Synthesis and antioxidant evaluation of novel silybin analogues

In this work, we evaluated the antioxidant properties of the eight novel silybin analogues for their capacity to scavenge free radicals including superoxide anion radicals and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals in vitro. Compound 7d demonstrated an excellent antioxidant effect in scavenging superoxide anion free radical with an IC₅₀ value of 26.5 μM, while the IC₅₀ of quercetin (the reference compound) was 38.1 μM. Compounds 7b, 7e, 7h showed certain scavenging activities for both types of free radicals.     

Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw

In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, alpha-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC(50)=16.1 microM), hydroxyl radical (IC(50)=12.8 microM), superoxide radical (IC(50)=34.0 microM) and peroxyl radical (IC(50)=32.6 microM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).     

杨桃提取物体外清除氧自由基作用

从杨桃果中提取得到三种提取物为匀浆提取物、蛋白提取物和多糖提取物。采用化学发光法测定这三种提取物清除氧自由基的活性,实验结果:匀浆提取物清除羟自由基(·OH)和H2O2的活性大小相近,而清除超氧阴离子自由基(O2–·)的活性较小,其IC50约为前两者的4倍。蛋白提取物清除O2–·和·OH的活性大小相近,而清除H2O2的活性明显小于前两者,IC50约为前两者的9倍。多糖提取物清除.OH的活性明显大于清除O2–·和H2O2的活性,其IC50约为O2–·的1/22,约为H2O2的1/65。结果表明,杨桃果具有清除O2–·、·OH和H2O2的作用,不同提取物对这些活性氧自由基的清除能力有所不同。     

Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion

To extract antioxidant peptide from hoki frame protein hydrolysate (APHPH), we employed six proteases (pepsin, trypsin, papain, alpha-chymotrypsin, Alcalase and Neutrase) for enzymatic hydrolysis, and the antioxidant activities of their hydrolysates were investigated using both lipid peroxidation inhibition assay and free radical scavenging assay by electron spin resonance spin-trapping technique. Among hydrolysates, peptic hydrolysate, having the highest antioxidant activity, further separated into four groups using ultrafiltration membranes and purified consecutive chromatographic methods. Finally, the purified peptide had a molecular mass of 1801 Da, and amino acid sequence was identified as Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn. APHPH inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radical: 1,1-diphenyl-2-pycryl-hydrazyl (IC(50)=41.37 microM), hydroxyl (IC(50)=17.77 microM), peroxyl (IC(50)=18.99 microM) and superoxide radicals (IC(50)=172.10 microM). Furthermore, APHPH decreased t-butylhydroperoxide-induced cytotoxicity on human embryonic lung fibroblasts and efficiently protected free-radical-induced DNA damage.     

Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems

Low molecular weight peptides obtained from ultrafiltration (UF) of giant squid (Dosidicus gigas) muscle protein were studied for their antioxidative effects in different in vitro oxidative systems. The most potent two peptides, Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-Glu-Gly-Leu-Ala (1307 Da) and Asn-Gly-Leu-Glu-Gly-Leu-Lys (747 Da), exhibited their antioxidant potential to act as chain-breaking antioxidants by inhibiting radical-mediated peroxidation of linoleic acid, and their activities were closer to highly active synthetic antioxidant, butylated hydroxytoluene. Addition of these peptides could enhance the viability of cytotoxic embryonic lung fibroblasts significantly (P<.05) at a low concentration of 50 microg/ml, and it was presumed due to the suppression of radical-induced oxidation of membrane lipids. Electron spin trapping studies revealed that the peptides were potent scavengers of free radicals in the order of carbon-centered (IC(50) 396.04 and 304.67 microM), hydroxyl (IC(50) 497.32 and 428.54 microM) and superoxide radicals (IC(50) 669.34 and 573.83 microM). Even though the exact molecular mechanism for scavenging of free radicals was unclear, unusually high hydrophobic amino acid composition (more than 75%) of giant squid muscle peptides was presumed to be involved in the observed activities.     

Antioxidant effects of an aqueous extract of Ilex paraguariensis

In this work we investigate the antioxidant properties of an aqueous extract prepared from an infusion of Ilex paraguariensis (Aquifoliaceae) using free radical-generating systems. The extract inhibited the enzymatic and nonenzymatic lipid peroxidation in rat liver microsomes in a concentration-dependent fashion, with IC(50) values of 18 microg/ml and 28 microg/ml, respectively. The extract also inhibited the H(2)O(2)-induced peroxidation of red blood cell membranes with an IC(50) of 100 microg/ml and exhibited radical scavenging properties toward superoxide anion (IC(50) = 15 microg/ml) and 2,2-diphenyl-1-picrylhydrazyl radical. In the range of concentrations used, the extract was not a scavenger of the hydroxyl radical. Our results suggest that ingestion of extracts of Ilex paraguariensis could contribute to increase the antioxidant defense of an organism against free radicals attack.     

芥子碱硫氰酸盐清除超氧阴离子自由基作用的研究

目的:研究芥子碱硫氰酸盐清除超氧阴离子自由基的作用。方法:用化学发光法测定芥子碱硫氰酸盐清除超氧阴离子自由基的能力,并以抗坏血酸的清除能力做为对照,以IC50值(清除率为50%时的浓度值)作为评价指标。结果:芥子碱硫氰酸盐和抗坏血酸的IC50值分别为0.135 mmol/L和18.74 mmol/L,后者的IC50值约为前者的140倍。结论:芥子碱硫氰酸盐具有良好的抗氧化作用,可以作为天然抗氧化剂进行开发。     

Identification of chemical structure and free radical scavenging activity of diphlorethohydroxycarmalol isolated from a brown alga, Ishige okamurae

To obtain a natural antioxidant from a marine biomass, this study investigated the antioxidative activity of methanolic extracts from the marine brown alga, Ishige okamurae collected off Jeju Island. A potent free radical scavenging activity was detected in the ethyl acetate fraction containing polyphenolic compounds, and the potent antioxidant elucidated as a kind of phlorotannin, diphlorethohydroxycarmalol, by NMR and mass spectroscopic data. The free radical scavenging activities of the diphlorethohydroxycarmalol were investigated in relation to 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, and hydroxyl radicals using an electron spin resonance (ESR) system. The diphlorethohydroxycarmalol was found to scavenge DPPH (IC50=3.41 microM) and alkyl (IC50=4.92 microM) radicals more effectively than the commercial antioxidant, ascorbic acid. Therefore, these results present diphlorethohydroxycarmalol as a new phlorotannin with a potent antioxidative activity that could be useful in cosmetics, foods, and pharmaceuticals.     

Synthesis and neuroprotective activity of bergenin derivatives with antioxidant activity

Norbergenin, which is the O-demethyl derivative of bergenin, the main component of Mallotus japonicus, has been found to show moderate antioxidant activity (IC(50) 13 microM in DPPH radical scavenging; 32 microM in superoxide anion scavenging). Modification of sugar part on norbergenin by coupling with a variety of fatty acids was employed for increasing its antioxidant activity. Selective esterification of hydroxyl groups on the sugar part enhanced greatly antioxidant activity. The most potent one is norbergenin 11-caproate, which not only exhibits stronger antioxidant activity than that of catechin but also prevents neuronal death at 10 microM on the primary culture of rat cortical neurons in DMEM supplemented with N2.     

Functional properties of dioscorin, a soluble viscous protein from Japanese yam (Dioscorea opposita thunb.) tuber mucilage Tororo

A soluble viscous protein was purified from yam (Dioscorea opposita Thunb.) tuber mucilage tororo by chromatographic steps, and its functional properties were estimated. The purified dioscorin having the molecular weight of about 200 kDa exhibited high scavenging activities against hydroxyl radicals (IC50 = 195.1 microg/ml) and superoxide anion radicals (IC50 = 92.7 microg/ml). Moreover, it showed extremely high angiotensin I-converting enzyme inhibitory activity (IC50 = 41.1 microg/ml). The results suggested that yam D. opposita tuber has a wide spectrum of strong antioxidative and antihypertensive activities and it could be utilized as a source of natural antioxidant.     

Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage

In this study, in vitro gastrointestinal digestion was employed to obtain potent antioxidative peptide from protein of oyster, Crassostrea gias. The protein was subjected to hydrolysate using consecutive chromatographic methods, on a Hiprep 16/10 diethylaminoethyl fast flow (DEAE FF) anion exchange column and octadecylsilane (ODS) C18 reversed phase column. Finally, the amino acid sequence of the peptide was determined. The peptide, having the amino acid sequence Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu (1.60 kDa), exhibited the higher activity against polyunsaturated fatty acid (PUFA) peroxidation than that of native antioxidant, alpha-tocopherol. The free radical scavenging assay conducted using electron spin resonance (ESR) spectroscopy, clearly exhibited that it scavenged hydroxyl radical and superoxide radical at IC50 values of 28.76 microM and 78.97 microM, respectively. Further, we investigated its antioxidant activities on cellular system, and the results showed that purified peptide significantly scavenged cellular radicals and protective effect on DNA damage caused by hydroxyl radicals generated. Furthermore (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assay showed no cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5) and mouse macrophages cell (RAW264.7), respectively. These results indicate that this peptide shows potent antioxidant.     

银耳碱提多糖的纯化、化学表征及其抗氧化作用的研究(英文)

研究银耳孢子发酵物中的多糖类化学成分,并探讨了分离得到的一个多糖组分的抗氧化活性。银耳孢子发酵粉用热水煮提除去水溶性组分后,再采用0.5 mol.L-1的氢氧化钠溶液提取,Sevage法除蛋白,用乙醇沉淀得到粗多糖。粗多糖经DEAE-32-纤维素和Sephadex G-200分离纯化得到分布均一的多糖TFBP-A。糖组成分析显示,TFBP-A单糖组成为:甘露糖:半乳糖:葡萄糖,摩尔比为90∶5∶5;HPGPC测定TFBP-A分子量为58962。TFBP-A的抗氧化活性实验显示:在H2O2引起的红细胞溶血试验中,以蒸馏水抑制率为0%计算,TFBP-A抑制率为78.6%;在超氧阴离子自由基的清除作用实验中,TFBP-A最高抑制率为53%;在清除羟基自由基实验中,TFBP-A的EC50为0.191 mg.mL-1。从银耳孢子发酵物中用碱液提取得到的多糖组分TFBP-A为酸性杂多糖,重均分子量为58962,且具有一定的抗氧化活性。     

Antioxidant potential of C-phycocyanin isolated from cyanobacterial species Lyngbya, Phormidium and Spirulina spp

The antioxidant activity of C-Phycocyanin (C-PC) isolated from three cyanobacterial species Lyngbya (marine), Phormidium (marine) and Spirulina (fresh water) was studied in vitro. The results demonstrate that C-PCs from Lyngbya, Phormidium and Spirulina spp. are able to scavenge peroxyl radicals (determined by crocin bleaching assay) with relative rate constant ratio of 3.13, 1.89 and 1.8, respectively. C-PCs also scavenge hydroxyl radicals (determined by deoxyribose degradation assay) with second order rate constant values of 7.87 x 10(10), 9.58 x 10(10) and 6.42 x 10(10), respectively. Interestingly, Lyngbya C-PC is found to be an effective inhibitor of peroxyl radicals (IC50 6.63 microM), as compared to Spirulina (IC50 12.15 microM) and Phormidium C-PC (IC50 12.74 microM) and is close to uric acid (IC50 2.15 microM). Further, the studies suggest that the covalently-linked tetrapyrrole chromophore phycocyanobilin is involved in the radical scavenging activity of C-PC. The electron spin resonance (ESR) spectra of C-PCs indicate the presence of free radical active sites, which may play an important role in its radical scavenging property. This is the first report on the ESR activity of native C-PCs without perturbations that can cause radical formation.     

Isolation and characterization of free radical scavenging activities peptides derived from casein

A peptide having the strong free radical scavenging activities was separated from casein protein hydrolysate by chromatographic analyses such as ion-exchange and gel filtration. SP-II fraction obtained by SP-Sephadex C-25 chromatography showed the most potent superoxide anion scavenging activity (SOSA), and it was further separated into a peptide using an octadecylsilano-high performance liquid chromatography. The amino acid sequence of the peptide was Tyr-Phe-Tyr-Pro-Glu-Leu (YFYPEL). The concentration of the test compound required to reduce the produced superoxide anion to one-half (IC(50)) value for SOSA was 79.2 microM using tetrazolium salt 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate method. The IC50 value for the 1,1-diphenyl-2-picrylhydrazyl radical and hydroxyl radical scavenging activities were 98 and 251 microM, respectively, based on the electron spin resonance method. We characterized SOSA of the C-terminal sequence using EL, PEL, YPEL, and FYPEL. The activities preferred sequences were EL>YFYPEL>FYPEL>YPEL>PEL, suggesting that the Glu-Leu sequence is important for the activity.     

Free radical scavenging properties of sulfinpyrazone

Sulfinpyrazone, a potent uricosuric drug, was tested in vitro for its scavenging action against oxygen free radicals. In this study, sulfinpyrazone was able to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with IC 50 value of 29.82 μg/ml compared to butylated hydroxytoluene (BHT, IC 50 value=20.15 μg/ml) and Trolox (IC 50 value=16.01 μg/ml). It was able to scavenge superoxide anion with IC 50 value of 27.72 μg/ml compared to Trolox (IC 50 value=22.08 μg/ml) and ascorbic acid (IC 50 value=14.65 μg/ml). The hydroxyl radical scavenging activity of sulfinpyrazone is in a concentration-dependent fashion. In the range of concentrations used, sulfinpyrazone was not a scavenger toward H 2 O 2 . However, the intracellular H 2 O 2 -induced 2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence in HL-60 cells was significantly reduced by sulfinpyrazone during 30-60 min of incubation. Finally, phorbol-12-myristate-13-acetate induced-lucigenin chemiluminescence in whole blood was markedly inhibited by sulfinpyrazone. Our results suggest a new direction for the pharmacological actions of sulfinpyrazone in free radical scavenging properties.     

A tyrosinase inhibitor, Daedalin A, from mycelial culture of Daedalea dickinsii

A chromene-type compound, daedalin A (1), was isolated from mycelial culture broth of Daedalea dickinsii. Based on spectroscopic data, the structure of 1 was found to be (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene. Daedalin A (1) strongly inhibited the activity of tyrosinase (IC(50): 194 muM). In addition, 1 also showed 1,1-diphenyl-2-picrylhydrazyl scavenging activity (IC(50): 16.9 microM) and superoxide anion scavenging activity (IC(50): 28.5 microM).     

Antioxidant activity of extract from Polygonum aviculare L

Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation, and DNA damage. It has been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum aviculare L. (Polygonaceae) has antioxidant activity is unknown. In this study, dried Polygonum aviculare L. was extracted by ethanol, and the extract was lyophilized. The antioxidant activities of extract powder were examined by free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays. The results show that the IC50 value of Polygonum aviculare L. extract is 50 microg/ml in free radical scavenging assays, 0.8 microg/ml in superoxide radical scavenging assays, and 15 microg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum aviculare L. extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 677.4 +/- 62.7 microg/g and 112.7 +/- 13 microg/g. The results indicate that Polygonum aviculare L. extract clearly has antioxidant effects.     

Stilbenes from the roots of Pleuropterus ciliinervis and their antioxidant activities

Two stilbene glycosides, pieceid-2"-O-gallate and pieceid-2"-O-coumarate, were isolated from the MeOH extract of the roots of Pleuropterus ciliinervis Nakai (Polygonaceae), together with two known compounds, resveratrol and pieceid. Their structures were determined spectroscopically, particularly by 2D NMR spectroscopic analysis. The antioxidant activities of stilbenes isolated were determined in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radicals and by determining their lipid peroxidation inhibitory activities. Among the compounds isolated, pieceid-2"-O-gallate had the most potent inhibitory scavenging effect on DPPH, superoxide radicals and upon lipid peroxidation inhibition with IC50 values of 16.5, 23.9 and 5.1 microM, respectively.     

褐蘑菇水溶性多糖抗氧化活性研究

采用水提醇沉以及sevag去蛋白的方法获得褐蘑菇水溶性多糖(WPPA)。通过测定还原力、超氧阴离子清除率、羟基自由基清除率和抑制H2O2诱导红细胞氧化溶血实验评价WPPA抗氧化活性。结果表明:WPPA具有较强的还原力,对O2-.和.OH具有较强的清除作用,IC50分别为527μg/mL、310μg/mL;对H2O2诱导红细胞氧化溶血及MDA生成有很强的抑制作用,IC50分别为700μg/mL和541μg/mL。说明WPPA在一定浓度内具有较强的抗氧化能力。     

翅果油树叶片中总生物碱抗氧化活性研究

采用80%乙醇浸提翅果油树叶片中总生物碱,硅胶柱层析纯化,并用碘量法测定翅果油树叶片中总生物碱对猪油抗氧化性能的影响,用番红花红O-Mn2 -H2O2光度法测定其对羟基自由基的清除效果,用NBT光还原法测定其对超氧阴离子的清除效果.结果表明:翅果油树叶片总生物碱可有效延缓猪油的脂质过氧化反应,对猪油氧化的抑制效果显著高于同浓度的维生素C;其具有较强的清除羟基自由基和超氧阴离子的能力,EC50值分别为0.236g/L和0.101g/L,当浓度为1g/L时,其对羟基自由基和超氧阴离子的清除率可达96.04%和90.05%,显著高于同浓度的维生素C.