Effect of natural phenolic acids on DNA oxidation in vitro

We examined the antioxidant activity of the following natural phenolic compounds present in food: 3-OH-benzoic acid (3-OH-BA); 4-OH-benzoic acid (4-OH-BA); 2,3-dihydroxybenzoic acid (2,3-diOH-BA); 3,4-dihydroxybenzoic acid (3,4-diOH-BA or protocatechuic acid); ferulic acid; caffeic acid; and 2-coumaric, 3-coumaric and 4-coumaric acids. We measured the inhibitory effect of these compounds on iron-dependent oxidative DNA damage in vitro [incubating herring sperm DNA with Fe(III)/GSH] or using cumene hydroperoxide (CumOOH) as a free-radical generating system; we also studied the interaction of these phenols with Fe(II) or Fe(III) spectrophotometrically. Among the tested compounds, 2,3-diOH-BA, 3,4-diOH-BA and caffeic acid interacted with Fe(II) and showed a potent inhibitory effect on iron-induced oxidative DNA damage. CumOOH-induced DNA oxidation was not modified by these compounds. On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids did not interact with iron but protected against oxidative DNA damage induced by Fe(III)/GSH and by CumOOH, indicating a direct free-radical scavenging activity of these compounds in both systems. The IC₅₀±S.E.M. of the three coumaric acids against CumOOH-induced DNA oxidation was 44.2±2.0, 54.7±2.0 and 33.1±1.0 μ , respectively. On the contrary, 3-OH-BA and 4-OH-BA did not have scavenging activity and 3-OH-BA actually enhanced oxidative DNA damage. In conclusion, some natural phenolic acids, commonly present in food, have interesting protective activity against DNA oxidation in vitro and deserve further consideration as effective antioxidants in vivo.     

甜叶菊叶中酚酸类成分样品制备方法及含量测定

目的 研究甜叶菊叶中酚酸类成分的最佳样品制备方法并进行含量测定.方法 通过正交实验设计法,对甜叶菊叶提取所需的乙醇浓度、提取方法、乙醇用量、提取时间及提取次数进行系统考察,分析比较各个因素及水平对酚酸总含量的影响,从而获得最佳的样品制备方法,并用此方法制备不同产地甜叶菊叶样品,检测其中酚酸成分含量.结果 绿原酸标准曲线的回归方程为Y=58.27X-51.38（r＝0.999 9）,在1.25～25.05 mg/L范围内呈现良好的线性关系.绿原酸精密度试验的峰面积分别为240.55、245.65、241.07、241.58、241.09、237.16,相对标准偏差为1.12%,表明精密度良好.稳定性试验测定得绿原酸峰面积分别为240.55、241.65、242.07、238.53、232.12、231.26,相对标准偏差为2.03%,表明供试品在24 h内基本稳定.没食子酸的标准曲线回归方程A= 8.61c＋0.26（r=0.999 5）,总酚酸含量在0.01～0.16 g/L范围内呈现良好线性关系.甜叶菊叶溶液在1 h内稳定,0、10、20、30、40、50、60 min的吸光度分别为0.940 7、0.951 8、0.938 1、0.931 1、0.938 3、0.928 7、0.916 7,相对标准偏差为1.18%.4种因素影响顺序依次为提取方法＞提取时间＞料液比＞提取次数,最后综合考虑各个因素,确定最佳提取工艺为加入20倍量的50%乙醇,超声提取30 min,超声2次.6次提取方法测定的总酚酸含量分别为12.10%、12.23%、12.18%、11.97%、11.42%和11.52%,相对标准偏差为2.93%,方法稳定可靠.分析检测得到不同产地的甜叶菊中绿原酸含量在0.66%～2.17%,总酚酸含量在8.60%～16.15%,绿原酸占总酚酸的比例在8.7%～16.2%.结论 所建立的方法准确、简便、重复性好,可用于甜叶菊叶中酚酸类成分的含量测定及其质量控制.     

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 3² factorial design in electrolytes consisting of sodium tetraborate buffer (STB) in the concentration range of 10–50 mmol L⁻¹ and methanol in the volume percentage of 5–20%. Derringer's desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L⁻¹ tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid–liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 μg mL⁻¹, R² > 0.999; LOD and LOQ from 1.32 to 3.80 μg mL⁻¹ and from 4.01 to 11.5 μg mL⁻¹, respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8–11% CV for peak area; recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50–47.3 μg g⁻¹ were found, with smaller amounts occurring as free phenolic acids.     

纳米二氧化硅致心肌线粒体损伤作用的研究

目的 研究纳米二氧化硅对心肌细胞线粒体的毒性作用及机制。方法 采用非暴露式气管内滴注的染 毒方式对 Balb/c 小鼠进行 3 种浓度（7、 21 和 35 mg/kg）粒径为 40 nm 左右的纳米二氧化硅暴露, 另设对照组滴注等 体积生理盐水。通过透射电子显微镜对小鼠心肌线粒体超微结构进行观察。通过对三磷酸腺苷 （ATP） 浓度的检测, 评价纳米二氧化硅对心肌线粒体功能的影响。通过对心肌组织抗 O2-·能力的检测, 评价心肌细胞线粒体抗氧化能 力。采用 Western blot 法对心肌组织中细胞色素 c 氧化酶 1 （COX1） 和琥珀酸脱氢酶 A （SDHA） 蛋白表达水平进行检 测, 从而阐明纳米二氧化硅对心肌细胞线粒体生物合成的影响。结果 与对照组相比, 高剂量的纳米二氧化硅可导 致线粒体结构的损伤, 主要表现为线粒体肿胀、 线粒体嵴排列紊乱甚至消失及线粒体融合。中、 高剂量的纳米二氧 化硅可导致心肌细胞线粒体功能下降。低、 中剂量的纳米二氧化硅可引起心脏组织抗 O2-·能力应激性升高, 而高剂 量的纳米二氧化硅则可导致心脏组织抗 O2-·能力下降。中剂量的纳米二氧化硅可应激性诱导线粒体的生物合成, 而 高剂量的纳米二氧化硅则抑制线粒体的生物合成。结论 高剂量的纳米二氧化硅可通过诱导线粒体内 O2-·的产生、 降低线粒体抗氧化能力, 从而导致线粒体结构和功能的损伤, 并抑制线粒体的生物合成。     

Taiwan cobra cardiotoxins induce apoptotic death of human neuroblastoma SK-N-SH cells mediated by reactive oxygen species generation and mitochondrial depolarization

Although Naja naja atra cardiotoxin 3 (CTX3) and cardiotoxin 4 (CTX4) showed different cytotoxicity toward human neuroblastoma SK-N-SH cells, the two toxins induced apoptotic death on SK-N-SH cells. The apoptosis signals of CTX3 and CTX3 included ROS generation, increase in mitochondrial permeability transition, cytochrome c release to the cytosol and activation of caspase-9 and -3. However, CTX3 quickly induced the effects with higher magnitude compared with CTX4. ROS production and subsequent apoptotic cell death in CTX-treated cells were partly blocked by the antioxidant 2,3-dihydroxybenzoic acid. Nevertheless, mitochondria alteration and cytosolic cytochrome c release were not significantly attenuated by the antioxidant. Cell death was not completely inhibited by caspase-3 inhibitor. Moreover, cyclosporine A, an inhibitor of mitochondrial permeability transition, slightly decreased CTX-induced ROS generation by approximately 15%. Taken together, our data indicate that N. naja atra CTXs induce ROS generation that is not wholly dependent on mitochondrial dysfunction, and that the cytotoxic potency of CTX3 and CTX4 on SK-N-SH cells is, at least in part, correlated with their capability in inducing ROS generation and mitochondrial alterations.     

Effect of GADD45a on olaquindox-induced apoptosis in human hepatoma G2 cells: Involvement of mitochondrial dysfunction

Olaquindox, a quinoxaline 1, 4-dioxide derivative, has been widely used as a feed additive for promoting animal growth in China. The aim of present study was to investigate the effect of grow arrest and DNA damage 45 alpha (GADD45a) on olaquindox-induced apoptosis in HepG2 cells. The result showed that olaquindox induced the decrease of cell viability in a dose dependent manner. Compared to the control group, olaquindox treatment at 400 and 800 μg/mL increased the expression level of GADD45a protein and reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and subsequently increased the expression of Bax while decreased the expression of Bcl-2, leading to the release of cytochrome c (Cyt c). However, knockdown of GADD45a enhanced olaquindox-induced ROS production, disrupted MMP and subsequently caused Cyt c release, then further increased olaquindox- induced cell apoptosis by increasing the activities of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). In conclusion, the results revealed that GADD45a played a critical role in olaquindox-induced apoptosis in HepG2 cells, which may embrace the regulatory ability on the mitochondrial apoptosis pathway.     

The effects of 3, 4 or 5 amino salicylic acids on manganese-induced neuronal death: ER stress and mitochondrial complexes

This study was designed to investigate the mechanisms that are associated with manganese (Mn) toxicity. In addition, the association between Mn toxicity and 3, 4, 5 amino salicylic acid (ASA), anti-oxidants, including N-acetyl cysteine (NAC), was examined by dopaminergic cell line, SK-M-NC. Our studies showed that Mn influenced the mitochondria dysfunction and endoplasmic reticulum stress (ER stress). It reduced the mitochondria complex I activity but did not affect the complex II, III, or IV activities. The presence of 3, 4, 5 ASA protected against Mn-induced-apoptosis, as did NAC. However, the salicylate analogues and the antioxidants did not mediate ER stress in this model. The salicylate analogues reduced reactive oxygen species (ROS) and reversed the deficient mitochondrial membrane potential that was induced by Mn. Taken together, the 3, 4, 5 ASA worked in a similar way, regulating the Mn-induced mitochondrial dysfunction and protecting cells.     

Antioxidant phenolic acids from the leaves of Armeniaca sibirica

Abstract

Phytochemical investigation of the leaves of Armeniaca sibirica (L.) Lam. led to the isolation of two new phenolic acids (1–2), together with eight known compounds (3–10) from the ethanol extracts of this plant. Structures of these compounds were elucidated through detailed spectroscopic analyses, using 1D-NMR and 2D-NMR in combination with HR-EI-MS techniques. All the compounds were evaluated for their antioxidant capabilities in vitro using 2, 2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1, 1′-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assays, and ferric-reducing antioxidant power (FRAP) methods.     

Toxic effects of indocyanine green on rat liver mitochondria.

Indocyanine green (ICG) (68 to 171 nmol/mg of protein) inhibited the oxygen consumption of rat liver mitochondria in vitro. In state IV respiration, an acceleration of oxygen consumption occurred prior to this inhibition. ICG induced mitochondrial swelling in an isotonic KCl and K⁺ acetate medium but not in a 0.27 m sucrose medium. The increase in the ⁴²K diffusion space of isolated mitochondria was significantly larger than that of the [¹⁴C]sucrose diffusion space, indicating that ICG renders the mitochondrial membrane permeable to K⁺ ions. At the same time, an efflux of H⁺ ions from mitochondria was observed. It is concluded that the inhibition of mitochondrial oxygen consumption could be the result of a decrease in substrate penetration, as suggested by the diminution in NAD reduction by glutamate or isocitrate in the presence of ICG, without change in the activity of the corresponding dehydrogenases. A nigericin-like-mediated K⁺ for H⁺ antiport is proposed to account for the effects of ICG upon mitochondrial membrane permeability.     

Simultaneous quantification of ten phenolic acids in Lonicerae Japonicae Flos by HPLC-DAD

A reverse-phase HPLC-DAD method was developed for simultaneous quantification of ten phenolic acids (caffeic acid, chlorogenic acid, neochlorogenic acid, 4-O-caffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid methyl ester, 3,4-O-dicaffeoylquinic acid methyl ester, and 4,5-O-dicaffeoylquinic acid methyl ester) in the dried flower buds of Lonicera japonica Thunb. (Lonicerae Japonicae Flos; LJF). An optimal sample preparation method was established as 30-min ultrasonication with 100 times 50% (v/w) ethanol aqueous solution based on the orthogonal test results. The chromatographic separation of the ten phenolic acids was achieved with an AQ-C18 column (4.6 mm×250 mm, 5 µm) and a gradient elution of acetonitrile, methanol and 0.1% formic acid aqueous solution within 55 min. All calibration curves showed good linearity (r²>0.999) within test ranges. The average recoveries were in the range of 98.57%-103.22% with RSD less than 3%. The method developed was accurate, sensitive and reproducible for determination of ten phenolic acids in LJF.     

土壤真菌07-11号菌株中的酚酸性成分

以稻瘟霉 (Pyriculariaoryzae)分生孢子及菌丝形态变化为指标 ,对从云南土壤中分离得到0 7 1 1真菌的发酵产物中活性成分追踪分离 ,从其乙酸乙酯层浸膏中得到 3个酚酸性成分 ,经光谱数据分析和理化鉴别 ,分别鉴定为邻羟基苯甲酸 (Ⅰ )、4 ,6 二羟基 5 甲基异苯并呋喃酮 (Ⅱ )及 2 甲酰基 3 ,5 二羟基 4 甲基苯甲酸 (Ⅲ ) ,它们对稻瘟霉分生孢子及菌丝体都有一定抑制作用     

Influence of phenolic acids on the storage and digestion stability of curcumin emulsions based on soy protein-pectin-phenolic acids ternary nano-complexes

Abstract

Aim: To design novel emulsifiers with the ability to improve the storage and digestion stability of curcumin emulsions, besides to investigate the influence of phenolic acids types on the emulsify ability of soy protein-pectin-phenolic acids complexes obtained by ultrasonication.

Methods: The ternary complexes were characterised by particle size, morphology, zeta potential, X-ray diffraction, Fourier transform infra-red and fluorescence spectroscopy. Additionally, changes in droplet size, charge, and microstructure were monitored as quantitative stability index of curcumin emulsions.

Results: Phenolic acid types significantly affected the formation of ternary complexes. Soy protein-pectin-ferulic acid complex (S-P-F) stabilised curcumin emulsion had the best emulsifying property, followed by soy protein-pectin- ellagic acid (S-P-E), and soy protein-pectin-tannic acid complexes (S-P-T). Moreover, S-P-F emulsion was found to retain efficiently cucumin within 30?days storage (77.35%) and simulated gastrointestinal tract (64.09%).

Conclusion: Protein-polysaccharide-phenolic acids emulsions are effective oral delivery systems for hydrophobic bioactives.     

Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey

Phenolic compounds of dark and clear honeys from Trás-os-Montes of Portugal were extracted with Amberlite XAD-2 and evaluated for their antioxidant and antimicrobial activities. The antioxidant effect was studied using the in vitro test capacity of scavenge the 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical and of reducing power of iron (III)/ferricyanide complex. The antimicrobial activity was screened using three Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Staphylococcus lentus) and three Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli). The results obtained from the partial identification of honey phenolic compounds by high-performance liquid chromatography with a diode array detector showed that p-hydroxibenzoic acid, cinnamic acid, naringenin, pinocembrin and chrysin are the phenolic compounds present in most of the samples analyzed. Antioxidant potential was dependent of honey extract concentration and the results showed that dark honey phenolic compounds had higher activity than the obtained from clear honey. In the biological assays, results showed that S. aureus were the most sensitive microrganisms and B. subtilis, S. lentus, K. pneumoniae and E. coli were each moderately sensitive to the antimicrobial activity of honey extracts. Nevertheless, no antimicrobial activity was observed in the test with P. aeruginosa.     

Rapid quantification of phenolic acids in Radix Salvia Miltrorrhiza extract solutions by FT-NIR spectroscopy in transflective mode

A rapid method for simultaneous determination of main phenolic acids in Radix Salvia Miltrorrhiza extract solutions was developed using Fourier transform near infrared spectroscopy in transflective mode and multivariate calibration and HPLC-UV as the reference method. Partial least squares (PLS) algorithm was conducted on the calibration of regression models. The multiplicative scatter correction, Norris derivative and second derivative were adopted for the spectral pre-processing, and the number of PLS factors were optimized by leave-one-out cross-validation. The performance of the final model was evaluated according to root mean square error of prediction (RMSEP) and correlation coefficient (R). The R values achieved in the prediction set were above 0.93. The developed models were used for analysis of unknown samples and routine monitoring with satisfactory results. This work demonstrated that NIR spectroscopy combined with PLS algorithm could be used for the rapid determination of the main phenolic acids of Salvia Miltrorrhiza extract solutions.     

共轭亚油酸对小鼠免疫功能的影响

研究共轭亚油酸的免疫调节作用。采用正常小鼠及腹腔注环磷酰胺(Cy)造成免疫低下小鼠模型，共轭亚油酸灌胃40d后，用MTT法测脾细胞增殖活性，二硝基氟苯(DNFB)诱发迟发性变态反应(DTH)，测血清溶血素水平(HC50)，测胸腺脾脏指数。共轭亚油酸能显著提高免疫低下及正常小鼠的淋巴细胞增殖水平。可增强免疫低下小鼠迟发变态反应，提高胸腺指数。但对脾脏指数及血清溶血素水平无影响。可见，共轭亚油酸对T淋巴细胞介导的细胞免疫具有一定的免疫调节作用。     

Synthesis of hydroxycinnamic acid derivatives as mitochondria-targeted antioxidants and cytotoxic agents

In order to develop agents with superior chemopreventive and chemotherapeutic properties against hepatocellular carcinomas, mitochondria-targeted hydroxycinnamic acids (MitoHCAs) were synthesized by conjugation with a triphenylphosphonium cation. These synthetic compounds were evaluated for their antioxidant activities in hepatic mitochondria, including against OH^∙− and ROO^∙− induced lipid peroxidation. H₂O₂ production was decreased significantly by increasing glutathione peroxidase and catalase activities. In addition, cell proliferation data from three cell lines (HepG2, L02 and WI38) indicated that the MitoHCAs were selective for cancer cells. Interestingly, the MitoHCAs both with or without Ca²⁺ triggered mitochondrial dysfunction by inducing mitochondrial swelling, collapsing the mitochondrial membrane potential and causing cytochrome c release. In particular, an inhibitor of the mitochondrial permeability transition pore (mPTP), cyclosporin A, attenuated mitochondrial damage and cell apoptosis, indicating that mPTP may be involved in the antiproliferative activity of MitoHCAs. Further studies focused on structural optimization of these compounds are onging.     

应用液相色谱-质谱联用技术检测血浆样品中丹参酚酸类成分的浓度

目的：研究丹参酚酸中丹参素（tanshinol，TSN）、原儿茶醛（protocatechualdehyde，PCA）、丹酚酸A（salvianolic acid A，SAA）、丹酚酸B（salvianolic acid B，SAB）及丹酚酸D（salvianolic acid D，SAD）的电喷雾串联质谱（ESI-MS／MS）检测特性，为开展复方丹参滴丸药代研究，建立灵敏可靠的定量分析血浆样品中丹参酚酸浓度的方法。方法：研究丹参酚酸的电离模式及效率、二级质谱的碎片离子及打碎效率；建立有效可靠的从血浆样品中提取被测丹参酚酸物质的前处理方法；优化色谱条件、减少分析干扰、促进质谱检测；围绕准确性、精密度和提取回收率等进行方法学验证。结果：电喷雾（ESI）负离子模式能有效实现丹参酚酸被测化合物的电离。以下离子对m／z197→135、137→108、493→295、717→519、417→197分别为被测化合物偈N、PCA、SAA、SAB、SAD提供最佳的选择离子对监测（SRM）检测条件。为取得可靠的分析结果，建立了两个分析方法：其一用于测定血浆样品中的TSN；其二用于测定血浆样品中的PCA、SAA、SAB和SAD。两个分析方法均以HCI酸化血样后用乙酸乙酯提取的方法处理血浆样品，其中，TSN的提取回收率在57．7％～61．6％之间；PCA、SAA、SAB和SAD的提取回收率在38．0％～58．9％之间。偈N的线性范围为2．7～2000．0ng／mL，相关系数r为0．999，最低检测限（LLOQ）为2．7ng／mL；PCA、SAA、SAB及SAD的线性范围为1．4～1000．0ng／mL或4．1～1000．0ng/mL，相关系数r〉0．99，LLOQ分别为1．4ng／mL（PCA）和4．1ng/mL（SAA、SAB和SAD）。两个分析方法的日内准确性和精密度分别为90％～112％和2．5％～14．5％；日间准确性和精密度分别为91％～110％和0．7％～9．8％。结论：成功建立了能够灵敏可靠地测定血浆样品中5种丹参酚酸化合物浓度的分析方法，此方法可用于复方丹参滴丸的药代动力学研究。