苦荞芦丁水解酶序列鉴定及在昆虫系统中的表达

为获得苦荞麦中芦丁水解酶基因(Fagopyrum tartaricum rutin-hydrolyzing enzyme gene,FtRHE),以云荞1号苦荞种子为材料,制备获得天然芦丁水解酶(rutin-hydrolyzing enzyme,RHE),通过基质辅助激光解吸电离飞行时间质谱鉴定天然RHE序列信息,结合转录组数据,筛选苦荞RHE基因FtRHE。结果显示,纯化的天然RHE,分子质量约为62 kDa,肽质量指纹图谱证明其为一种β-葡萄糖苷酶,质荷比为914.428 6的肽段FSISWSR为其特征肽段。在苦荞种子转录组数据中筛选获得FtRHE基因序列基础上,通过反转录聚合酶链式反应获得了苦荞FtRHE基因。FtRHE开放阅读框由1 539个碱基组成,编码512个氨基酸,1~29位氨基酸为其信号肽。多重序列比对发现RHE与不同物种来源的β-葡萄糖苷酶氨基酸保守性不高,同源性普遍集中在54%~62%之间。构建Bacmid-FtRHE杆粒,转染昆虫细胞Sf9后,成功表达了具有较高活性的重组RHE。结果表明,获得的FtRHE即为苦荞中RHE基因。该研究为高含量芦丁及无苦涩味荞麦育种以及由芦丁生物转化生产槲皮素等的应用提供了理论支持。     

基于循环伏安法研究槲皮素醌与亲核物质的相互作用

多酚氧化形成的醌类物质作为强亲电体可能与食品体系中多种亲核物质作用,进而引发系列营养与安全问题。利用电化学工作站研究了酸性(pH 5.0)、中性(pH 7.0)和碱性(pH 8.0)条件下槲皮素醌与5种氨基化合物、2种巯基化合物以及牛血清白蛋白(BSA)的相互作用。结果表明:槲皮素醌在pH 7.0或pH 8.0条件下与8种亲核物质的互作效率显著高于pH 5.0;相同作用条件下,槲皮素醌与巯基化合物互作效率显著优于氨基化合物;在pH 7.0、扫描速率10 mV/s的条件下,槲皮素醌(0.08 mmol/L,以槲皮素计)与5种氨基化合物(16.0 mmol/L)互作效率排序为:L-赖氨酸(55.13%±5.37%)>L-精氨酸(39.58%±4.64%)>L-甲硫氨酸(17.80%±2.72%)>甘氨酸(~0.00%)≈Nα-乙酰-L-赖氨酸(~0.00%),槲皮素醌(0.08 mmol/L,以槲皮素计)与2种巯基化合物及BSA(0.048 mmol/L)互作效率排序为:L-半胱氨酸(63.17%±1.51%)>Nα-乙酰-L-半胱氨酸(54.28%±2.60%)>BSA(37.55%±5.98%)。研究表明:槲皮素氧化形成的醌类物质可以化学改性蛋白质,消耗体系必需氨基酸。     

Isolation and characterization of components responsible for neuroprotective effects of Allium cepa outer scale extract against ischemia reperfusion induced cerebral injury in mice

The antioxidant-mediated neuroprotective effect of Allium cepa outer scale extract (ACE) in mice with cerebral ischemia-reperfusion (I-R) injury was demonstrated in our earlier work. The current investigation aimed at establishing the bioactive component(s) responsible for this activity. Thus ACE was fractionated into ethyl acetate (EF) and aqueous (AF) fractions. These fractions were evaluated against cerebral I-R injury in mice. I-R injury in mice was induced by bilateral common carotid artery occlusion followed by 24 hr reperfusion. Memory, sensorimotor functions, cerebral infarct size, and oxidative stress were measured to address the neuroprotective mechanism of test substances. EF showed marked improvement of memory and sensorimotor functions by reducing brain oxidative stress and infarct size in mice after I-R injury. The bioactive EF was subjected to chromatographic (HPLC-PDA, HPLC-MS, preparative HPLC) and spectroscopic studies to isolate and identify the neuroprotective compounds. This lead to separation of three components, namely quercetin, quercetin 4′-O-glucoside, and the remaining fraction, from EF. The separated components were biologically evaluated. These components showed improvement in mice with I-R injury. But, EF displayed more marked neuroprotective effects as compared to the isolated components. The distinct neuroprotective outcome of EF may be credited to the synergistic action of compounds present in EF. Further studies such as evaluation of neurotoxic effects and other possible neuroprotective mechanisms are required to develop EF as a neuroprotective drug.     

Designing amorphous formulations of polyphenols with naringin by spray-drying for enhanced solubility and permeability

Naringin (NAR), a major flavanone (FVA) glycoside, is a component of food mainly obtained from grapefruit. We used NAR as a food additive to improve the solubility and permeability of hydrophobic polyphenols used as supplements in the food industry. The spray-dried particles (SDPs) of NAR alone show an amorphous state with a glass transition temperature (T_g) at 93.2 °C. SDPs of hydrophobic polyphenols, such as flavone (FVO), quercetin (QCT), naringenin (NRG), and resveratrol (RVT) were prepared by adding varying amounts of NAR. All SDPs of hydrophobic polyphenols with added NAR were in an amorphous state with a single T_g, but SDPs of hydrophobic polyphenols without added NAR showed diffraction peaks derived from each crystal. The SDPs with NAR could keep an amorphous state after storage at a high humidity condition for one month, except for SDPs of RVT/NAR. SDPs with NAR enhanced the solubility of hydrophobic polyphenols, especially NRG solubility, which was enhanced more than 9 times compared to NRG crystal. The enhanced solubility resulted in the increased membrane permeability of NRG. The antioxidant effect of the hydrophobic NRG was also enhanced by the synergetic effect of NAR. The findings demonstrated that NAR could be used as a food additive to enhance the solubility and membrane permeability of hydrophobic polyphenols.     

Improved aqueous solubility,bioaccessibility and cellular uptake of quercetin following pH-driven encapsulation in whey protein isolate

The purpose of this study was to increase the water solubility and potential bioavailability of quercetin by encapsulation in whey protein isolate (WPI) based on a green, efficient pH-driven method. According to the results, the water solubility of quercetin increased by 346.9: times after loading into WPI nanoparticles. When the initial quercetin concentration was 0.25 mg mL⁻¹ and WPI was 2% w/v, the encapsulation efficiency reached 94.1%, the Z-average diameter was 36.63 nm, and the zeta potential was −36.4 mV at pH 7.0. The fluorescence spectroscopy assay suggested the molecular complexation of quercetin and WPI at pH 12.0. X-ray diffraction assay indicated the enclosure of amorphous quercetin in WPI. Correspondingly, the bioaccessibility increased from 2.76% to 31.23% and the Caco-2 cell monolayer uptake increased from 0% to 2.12% after nanoencapsulation. This work confirmed that the pH-driven method is an effective approach to prepare WPI–quercetin nanocapsules to improve physical and potentially biological properties of quercetin.     

超声波提取赶黄草中槲皮素方法的研究

研究了赶黄草中槲皮素的最佳提取条件。采用超声提取,用紫外分光光度法,以芦丁作为对照,在370 nm处测定槲皮素的含量。结果显示:赶黄草最佳提取条件为以浓度为60%乙醇,超声功率50 W,超声温度55℃,料液比1∶25,超声时间60 min,槲皮素浸出率最高。超声提取工艺稳定、简单可行。     

离子液体修饰碳糊电极测定海红果中的槲皮素

用离子液体1-甲基-3-丁基咪唑六氟磷酸盐([bmim]PF6)作修饰剂制备了离子液体修饰碳糊电极(IL/CPE)。在0.2 mol/L B-R(p H 3.2±0.1)缓冲溶液中,采用循环伏安法研究了槲皮素在该修饰电极上的电化学行为,建立了测定槲皮素的新方法。研究表明,槲皮素在IL/CPE电极上的氧化、还原峰电位差比在裸碳糊电极(CPE)上的小,而峰电流却显著增加,说明IL/CPE对槲皮素有电催化作用;在循环伏安曲线上,槲皮素的峰电流与其浓度在0.1~50μmol/L呈良好的线性关系,其线性方程为ip(μA)=3.941 3+0.211 9 c(μmol/L),线性相关系数为0.999 4,检出限为3.0×10-8mol/L。该法用于测定海红果中的槲皮素取得了较好的结果。