向日葵茎芯多糖的提取工艺优化及其体外抗肿瘤活性研究

研究了向日葵茎芯中主要活性物质多糖的提取工艺,并对此工艺进行了优化,选取的提取方法为水提醇沉法,以多糖含量作为指标,采用单因素试验研究了提取次数、原料颗粒的大小（目数）、料液比、提取时间、提取温度对向日葵茎芯多糖含量的影响。用苯酚-硫酸法测定提取液中多糖的含量,得出向日葵茎芯中多糖的最佳提取工艺条件为：提取次数2次,原料颗粒的大小（目数）60~80目,料液比（g·mL^-1）1：50,提取时间3.0 h,提取温度90℃,在最优提取条件下,多糖的提取得率为6.56%,多糖的含量为266.03 mg·g^-1。本文也对多糖的体外抗肿瘤活性进行了研究,结果表明向日葵茎芯多糖的体外抗肿瘤活性较弱。这些条件的确定为向日葵茎芯的深入研究奠定了基础。     

猴头菌子实体超临界流体技术萃取活性物质初探

以猴头菌子实体为原料,萃取物得率作为指标,采用CO₂超临界流体萃取技术,以萃取压力和CO₂流量等参数为考察因素,结合正交试验获得优化的萃取工艺：萃取压力为30MPa,温度为45℃,时间为1.5h,CO₂流量为20g/min,夹带剂（乙醇）与猴头菌子实体的物料比为5:1（mL:g）,在此条件下,萃取物得率为2.78%。与猴头菌子实体的醇提物相比,猴头菌子实体超临界萃取物具有更好的体外抗氧化能力和抗肿瘤活性,本研究结果为合理地开发和利用猴头菌子实体超临界萃取物产品提供科学的数据。     

响应面优化细叶杜香提取工艺及抗氧化活性研究

研究细叶杜香抗氧化物质的最佳提取工艺及提取物的抗氧化性能。在单因素试验基础上,选择提取时间、提取温度及料液比为影响因子,应用Box-Benhnken中心组合法进行3因素3水平试验设计,以1,1-二苯基-2-三硝基苯肼(DPPH)清除率为响应值,进行响应面分析,并研究提取物的体外抗氧化活性。结合实际操作,得到最佳提取条件为提取时间3.3 h、提取温度69℃、料液比为1∶23,在该条件下提取液的DPPH自由基清除率为84.37±0.17%,接近预测值。抗氧化物质得率为4.68±0.24 g/100 g,总黄酮含量为236.17±2.16 mg/g,多酚含量为73.97±3.18mg/g。抗氧化活性实验结果表明,细叶杜香提取物具有较好的抗氧化活性。     

响应面法优化藜麦麸皮皂苷最佳提取工艺及其α-葡萄糖苷酶抑制活性

采用响应面法优化了藜麦麸皮超声提取皂苷的最佳工艺,并考察了最佳提取工艺条件下提取物对α-葡萄糖苷酶的抑制活性。同时建立酶反应动力学方程对其抑制动力学进行分析,探讨了藜麦麸皮提取物对α-葡萄糖苷酶的抑制机制。实验结果表明,藜麦麸皮超声提取皂苷的最佳工艺条件为液料比15 mL/g, 75%乙醇超声提取,超声时间1.5 h,超声温度45℃,在此条件下藜麦麸皮皂苷的提取得率为2.370%±0.022%。α-葡萄糖苷酶抑制活性实验表明提取物的抑制作用明显强于阳性药阿卡波糖(P0.05),其IC_(50)值为0.620±0.057 mg/mL。酶动力学研究表明藜麦麸皮总皂苷提取物是一种可逆的混合性抑制类型。本研究为明确藜麦麸皮提取物的降低餐后血糖作用机理提供了参考,为藜麦麸皮的开发与利用提供了依据。     

响应面法优化黄绿卷毛菇子实体多糖的提取工艺及其体外抗氧化活性研究

以采自四川石渠的黄绿卷毛菇Floccularia luteovirens为研究对象,采用水提醇沉的经典方法以及单因素试验,并利用Box-Benhnken Design（BBD）中心组合试验设计原理,以提取温度、时间以及料液比3个影响因素作为自变量,多糖提取率为考察指标,设计3因素3水平的响应面试验,优化黄绿卷毛菇子实体多糖（FLPs）的提取工艺,并通过?OH和O₂ ^-?自由基清除能力考察FLPs的抗氧化能力。最终确定最优水提工艺为提取温度89.31℃,提取时间5.08h,料液比1:48.54（g/mL）,且FLPs具有良好的?OH和O₂ ^-?自由基清除能力,具有较强的体外抗氧化活性。同时,验证试验证明了该设计方法和模型的准确性和可行性,且在该工艺条件下,多糖提取率有所提高,可为其多糖功能性食品和药品的开发提供理论依据。     

蕨菜多糖超声波辅助提取及其药理活性初步研究

为优化蕨菜多糖的提取工艺,同时检测蕨菜多糖的药理学活性。实验采用超声波辅助提取法,在单因素试验的基础上,考察液料比、浸提次数、超声浸提时间、超声功率四因素对蕨菜多糖提取得率的影响。运用Design Expect 10.0软件分析,通过响应面分析法(RSM)优化提取条件,对蕨菜多糖促进小鼠脾细胞增殖能力和抑制结肠癌细胞(HTC-8)增殖能力进行分析。结果表明:蕨菜多糖的优化提取工艺为:液料比:36.2∶1;浸提次数:4次;超声浸提时间:43.1 min;超声波功率:240 W,在此条件下,蕨菜多糖提取效果最好,提取得率达8.60%。各因素对多糖提取得率的影响程度:浸提次数>液料比>超声浸提时间>超声功率。通过药理活性研究表明,本实验获得的蕨菜多糖具有脾细胞免疫增值和抑制结肠癌细胞增殖活性。     

五步蛇毒和蝮蛇毒透析前后的某些性质及五步蛇毒热稳定性的研究

五步蛇（Agkistrodon acutus）和蝮蛇（Agkistrodon halys）蛇毒透析前后含量、毒性和抗原成分的变化,以及温度对五步蛇毒致死活性的影响等,尚未见有资料。本文就浙江产五步蛇毒和腹蛇毒在透析前后的某些性质及五步蛇毒的热稳定性研究作初步报道。     

超声-负压法提取藜麦皂苷及其活性研究CSCD

本文以藜麦皂苷为研究对象,基于单因素试验,以皂苷提取率为指标,通过响应面法优化,结合藜麦粉末SEM表征,以获得皂苷的超声-负压提取最佳工艺参数。藜麦皂苷粗提物经正丁醇萃取后,探讨其体外抗氧化活性及相关机理。实验结果表明:超声-负压法提取藜麦皂苷最佳工艺参数:超声时间20 min、超声温度59℃、负压0.064 MPa,该条件下皂苷得率(预测值12.37 mg/g,实验值11.95±0.034 mg/g)最高,三因素对皂苷得率的影响顺序:负压>超声温度>超声时间。藜麦皂苷在浓度范围(0~0.25 mg/mL)内有较强的还原力和清除DPPH自由基的能力,抗氧活化活性与其浓度呈正相关。     

不同种植模式对土壤氮素转化及酶活性的影响

采用两年大田试验,研究了小麦-大豆（A₁）、小麦-甘薯（A₂）、玉米（A₃）、小麦/玉米/大豆（A₄）和小麦/玉米/甘薯（A₅）5种种植模式下,小麦/玉米/大豆套作体系的土壤酶活性变化特征及其对土壤氮素转化的影响.结果表明：与A₁、A₂、A₃和A₅相比,A₄提高了各作物在开花期（或吐丝期）和成熟期的土壤总氮含量及脲酶和蛋白酶活性,处理间表现为套作＞单作、大豆茬口＞甘薯茬口、边行＞中行;降低了小麦、玉米土壤的NO₃^--N、NH₄⁺-N含量及小麦土壤的硝酸还原酶活性.玉米土壤的硝酸还原酶活性在玉米、大豆共生前的拔节期为单作＞套作,在玉米、大豆共生后的吐丝期和成熟期为套作＞单作、大豆茬口＞甘薯茬口;大豆土壤的NO₃^--N、NH₄⁺-N含量及硝酸还原酶活性在分枝期为单作＞套作,在开花期和成熟期为套作＞单作、大豆茬口＞甘薯茬口、中行＞边行.     

黑木耳中抑制胰脂肪酶活性物质的提取工艺及体外抑制效果

本文以黑木耳醇提物和水提物为研究对象,以对胰脂肪酶活性的抑制率为指标,分别对其提取工艺进行了单因素和正交试验,选取优化后抑制率高的提取物进行抑制类型和3T3-L1前脂肪细胞方面的研究。结果表明醇提物的最佳提取工艺为提取温度70℃、提取时间1h、乙醇浓度90%、料液比1:20,抑制胰脂肪酶的IC₅₀=681.56μg/mL。水提物的最佳提取工艺为提取温度70℃、提取时间2h、料液比1:40,抑制胰脂肪酶的IC₅₀=850.59μg/mL,醇提物的抑制效果优于水提物。醇提物对胰脂肪酶的抑制类型为非竞争性抑制,抑制常数为4.69mg/mL;醇提物浓度低于1mg/mL时,对3T3-L1前脂肪细胞的活性无影响;浓度高于400μg/mL时即可显著抑制前脂肪细胞的分化。     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Preparation and characterization of immobilized phospholipase A2 on chitosan beads for lowering serum cholesterol concentration

Phospholipase A₂ (PLA₂) from cobra venom, which can hydrolyze the S_N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10⁻³ to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA₂ was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA₂ could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA₂ should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.     

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A₂ (PLA₂) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA₂ or PLA₂-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.     

Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake

Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22E,24R)-ergosta-5,7,22-dien-3β-ol (ergosterol, 1); 5,8-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (ergosterol peroxide, 2); 5,8-epidioxy-(24S)-ergosta-6-en-3β-ol (3); and (22E,24R)-ergosta-7,22-dien-3β,5,6β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A₂ (PLA₂) enzyme, but not against Apis mellifcra bee venom PLA₂. The antiphospholipase A₂ activity of compounds 2 and 3 are reported here for the first time.     

Production of phosphatidylcholine containing conjugated linoleic acid mediated by phospholipase A2

Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A₂ (PLA₂). PLA₂ only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA₂ was affected by the amount of substrate CLA and PLA₂ in the reaction system. Under optimal reaction conditions using 11 mg LPC, 18 mg CLA, 550 mg glycerol, 50 μL formamide, 3.3 × 10⁴ U PLA₂, and 0.3 μmol CaCl₂ at 37 °C for 6 h, the reaction yield of CLA-PC reached 65 mol%. Furthermore, addition of protein such as albumin and casein suppressed the decrease of CLA-PC yield after 6 h. PLA₂ exhibited the highest activity for the 10t,12c-CLA isomer among four CLA isomers (9c,11t-CLA, 9c,11c-CLA, 9t,11t-CLA and 10t,12c-CLA), whereas that for 9c,11c-CLA was the lowest. These results showed that the present esterification system for LPC and CLA by PLA₂ is effective for producing CLA-PC.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

The level of pancreatic PLA2 receptor is closely associated with the proliferative state of rat uterine stromal cells

Rat uterine stromal cells (U_III) express pancreatic type PLA₂ (PLA₂-I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA₂-I. There is a dramatic decline in PLA₂-I binding in U_III cells as they progress from a nonconfluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA₂-I changed with the alteration in binding, suggesting that regulation in the PLA₂ binding capacity may have important implications in growth control mechanisms.     

Properties of the Group IV phospholipase A2 family

The Group IV phospholipase A₂ family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A₂ (cPLA₂) , cPLA₂β, cPLA₂γ, cPLA₂δ, cPLA₂ε and cPLA₂ζ. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA₂s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA₂s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA₂. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA₂, PLA₁, lysophospholipase and transacylase activities). cPLA₂, the most extensively studied Group IV PLA₂, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA₂ and lysophospholipase activities and is the only PLA₂ that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA₂ activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA₂s suggests they may have distinct mechanisms of regulation and functional roles.     

五步蛇毒对小鼠免疫功能的影响

目的探索五步蛇毒对正常小鼠免疫功能的影响。方法将KM小鼠随机分为5组,分别为空白组、五步蛇毒安全范围内低剂量组(30 mg/kg)、五步蛇毒安全范围内中剂量组(60 mg/kg)、五步蛇毒安全范围内高剂量组(120 mg/kg)(以下简称五步蛇毒低、中、高剂量组)以及西洋参组(30 mg/kg),每组10只。各组小鼠每日定时灌胃1次,连续14天。观察小鼠的呼吸、精神状态等生命体征。取小鼠脾脏和胸腺组织称重后,计算小鼠脾脏、胸腺指数;制备脾细胞悬液,采用CCK-8法检测小鼠脾淋巴细胞增殖率,碳粒廓清法检测小鼠吞噬系数α,中性红法检测小鼠腹腔巨噬细胞吞噬率;同时采用二硝基氟苯诱导小鼠迟发型变态反应,检测小鼠耳廓肿胀度。结果小鼠的吞噬系数α在五步蛇毒高剂量组最高(5.56±0.46),高于其余四组,差异均有统计学意义(均P<0.05)。小鼠腹腔巨噬细胞吞噬率(%)在五步蛇毒中剂量组和西洋参组中较高,分别为(119.07±19.31)%、(124.79±24.93)%,差异有统计学意义(均P<0.05)。小鼠的B淋巴细胞增殖率(%)在五步蛇毒中剂量组和西洋参组中较高,分别为(105.95±14.59)%、(108.14±10.58)%,差异有统计学意义(均P<0.05)。小鼠的T淋巴细胞增殖率(%)在五步蛇毒中、高剂量组中较高,分别为(119.30±20.07)%、(110.85±23.13)%,差异有统计学意义(均P<0.05)。与空白组比较,五步蛇毒中、高剂量组和西洋参组均能显著提高小鼠的耳廓肿胀度,差异有统计学意义(均P<0.05)。与空白组比较,五步蛇毒各剂量组和西洋参组均能显著提高小鼠脾脏指数(均P<0.05)。与空白组比较,各实验组胸腺指数均无明显差异(均P>0.05)。结论口服低于120 mg/kg的五步蛇毒对正常小鼠的免疫功能具有一定的增强作用。