Evaluation of the antioxidant capacity of limonin, nomilin, and limonin glucoside

The antioxidant capacity (AOC) of three representative citrus limonoids, limonin, nomilin, and limonin glucoside, was examined by the oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), beta-carotene-linoleic acid bleaching, and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assays. Pure compounds and proper negative (cinnamic acid) and positive (2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid) controls were used to remove any ambiguity in interpreting results. In all cases, limonin and nomilin gave results equivalent to those of cinnamic acid, indicating that they do not possess any inherent AOC and should not be considered antioxidants. Similar results were observed for limonin glucoside, with the exception of an anomalous result obtained from the beta-carotene-linoleic acid bleaching assay. Limonin glucoside was deemed not to be an antioxidant on the basis of the three unequivocal assays.     

Supercritical fluid extraction of limonoid glucosides from grapefruit molasses

Limonoid glucosides (primarily limonin 17-beta-D-glucopyranoside, LG) were extracted from grapefruit molasses by supercritical fluid extraction using a supercritical carbon dioxide-ethanol (SC CO(2)-ethanol) system. Extraction conditions to maximize the yield of LG were determined by varying pressure, temperature, ethanol concentration, and extraction time. The highest yield of LG at 0.61 mg/g molasses was obtained at a pressure 48.3 MPa, a temperature of 50 degrees C, 10% ethanol (X(Eth) = 0.1), and 40 min of extraction time at a flow rate of 5.0 L/min. The results demonstrated that SC CO(2) extraction of limonoid glucosides from grapefruit molasses has practical significance for commercial production.     

Inhibition of low-density lipoprotein oxidation and up-regulation of low-density lipoprotein receptor in HepG2 cells by tropical plant extracts

Twelve edible plant extracts rich in polyphenols were screened for their potential to inhibit oxidation of low-density lipoprotein (LDL) in vitro and to modulate LDL receptor (LDLr) activity in cultured HepG2 cells. The antioxidant activity (inhibition of LDL oxidation) was determined by measuring the formation of conjugated dienes (lag time) and thiobarbituric acid reagent substances (TBARS). Betel leaf (94%), cashew shoot (63%), Japanese mint (52%), semambu leaf (50%), palm frond (41%), sweet potato shoot, chilli fruit, papaya shoot, roselle calyx, and maman showed significantly increased lag time (>55 min, P < 0.05) and inhibition of TBARS formation (P < 0.05) compared to control. LDLr was significantly up-regulated (P < 0.05) by Japanese mint (67%), semambu (51%), cashew (50%), and noni (49%). Except for noni and betel leaf, most plant extracts studied demonstrated a positive association between antioxidant activity and the ability to up-regulate LDL receptor. Findings suggest that reported protective actions of plant polyphenols on lipoprotein metabolism might be exerted at different biochemical mechanisms.     

Soy and alfalfa phytoestrogen extracts become potent low-density lipoprotein antioxidants in the presence of acerola cherry extract

Postmenopausal women have an increased risk of coronary heart disease. Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis, and the presence of modified LDL (LDL(-)) in plasma appears to represent LDL oxidation in vivo. Because previous studies have demonstrated a strong antiatherogenic effect of estrogen due to its antioxidant activity and similar antioxidant activity was found for specific isoflavones derived from soy extract, the antioxidant activity of a phytoestrogen extract derived from soy and alfalfa was studied. Copper-mediated LDL oxidation was inhibited in the presence of soy and alfalfa extracts, and this effect was further enhanced in the presence of acerola cherry extract, which is rich in ascorbic acid. Male rabbit aortic endothelial cells pretreated with soy extract were resistant to the toxic effects of high levels of LDL and LDL(-), and a lesser, but significant protection, was also afforded by alfalfa extract. Cell-mediated oxidation of LDL, measured by LDL(-) formation, was inhibited in the presence of soy extract but not alfalfa extract. However, in the presence of acerola cherry extract, both soy and alfalfa extracts potently inhibited the formation of LDL(-). These findings show that acerola cherry extract can enhance the antioxidant activity of soy and alfalfa extracts in a variety of LDL oxidation systems. The protective effect of these extracts is attributed to the presence of flavonoids in soy and alfalfa extracts and ascorbic acid in acerola cherry extract, which may act synergistically as antioxidants. It is postulated that this synergistic interaction among phytoestrogens, flavonoids, and ascorbic acid is due to the "peroxidolitic" action of ascorbic acid, which facilitates the copper-dependent decomposition of LDL peroxides to nonradical products; this synergy is complemented by a mechanism in which phytoestrogens stabilize the LDL structure and suppress the propagation of radical chain reactions. The combination of these extracts markedly lowers the concentrations of phytoestrogens required to achieve significant antioxidant activity toward LDL.     

Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 microM, all of the prenylchalcones tested inhibited the oxidation of LDL (50 microg protein/ml) induced by 2 microM copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones, both at 5 and 25 microM. At 25 microM, the nonprenylated chalcone, chalconaringenin (CN), and the nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high antioxidant activity in inhibiting LDL oxidation, higher than alpha-tocopherol and the isoflavone genistein but lower than the flavonol quercetin. When combined, XN and alpha-tocopherol completely inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.     

Screening for antioxidant activity in edible plant products: comparison of low-density lipoprotein oxidation assay, DPPH radical scavenging assay, and Folin-Ciocalteu assay

Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis. Antioxidants that prevent LDL from oxidizing may reduce atherosclerosis. This study investigated LDL antioxidant activity in edible plant products for development of dietary supplementation to prevent atherosclerosis. Fifty-two kinds of edible plants were extracted using 70% aqueous ethanol solution, and the antioxidant activity of the extracts, which inhibit human LDL oxidation induced by copper ion, was determined on the basis of the oxidation lag time and represented as epigallocatechin 3-gallate equivalent. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content were also measured for comparisons with antioxidant activity in LDL. Plant products showing the greatest activity in LDL oxidation assay were akamegashiwa (Mallotus japonicus) leaf, Japanese privet (Ligustrum japonicum) leaf, green tea [Camellia sinensis (L.) O. Kuntze], and astringent persimmon (Diospyros kaki). The present study revealed high levels of LDL antioxidant activity in plant products for which such activity levels are underestimated in the DPPH radical scavenging assay and Folin-Ciocalteu assay.     

Comparison of the antioxidant activity of commonly consumed polyphenolic beverages (coffee, cocoa, and tea) prepared per cup serving.

In this study, the in vitro low-density lipoprotein oxidation model was used to assess the relative antioxidant activity of the polyphenolic beverages tea, coffee, and cocoa on a cup-serving basis. The beverages were prepared as 0.7-2.5% soluble coffee and 1.5-3.5% cocoa; teas (green, black, or herbal) were prepared as one tea bag infused over 5 min in 220 mL of hot water. Under these standard cup serving conditions, the antioxidant activity as determined by the lag time was in the range of 292-948 min for coffee, 217-444 min for cocoa, 186-338 min for green tea, 67-277 min for black tea, and 6-78 min for herbal tea. Addition of milk did not alter the antioxidant activity. The influence of coffee bean source and degree of roasting was further investigated. Green coffee beans of Robusta coffee exhibited a 2-fold higher antioxidant activity than Arabica coffee, but after roasting this difference was no longer significant. In conclusion, these commonly consumed beverages have a significant antioxidant activity, the highest being soluble coffee on a cup-serving basis.     

苦菜总黄酮超声波辅助提取及抗氧化能力研究

为开发和利用苦菜中的黄酮资源,采用超声波辅助法提取苦菜总黄酮,通过响应面分析法对苦菜总黄酮超声波辅助提取工艺条件进行优化,并对最佳提取条件下苦菜总黄酮提取液的抗氧化能力进行分析。结果表明,苦菜总黄酮超声泼辅助提取最优工艺参数为:乙醇浓度49%、浸提温度69℃、浸提时间162 min、料液比1∶62(m/v),此条件下苦菜黄酮得率为3.73%±0.03%。最优提取条件下,苦菜总黄酮提取液清除DPPH自由基、羟基自由基、超氧阴离子自由基的IC₅₀分别为0.005 9、0.153、0.024 8 mg·mL^-1,表现出较强的抗氧化能力。本研究结果为苦菜黄酮的开发应用提供了理论基础。     

Inhibitory effect of natto,a kind of fermented soybeans,on LDL oxidation in vitro

The oxygen radical scavenging activity of natto (fermented soybeans) and its inhibitory effect on the oxidation of rat plasma low-density lipoprotein (LDL) in vitro were investigated to evaluate the usefulness of the antioxidant properties of natto, which has been shown to have antioxidant activity. Natto was separated into three water-soluble fractions: high-molecular-weight viscous substance (HMWVS; Mw > 100 000), low-molecular-weight viscous substance (LMWVS; Mw < 100 000), and soybean water extract (SWE). LMWVS had the strongest radical scavenging activity for hydroxyl and superoxide anion radicals, as assessed by electron spin resonance. The increase of conjugated dienes in LDL oxidized by copper and an azo pigment was depressed by the addition of LMWVS and SWE. These results demonstrate that natto fractions have inhibitory effects on LDL oxidation as a result of their radical scavenging activity.     

LDL-antioxidant pterocarpans from roots of Glycine max (L.) Merr

The methanolic root extract of Glycine max (L.) Merr. was chromatographed, which yielded 10 flavonoids, including three isoflavones 1-3, five pterocarpans 4-8, one flavonol 9, and one anthocyanidin 10. All isolated compounds were examined for LDL-antioxidant activities using four different assay systems on the basis of Cu2+-mediated oxidation. Among them, seven compounds showed potent LDL-antioxidant activities in the thiobarbituric acid reactive substances (TBARS) assay, the lag time of conjugated diene formation, relative electrophoretic mobility (REM), and fragmentation of apoB-100 on copper-mediated LDL oxidation. Three pterocarpans 4, 6, and 7, never reported as LDL-antioxidant, showed potent activities with IC50 values of 19.8, 0.9, 45.0 microM, respectively, in comparison with probucol (IC50 = 5.6 microM) as positive control. Interestingly, coumestrol 6 (IC50 = 0.9 microM) showed 20 times more activity in the TBARS assay than genistein (IC50 = 30.1 microM) and daidzein (IC50 = 21.6 microM), representative antioxidants in soybean. Moreover, coumestrol 6 had an extended lag time of 190 min at 3.0 microM in measuring conjugated diene formation, while both genistein (120 min) and daidzein (93 min) lag times were extended to less than 120 min at the same concentration.     

柠檬籽粒中柠檬苦素离子液体双水相提取体系的优化与抗氧化活性分析

为更好地开发和利用柠檬籽粒中的功能性成分柠檬苦素,本试验研究了离子液体双水相对柠檬苦素提取的最优体系,并通过响应面分析法对最优体系的提取工艺进行了优化。结果表明,1.50 mol·L^-1 [TMG][Cl]/1.35 mol·L^-1 NaH₂PO₄离子液体双水相萃取体系中柠檬苦素的提取量最高,萃取率达97.59%;响应面优化试验得出最优工艺参数:提取时间90.74 min、乙醇浓度59.40%、提取温度74.22℃、液料比18.08∶1条件下,预测柠檬苦素最大提取量为12.126 mg·g^-1FW,校正工艺下柠檬苦素实际提取量为12.381 mg·g^-1 FW,方差分析显示两数值差异不显著,表明有关柠檬苦素提取量的四元二次方程模型预测准确度高。此外,柠檬苦素可有效清除活性氧自由基并抑制不饱和脂肪酸的过氧化,具有较强的抗氧化活性。本研究结果为柠檬资源的充分开发和实际应用提供了理论基础。     

Nitrogen effects on total flavonoids,chlorogenic acid,and antioxidant activity of the medicinal plant Chrysanthemum morifolium

Chrysanthemum morifolium (Ramat.) has a long history of cultivation and use as a traditional medicine and tea plant in China. A greenhouse experiment with potted soil–quarz mixture studied the effects of nitrogen supply (0, 56, 112, 167, 224, 334, 501, 556, and 668 mg N kg^–1) on concentrations and ratios of total flavonoids and chlorogenic acid in the flowers of C. morifolium using spectrophotometric and HPLC methods. The antioxidant activity of the flowers was determined as the radical scavenging activities of hydroxyl, superoxide anion, and 2,2‐diphenyl‐1‐picrylhydrazyl hydrate (DPPH) free radicals. A high N supply decreased the concentrations of total flavonoids by 18%–35% and that of chlorogenic acid by 8%–60% compared to a low N‐supply rate. At the same time, increasing N supply significantly decreased the antioxidant activity of the flowers. The antioxidant activity of C. morifolium flowers was significantly positively correlated with the concentrations of total flavonoids and chlorogenic acid. We conclude that an N supply in excess of 300 mg (kg soil)^–1 will negatively affect the antioxidant activity and thereby reduce the quality of C. morifolium flowers.     

Phytochemicals of apple peels: isolation, structure elucidation, and their antiproliferative and antioxidant activities

Bioactivity-guided fractionation of Red Delicious apple peels was used to determine the chemical identity of bioactive constituents, which showed potent antiproliferative and antioxidant activities. Twenty-nine compounds, including triterpenoids, flavonoids, organic acids and plant sterols, were isolated using gradient solvent fractionation, Diaion HP-20, silica gel, and ODS columns, and preparative HPLC. Their chemical structures were identified using HR-MS and 1D and 2D NMR. Antiproliferative activities of isolated pure compounds against HepG2 human liver cancer cells and MCF-7 human breast cancer cells were evaluated. On the basis of the yields of isolated flavonoids (compounds 18- 23), the major flavonoids in apple peels are quercetin-3-O-beta-D-glucopyranoside (compound 20, 82.6%), then quercetin-3-O-beta-D-galactopyranoside (compound 19, 17.1%), followed by trace amounts of quercetin (compound 18, 0.2%), (-)-catechin (compound 22), (-)-epicatechin (compound 23), and quercetin-3-O-alpha-L-arabinofuranoside (compound 21). Among the compounds isolated, quercetin (18) and quercetin-3-O-beta-D-glucopyranoside (20) showed potent antiproliferative activities against HepG2 and MCF-7 cells, with EC 50 values of 40.9 +/- 1.1 and 49.2 +/- 4.9 microM to HepG2 cells and 137.5 +/- 2.6 and 23.9 +/- 3.9 microM to MCF-7 cells, respectively. Six flavonoids (18-23) and three phenolic compounds (10, 11, and 14) showed potent antioxidant activities. Caffeic acid (10), quercetin (18), and quercetin-3-O-beta-D-arabinofuranoside (21) showed higher antioxidant activity, with EC 50 values of <10 microM. Most tested flavonoids and phenolic compounds had high antioxidant activity when compared to ascorbic acid and might be responsible for the antioxidant activities of apples. These results showed apple peel phytochemicals have potent antioxidant and antiproliferative activities.     

Changes in the antioxidative property of herring (Clupea harengus) press juice during a simulated gastrointestinal digestion

The aqueous fraction (press juice, PJ) from herring muscle was recently shown to inhibit hemoglobin-mediated oxidation of washed fish mince lipids during ice storage. As a first step to evaluate potential in vivo antioxidative effects from herring PJ, the aim of this study was to investigate whether herring PJ retains its antioxidative capacity during a simulated gastrointestinal (GI) digestion. Press juice from whole muscle (WMPJ) and light muscle (LMPJ) was mixed with pepsin solution followed by stepwise pH adjustments and additions of pancreatin and bile solutions. Digestive enzymes were removed from samples by ultrafiltration (10 kDa). Before, during, and after digestion, samples were analyzed for their peptide content and for antioxidative properties with the oxygen radical absorbance capacity (ORAC) and the low-density lipoprotein (LDL) oxidation assays. From 0 to 165 min of digestion, the content of <10 kDa peptides in WMPJ and LMPJ samples increased 12- and 7-fold, respectively. Further, both samples got approximately 12.5 times higher ORAC values and gave rise to approximately 1.3-fold increased lag phase in Cu2+-induced LDL oxidation. The largest changes in peptide content, ORAC values, and LDL oxidation inhibition occurred between 30 and 75 min of digestion, indicating that these parameters might be interrelated. When comparing analytical data obtained after 165 min of digestion with data obtained from analyses of native nondigested PJs, it was found that the data on peptide content, ORAC, and LDL oxidation from digested PJs were 64-69%, 121-161%, and 112-115%, respectively, of those of nondigested PJs. The study thus showed that enzymatic breakdown of PJ proteins under GI-like conditions increases the peroxyl radical scavenging activity and the potential to inhibit LDL oxidation of herring PJs. These data provide a solid basis for further studies of uptake and in vivo activities of herring-derived aqueous antioxidants.     

New insights into controversies on the antioxidant potential of the olive oil antioxidant hydroxytyrosol

In the present study, the antioxidant profile of olive oil antioxidants was investigated. Hydroxytyrosol and oleuropein are potent scavengers of hydroxyl radicals (OH*), peroxynitrite (ONOOH), and superoxide radicals (O(2)*(-)). Homovanillic alcohol, one of the main metabolites of hydroxytyrosol, and tyrosol are less potent scavengers of these reactive species. None of the olive oil antioxidants are good hypochlorous acid (HOCl) or hydrogen peroxide (H(2)O(2)) scavengers. Hydroxytyrosol efficiently protects against LDL oxidation in vitro and in vivo. However, no protective effect of hydroxytyrosol is usually demonstrated ex vivo against the oxidation of LDL isolated from humans after hydroxytyrosol consumption. The present study shows that this controversy is due to the isolation of LDL, which greatly reduces the protective effect of hydroxytyrosol against LDL oxidation. Hydroxytyrosol is an efficient scavenger of several free radicals. The physiological relevance of the high intrinsic antioxidant activity of hydroxytyrosol is illustrated by its protection against LDL oxidation.     

广东石豆兰不同溶剂提取物抗氧化及与总黄酮、总酚含量的关系

为明确广东石豆兰的抗氧化能力及抗氧化成分,以鳞茎为材料,以甲醇、乙醇、丙酮和乙酸乙酯为提取剂得到4种提取物,测定了4种提取物中总黄酮、总酚含量及其抗氧化活性,并分析了两者之间的关系。结果表明,石豆兰总酚含量高于总黄酮,乙醇为总酚和总黄酮2种物质的最佳提取溶剂;甲醇提取物对羟基自由基清除力和对Fe²⁺的螯合力最强,对DPPH自由基清除效果最差;丙酮提取物具有最强的总还原力和清除超氧阴离子的能力,而清除羟基自由基能力最弱;乙酸乙酯提取物清除DPPH自由基的能力最强,但总还原力、清除超氧阴离子的能力、Fe²⁺的螯合力最弱。此外,除了DPPH自由基外,其他4个抗氧化指标与总黄酮或总酚含量呈正相关(0.185<r<0.969),总还原力与总黄酮含量呈显著相关。本研究结果为广东石豆兰抗氧化物质的提取及进一步开发利用提供了参考依据。     

Extraction parameters and capillary electrophoresis analysis of limonin glucoside and phlorin in citrus byproducts.

Limonin glucoside (LG) and phlorin were extracted from citrus fruit tissues and assayed by capillary electrophoresis (CE). LG was determined in dried [1.20 +/- 0.10 mg of dry weight (dw)] and wet peel residues (1.16 +/- 0.04 mg of dw), orange juice finisher pulp (0.58 +/- 0.03 mg of dw), dried grapefruit seeds (2.70 +/- 0.15 mg of dw), and 50 degrees Brix molasses (2225 +/- 68 mg/L). Phlorin was purified from orange peel residue and grapefruit albedo, and concentrations were determined in some citrus products. Phlorin and LG were extracted from residues with water/pectinase or with water solutions of methanol and ethanol. Efficient LG extraction from grapefruit seeds (2.40 +/- 0.15 mg/g) was achieved with 50-65% methanol, solvent polarity P' approximately equal to 7-8. Extracts were purified and concentrated by adsorptive resins and HPLC to obtain 95% pure compounds of LG and phlorin. CE analysis did not require extract purification beyond filtration. LG and phlorin migrated as anions in electropherograms containing peaks representing other citrus flavonoids and limonoid glucosides.     

In vitro antioxidant activity and antigenotoxicity of 5-n-alkylresorcinols

Incubation with 5-n-alkylresorcinols (chain lengths C15:0, C17:0, C19:0, C21:0, and C23:0) increased the self-protection capacity of HT29 human colon cancer cells against DNA damage induced by hydrogen peroxide and genotoxic fecal water samples using comet assay (single-cell gel electrophoresis assay). The alkylresorcinols did not exert potent antioxidant activity in the FRAP (ferric reduction ability of plasma) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical assays. However they were able to significantly inhibit copper-mediated oxidation of human LDL (low-density lipoprotein) in vitro, and pentadecylresorcinol at 25 micromol/L increased lag time by 65 min. The results show that alkylresorcinols have antigenotoxic and antioxidant activity under in vitro conditions.     

超声波微波协同提取沙棘叶黄酮及其组成和活性研究

沙棘叶中富含黄酮类化合物等多种活性成分,为提高其提取率和利用率,本研究采用常规溶剂萃取、超声辅助、微波辅助、超声波微波协同提取4种方法对沙棘叶黄酮进行提取,测定沙棘叶黄酮得率并观察沙棘叶的微观组织结构,比较筛选沙棘叶黄酮的最佳提取方法。并采用响应面法对最佳提取方法进行工艺优化,同时测定沙棘叶黄酮组成和体外抗氧化活性。结果表明,超声波微波协同提取法是提取沙棘叶黄酮的最佳方法,黄酮得率较常规溶剂萃取法提高了42.54%(P<0.05),沙棘叶细胞损伤最严重。超声波微波协同提取沙棘叶黄酮的最佳工艺为乙醇体积分数61%、提取时间18 min、微波功率446 W,此时黄酮得率为42.09 mg·g^-1。沙棘叶黄酮提取液中共鉴定出6种黄酮类成分,分别为儿茶素、丁香酸、山萘酚、槲皮素、异鼠李素、杨梅素,其中儿茶素含量最高,为1.474 8 mg·g^-1,其余5种的含量均在0.1~0.3 mg·g^-1之间,山萘酚最低,为0.125 2 mg·g^-1;沙棘叶黄酮提取液具有较强的还原力及较高的1,1-二苯基-2-三硝基苯肼(DPPH)、2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)和羟基自由基清除率,抗氧化活性较高。本研究为沙棘叶黄酮的工业化生产提供了科学依据。