases from pyloric caecaPyloric caeca from brownstripe red snapper was collected and powdered in liq-uid nitrogen.Thereafter,the pyloric caeca extract was prepared according to the method of Khantaphant and Benjakul[20].Pyloric caeca powder was suspended in ten volumes of extraction buffer(50mmol L−1Tris–HCl buffer,pH8.0,contain-ing10mmol L−1CaCl2.The mixture was homogenized at11000rpm for2min.The homogenate was continuously stirred for30min at4◦C and centrifuged at8000×g for30min at4◦C.The supernatant wasﬁltered through a Whatmanﬁlter paper No.1(Schleicher&Schuell,Maidstone,England).Theﬁltrate obtained was further subjected to40–60%saturation ammonium sulfate precipitation.After stirring at 4◦C for30min,the mixture was centrifuged at8000×g for30min at4◦C and the pellet obtained was dissolved in50mmol L−1Tris–HCl buffer,pH8.0followed by dialysis against20volumes of the extraction buffer overnight at4◦C with three changes of dialysis buffer.The dialysate was kept in ice and referred to as‘pyloric caeca protease;PCP’.The proteolytic activity of PCP was determined using casein as a substrate under optimal condition(60◦C and pH8.5)2.4.Preparation of protein hydrolysate from brownstripe red snapper2.4.1.One-step hydrolysis using different single proteasesPretreated brownstripe red snapper mince with a protein content of95.42%pro-tein(dry basis)determined by Kjeldahl method[23]was mixed with50mmol L−1 Tris–HCl buffer with pH of7.0,8.0and8.5for hydrolysis by Flavourzyme,Alcalase and PCP,respectively,to obtain aﬁnal protein concentration of20g protein L−1.The mixtures were homogenized at a speed of11,000rpm for1min and the pH was rechecked and readjusted using1mol L−1NaOH or1mol L−1HCl.The homogenates were pre-incubated for10min at50◦C for Alcalase and Flavourzyme and at60◦C for PCP[20].To start the hydrolysis,the different levels of enzymes were added into the mixture to obtain the desirable degree of hydrolysis(DH)of20,30and 40%following the method of Benjakul and Morrissey[24].After2h of hydrolysis, the reactions were inactivated by placing the mixture in boiling water for10min. Thereafter,the mixture was centrifuged at2000×g at4◦C for10min.The super-natant was collected and referred to as protein hydrolysate prepared using Alcalase (HA),Flavourzyme(HF)or PCP(HP).All protein hydrolysates were determined for antioxidative activities.2.4.2.Two-step hydrolysis using different proteasesAfter2h of theﬁrst hydrolysis,the mixtures with DH of40%,which had the high-est antioxidative activities,were heated for10min in boiling water and adjusted to the desirable pH using1mol L−1NaOH or1mol L−1HCl for proteases used for the second step.Those proteases included Alcalase,Flavourzyme and PCP.The same amount of proteases used in theﬁrst step was added into the pre-incubated mix-ture with optimal temperature of each protease.Reaction was conducted for1,2, 3and5h.At the time designated,the reaction was terminated by submerging the mixture in boiling water for10min.The mixture was then centrifuged at2000×g at4◦C for10min and the supernatant was collected and adjusted to neutral pH. The neutral solution was referred to as‘protein hydrolysates’and lyophilized to obtain hydrolysate powder.The hydrolysates obtained from HP with further hydrol-ysis using Alcalase(HPA)or Flavourzyme(HPF),hydrolysate from HA using PCP or Flavourzyme for the second step(HAP and HAF)and hydrolysate from HF using PCP or Alcalase for the second step(HFP and HFA)were determined for their antioxida-tive and ACE inhibitory activities.2.5.Determination of antioxidative activities2.5.1.DPPH radical scavenging activityDPPH radical scavenging activity was determined according to the method of Blois[25]with a slight modiﬁcation.Sample solution(1.5mL)was added with1.5mL of0.1mmol L−1DPPH in950mL L−1ethanol.The mixture was allowed to stand for 30min in dark at room temperature.The resulting solution was measured at517nm. The control was prepared in the same manner except that distilled water was used instead of the sample.The DPPH radical scavenging activity was calculated from Trolox standard curve(0–60␮mol L−1)and expressed as␮mol Trolox equivalents (TE)g−1protein.2.5.2.ABTS radical scavenging activityABTS radical scavenging activity was determined as described by Re et al.[26]. ABTS radical(ABTS•+)was produced by reacting ABTS stock solution(7.4mmol L−1 ABTS)with2.6mmol L−1potassium persulfate at the ratio of1:1(v/v).The mixture was allowed to react in the dark for12h at room temperature.Prior to assay,the ABTS•+solution was diluted with methanol to obtain an absorbance of1.1(±0.02) at734nm.To initiate the reaction,150␮L of sample was mixed with2.85mL of ABTS•+solution.The mixture was incubated at room temperature for2h in dark. The absorbance was then read at734nm.A Trolox standard curve(0–200␮mol L−1) was prepared.Distilled water was used instead of the sample and prepared in the same manner to obtain the control.ABTS radical scavenging activity was expressed as␮mol TE g−1protein.2.5.3.Ferric reducing antioxidant power(FRAP)The ability of samples to reduce ferric ion(Fe3+)was evaluated by the method of Benzie and Strain[27].FRAP reagent(a freshly prepared mixture of 10mmol L−1TPTZ solution in40mmol L−1HCl,20mmol L−1FeCl3.6H2O solution and300mmol L−1acetate buffer,pH3.6(1:1:10,v/v/v))(2.85mL)was incubated at 37◦C for30min prior to mixing with150␮L of sample.The reaction mixture was allowed to stand in dark for30min at room temperature.Absorbance at593nm was read and FRAP was calculated from the Trolox standard curve(0–60␮mol L−1)and expressed as␮mol TE g−1protein.The control was prepared in the same manner except that distilled water was used instead of the sample.2.5.4.Ferrous chelating activityChelating activity of samples towards ferrous ion(Fe2+)was measured by the method of Benjakul et al.[28]with a slight modiﬁcation.Sample(200␮L)was mixed with800␮L of distilled water.Thereafter,0.1mL of2mmol L−1FeCl2and0.2mL of 5mmol L−1ferrozine were added.The mixture was allowed to react for20min at room temperature.The absorbance was then read at562nm.The standard curve of EDTA(0–1.0mmol L−1)was prepared.The control was prepared in the same manner except that distilled water was used instead of the sample.Ferrous chelating activity was expressed as␮mol EDTA equivalents g−1protein.2.6.Determination of ACE inhibitory activityThe angiotensin I-converting enzyme inhibitory activity was determined as described by Hayakari et al.[29]with slight modiﬁcations.Sample(0.3mL)was incubated with725unit ACE L−1(50␮L)at37◦C for5min.Thereafter,the incubated mixture was added into the assay mixture(0.2mL)which was100mmol L−1K2HPO4 buffer(pH8.3)containing600mmol L−1NaCl and3mmol L−1hippuryl-l-histidyl-l-leucine(HHL).The mixture was incubated at37◦C for15min.To terminate the reaction,1.5mL of30g L−12,4,6-trichloro-1,3,5-triazine(dissolved in dioxane)and 3mL of0.2mol L−1K2HPO4buffer(pH8.3)were added and mixed thoroughly.The mixture was left for10min until the solution was clear and the absorbance at382nm was determined.Sample blank was prepared in the same manner except that HHL was added after the reaction mixture was terminated.The control was prepared320S.Khantaphant et al./Process Biochemistry46(2011)318–327by using distilled water instead of the sample,whereas the control blank was donein the same manner with the control but HHL was added after the termination.%inhibition of ACE was determined using the following equation:%Inhibition=A C−A SA C×100where A C=A control−A control blank and A S=A sample−A sample blank.2.7.Determination of antioxidative activity in different model systems2.7.1.ˇ-Carotene linoleic acid emulsion model systemThe antioxidative activity of the sample in the␤-carotene linoleic acid emulsion model system was determined as described by Binsan et al.[30].␤-Carotene(1mg) was dissolved in10mL of chloroform.Thereafter,the solution(3mL)was added to 20mg linoleic acid and200mg Tween40.Chloroform was then removed by purg-ing with nitrogen.Fifty milliliters of oxygenated distilled water were added to the ␤-carotene emulsion and mixed well.Hydrolysate(200␮L)was then mixed with 3mL of oxygenated␤-carotene emulsion to obtain theﬁnal concentrations of500 and1000ppm.The oxidation of␤-carotene emulsion was monitored spectrophoto-metrically at470nm after0,10,20,3040,60,90and120min of incubation at50◦C in dark.BHT at levels of100ppm was also used.The control was prepared by using distilled water instead of hydrolysate in the assay system.2.7.2.Lecithin liposome model systemThe antioxidative activity of protein hydrolysates in a lecithin liposome system was determined according to the method of Frankel et al.[31]slightly modiﬁed by Thiansilakul et al.[8].Lecithin liposome system was prepared by suspending lecithin in deionized water at a concentration of8g L−1.The mixture was stirred with a glass rod followed by soniﬁcation for30min in a sonicating bath(Elma Model S30H, Singen,Germany).Protein hydrolysate(3mL)was added to the lecithin liposome system(15mL)to obtain aﬁnal concentration of1000ppm.The mixture was son-icated for2min.To initiate the reaction,20mL of0.15mol L−1cupric acetate were added.The mixture was shaken in the dark at120rpm using a shaker(Heidolph Model Unimax1010,Schwabach,Germany)at37◦C.The systems containing25or 100ppm BHT were also prepared.The control was prepared in the same manner, except that distilled water was used instead of sample.Oxidation in lecithin lipo-some systems was monitored at6h intervals by determining the formation of TBARS and conjugated dienes.2.8.Determination of thiobarbituric acid reactive substances(TBARS)Thiobarbituric acid reactive substances(TBARS)were determined as described by Buege and Aust[32]with a slight modiﬁcation.Sample was homogenized with TBARS solution(3.75g L−1TBA,150g L−1TCA and0.25mol L−1HCl)with a ratio of 1:4(w/v).The mixture was heated in boiling water for10min to develop the pink color.Then the mixture was cooled with running water and centrifuged at5000×g for10min at room temperature using Hettich centrifuge(Hettich Model MIKRO-20,Tuttlingen,Germany).The supernatant was collected and measured at532nm using a UV-1800Spectrophotometer(Shimadzu,Kyoto,Japan).TBARS was calcu-lated from a standard curve of malonaldehyde(MDA)(0–10mg L−1)and expressed as mg MDA kg−1sample.2.9.Determination of conjugated dieneConjugated diene formed in the sample was measured according to the method of Frankel et al.[31].Sample(0.1mL)was dissolved in methanol(5.0mL)and con-jugated dienes were measured as the increase in absorbance at234nm.2.10.Preparation of gastrointestinal tract model system(GIMs)Gastrointestinal tract model system was prepared according to the method of Lo et al.[33]with slight modiﬁcation.Hydrolysate powder was dissolved in distilled water to obtain a concentration of50g protein L−1.The solution was adjusted to pH 2.0with1mol L−1HCl and pepsin dissolved in0.1mol L−1HCL was added to obtain theﬁnal concentration of40g pepsin kg−1protein.The mixture was incubated at 37◦C for1h with continuous shaking(Memmert Model SV1422,Schwabach,Ger-many).Thereafter,the pH of reaction mixture was raised to5.3with1mol L−1NaOH before adding20g pancreatin kg−1protein and the pH of mixture was adjusted to 7.5with1mol L−1NaOH.The mixture was incubated at37◦C for3h with continuous shaking.The digestion was terminated by submerging the mixture in boiling water for10min.During digestion,the mixture was randomly taken and determined for antioxidative activities at0,20,40,60,80,100,120,150,180,210and240min.2.11.Proximate analysisHAP and HFP were determined for protein,fat,ash and moisture contents according to the methods of AOAC[23]with the analytical number of992.15,991.36, 942.05and950.46,respectively.2.12.Amino acid analysisHAP and HFP were hydrolyzed under reduced pressure in4.0M methanesul-fonic acid containing0.2%(v/v)3-2(2-aminoethyl)indole at115◦C for24h.The hydrolysates were neutralized with3.5M NaOH and diluted with0.2M citrate buffer (pH2.2).An aliquot of0.4mL was applied to an amino acid analyzer(MLC-703;Atto Co.,Tokyo,Japan).2.13.Determination of protein concentrationProtein concentration was measured by the method of Lowry et al.[34]using bovine serum albumin as a standard.2.14.Statistical analysisExperiments were run in triplicate.All data were subjected to analysis of vari-ance(ANOVA)and differences between means were evaluated by Duncan’s Multiple Range Test.For pair comparison,T-test was used[35].SPSS Statistic Program(Ver-sion10.0)(SPSS Inc,Chicago,IL,USA)was used for data analysis.3.Results and discussion3.1.Antioxidative activities of brownstripe red snapper protein hydrolysates prepared with single step of hydrolysis usingdifferent proteasesProtein hydrolysates from brownstripe red snapper muscle prepared using protease from pyloric caeca of brownstripe red snapper,Alcalase and Flavourzyme referred to as HP,HA and HF, respectively,with different DH(20,30and40%DH)showed varying antioxidative activities(Fig.1).3.1.1.DPPH radical scavenging activityThe DPPH radical scavenging activity of HA increased with increasing DH(p<0.05)(Fig.1(a)).HF showed the similar activity at all DH used(p>0.05),whereas HP showed no further increase in activity when DH was higher than30%(p>0.05).At all designated DH,HA and HF showed the higher activity than did HP(p<0.05). The result suggested that the peptides in different hydrolysates might be different in term of chain length and amino acid sequence, which contributed to varying capabilities of scavenging DPPH rad-icals.The increase in DPPH radical scavenging activity of HA was in agreement with Thiansilakul et al.[8]who reported the increases in DPPH radical scavenging activity as the DH of the hydrolysate from round scad muscle protein prepared using Flavourzyme and Alcalase increased.On the other hand,Klompong et al.[9]found that DPPH radical scavenging activity of protein hydrolysate pre-pared from the muscle of yellow stripe trevally using Flavourzyme and Alcalase decreased when DH increased.Invert correlation between DH and DPPH radical scavenging activity was obtained for protein hydrolysates prepared from alkaline-aided channel catﬁsh protein isolates using Protamex[36].You et al.[37]reported that loach protein hydrolysate showed the greater DPPH radical scav-enging activity when DH increased.DPPH is a stable free radical and can be scavenged with a proton-donating substance,such as an antioxidant[25].Therefore,protein hydrolysates from brown-stripe red snapper muscle more likely contained peptides acting as hydrogen donors,thereby scavenging free radicals by converting them into more stable products.3.1.2.ABTS radical scavenging activityIn general,ABTS radical scavenging activities of protein hydrolysates increased as DH increased(p<0.05)(Fig.1(b)).The highest activity was observed in HP and HA with40%DH(p<0.05). However,no difference in activity was found in HF with all DH used(p>0.05).Among all hydrolysates,HA had the lowest activity for all DH tested(p<0.05).At DH of20%,HF showed higher ABTS radical scavenging activity than did HP(p<0.05).Conversely,HPS.Khantaphant et al./Process Biochemistry46(2011)318–327321Fig.1.Antioxidative activities of protein hydrolysate from brownstripe red snapper muscle prepared using pyloric caeca protease(PCP)from brownstripe red snapper (HP),Alcalase(HA)and Flavourzyme(HF)with different DHs.Bars represent the standard deviation(n=3).Different capital letters within the same enzyme used indicate signiﬁcant differences(p<0.05).Different letters within the same DH indicate signiﬁcant differences(p<0.05).had the highest activity when DH of40%was used(p<0.05).Pro-tein hydrolysate from alkali-solubilized tilapia protein prepared using various proteases showed a sharp increase in ABTS radical scavenging activity when DH increased from18to23%[11].Loach protein hydrolysate showed a similar result,in which ABTS radical scavenging activity increased with increasing DH[37].For both DPPH and ABTS assays,HF showed no differences in activities with all DH tested(p>0.05).ABTS radical assay is used for determining the antioxidative activity,in which the rad-ical is quenched to form ABTS-radical complex[26].Generally, all hydrolysates contained peptides,which were able to scav-enge ABTS radicals,leading to the termination of radical chain reaction.3.1.3.Ferric reducing antioxidant power(FRAP)Ferric reducing antioxidant power(FRAP)measures the reduc-ing ability against ferric ion(Fe3+),indicating the ability of hydrolysates to transfer an electron to the free radical[27].FRAP of different hydrolysates with varying DH is depicted in Fig.1(c). An increase in FRAP was observed in all hydrolysates when DH increased(p<0.05).FRAP of HA was generally higher than those of HP and HF at all DHs tested(p<0.05).The result suggested that HA had the greater reducing power than did others,leading to the greater efﬁcacy in prevention and retardation of propagation in lipid oxidation.However,protein hydrolysates prepared from alkaline-aided channel catﬁsh protein isolates showed the decrease in reducing power with increasing DH[36].Raghavan et al.[11] reported that alkaline-solubilized tilapia protein hydrolysate pre-pared using Flavourzyme showed the increase in reducing ferric ion when DH increased.The hydrolysis most likely increased the reducing power,especially when the cleavage of peptides increased as indicated by the increase in DH.The result indicated that pep-tides generated from the hydrolysis by different proteases had the different capacities of providing electron to the radicals.3.1.4.Ferrous chelating activityFerrous chelating activities of hydrolysates prepared using dif-ferent proteases with different DHs are shown in Fig.1(d).Chelating activity against Fe2+of HP slightly increased when DH increased up to30%(p<0.05).For HA and HF,the decreases in ferrous chelating activities were found as DH increased(p<0.05).Ferrous ion(Fe2+) is the most powerful pro-oxidant among metal ions[38],leading to the initiation and acceleration of lipid oxidation by interaction with hydrogen peroxide in a Fenton reaction to produce the reactive oxy-gen species,hydroxyl free radical(OH•)[39].Therefore chelation of metal ions by peptides in hydrolysates could retard the oxida-tive reaction.The result indicated that a higher DH rendered HA and HF with lower metal-chelating activities.The shorter chain of peptides might lose their ability to form the complex with Fe2+. At DH of20%,HF showed the highest chelating activity(p<0.05), followed by HA and HP,respectively.Peptides in HF could effec-tively chelate the Fe2+,leading to the retardation of initiation stage. The result indicated that the limited hydrolysis of muscle protein resulted in the enhanced ferrous chelating activity,compared with the excessive hydrolysis.The higher chelating activity of HP was coincidental with the higher DPPH and ABTS radical scavenging activity and FRAP,as the DH increased.Fe2+chelating activity of round scad protein hydrolysate prepared using Alcalase showed the increase in chelating activity with increasing DH,but those treated with Flavourzyme showed no difference in activity at all DH tested[8].With the same enzymes used,chelating activity of pro-tein hydrolysate prepared from the muscle of yellow stripe trevally322S.Khantaphant et al./Process Biochemistry46(2011)318–327using Flavourzyme and Alcalase increased with increasing DH[9]. Higher ferrous chelating activity was reported for hydrolysate of sil-ver carp using Alcalase and Flavourzyme when DH increased[12]. Apart from Fe,other transition metals,such as Cu and Co can affect the rate of lipid oxidation and decomposition of hydroperoxide. Theodore et al.[36]reported that Cu2+chelating activity of catﬁsh protein hydrolysate increased with increasing DH.Some proteins and peptides can chelate metal ions like Fe2+due to the presence of carboxyl and amino groups in the side chains of acidic and basic amino acids[10,40].Alcalase is endopeptidase capable of hydrolyzing proteins with broad speciﬁcity for peptide bonds and prefers for the uncharged residue,whereas Flavourzyme is a mixture of endo-and exopeptidase enzyme,which can produce both amino acids and peptides[41].Hydrolysates showing differ-ent antioxidative activities might be attributed to the differences in the exposed side chains of peptides as governed by the speciﬁcity of different proteases towards peptide bonds in the proteins[42]. DH also greatly inﬂuenced the peptide chain length.The higher DH,the more cleavage of peptide chains took place.Peptides with various sizes and compositions had different capacities of scaveng-ing or quenching free radicals[8,9].PCP,Alcalase and Flavourzyme more likely cleaved the peptide bonds in brownstripe red snap-per muscle at different positions,resulting in the different products with varying antioxidative activities.With40%DH,all hydrolysates functioned more effectively as primary antioxidant,whereas the secondary antioxidative activity,chelating ability,was lowered.To enhance the antioxidative activity of peptides,especially as the pri-mary antioxidant,the hydrolysate with40%DH was prepared for theﬁrst step of hydrolysis.3.2.Antioxidative and ACE inhibitory activities of brownstripered snapper protein hydrolysate prepared with two-stephydrolysis using different proteases3.2.1.DPPH radical scavenging activityDPPH radical scavenging activity of protein hydrolysates with various hydrolysis times in the second step of hydrolysis using another protease is shown in Fig.2(a).For HAF,HAP,HFA and HFP, the activities increased with increasing time up to2h(p<0.05). Thereafter,the gradual decrease was observed when hydrolysis times were3and5h(p<0.05).No changes in DPPH radical scav-enging activity were observed for HPF,while HPA showed the maximal activity at3h of hydrolysis(p<0.05).The results sug-gested that scavenging activity against DPPH radical was enhanced by additional hydrolysis using another enzyme with an appropriate time.Generally,hydrolysates prepared by further hydrolyzing the hydrolysate obtained from theﬁrst step of hydrolysis with another protease for2h had a greater scavenging ability against DPPH rad-ical.3.2.2.ABTS radical scavenging activityABTS radical scavenging activity of protein hydrolysates pre-pared by two-step hydrolysis is shown in Fig.2(b).Only HAF and HAP showed the increases in ABTS radical scavenging activity,com-pared with their parent counterparts,HA.HAP with the hydrolysis time of2h for the second step had the highest activity(p<0.05). This suggested the enhancement of ABTS radical scavenging activ-ity when PCP was used for further hydrolysis of HA.However,the further hydrolysis of HF with another protease had no impact on the increases in activity(p>0.05).For HP,the decrease in activities was obtained when the second step of hydrolysis was performed (p<0.05).The results suggested that the second step of hydrolysis could slightly increase ABTS radical scavenging activity,depending on theﬁrst hydrolysate as well as the types of protease used for the second step.It was noted that the abilities of scavenging ABTS and DPPH radicals by hydrolysates were different.This might be due to the differences in ability of scavenging the different radicals,ABTS and DPPH,by the same peptide.3.2.3.Ferric reducing antioxidant power(FRAP)Fig.2(c)shows FRAP of protein hydrolysates prepared using two-step hydrolysis.The increases in FRAP were observed for all hydrolysates,when the second step of hydrolysis was applied.For HF and HP,when Alcalase was used for the second step of hydroly-sis,the continuous increases in FRAP were observed(p<0.05).For other hydrolysates,the hydrolysis time was the factor affecting the FRAP of resulting hydrolysate,depending on theﬁrst hydrolysate and the types of protease used for the second step of hydrolysis.The increase in FRAP with increasing hydrolysis time was coincidental with the increase in DPPH radical scavenging activity(Fig.2(a)). For HAF,HAP and HPF,the activities were increased with increas-ing hydrolysis time up to2h(p<0.05).For HFP,the hydrolysis time more than1h had the negative effect on FRAP(p<0.05).Gener-ally,further hydrolysis with another protease led to the increase in FRAP,but the activities of resulting hydrolysate were governed by hydrolysis time.3.2.4.Ferrous chelating activityFerrous chelating activities of hydrolysates prepared by two-step hydrolysis are shown in Fig.2(d).HFA and HPA showed the marked decrease in chelating activities when the second hydroly-sis was conducted(p<0.05).Alcalase used in the second step might generate peptides with the lower ability in Fe2+chelating.The abil-ities of HAF,HAP,HFP and HPF in chelating Fe2+ion were more pronounced,compared with their parent hydrolysate counterparts. The increases were observed when a certain time of the second hydrolysis was used.Among all protein hydrolysates,HFP showed the highest ferrous chelating activity,especially when the second step hydrolysis time of1h was used(p<0.05).3.3.ACE inhibitory activityThe inhibitory effect of all hydrolysates prepared using two-step hydrolysis against angiotensin I-convering enzyme(ACE)was determined as shown in Fig.2(e).For theﬁrst hydrolysate prepared using the different single proteases(0h),HF showed the highest ACE inhibition(11.44%)(p<0.05).Flavourzyme might produce the peptides with ACE inhibitory activity.Raghavan and Kristinsson [19]used Cryotin and Flavourzyme for hydrolysis of tilapia protein and found the higher ACE inhibition by hydrolysate prepared using Flavourzyme.When the second step of hydrolysis using another protease was performed,HFA and HFP showed the higher ACE inhi-bition when hydrolysis time of2and3h was used,respectively.HAP also showed high ACE inhibitory activity.Wu et al.[43]reported that shark meat treated with enzyme showed the higher ACE inhi-bition than that of untreated one.ACE inhibition by tilapia protein hydrolysate was reported,especially with increasing DH[19].Fur-thermore,smaller peptides are the better ACE inhibitors than the larger counterpart[19].The hydrolysis might release ACE inhibitory peptides in hydrolysate[43].Hydrolysates from muscle of differ-entﬁsh have been reported to possess ACE inhibitory activity,e.g. hydrolysates from tilapia protein[19],freshwater clam muscle[44], shark meat[43],Atlantic salmon,Coho salmon,Alaska pollack and southern blue whiting muscle[45].From the results,the use of Alcalase for theﬁrst hydrolysis and the use of PCP in the second hydrolysis for2h(HAP)yielded the resulting hydrolysate with the higher DPPH and ABTS radical scavenging activities and FRAP,compared to other hydrolysates (p<0.05).However HF hydrolyzed with PCP in the second hydrol-ysis step for1h(HFP)showed much higher chelating activity, compared with others(p<0.05).Therefore,HFP and HAP prepared with1and2h for the second step of hydrolysis,respectively,were。

银杏肽锌螯合物的制备、体外消化及抗氧化活性分析

郑义，李诗颖，李闯，等. 银杏肽锌螯合物的制备、体外消化及抗氧化活性分析[J]. 食品工业科技，2023，44（17）：420−427. doi:10.13386/j.issn1002-0306.2022110135ZHENG Yi, LI Shiying, LI Chuang, et al. Preparation, in Vitro Gastrointestinal Digestion and Antioxidant Activity of Ginkgo biloba Peptides-Zinc Chelate[J]. Science and Technology of Food Industry, 2023, 44(17): 420−427. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2022110135· 营养与保健 ·银杏肽锌螯合物的制备、体外消化及抗氧化活性分析郑　义1,2，李诗颖1，李　闯1，周小芮1，陈亚楠1，张秀芸1，张银雨1（1.徐州工程学院食品与生物工程学院，江苏徐州 221018；2.江苏省食品资源开发与质量安全重点建设实验室，江苏徐州 221018）摘　要：本文优化了银杏肽锌螯合物（Ginkgo biloba peptides-zinc chelate, Zn-GBP ）的制备工艺，分析了Zn-GBP 的体外消化特性及抗氧化活性。

采用单因素实验及响应面法优化了Zn-GBP 的制备工艺；采用体外模拟胃肠道消化测定了Zn-GBP 中锌离子的生物利用率；以DPPH 自由基清除能力、ABTS +自由基清除能力、还原能力为指标，评价了Zn-GBP 的体外抗氧化活性。

结果表明，Zn-GBP 的最佳制备工艺条件为：银杏肽与锌质量比3:1、螯合pH 8.2、螯合温度70 ℃、螯合时间2 h ；在此条件下，螯合率为49.23%±0.35% ，螯合物得率为42.34%±0.45%。

中国弯颈霉多糖抗氧化和抑制氧化应激活性研究

白明健，周颖，程昊，等. 中国弯颈霉多糖抗氧化和抑制氧化应激活性研究[J]. 食品工业科技，2024，45（2）：333−341. doi:10.13386/j.issn1002-0306.2023030199BAI Mingjian, ZHOU Ying, CHENG Hao, et al. Antioxidant and Oxidative Stress Inhibitory Activities of Tolypocladium sinense Polysaccharide[J]. Science and Technology of Food Industry, 2024, 45(2): 333−341. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023030199· 营养与保健 ·中国弯颈霉多糖抗氧化和抑制氧化应激活性研究白明健，周　颖，程　昊，边　聪，李名正，李　林*，张春晶*（齐齐哈尔医学院医学技术学院，黑龙江齐齐哈尔 161006）O −2摘　要：目的：探究中国弯颈霉菌丝体多糖（Tolypocladium sinense polysaccharide ，TSP ）的体外抗氧化活性和抑制过氧化氢诱导小鼠胰岛MIN6细胞氧化应激导致的细胞凋亡。

方法：采用热水浸提法提取中国弯颈霉菌丝体多糖，随后测定其超氧阴离子自由基（superoxide anion ，·）、羟自由基（hydroxy radical ，·OH ）、对1,1-二苯基苦基苯肼自由基（P-1,1-diphenylpicryl phenylhydrazine radical ，DPPH·）清除能力；采用200 μmol/L 过氧化氢（hydrogen peroxide ，H 2O 2）诱导小鼠胰岛MIN6细胞氧化应激，给予高剂量和低剂量TSP （0.625、0.156 mg/mL ）进行保护，MTT 法测定MIN6细胞生存率；倒置显微镜观察细胞形态；采用试剂盒测定培养基中乳酸脱氢酶（lactate dehydrogenase ，LDH ）水平，细胞内超氧化物歧化酶（superoxide dismutase ，SOD ）活性和丙二醛（malondialdehyde ，MDA ）含量；流式细胞术检测细胞凋亡；Western Blot 检测核因子E2相关因子2（nuclear factor E2-related factor2，Nrf-2）和磷酸化c-Jun 氨基末端激酶（phosphorylated c-jun N-terminal kinase ，pJNK ）相对表达量。

氧化应激与肝脏损伤

氧化应激与肝脏损伤氧化应激与肝脏损伤吴娜, 蔡光明, 何群吴娜,湖南中医药⼤学湖南省长沙市410208吴娜, 蔡光明,中国⼈民解放军302医院中药研究所北京市100039何群,湖南中医药⼤学药剂教研室湖南省长沙市410208作者贡献分布:本⽂选题, 参考⽂献检索及撰写由吴娜完成; 论⽂修改由蔡光明与何群完成.通讯作者:蔡光明, 100039, 北京市, 中国⼈民解放军302医院中药研究所.cgm1004@/doc/0f50be76a417866fb84a8e84.html电话: 010-********收稿⽇期: 2008-08-14 修回⽇期: 2008-09-21接受⽇期: 2008-09-22 在线出版⽇期: 2008-10-18Oxidative stress and hepatic injuryNa Wu, Guang-Ming Cai, Qun HeNa Wu,Hunan University of Traditional Chinese Medicine, Changsha 410208, Hunan Province, ChinaNa Wu, Guang-Ming Cai,Institute of Chinese Materia Medica, the 302th Hospital of Chinese PLA, Beijing 100039, China Qun He, Pharmaceutical Division, Hunan University of Traditional Chinese Medicine, Changsha 410208, Hunan Province, ChinaCorrespondence to:Guang-Ming Cai, Institute of Chinese Materia Medica, the 302th Hospital of Chinese PLA, Beijing 100039, China. cgm1004@/doc/0f50be76a417866fb84a8e84.htmlReceived: 2008-08-14 Revised: 2008-09-21Accepted:2008-09-22 Published online: 2008-10-18AbstractOxidative stress, initiated by reactive oxygen species, is the collective pathophysiological mechanism of many hepatopathies. Oxidative stress results in hepatic injury mainly by priminglipid peroxidation to change the function of biological membrane, covalent immobilization of biomacromolecules and destroying the enzyme activity considering cytokine (TNF-α and NF-κB) interaction. The role of oxidative stress in many hepatopathies such as fatty liver desease, viral hepatitis, hepatic fibrosis is innegligible.Key Words: Oxidative stress; Reactive oxygen species; Hepatic injury; Lipid peroxidation; CytokineWu N, Cai GM, He Q. Oxidative stress and hepatic injury. Shijie Huaren Xiaohua Zazhi 2008; 16(29): 3310-3315摘要活性氧⾃由基引发的氧化应激是多种肝病发病的共同病理⽣理基础. 氧化应激主要通过启动膜脂质过氧化改变⽣物膜功能、与⽣物⼤分⼦共价结合及破坏酶的活性等在细胞因⼦(如TNF-α、NF-κB)的共同作⽤下引起不同程度的肝损伤. 氧化应激在脂肪肝、病毒性肝炎、肝纤维化等肝病中可产⽣不容忽视的作⽤.关键词:氧化应激; 活性氧; 肝损伤; 脂质过氧化; 细胞因⼦吴娜, 蔡光明, 何群. 氧化应激与肝脏损伤. 世界华⼈消化杂志2008; 16(29): 3310-3315/doc/0f50be76a417866fb84a8e84.html /1009-3079/16/3310.asp0 引⾔胃⽣物体内的能量代谢将氧⽓作为有氧代谢过程中的电⼦接受体, 不可避免地产⽣活性氧(reactive oxygen species, ROS)⾃由基. ROS具有双重效应, 与某些⽣理活性物质的调控和炎症免疫过程密切相关, 但是过量的ROS容易导致氧化应激(oxidative stress, OS)状态[1]. 线粒体呼吸链复合体利⽤电⼦传递⽣产ATP, 是ROS的主要来源[2], 肝脏含有丰富的线粒体, 因此也是ROS 攻击的主要器官. OS可能是肝病的共同发病机制. 因此, 本⽂就近年来关于OS在不同肝损伤机制中的作⽤进⾏综述.1 氧化应激与脂肪性肝病脂肪性肝病指脂肪在肝细胞中的异常沉积, 可分为⾮酒精性脂肪肝病(nonalcoholic fatty liver disease, NAFLD)和酒精性脂肪肝病(alcoholic fatty liver disease, AFLD). 在复杂的脂肪肝发病机制中OS起关键作⽤, ⽬前对脂肪肝发病机制⼴泛接受的理论是1998年Day et al[3]提出的“⼆次打击”学说. 第⼀次打击主要是胰岛素抵抗和脂肪代谢的失衡导致的脂肪在肝细胞中的沉积, 尤其是脂肪酸和⽢油三酯, 最终引起单纯性肝脂肪变性; 第⼆次打击为环境应激物(如饮⾷成分)及代谢应激物(如⾼⾎糖)主要通过对肝细胞线粒体的损伤产⽣OS, 在细胞因⼦等共同作⽤下, 引起脂肪性肝炎, 进⼀步形成脂肪性肝纤维化和脂肪性肝硬化.1.1 氧化应激与NAFLD随着肥胖及Ⅱ型糖尿病患病率的增加, NAFLD已成为当今医学领域的⼀个难题. NAFLD包含两种组织学损伤: 单纯性⾮酒精性脂肪肝(nonalcoholic fatty liver, NAFL)及⾮酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH). NAFLD的发病机制较复杂, 迄今尚未完全阐明, 过量的ROS使肝内抗氧化系统遭到严重破坏. ⾕胱⽢肽(glutathione, GSH)是细胞内重要的肽类抗氧化剂, 是诸多ROS清除酶的还原底物, 反应后⽣成具有潜在⾼细胞毒素的氧化型⾕胱⽢肽(oxidative glutathione, GSSG). Nobili et al[4]以GSSG/GSH的⽐值评估体内OS时发现NASH患者⾎液中GSSG增长了1.5倍, 导致GSSG/GSH发⽣显著性变化. 此外, 肝内其他重要的抗氧化物质, 如辅酶Q10, CuZn-SOD及过氧化氢酶(CAT)等的不断衰竭在NAFLD患者中也得到了证实[5].NASH患者的肝细胞以异常的线粒体为特征, 线粒体损伤是肝细胞损伤的重要原因之⼀, ⽽线粒体中过量游离脂肪酸(FFA)的氧化是导致线粒体损伤的重要原因: ⼀⽅⾯, FFA的β-氧化过程产⽣的ROS使线粒体膜发⽣脂质过氧化(lipid peroxidation, LPO),导致进⼀步的解偶联作⽤, 即抑制氧化磷酸化的作⽤, 同时也增强了线粒体的OS[6]. ROS的增多促使肿瘤坏死因⼦(tumor necrosis factor-α, TNF-α)的⽣成, 使其通过⼲预线粒体呼吸链并形成超氧阴离⼦(O2-)⽽加剧线粒体损伤, 并增加了线粒体膜的通透性[7]. 另⼀⽅⾯, ROS能导致mtDNA碱基的氧化, Seki et al[8]研究发现在17例NASH患者中, 有11例患者(64.7%)的肝细胞中表达了8-羟基鸟苷酸(8-OHdG)(mtDNA损伤的⼀种标志).ROS还能启动多种细胞因⼦, 如⽣长转化因⼦β(transforming growth factorβ, TGFβ)、⽩细胞介素-8(interleukin-8, IL-8)、NF-κB等. NF-κB、TGF-β、IL-8和LPO产物4-OH壬烯酮(4-hydroxynonenal, HNE)在⼀定程度上还可以导致中性粒细胞的浸润, 使促炎症反应加强, 最终导致肝细胞凋亡.OS与主要存在于肝实质细胞中的过氧化物酶体增殖物启动受体-α(peroxisome proliferator activated receptor alpha, PPARα)有关[9], PPARα主要对肝内脂肪酸氧化相关基因表达进⾏调控, 如脂酰辅酶A氧化酶(acyl-CoA oxidase, AOX)等[10].1.2 氧化应激与AFLD在AFLD的发病机制中OS同样受到格外关注. 细胞⾊素P450 2E1(CYP2E1)和NADPH氧化酶是⼄醇代谢过程中产⽣ROS的重要酶体系[11]. ⼄醇的代谢产物在CYP2E1及Fe3+参与下的氧化作⽤, 会产⽣⼤量的OS产物, 如OH·、O2-、H2O2等.ROS的增多将会引起细胞内ATP衰竭, 使线粒体氧化容量受损, 进⼀步影响⼄醛的氧化, 使⼄醛在肝脏中不断蓄积, 并可能通过抑制AMP激活的蛋⽩激酶AMPK和sirtuin 1蛋⽩活化肝脏的固醇调节组件结合蛋⽩-1(sterol regulatory element binding protein-1, SREBP-1)增加⽣脂酶基因的表达, 加速脂肪酸的合成[12]; ⼄醇的代谢受阻, 更易导致肠黏膜通透性增加, 肠源性内毒素可通过增强Kupffer细胞内毒素受体CD14和Toll样受体4的表达, 诱导Kupffer细胞中产⽣⼤量的以TNF-α为主的细胞因⼦, 加剧炎症反应, 进⼀步引起肝细胞的坏死、凋亡[13].在⼄醇介导的肝损伤中LPO产物衍⽣的抗原类引起的免疫反应可能起重要作⽤. 丙⼆醛(MDA), HNE可与⼈⾎清⽩蛋⽩结合(HSA), 分别形成MDA-HSA和HNE-HSA, 引起免疫反应[14]. 此外, MDA还能与⼄醛⽣成加合物MAA, 同样具有很强的致免疫特性[15].另外, PPARα也参与了⼄醇介导的肝损伤过程, 是其发病机制的重要原因之⼀, PPARα基因的失活将导致肝脏⼤量的脂质聚集,使脂质代谢机制紊乱[16]图1 氧化应激在NAFLD/AFLD中的损伤机制2 氧化应激与药物性肝病随着临床药物和⾮处⽅药种类的增多及联合⽤药的⼴泛应⽤, 药源性肝损害的发⽣率逐年增⾼. 根据2007年美国疾控中⼼的最新调查数据显⽰每年⼤约有由药物引起的1600例急性肝功能衰竭的案例[17]. 引起肝损害的常见药物有对⼄酰氨基酚(解热镇痛抗炎药)、阿莫西林(青霉素类抗⽣素)、胺碘酮(抗⼼律失常药), 他莫西芬(抗癌药), 司他夫定(抗病毒药)等[18].药物代谢反应与众多酶系有关, 不仅包括CYP450, 还包括含黄素单氧化酶(flavin-containing monooxygenase, FMO), 环氧化物⽔解酶(epoxide hydrolase, EH), 其中CYP450起重要作⽤, 如CYP1A1、CYP1A2、CYP1B1、CYP2E1等. Gonzalez[19]研究发现在不同剂量的⼄酰氨基酚(acetaminophen, AP)产⽣肝毒性条件下, 缺失CYP2E1的⼩⿏存活率明显⾼于野⽣型⼩⿏, 缺失CYP2E1及CYP1A2的⼩⿏在最⾼剂量的AP(1200 mg/kg)条件下, 存活率仍约为90%.药物代谢可以通过多种途径引起的不同程度的肝损伤. 如AP经CYP450(主要为CYP2E1)代谢产⽣的活性中间体N-⼄酰-对苯醌亚胺(NAPQI), 与肝细胞⼤分⼦结合导致肝毒性及GSH的耗竭[20], 加剧LPO; 司他夫定, 通过抑制DNA pol-γ(多聚酶中唯⼀能复制mtDNA的酶类), 最终导致乳酸性酸中毒[21]; 曲格列酮能明显改变70种线粒体蛋⽩质, 并检测出Lon蛋⽩酶、CA T等含量增加[22].药物代谢过程中CYP2E1激活产⽣的ROS可能通过表⽪⽣长因⼦受体(epidermal growth factor receptor, EGRF)/c-Raf的信号增强细胞外信号调节激酶1/2(extracelluar signal-regulated kinase 1/2, ERK 1/2)的磷酸化导致细胞凋亡和坏死基因的激活或改变[23].图2 氧化应激在肝纤维化中的损伤机制.3 氧化应激与病毒性肝炎病毒性肝炎(viral hepatitis, VP)的发病机制⾄今尚未完全阐明, 越来越多的研究表明OS是重要的影响因素. VP患者的肝脏中促氧化/抗氧化系统处于严重失衡状态[24].⽬前, 研究肝炎病毒与OS的关系多集中在HBV(hepatitis B virus)、HCV(hepatitis C virus). Levent et al[25-26]发现HBV、HCV 患者⾎清中抗氧化酶CuZn-SOD、GSX-Px及GSH、β-胡萝⼘素(抑制单线氧活性)明显低于健康者. HBV、HCV蛋⽩的表达可以通过Ca2+信号产⽣OS[27-28]; OS 能导致GSH-Px(⼀种极少见的硒依赖酶)的耗竭使硒含量(主要以硒代半胱氨酸的型式存在)减少, 使免疫反应受到抑制, 并能促进病毒的复制[29]. HBV、HCV患者⾎清中MDA、共轭⼆烯(CD)含量也明显升⾼, 在丙肝患者表现得尤为突出, 这不仅与直接引起细胞溶解的病毒有关, 还与O2-引发的细胞膜损伤有关[30]. 另外, ROS还能激活NF-κB 及转录信号转导⼦与激活⼦-3(Signal Transducer and Activator of Transcription-3, STAT-3), ROS在NF-κB介导下可进⼀步激活环氧化酶-2(Cox-2), Cox-2与STAT-3在细胞增殖、分化、肿瘤形成中起重要作⽤[31-32].4 氧化应激与肝纤维化现代医学认为减少胶原纤维的⽣成和增强其降解, 肝纤维化是可以逆转的. 肝星状细胞(heptic stellate cells, HSCs)的激活转化为肌纤维母细胞, 并⼤量分泌细胞外基质[33](extracellular matrix, ECM)是肝纤维化发⽣和发展的核⼼病理环节, 发病机制复杂, OS引发的LPO是HSC活化、增殖、及胶原合成的重要原因.Wang et al[34]在研究褪⿊素对四氯化碳(CCl4)导致肝纤维化的保护作⽤时发现6个星期后, CCl4模型组⼤⿏的羟脯氨酸(胶原代谢的⽣化指标)、MDA、促炎症因⼦TNF-α及IL-1β显著性升⾼, ⽽抗氧化物酶GSH-Px及SOD的活性显著性降低. 另外, 平滑肌肌动蛋⽩(alpha-smooth muscle actin, α-SMA)、8-OhdG、转化⽣长因⼦β1(TGFβ1)mRNA、基质⾦属蛋⽩酶(matrix metalloproteinase, MMP-2)⽔平在肝纤维化模型中也明显升⾼[35-36].TGFβ1是⽬前研究发现的促纤维增⽣的最重要的细胞因⼦之⼀, ROS能促进HSC通过上调核转录因⼦KLF6(Kruppel-like factor 6, KLF6)分泌TGFβ1[37], 并激活HSC转化为肌纤维母细胞[38]. TGFβ1的激活与丝裂原活化蛋⽩激酶(mitogen activated protein kinase, MAPK)和Akt信号有关[35]. TGFβ1还能释放F2-异前列烷(F2-isoprostanes, F2-IsoPs), F2-IsoPs是肝细胞中LOP 反应⽣成的⼀类前列腺素F2样产物, 被认为是反应LPO⽔平最可靠的标志[39], 能介导HSC的分化和促进胶原的过度形成[40].对氧磷酶-1(paraoxonase-1, PON-1)对OS的调节和肝纤维化的形成起重要作⽤, PON-1是⼀类钙离⼦依赖性⾼密度脂蛋⽩(high-density lipoproteins, HDL)的酯酶, 在脂类代谢中具有重要的抗氧化活性. Ferre et al[41]发现在肝硬化患者的⾎清和肝脏中PON-1表达显著性增加, 但其活性显著性降低. 原因可能是PON-1在⽔解脂质过氧化物后活性降低或HDL的结构发⽣变化导致PON-1活性降低.另外, 过氧化物酶体增殖物启动受体-γ(peroxisome proliferator activated receptor gamma, PPARγ)活性[42]、ERK 1/2信号及Na+/H+交换[43]在激活HSC和胶原的合成中都起作⽤.5 氧化应激与肝癌OS在肝癌细胞增殖、凋亡机制中的作⽤也是不容忽视的. ROS在特定细胞内氧化还原环境下可通过激活蛋⽩激酶B(protein kinase B, PKB)途径传递信号, 促使激活蛋⽩-1(activator protein, AP-1)转录因⼦组成成员c-fos/c-jun基因的mRNA表达, 最终促进肝癌细胞⽣长[44]. 肝癌细胞凋亡与线粒体突变是密不可分的. 李国平et al[45]研究了OS诱导HepG2肝癌细胞凋亡及其机制时发现HepG2暴露于2 mmol/L的H2O2可以产⽣OS, OS作⽤后4 h, 细胞线粒体膜电位明显下降;作⽤8、12 h后细胞凋亡蛋⽩酶3、9(Caspase-3、Caspase-9)分别升⾼6.7和3.6倍; 作⽤12 h后细胞开始凋亡, 提⽰OS诱导HepG2肝癌细胞的凋亡与线粒体通路及Caspase启动有关. OS引起的mtDNA 突变也是肝癌细胞凋亡的⼀个重要原因. 张国强et al[46]⽤溴化⼄锭诱导建⽴mtDNA缺失肝癌细胞(ρ0SK-Hep1)模型, 探讨ROS及线粒体跨膜电位在ρ0SK-Hep1肝癌细胞中的改变时发现mDNA缺失后, 细胞内ROS荧光强度明显增强, 细胞膜电位下降, ROS的增加进⼀步加重线粒体膜的损伤.另外, 端粒酶的启动可能是细胞癌变的⼀个共同通路. 端粒酶的激活是原发性肝癌(hepatocellular carcinoma, HCC)的早期事件, Liu et al[47]发现端粒酶的活性与MDA的含量成正相关, OS对端粒还能引起端粒的损伤或缩短加速[48].6 结论作为细胞信号系统中第⼆信使⾓⾊的ROS, 具有独特的⽣理作⽤, 但过量的ROS通过多种途径在细胞因⼦等共同作⽤下引起不同程度的肝损伤.了解氧化应激在不同肝病中的损伤机制, 对今后肝病的预防和治疗⼯作、开发和利⽤⾼效低毒的抗氧化活性物质具有重要意义.背景资料⼈体内95%的⾃由基属于氧⾃由基, 他往往是其他⾃由基产⽣的起因. 过量的氧⾃由基容易引起细胞的损伤和死亡, 与多种疾病有密切的关系, 如衰⽼、肿瘤及冠⼼病等.同⾏评议者王蒙, 副教授, 中国⼈民解放军第⼆军医⼤学附属东⽅肝胆外科医院肝外综合治疗⼀科; 谭学瑞, 教授, 汕⼤医学院第⼀附属医院院长室研发前沿肝病的发病机制⼗分复杂, 氧⾃由基引起的氧化性损伤是其中⼀个⼗分重要的原因. ⽬前研究的热点是天然抗氧化性药物及其作⽤机制.应⽤要点本⽂综合阐述了氧化应激在多种肝病发病机制中的损伤机制, 为临床肝病的治疗中筛选⾼效的抗氧化活性药物提供了⼀定的理论依据.同⾏评价本⽂主题明确, 重点突出, 层次分明, 逻辑性强, 是⼀篇较⾼学术价值的综述.7参考⽂献1 张庆柱. 分⼦药理学. 第1版. 北京: ⾼等教育出版社,2007: 1902 光吉博则, ⾕仁烨. 氧化应激的病理⽣理作⽤. ⽇本医学介绍2007; 28: 150-1523 Day CP, James OF. Steatohepatitis: a tale of two "hits"? 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Reverse transformation of hepaticmyofibroblast-like cells by TGFbeta1/LAP. Biochem Biophys Res Commun2003; 311: 959-965 PubMed DOI39 Zhang L, Wei W, Xu J, Min F, Wang L, Wang X, Cao S, Tan DX, Qi W, Reiter RJ. Inhibitoryeffect of melatonin on diquat-induced lipid peroxidation in vivo as assessed by the measurement of F2-isoprostanes. J Pineal Res2006; 40: 326-331 PubMed DOI40 Comporti M, Arezzini B, Signorini C, Sgherri C, Monaco B, Gardi C. F2-isoprostanesstimulate collagen synthesis in activated hepatic stellate cells: a link with liver fibrosis? Lab Invest 2005; 85: 1381-1391 PubMed DOI41 Ferre N, Marsillach J, Camps J, Mackness B, Mackness M, Riu F, Coll B, Tous M, Joven J.Paraoxonase-1 is associated with oxidative stress, fibrosis and FAS expression in chronic liver diseases. J Hepatol2006; 45: 51-59 PubMed DOI42 Ghosh AK, Wei J, Wu M, Varga J. 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基于线粒体功能障碍和内质网应激探讨非酒精性脂肪性肝病发病机制

· 综述·基于线粒体功能障碍和内质网应激探讨非酒精性脂肪性肝病发病机制*薛春燕　饶晨怡　吴　玲　黄晓铨　陈世耀　李　锋#复旦大学附属中山医院消化科（200032）摘要　非酒精性脂肪性肝病（NAFLD ）是一种肝脏细胞脂肪异常堆积引起的慢性肝病，其患病率在全世界范围内呈上升趋势，已成为慢性肝病的最常见原因。

NAFLD 发病机制复杂多样，胰岛素抵抗、遗传和表观遗传因素、慢性全身性炎症、线粒体功能障碍、内质网应激、饮食和肠道菌群等均是NAFLD 发生、发展的重要因素。

本文主要讨论了线粒体功能障碍和内质网应激等参与NAFLD 形成的机制，旨在为NAFLD 防治提供新的认识和治疗思路。

关键词　非酒精性脂肪性肝病；　线粒体功能障碍；　内质网应激；　氧化性应激；　非折叠蛋白质应答Exploring Pathogenic Mechanisms of Non⁃alcoholic Fatty Liver Disease Based on Mitochondrial Dysfunction and Endoplasmic Reticulum Stress XUE Chunyan, RAO Chenyi, WU Ling, HUANG Xiaoquan, CHEN Shiyao, LI Feng. Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai (200032)Correspondence to: LI Feng, Email: li.feng2@zs⁃Abstract Non⁃alcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by abnormal accumulation offat in the hepatocytes. Its prevalence is rising globally and has become the most common cause of chronic liver disease worldwide. The pathogenesis of NAFLD is multifaceted, involving insulin resistance, genetic and epigenetic factors, chronic systemic inflammation, mitochondrial dysfunction, endoplasmic reticulum stress, diet, gut microbiota, and other significantcontributors. This article primarily delves into the mechanisms of mitochondrial dysfunction and endoplasmic reticulum stress in the development of NAFLD, aiming to provide new insights and therapeutic strategies for NAFLD.Key words Non⁃Alcoholic Fatty Liver Disease;　Mitochondrial Dysfunction;　Endoplasmic Reticulum Stress;　Oxidative Stress;　Unfolded Protein ResponseDOI ： 10.3969/j.issn.1008⁃7125.2022.12.007*基金项目：复旦大学附属中山医院科研基金⁃308（2019ZSFZ09）#本文通信作者， Email: li.feng2@zs⁃非酒精性脂肪性肝病（non⁃alcoholic fatty liver disease, NAFLD ）指在无酒精作用下，以肝内细胞脂肪过度沉积为特征的慢性渐进性肝病。

Y. Geometric stability switch criteria in delay differential systems with delay dependent p

EDOARDO BERETTA† AND YANG KUANG‡ Abstract. In most applications of delay diﬀerential equations in population dynamics, the need of incorporation of time delays is often the result of the existence of some stage structure. Since the through-stage survival rate is often a function of time delays, it is easy to conceive that these models may involve some delay dependent parameters. The presence of such parameters often greatly complicates the task of an analytical study of such models. The main objective of this paper is to provide practical guidelines that combine graphical information with analytical work to eﬀectively study the local stability of some models involving delay dependent parameters. Speciﬁcally, we shall show that the stability of a given steady state is simply determined by the graphs of some functions of τ which can be expressed explicitly and thus can be easily depicted by Maple and other popular software. In fact, for most application problems, we need only look at one such function and locate its zeros. This function often has only two zeros, providing thresholds for stability switches. The common scenario is that as time delay increases, stability changes from stable to unstable to stable, implying that a large delay can be stabilizing. This scenario often contradicts the one provided by similar models with only delay independent parameters. Key words. delay diﬀerential equations, stability switch, characteristic equations, stage structure, population models AMS subject classiﬁcations. 34K18, 34K20, 92D25 PII. S0036141000376086

二乙烯三胺／一氧化氮聚合物诱导的奶牛乳腺上皮细胞损伤模型的建立

二乙烯三胺／一氧化氮聚合物诱导的奶牛乳腺上皮细胞损伤模型的建立郭咏梅;张博綦;石惠宇;闫素梅;史彬林;郭晓宇【摘要】Due to exuberant metabolism, dairy cows produce a great deal of nitric oxide ( NO) during lacta⁃tion, which leads to oxidative stress, and then results in cell toxicity. This experiment was conducted to investi⁃gate the suitable condition for oxidative damage model of bovine mammary epithelial cells ( BMEC) induced by diethylenetriamine/nitric oxide adduct ( DETA/NO) and establish an oxidative damage model. The oxida⁃tive damage model of BMEC was established using DETA/NO as the stimulation source. BMEC was exposed in DETA/NO with different medium concentrations (0, 10, 30, 100, 500 and 1 000μmol/L) and for differ⁃ent times (2, 4, 6, 8, 12 and 24 h). According to the analysis of cell survival rate and antioxidative parame⁃ters, inflammatory cytokines and nitric oxide ( NO) contents, as well as inducible nitric oxide synthase ( iN⁃OS) activity, the suitable treatment time and concentration of DETA/NO were screened. The results showed that the survival rate decreased to 74.27%, and the activitise of superoxide dismutase, catalase and glutathione peroxidase also significantly reduced ( P<0.05) after that BMEC was cultured with 1 000 μmol/L DETA/NO for 6 h. However, the activity of iNOS and the contents of NO, interleukin⁃1, interleukin⁃6, tumor necrosis factor⁃α and malondialdehyde showed opposite changes and increased significantly (P<0.05). In summary, the treatment of 1 000 μmol/L DETA/NO for 6 h issuitable for establishment of oxidative damage model.%泌乳期间的奶牛由于代谢旺盛，往往会产生大量一氧化氮（ NO），诱导发生氧化应激，进而对细胞产生毒害作用。

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reversible metabolic changes
c.ROS level
reversible metabolic changes
d.Mitochondrial mass and membrane potential
These data raised the possibility that mitochondrial function is reduced in circulating melanoma cells in an effort to reduce ROS generation.
Q2.Whether oxidative stress limits melanoma metastasis?
Oxidative stress therefore limits the metastasis of melanoma cells in vivo.
Treatment with the antioxidant NAC increased tumour formation by intravenously transplanted melanoma cells.
Oxidative stress inbibits distant metastasis by human melanoma cells
University of Texas Southwestern Medical Center University of Michigan
Background
• Solid cancer cells commonly enter the blood and
3
Oxidative stress limits distant metastasis
3.Oxidative stress limits distant metastasis
Q1.Whether successfully metastasizing cells undergo reversible metabolic changes that increase their capacity to withstand oxidative stress?
A
1.Blood and viscera are hostile to metastasis
Q1.Whether the main difference between efficiently and inefficiently metastasizing melanomas is the ability to enter the circulation?
• Q2.Whether oxidative stress limits melanoma metastasis?
reversible metabolic changes
a.unsupervised clustering of metabolomics
reversible metabolic changes
Melanoma cells undergo reversible changes in tropism during metastasis
1.Blood and viscera are hostile to metastasis
• Q1.Whether the main difference between efficiently and inefficiently metastasizing melanomas is the ability to enter the circulation?
3.Oxidative stress limits distant metastasis
4.Metabolic adaptations during metastasis

Blood and viscera are hostile to metastasis
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• Q2.Whether distant metastasis is limited mainly by survival in the blood?
2
Reversible tropic changes during metastasis
2.Reversible tropic changes during metastasis
Q2.Whether the changes were irreversible (for example, genetic) or reversible (for example, epigenetic or metabolic)?
The changes in tumorigenic tropism during metastasis are thus reversible. Since these reversible changes were observed very consistently, even in small numbers of melanoma cells, these changes cannot reflect selection for rare genetic events during metastasis followed by reversion mutations after subcutaneous re-transplantation.
• LC-MS/MS ;lentiviral transduction ;isotope
trace;western blotting
Content
1.Blood and viscera are hostile to metastasis 2.Reversible tropic changes during metastasis
Q2.Whether distant metastasis is limited mainly by survival in the blood?
The ability to survive in the circulation is therefore not the only factor that limits distant metastasis. The spleen is also a relatively hostile environment for melanoma cells.
2.Reversible tropic changes during metastasis
Q2.Whether the changes were irreversible (for example, genetic) or reversible (for example, epigenetic or metabolic)?
reversible metabolic changes
e.Changes are reversible
3.Oxidative stress limits distant metastasis
Q2.Whether oxidative stress limits melanoma metastasis?
Q1.Whether melanoma cells undergo changes in properties during metastasis?
Q1.Whether melanoma cells undergo changes in properties during metastasis?
This suggests melanoma cells adapt to the sites of metastasis as they metastasize.
Materials and methods
• melanoma cell→enzymatic dissociation • labelling and sorting:flow cytometry, bioluminescence imaging(DsRed, luciferase) • NOD-SCID-Il2rg-/- (NSG) mice
Method:we subcutaneously transplanted efficiently metastasizing melanoma cells derived from three patients into NSG mice and treated the mice with daily subcutaneous injections of the antioxidant Nacetyl-cysteine (NAC; 200 mg kg21 day21).
disseminate systemically, but are highly inefficient at
forming distant metastases for poorly understood reasons. • Whether additional adaptations are required during metastasis? • Raising the possibility that ROS promotes metastasis.
2.Reversible tropic changes during metastasis
• Q1.Whether melanoma cells • Q2.Whether the changes were irreversible (for undergo changes in example, genetic) or properties during metastasis? reversible (for example, epigenetic or metabolic)?
reversible metabolic changes