超声辅助酶解提取五味子多糖及其抗细胞氧化应激研究

以五味子(Schisandra chinensis)干果为原料,采用超声辅助纤维素酶酶解法提取五味子多糖,研究五味子多糖的超声辅助酶解工艺与抗细胞氧化应激活性。以多糖得率为指标,采用单因素试验和Box-Behnken响应面试验研究加酶量、超声功率、提取时间和料液比对五味子多糖得率的影响,优化得到五味子多糖最佳超声辅助酶解提取工艺条件;建立脂多糖(LPS)诱导的HepG2细胞氧化应激损伤模型,以丙二醛(MDA)、超氧化物歧化酶(SOD)和活性氧(ROS)水平为指征,研究五味子多糖的抗细胞氧化应激能力。结果表明：五味子多糖的最佳提取工艺条件为加酶量1 420 U·g^-1,超声功率500 W,提取时间46 min,料液比1 g:32 mL,该条件下五味子多糖得率为22.25%;此外,所提取得到的五味子多糖可提高LPS诱导HepG2细胞中的SOD水平,并可降低细胞中MDA的积累量和ROS荧光强度,有效缓解了LPS诱导的HepG2细胞氧化应激反应。优化确立了超声辅助纤维素酶提取五味子多糖最佳工艺条件,发现所提取得到的五味子多糖有良好抗氧化应激能力。     

壳寡糖缓解甲萘醌诱导巨噬细胞损伤机制初探

目的:研究壳寡糖对甲萘醌诱导的巨噬细胞氧化损伤的保护作用.方法:通过MTT实验检测相应处理的细胞活力,并通过相应试剂盒检测细胞氧化还原体系中某些相关酶的活力及相应产物含量.结果:壳寡糖能够缓解甲萘醌诱导的细胞损伤,并且发现壳寡糖可以缓解甲萘醌导致的胞内超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-PX)活力的降...     

顽拗植物澳洲坚果成熟叶片DNA提取方法比较

目的:针对澳洲坚果成熟叶片中富含多糖、多酚等杂质的特点,建立澳洲坚果成熟叶片中提取高质量 DNA 的方法.方法:采用改良CTAB法和改良SDS法提取澳洲坚果样品的总DNA,并对产物进行紫外、电泳及PCR扩增检测.结果:改良CrAB法的平均产率为13.6μg/g,略低于改良SDS法18.5μg/g,但改良CTAB法可有效去除多糖等杂质,获得的基因组DNA质量高.OD260/OD280均在1.7～1.9之间.进行ISSR扩增可获得清晰、多态性好的条带.结论:改良CrAB法较之改良SDS法更适合于从澳洲坚果成熟叶片中提取高质量DNA.     

正交设计优选五味子果实中总木脂素提取工艺

目的:比较不同方法对北五味子总木脂素提取的影响,优选最佳提取工艺方法。研究五味子木脂素抗微波辐射损伤作用。方法:以干燥北五味子果实为原料,分别采用传统加热回流法、超声提取法、微波提取法、微波—超声提取法提取北五味子总木脂素,对这四种提取方法分别进行正交试验分析,以确定最佳提取工艺。结果:通过结果比较分析,以微波提取工艺结果最佳,在微波提取功率为200W,乙醇体积分数为80%,提取时间为40min,料液比为1:12的提取条件下,北五味子总木脂素的产量达到了16.44mg/g,使得木脂素的提取产量较传统加热回流法、超声提取法、微波—超声提取法得到了显著的提高。     

地鳖多糖提取物的体内外抗氧化作用

目的研究地鳖多糖提取物体内外抗氧化作用。方法体外试验采用分光光度法、邻苯三酚自氧化法和Fe2+-邻二氮菲法分别检测地鳖多糖对二苯代苦味酰基自由基(DPPH·)、超氧阴离子自由基(O-2·)和羟自由基(·OH)的清除,检测体外红细胞氧化溶血和肝线粒体肿胀程度。体内试验小鼠连续灌胃不同剂量(0、40、80、160 mg/kg)地鳖多糖提取物20 d,硫代巴比妥酸(TBA)比色法测定小鼠组织中丙二醛(MDA)含量,分光光度法检测抗氧化酶(SOD、CAT、GSH-Px)活力。结果与对照组比较,地鳖多糖组对自由基清除率随多糖浓度升高而显著提高;体外红细胞氧化溶血和线粒体肿胀显著降低。多糖组小鼠肝肾中MDA含量降低,SOD、CAT、GSH-Px抗氧化酶活力提高并明显高于对照组。结论地鳖多糖提取物能够清除自由基,降低自由基引起细胞氧化损伤,抑制组织中过氧化物生成,提高抗氧化酶活力,具有显著的体内外抗氧化作用。     

超声波提取加拿大蓬总黄酮的研究

目的:研究加拿大蓬中总黄酮提取方法及最佳工艺.方法:采用超声提取法,通过单因素试验和正交试验筛选最佳工艺条件.结果:最佳提取条件为60%乙醇、料液比1:20、60 kW下超声45 min.得率为31.076 mg/g.结论:该方法提取提取总黄酮快速、简便,可适用于生产中加拿大蓬总黄酮类物质的检测.该方法是提取加拿大蓬总...     

正交试验法考察参附丹强心颗粒的制备工艺

目的:优选参附丹强心颗粒的提取工艺。方法:采用正交试验,以出膏率和丹参素钠含量考察水提工艺条件。结果:水提取的最佳工艺为药材用8倍量水提取3次,每次3h。加入乙醇使含醇量达70%,搅匀,静置24小时,滤过,回收乙醇至无醇味。3批验证结果,出膏率18.81%,丹参素钠含量23.06mg/g。结论:上述实验结果可作为参附丹强心颗粒的提取工艺。     

水葫芦叶蛋白提取工艺优化研究

本文选用水葫芦进行叶蛋白提取工艺优化研究,以叶蛋白提取率、叶蛋白中粗蛋白含量为指标,探讨温度、p H值、加盐量和料水比(鲜叶与水的重量比,m/m)等四个因素对水葫芦叶蛋白提取率的影响,并结合正交试验,以期获得叶蛋白提取的优化工艺参数。正交试验结果表明,水葫芦叶蛋白最佳提取工艺组合为A_3B_1C_2D_1,即温度为80℃、p H值为3.0、加盐量2.0%、料水比1:2.0,最佳工艺条件下,水葫芦叶蛋白中粗蛋白含量为43.69%,提取率为14.55%。方差分析结果表明,影响水葫芦叶蛋白提取率的主次顺序为p H值絮凝温度料水比加盐量,四个因素中温度对叶蛋白提取率的影响差异极显著(P=0.001),p H对叶蛋白提取率的影响差异显著(P=0.017)。加盐量和料水比对叶蛋白提取率的影响差异不显著(P0.05)。本研究为水葫芦叶蛋白的开发应用提供了理论依据。     

甘草次酸通过PI3K-AKT途径抑制H2O2所致大鼠心肌细胞氧化损伤

目的:研究甘草次酸对H_2O_2所致大鼠心肌细胞氧化损伤的影响及其可能机制。方法:采用H_2O_2处理建立H9c2大鼠心肌细胞氧化损伤模型后,比较模型中ROS生成和细胞凋亡比例,使用不同浓度的甘草次酸孵育H9c2细胞24、48h后,通过流式细胞仪检测ROS的生成量,Annexin V-FITC/PI双标记流式细胞术检测凋亡率,蛋白质印迹法检测检PI3K、AKT1、p-AKT蛋白的表达。结果:H9c2大鼠心肌细胞氧化损伤模型中ROS生成和细胞凋亡率分别为(49.33±3.23)%和(33.89±1.45)%,与对照组相比有显著差异;100μmol/L和200μmol/L的甘草次酸作用24 h后,H9c2大鼠心肌细胞氧化损伤模型中表达ROS细胞的比例(35.39±1.24)%和(30.46±0.95)%,凋亡细胞比例分别为(29.47±3.15)%和(23.17±1.46)%,当作用48 h后,H9c2大鼠心肌细胞氧化损伤模型中表达ROS细胞的比例(42.67±1.89)%和(35.49±1.63)%,凋亡细胞比例分别为(40.22±3.06)%和(35.26±2.78)%,与对照组相比有显著性差异;使用渥曼青霉素后,各甘草次酸组的与对照组无显著性差异。结论:甘草次酸可能通过PI3K-AKT途径抑制H_2O_2所致大鼠心肌细胞氧化损伤。     

褪黑素缓解空肠黏膜上皮细胞氧化损伤的效果研究

肠道黏膜氧化损伤与畜禽的生长发育和腹泻等疾病的发生密切相关，而褪黑素作为一种吲哚胺类化合物，具有明显抗氧化保护作用。为探究褪黑素对肠黏膜上皮细胞氧化损伤时的保护作用，本研究选取原代小鼠空肠黏膜上皮细胞，利用硫酸亚铁和过氧化氢处理建立氧化损伤模型，并通过添加不同浓度的褪黑素预处理上皮细胞，检测细胞损伤、氧化产物、抗氧化酶和炎性相关细胞因子的表达变化。结果发现，硫酸亚铁和过氧化氢处理后，黏膜上皮细胞明显受损，丙二醛含量显著增加、抗氧化酶表达水平降低。而褪黑素能明显减轻细胞氧化损伤程度、抑制丙二醛的产生并增加抗氧化酶的表达。同时，褪黑素还能显著减少炎性因子白介素-6的表达，并增加趋化因子白介素-8的表达。因此，褪黑素可以缓解自由基增加诱发的空肠黏膜上皮细胞氧化损伤，并能够减轻细胞损伤引起的炎症反应。     

Chlorophyllin: a potent antimutagen against environmental and dietary complex mixtures

Chlorophyllin, the sodium and copper salt of chlorophyll, was tested for its ability to inhibit the mutagenic activity of a variety of complex mixtures--extracts of fried beef, fried shredded pork, red grape juice, red wine, cigarette smoke, tobacco snuff, chewing tobacco, airborne particles, coal dust and diesel emission particles--in strain TA98 of Salmonella typhimurium. Chlorophyllin was highly effective against the mutagenicity (90-100% inhibition) of 8 of these 10 mixtures. The mutagenicity of the other 2 mixtures was inhibited 75-80% at the highest concentration of chlorophyllin studied. Control and reconstruction experiments showed that chlorophyllin was not toxic to Salmonella at the concentrations used. The antimutagenic activity of chlorophyllin was heat-stable. The mechanism of the antimutagenicity of chlorophyllin in these experiments is not known; however, chlorophyllin is an antioxidant. Scavenging of radicals and/or interaction with the active group of mutagenic compounds may be responsible for its antimutagenic activity. The data reported here indicate that chlorophyllin is potentially useful as an antimutagenic agent.     

Phytoremediative Capacity of Plants Enriched with Melatonin

Melatonin is an environmentally friendly-molecule with broad spectrum antioxidant capacity. Melatonin is widely present in the plant kingdom. High levels of melatonin exist in an aquatic plant, the water hyacinth, which is highly tolerant of environmental pollutants. Elevated levels of melatonin probably help plants to protect against environmental stress caused by water and soil pollutants. To investigate the potential relationships between melatonin supplementation and environmental tolerance in plants, pea plants were treated with high levels of copper in the soil. The results show that copper contamination kills pea plants; however, melatonin added to the soil significantly enhanced their tolerance to the copper contamination and, therefore, increased their survival. Based on the theory and these preliminary data, we speculate that melatonin could be used to improve the phytoremediative efficiency of plants against different pollutants. Since melatonin is safe to animals and humans as well as being inexpensive, it may be a feasible and cost-effective approach to clean environmental contaminations.Key Words: phytoremediation, plants, melatonin, antioxidant     

Comparative study on chemical pretreatments to accelerate enzymatic hydrolysis of aquatic macrophyte biomass used in water purification processes

In this study, enzymatic hydrolysis of two floating aquatic plants which are suitable for water purification, water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes L.), was performed to produce sugars. Twenty chemical pretreatments were comparatively examined in order to improve the efficiency of enzymatic hydrolysis. As a result, the alkaline/oxidative (A/O) pretreatment, in which sodium hydroxide and hydrogen peroxide were used, was the most effective pretreatment in terms of improving enzymatic hydrolysis of the leaves of water hyacinth and water lettuce. The amount of reducing sugars in enzymatic hydrolysate of water lettuce leaves was 1.8 times higher than that of water hyacinth leaves, therefore water lettuce seems to be more attractive as a biomass resource than water hyacinth. Although roots of these plants contained large amounts of polysaccharides such as cellulose and hemicellulose, they generated less monosaccharides than from leaves, no matter which chemical pretreatment was tested.     

Deciphering the protective role of nitric oxide against salt stress at the physiological and proteomic levels in maize

Saline stress is a major factor that limits crop yield. Nitric oxide (NO) is functional during plant growth, development, and defense responses. In the present study, the protective role of NO in alleviating saline stress in maize at the physiological and proteomic levels was examined. Our results showed that salt treatment quickly induced NO accumulation and addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) efficiently eliminated the inhibitory effect of salt on shoot growth and photosynthesis and inhibited salt-inducible H2O2 accumulation. These effects could be reversed by NO metabolic scavengers and inhibitors. Further proteomic and Western blotting analysis revealed that NO induced G-protein-associated protein accumulation and antioxidant enzymes activities, in addition to activation of defense proteins, energy metabolism, and cell structure/division in salt-treated maize seedlings. Controlling the G-protein status with G-protein activators or inhibitors also affected NO generation and root and stem growth in maize seedlings after saline stress. On the basis of these results, we propose that NO enhances salt tolerance in maize seedlings by enhancing antioxidant enzyme activities and controlling H2O2 levels, and these effects are accompanied by diverse downstream defense responses. During this process, G-protein signaling is an early event that works upstream of NO biogenesis.     

Effect of nitric oxide and putrescine on antioxidative responses under NaCl stress in chickpea plants

Chickpea plants were subjected to salt stress for 48 h with 100 mM NaCl, after 50 days of growth. Other batches of plants were simultaneously treated with 0.2 mM sodium nitroprusside (NO donor) or 0.5 mM putrescine (polyamine) to examine their antioxidant effects. Sodium chloride stress adversely affected the relative water content (RWC), electrolyte leakage and lipid peroxidation in leaves. Sodium nitroprusside and putrescine could completely ameliorate the toxic effects of salt stress on electrolyte leakage and lipid peroxidation and partially on RWC. No significant decline in chlorophyll content under salt stress as well as with other treatments was observed. Sodium chloride stress activated the antioxidant defense system by increasing the activities of peroxidase (POX), catalase (CAT) superoxide dismutase (SOD) and ascorbate peroxidase (APX). However no significant effect was observed on glutathione reductase (GR) and dehydro ascorbate reductase (DHAR) activities. Both putrescine and NO had a positive effect on antioxidant enzymes under salt stress. Putrescine was more effective in scavenging superoxide radical as it increased the SOD activity under salt stress whereas nitric oxide was effective in hydrolyzing H₂O₂ by increasing the activities of CAT, POX and APX under salt stress.     

Facilitation and Competition among Invasive Plants: A Field Experiment with Alligatorweed and Water Hyacinth

Ecosystems that are heavily invaded by an exotic species often contain abundant populations of other invasive species. This may reflect shared responses to a common factor, but may also reflect positive interactions among these exotic species. Armand Bayou (Pasadena, TX) is one such ecosystem where multiple species of invasive aquatic plants are common. We used this system to investigate whether presence of one exotic species made subsequent invasions by other exotic species more likely, less likely, or if it had no effect. We performed an experiment in which we selectively removed exotic rooted and/or floating aquatic plant species and tracked subsequent colonization and growth of native and invasive species. This allowed us to quantify how presence or absence of one plant functional group influenced the likelihood of successful invasion by members of the other functional group. We found that presence of alligatorweed (rooted plant) decreased establishment of new water hyacinth (free-floating plant) patches but increased growth of hyacinth in established patches, with an overall net positive effect on success of water hyacinth. Water hyacinth presence had no effect on establishment of alligatorweed but decreased growth of existing alligatorweed patches, with an overall net negative effect on success of alligatorweed. Moreover, observational data showed positive correlations between hyacinth and alligatorweed with hyacinth, on average, more abundant. The negative effect of hyacinth on alligatorweed growth implies competition, not strong mutual facilitation (invasional meltdown), is occurring in this system. Removal of hyacinth may increase alligatorweed invasion through release from competition. However, removal of alligatorweed may have more complex effects on hyacinth patch dynamics because there were strong opposing effects on establishment versus growth. The mix of positive and negative interactions between floating and rooted aquatic plants may influence local population dynamics of each group and thus overall invasion pressure in this watershed.     

Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions. Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls. Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances. The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride. Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM. The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol. When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis. To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen. The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1). In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.     

Influence of environment on piroxicam polymorphism: vibrational spectroscopic study

FTIR and FT-Raman spectroscopies were used to evaluate the mechanism of transformation of piroxicam into its different forms (alpha, beta, and monohydrate), depending on the environment. These vibrational techniques allowed us to identify the forms of piroxicam that crystallize from different solvents at different cooling rates and the conformation of the drug in some of its derivatives: piroxicam hydrochloride, piroxicam thallium and sodium salt hemihydrates, and piroxicam sodium salt. The usefulness of Raman spectroscopy in characterizing piroxicam:beta-cyclodextrin (PbetaCD) inclusion compounds was described. The Raman spectrum of 1:2 PbetaCD was discussed in comparison with that of the corresponding piroxicam sodium salt containing inclusion compound (1:2 PNabetaCD) in order to study the influence of the piroxicam derivative used on the structure of the inclusion compound. The Raman results showed that in both of the inclusion compounds the piroxicam mainly assumes the zwitterionic structure typical of a monohydrate; therefore, the kind of derivative used does not affect the conformation of the drug in its inclusion compound. The effect of the method of synthesis utilized (freeze-drying or freeze-thaw cycling) to obtain 1:2.5 PbetaCD was investigated. The inclusion compound obtained by freeze-thaw cycling proved to be more crystalline and to contain a higher amount of the beta form than the freeze-dried inclusion compound. Raman spectroscopy proved to be a useful technique for evaluating the effectiveness of the manufacturing process in relation to the pharmaceutical properties of the drug and to the nondestructive and noninvasive on-line quality control of the industrial products.     

Experimental and kinetic modelling studies on the acid-catalysed hydrolysis of the water hyacinth plant to levulinic acid

A comprehensive experimental and modelling study on the acid-catalysed hydrolysis of the water hyacinth plant (Eichhornia crassipes) to optimise the yield of levulinic acid (LA) is reported (T=150-175 degrees C, [Formula: see text] , water hyacinth intake=1-5wt%). At high acid concentrations (>0.5M), LA was the major organic acid whereas at low acid concentrations (<0.1M) and high initial intakes of water hyacinth, the formation of propionic acid instead of LA was favoured. The highest yield of LA was 53mol% (35wt%) based on the amount of C6-sugars in the water hyacinth (T=175 degrees C, [Formula: see text] , water hyacinth intake=1wt%). The LA yield as a function of the process conditions was modelled using a kinetic model originally developed for the acid-catalysed hydrolysis of cellulose and good agreement between the experimental and modelled data was obtained.     

The metabolic activation of 4-nitro-o-phenylenediamine by chlorophyll-containing plant extracts: The relationship between mutagenicity and antimutagenicity

Chlorophyllin, the sodium and copper salt of chloropyll, and chlorophyll a, and chlorophyll b were tested for their ability to inhibit the mutagenic activity of the direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) and its plant-activated mutagenic enhancement. All three forms of chlorophyll were antimutagenic against both NOP and its plant-activated product, with chlorophyllin proving most effective. Chlorophyll-containing plant extracts, however, proved very efficient at activating NOP into a mutagen of greater potency. When these extracts were assayed for total chlorophyll content it was found that they contained far less chlorophyll than was required for an antimutagenic effect to occur. Thus, the balance between chemical mutagen activation and/or enhancement by chlorophyll-containing plant extracts and the potential antimutagenicity of these plant extracts is a function of chlorophyll concentration. The data presented here indicate that this balance must be taken into consideration in future studies investigating the efficacy of complex natural plant extracts as antimutagenic substances.