薄层层析-紫外分光光度法测定葡萄果皮中白藜芦醇及白藜芦醇苷

建立了葡萄果皮中反式白藜芦醇及反式白藜芦醇苷含量的薄层层析-紫外分光光度测定法.以甲醇提取目标组分,以高效硅胶GF254为固定相,甲苯:乙酸乙酯:甲酸(5:4:1)为展开剂,对反式白藜芦醇及反式白藜芦醇苷进行分离纯化.并用紫外分光光度法进行定量,波长为306 nm.结果表明,该方法测定反式白藜芦醇及反式白藜芦醇苷含量的线性关系良好,相关系数为0.9997;平均回收率为98.12%、97.23%;方法的精密度较高.并用该方法测定了酿酒红葡萄品种蛇龙珠不同生长时期的反式白藜芦醇和反式白藜芦醇苷含量.     

凝胶柱层析分离虎杖中白藜芦醇的研究

本论文初步探讨了虎杖中白藜芦醇分离纯化的工艺条件,比较了不同溶剂、不同浓度、不同流速洗脱条件下白藜芦醇样品在LH-20凝胶层析柱上的分离效果,以及虎杖的乙醇提取液通过氯仿、乙酸乙酯萃取,凝胶柱层析分离后的成分变化,实验表明,当以甲醇为洗脱液,浓度为60%,流速为0.5 mL/min时,白藜芦醇样品的分离效果最好.采用中压液相色谱检测收集到的白藜芦醇纯度(以白藜芦醇峰面积占总峰面积计算)可达80.34%,回收率达86.16%.     

白藜芦醇抑制大鼠下丘脑脑片室旁核神经元放电

本文旨在研究白藜芦醇(resveratrol)对下丘脑脑片室旁核神经元放电的影响.应用玻璃微电极细胞外记录单位放电技术,在下丘脑脑片上观察白藜芦醇对静息状态下室旁核神经元放电的影响.结果如下:(1)在29张下丘脑脑片室旁核神经元放电单位给予白藜芦醇(O.05,0.5,5.0 μmol/L)2 min,有28张脑片(96.6%)放电频率显著降低,且呈剂量依赖性;(2)预先用0.2mmol/L的L.glutamate灌流8张下丘脑脑片,8张脑片(100%)放电频率显著增加,表现为癫痫样放电,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制:(3)预先用L型钙通道开放剂Bay K8644(0.1μmol/L)灌流8张下丘脑脑片,8张脑片(100%)放电频率显著增加,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制;(4)用一氧化氮合酶抑制剂Nω-nitro.L-arginine methyl ester(L-NAME)50μmol/L灌流8张下丘脑脑片,7张脑片(87.5%)放电频率显著增加,该放电可被白藜芦醇(5.0 μmol/L)灌流2 min抑制.以上结果提示,白藜芦醇抑制下丘脑室旁核神经元自发放电,可能通过降低心血管中枢的活动性而产生中枢保护作用.这种抑制作用可能与白藜芦醇抑制L型钙通道、减少钙内流有关,与NO释放无关.     

虎杖中白藜芦醇的酶法制备

以白藜芦醇得率为指标,通过对酶制剂的筛选和酶解条件的考察,分别选出酶法提取和酶法转化制备虎杖中白藜芦醇的最佳条件,并对两种酶法进行比较.结果显示:(1)酶法提取最佳酶解条件为:酶解温度45℃,酶解时间60 min,最适pH 5.0,底物浓度17%;酶法转化最佳酶解条件为:酶解温度40℃,酶解时间48 h,最适pH 5.0,底物浓度15%.(2)与空白样品(不加酶)相比,酶法提取白藜芦醇得率增加了3.12 mg/g;酶法转化白藜芦醇得率增加了12.72 mg/g.研究表明,两种方法与不加酶提取相比均提高了白藜芦醇的得率,但酶法转化效果更显著,可用于白藜芦醇的制备.     

大孔吸附树脂分离虎杖中白藜芦醇的研究

目的:采用大孔吸附树脂对虎杖粗提物中白藜芦醇进行初步富集、分离和纯化.方法:考察18种树脂对白藜芦醇的吸附量和解吸率,选择吸附量大、解吸率高的数种树脂进行吸附动力学研究,确定最佳的脱附工艺.结论:HPD-500树脂对白藜芦醇的吸附量可达58.67mg/g,解吸率为92.6%,经大孔吸附树脂的吸附与解吸,白藜芦醇的含量由粗提物中9.25%提高至39.5%.     

白藜芦醇微粉的制备、表征与抗氧化功能评价

实验利用超临界SAS法使白藜芦醇在超临界状态下反溶剂重结晶,来制备白藜芦醇微粉借助超临界反溶剂法(SAS)来减小白藜芦醇的粒径从而提高其抗氧化能力。制备的条件为:反应温度为50℃,反应压力为20MPa,白藜芦醇乙醇溶液的浓度为9 mg·m L-1,给药速度为1 m L·min-1。利用激光粒度仪和扫描电镜检测白藜芦醇微粉,结果显示白藜芦醇粉体较原粉粒径大大降低,平均粒径为0.68±0.03μm。而后通过了傅里叶红外光谱、X衍射仪、高效液相色谱和质谱检测白藜芦醇微粉和原粉的各项特性。结果显示SAS过程并没有改变白藜芦醇的化学结构,但白藜芦醇微粉的结晶度大大降低,白藜芦醇的纯度也从原粉的98.5%纯化为微粉的99.2%。利用果蝇抗衰老实验来检测白藜芦醇的抗氧化能力。结果表明在白藜芦醇溶液浓度为,2.5、5和10 mg·m L-1时,白藜芦醇微粉的抗氧化能力均高于原粉和空白实验。这说明白藜芦醇的不但具有抗氧化能力,而且白藜芦醇微粉在效果上优于白藜芦醇原粉。这为提高白藜芦醇品质提供了依据。     

HPLC检测三叶青块根中白藜芦醇和白藜芦醇苷含量的研究

目的：建立三叶青地下部分白藜芦醇、白藜芦醇苷以及总黄酮的提取分离和含量测定方法。方法：白藜芦醇提取分离选取溶剂为70%乙醇，白藜芦醇苷的提取分离选取溶剂为40%乙醇。含量测定采用高效液相色谱法，色谱柱选择Agilent ZORBAX SB-C18色谱柱(250 mm×4.6 mm,5μm)，流动相为甲醇-水梯度洗脱，流速为0.8 mL/min，检测波长为304 nm，柱温25℃。结果：白藜芦醇的线性关系方程为Y=113.836X+0.911 5，在0.02～2μg/mL范围内的峰面积与进样浓度线性关系良好(R²=0.997 6)。采用70%乙醇提取三叶青根茎中白藜芦醇的平均含量为0.59μg/g。白藜芦醇苷的线性关系方程为Y=52.515X-0.497 7，在0.02～2μg/mL范围内的峰面积与进样浓度线性关系良好(R²=0.994 4)。采用40%乙醇提取三叶青根茎中白藜芦醇的平均含量为1.76μg/g。结论：建立了三叶青地下部分白藜芦醇、白藜芦醇苷以及总黄酮的提取分离和含量测定方法。建立的高效液相色谱法方法简便，准确，重复性好，可应用于...     

HPLC法测定小叶山葡萄叶中白藜芦醇含量

建立一种高效液相色谱法(HPLC)测定小叶山葡萄Vitis thunbergii var.taiwaniana叶中白藜芦醇含量的检测方法。Waters高效液相色谱仪:Hypersil C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇-水(72:28);流速:1 mL·min^-1;检测波长306 nm。结果表明,在上述色谱条件下,白藜芦醇含量在10~200μg·mL^-1范围内线性关系良好,相关系数r为0.9999。精密度、重现性、稳定性的RSD(n=5)分别为0.77%、0.41%、0.21%;平均加标回收率为99.92%,相对标准偏差为0.39%。该方法准确、灵敏、可靠,可用于白藜芦醇的定量和定性分析。     

白藜芦醇对人胶质瘤细胞迁移能力的影响

目的:探讨白藜芦醇对人胶质瘤U251细胞迁移能力的影响。方法:常规培养人胶质瘤U251细胞,取对数生长期细胞进行实验。采用细胞划痕实验检测经终浓度为50和100μg/ml的白藜芦醇刺激后的人胶质瘤U251细胞的迁移距离。结果:细胞划痕实验结果显示,对照组、50μg/ml白藜芦醇组和100μg/ml白藜芦醇组刺激的U251细胞迁移率分别为(25.7±2)%、(19.8±7)%和(8.3±2)%,均有显著性差异(p0.05)。结论:白藜芦醇具有抑制人胶质瘤U251细胞迁移的作用。     

HPLC法测定芦笋茎秆中芦丁、槲皮素及白藜芦醇含量

为了建立高效液相色谱法对芦笋茎秆中芦丁、槲皮素和白藜芦醇含量的测定方法。采用不同方法提取芦笋茎秆中的芦丁、槲皮素和白藜芦醇,得出甲醇法提取芦丁、甲醇-HCl法提取槲皮素以及乙醇法提取白藜芦醇得率最高;确定了高效液相色谱法对3种物质的最佳色谱条件为流动相:芦丁为0.2 mol/L乙酸钠-甲醇(Me-OH∶H2O=35∶65)溶液(用磷酸调p H 2.80)、槲皮素为无水甲醇-0.4%磷酸溶液(55∶45,V/V),等梯度洗脱,白藜芦醇为水和甲醇,二元线性梯度洗脱;检测波长分别为:254 nm、360 nm和306 nm;流速:0.8 m L/min、1.0 m L/min、0.8 m L/min。通过该方法测得芦丁、槲皮素及白藜芦醇的平均回收率分别为:98%、97%、98%。     

Moderate exercise training is more effective than resveratrol supplementation for ameliorating lipid metabolic complication in skeletal muscle of high fat diet-induced obese mice

[Purpose]

The aim of this study was to compare the effectiveness of moderate exercise training or resveratrol supplementation with a low fat diet on lipid metabolism in the skeletal muscle of high fat diet-induced obese mice.

[Methods]

C57BL/6J mice (5 weeks old, n = 30) were fed a high fat diet (45% fat) for 8 weeks first to make them obese. Afterward, all the mice were fed a low fat diet during 8 weeks of intervention with moderate exercise training and resveratrol supplementation. Before the intervention, the mice were separated into 3 groups: low-fat diet control (HLC; n = 10), low fat diet with resveratrol (HLR; n = 10) or low fat diet with exercise (HLE n = 10). The exercise group (HLE) performed treadmill running for 30-60 min/day at 10-22 m/min, 0% grade, 5 times/week for 8 weeks, while the resveratrol group (HLR) received a daily dose of resveratrol (10 mg/kg of body weight), 5 days/week for 8 weeks.

[Results]

Body weight was significantly reduced in HLE. Further, the lipogenesis marker SREBP and the inflammatory cytokine TNF-α were significant reduced in HLE. However, there was no significant effect from resveratrol supplementation with a low fat diet. Taken together, exercise training with a low fat diet has the positive effect of ameliorating lipid disturbance in the skeletal muscle of high fat diet-induced obese mice.

[Conclusion]

These findings suggest that exercise training with a low fat diet is most effective to improve lipid metabolism by reducing lipogenesis and inflammation in the skeletal muscle of high fat diet-induced obese mice.     

Red wine triggers cell death and thioredoxin reductase inhibition: Effects beyond resveratrol and SIRT1

Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 μM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (≥ 10 μM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v) - but not white wine - induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.     

Biocatalytic synthesis of 3,4,5,3′,5′-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC

HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3′,5′-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100?mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9?mM (1.8?g L^?1) in 48?h.     

白藜芦醇抑制小鼠肥胖的功效及相关机制探讨

目的:研究白藜芦醇抑制高脂引起的肥胖的作用机制。方法:将18只C57小鼠随机分为3组,分别为对照组、高脂以及高脂+白藜芦醇小鼠模型,给小鼠喂养一定剂量白藜芦醇(100 mg/kg/d),喂养12周。提取小鼠皮下脂肪细胞,分化成熟,加入白藜芦醇,采用q RT-PCR以及Western blot等方法检测HO-1以及棕色脂肪标志基因的表达。通过q RT-PCR检测小鼠脂肪组织炎症因子、UCP-1以及HO-1的表达。结果:白藜芦醇在体内可以明显抑制高脂引起的肥胖,糖耐量异常,同时促进棕色脂肪标志基因UCP-1,PGC-1以及PRDM16的表达。白藜芦醇还可抑制肥胖小鼠脂肪组织炎症因子的增加以及抗炎蛋白HO-1的表达。在体外分化的成熟的皮下脂肪细胞中,白藜芦醇同样可以促进棕色脂肪标志基因UCP-1,PGC-1以及PRDM16的表达。白藜芦醇通过促进抗炎蛋白HO-1的表达抑制高脂引起的脂肪炎症反应。结论:白藜芦醇可以通过促进白色脂肪棕色化以及抑制慢性低度炎症抑制高脂引起的肥胖、糖耐量异常以及改善胰岛素敏感性。     

Design, synthesis and spectroscopic studies of resveratrol aliphatic acid ligands of human serum albumin

As one of the natural polyphenols, resveratrol possesses hydroxyl substituted trans-stilbene structure and exerts impact on health by inhibiting multiple human enzymes, such as cyclooxygenase, F1 ATPase, and tyrosinase. Resveratrol has to be bound by human serum albumin (HSA) to keep a high concentration in serum, since its solubility is low in water. To improve water solubility and bioavailability, two resveratrol aliphatic acids and their esters have been designed and synthesized. The solubilities of the resveratrol and its derivatives have been measured using a standard procedure. The two aliphatic acids showed better solubilities in pure water and phosphate buffer (pH 7). The binding affinities of resveratrol derivatives for HSA were also measured, and the drug-protein interaction mechanism was investigated using fluorescence, UV-vis, and NMR spectroscopies. Interestingly, resveratrol hexanoic acid (5) was found to be a much better ligand (K(a)=(6.70+/-0.10)x10(6) M(-1)) for HSA than resveratrol (K(a)=(1.64+/-0.07)x10(5) M(-1)), and there was 41-fold improvement for the binding affinity. It was the first time that the increase of fluorescence of resveratrol moiety was observed during the binding to HSA, suggesting that 5 should be bound tightly by HSA. The UV-vis absorption spectroscopy revealed a maximum absorption shift from 318 to 311 nm with decreasing intensity by 20% upon complexation, suggesting that the pi-pi conjugation of the stilbene structure was impaired during the binding. Although HSA was reported to have only one binding site for resveratrol, the Job's and molar ratio plots suggested that HSA should bind two molecules of 5. NMR study suggested that phenyl group (B ring) in the center of the molecule of 5 should be involved in the pi-pi stacking interactions with HSA aromatic amino acid residues. Molecular geometry calculation of 5 with Spartan software showed that the stilbene structure had two conformers, orthogonal and planar ones. The former (E=-1.432 KJ/mol) was more stable than the latter (E=-0.128 KJ/mol), suggesting that the former should be the conformer of 5 in the complexation with HSA.     

白藜芦醇后处理对大鼠脑缺血再灌注损伤海马CA1区Bax、Bcl-2的影响

摘要 目的：探讨白藜芦醇后处理对大鼠脑缺血再灌注损伤Bax、Bcl-2表达的影响。方法：清洁级雄性SD大鼠60只随机分为假手术组（n=12）、I/R组（n=12）、白藜芦醇组（n=36）,白藜芦醇组按不同剂量分为低剂量、中剂量、高剂量组（10 mg/kg、20 mg/kg、40 mg/kg）,每组12只。假手术组：仅暴露大鼠颈外动脉,不做缺血处理;I/R组：采用改良线栓法制备大鼠大脑中动脉缺血再灌注损伤模型（缺血2 h,再灌注24 h）;白藜芦醇组：造模方法同I/R组,在大鼠缺血2h后,将不同剂量白藜芦醇腹腔注射入大鼠体内,比较各组SD大鼠神经功能缺损评分、采用Western blotting法、免疫组化法对大鼠脑组织缺血侧海马CA1区Bax和Bcl-2表达进行比较。结果：白藜芦醇低、中、高剂量组神经功能缺损评分均低于I/R组,随着白藜芦醇剂量的增加,神经功能缺损评分逐渐降低,其中白藜芦醇高剂量组神经功能缺损评分降低最为明显;白藜芦醇组与I/R组相比,不同剂量白藜芦醇组Bax表达逐渐减少,而Bcl-2表达明显增加,其中以白藜芦醇高剂量组改变最为明显。结论：高剂量白藜芦醇可以降低大鼠神经功能缺损评分,减轻脑缺血再灌注损伤,对大鼠脑缺血再灌注损伤具有保护作用,其机制与Bax、Bcl-2的表达有关。     

Resveratrol attenuates oxidative stress and prevents steatosis and hypertension in obese rats programmed by early weaning

We hypothesized that resveratrol, a natural phytoalexin found in grapes, can prevent oxidative stress, obesity and its related disturbances in obese rats programmed by early weaning. Lactating Wistar rats were separated into two groups: early weaning (EW) — dams who were wrapped with a bandage to interrupt the lactation in the last 3 days of lactation; control — dams whose pups had free access to milk during all lactation. At the 150th day, EW offspring were randomly subdivided into EW+resveratrol (EW+Res) — resveratrol (30 mg/kg/day); EW+vehicle (EW) — rats that received 0.5% (w/v) aqueous methylcellulose. The control group received vehicle. Rats were treated by gavage daily for 30 days. EW offspring developed hyperphagia, higher body weight, visceral obesity, higher systolic (SBP) and diastolic blood pressure (DBP) (+15% and +20%, respectively; P<.05) and higher serum triglycerides (TG) and low-density lipoprotein but lower high-density lipoprotein (+55%, +33% and ?13%, respectively; P<.05). Resveratrol normalized food intake, SBP and DBP and prevented obesity and dyslipidemia in EW+Res. EW rats had higher plasma and liver thiobarbituric-acid-reactive substances (TBARS) and lower plasma superoxide dismutase (SOD) and liver glutathione peroxidase activities (+51%, +18%, ?58%, ?31%, respectively; P<.05), and resveratrol normalized both plasma and liver TBARS and increased the activity of SOD and catalase in plasma. EW rats presented liver steatosis and higher liver TG, and resveratrol prevented these hepatic alterations. In conclusion, this study demonstrated a potential therapeutic use of resveratrol in preventing obesity and oxidative stress and reducing the risk of hypertension, dyslipidemia and steatosis in adult rats programmed by early weaning.     

High-performance liquid chromatography spectrometric analysis of trans-resveratrol in rat plasma

A HPLC method for determination of trans-resveratrol concentrations in rat plasma was developed. Plasma samples were treated with acetonitrile to deposit proteins. The analysis used a Hypersil ODS(2) C(18) column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow-rate=1 mL/min). The UV detection wavelength was 303 nm, and chlorzoxazone was used as the internal standard. The calibration curve was linear over the range of 0.02-40 microg/mL with a correlation coefficient of 0.9997. This concentration range corresponds well with the plasma concentrations of resveratrol in pharmacokinetic studies. There was 98.7%, 91.3% and 84.4% recovery from 0.02, 0.4 and 40 microg/mL plasma samples respectively. The R.S.D. of intra- and inter-day assay variations were all less than 12%. This HPLC assay is a quick, precise and reliable method for the analysis of resveratrol in pharmacokinetic studies.