UPLC-MS/MS同时测定白及中6个指标成分的含量

建立UPLC-MS/MS同时测定白及药材中的α-异丁基苹果酸(B6)、4-(葡萄糖氧基)-肉桂酸葡萄糖氧基苄酯(B12)、1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯(B14)、1-[4-(葡萄糖氧)苄基]-2-异丁基苹果酸(B17)、二氢菲1(B19)和1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯-2-[4-O-肉桂酰基-6-O-乙酰基]葡萄糖苷(B23)含量的分析方法,采用Acquity UPLC BEH C18柱(2.1 mm×50 mm,1.7μm);流动相0.1%甲酸乙腈溶液(A)-0.1%甲酸水溶液(B)为流动相,梯度洗脱,采用电喷雾离子源(ESI),多反应离子监测(MRM)模式进行正负离子同步监测。各指标成分之间分离度良好,在选定的浓度范围内线性关系良好(r≥0.9974),平均加样回收率为98.07%~103.19%,RSD3.04%为,重复性、精密度和稳定性良好。此方法操作简便,快速,灵敏度高,可用于白及药材中主要活性成分的含量测定,并为白及药材的质量控制提供了新方法。     

基于大鼠在体肠灌流模型研究白及有效部位在肠道的可吸收及代谢成分

为了考查白及有效部位在肠道的可吸收成分及其代谢特征。基于在体肠灌流模型,采用超高效液相色谱-四极杆-飞行时间质谱仪(UPLC-Q-TOF/MS)对收集到的健康SD大鼠循环肠灌流液、血清、胆汁进行分析检测,并结合对照品、质谱碎片信息和Masslynx V4.1工作站中的Single Mass Analysis功能,初步推测吸收和代谢产物的结构式。在大鼠血清和胆汁中,初步鉴定出1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸、4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、α-异丁基苹果酸酯原型产物。在大鼠循环肠灌流液、血清和胆汁中,共鉴定出4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯的脱糖后硫酸化代谢产物和二氢菲5的葡萄糖醛酸化代谢产物,其代谢产物主要生水解和葡萄糖醛酸化反应。该方法初步探究了白及有效部位在大鼠循环肠灌流液中可吸收成分和代谢特征,为阐释白及药材的药效物质基础提供实验依据。     

米碎花的酚苷类化合物及其鼻咽癌细胞毒活性

为了解米碎花(Eurya chinensis)的化学成分及其生物活性,运用多种色谱技术从其乙醇提取物分离得到11个化合物,并对化合物进行体外抗鼻咽癌细胞增殖活性评价。经波谱数据分析,分别为异落新妇苷(1)、3,5,7-三羟基色原酮-3-O-α-L-鼠李糖苷(2)、1-O-反式-桂皮酰基-β-D-葡萄糖(3)、1-O-(4-羟基苯乙基)-6-O-反式-桂皮酰基-β-D-葡萄糖(4)、eutigoside D (5)、1-O-(3,4-二羟基苯乙基)-6-O-反式-香豆酰基-β-D-葡萄糖(6)、eutigoside A (7)、1-O-(4-羟基苯乙基)-6-O-反式-咖啡酰基-β-D-葡萄糖(8)、grayanoside A (9)、1-O-(4-羟基苯乙基)-6-O-(4-羟基苯甲酰基)-β-D-葡萄糖(10)、3-O-β-D-葡萄糖基-4-羟基-苄基苯甲酸酯(11)。其中,化合物4为首次从天然来源获得,化合物2～4和8～11均为首次从该属植物中分离得到。MTT法表明,化合物10具有中等抑制5-8F细胞增殖活性。     

白及属药用植物化学成分及药理作用研究进展

结合国内外文献综述了白及属植物化学成分的研究进展,并对该属药用植物的药理活性进行了总结。白及属植物含有多种化学成分,主要有联苄类、二氢菲类、菲类、黄酮类、2-异丁基苹果酸葡萄糖氧基苄酯类、多酚类等。该属药用植物具有显著收敛止血、消肿生肌的功效。     

体循环肠灌流法研究红禾麻提取物在类风湿性关节炎大鼠与正常大鼠体内的肠吸收差异

比较红禾麻提取物在AA大鼠与正常大鼠体内的肠吸收差异。实验采用体循环肠灌流法,建立大鼠RA模型,应用UPLC-MS/MS检测肠灌流液中新绿原酸、绿原酸、隐绿原酸、芦丁、异槲皮苷、槲皮苷、山奈酚-3-O-芸香糖苷、木犀草苷的含量,探究各成分在不同因素(pH值、药物浓度、肠段、胆汁和P-gp抑制剂)下的肠吸收特性。结果表明,槲皮苷的吸收方式为主动转运,其余7个成分的吸收方式为被动扩散。除绿原酸、隐绿原酸外,正常状态下各成分以回肠吸收最好,病理状态下以十二指肠吸收最好,推测药物吸收的特定部位会受疾病状态的影响而发生改变。各成分的吸收均受pH、胆汁和P-gp的影响,其中,槲皮苷可能是P-gp的底物。RA能影响红禾麻提取物在大鼠体内的肠吸收,其作用机制尚需进一步研究。     

拟诺卡氏放线菌YIM90087的代谢产物研究北大核心CSCD

对拟诺卡氏菌YIM90087的固体发酵提取物进行了化学成分研究,从中分离得到10个化合物。通过波谱数据分析,鉴定其结构分别为:(3S)-3-苄基-2,5-哌嗪二酮(1)、(3S,6S)-3-(2-丁基)-6-(1-羟乙基)-2,5-哌嗪二酮(2)、(3S,6S)-3-(2-丁基)-6-羟甲基-2,5-哌嗪二酮(3)、(4S)-4-苄基-1,3-恶唑-2,5-二酮(4)、6'-羟基-4,2',3',4'-四甲氧基-对三联苯(5)、(3S,6S)-3-(4-羟苄基)-6-异丁基-2,5-哌嗪二酮(6)、(3R,8aS)-3-异丙基-六氢吡咯并[1,2-a]吡嗪-1,4-二酮(7)、(3S,6S)-3-异丁基-6-甲基-2,5-哌嗪二酮(8)、(3S,6S)-3-苄基-6-(1-羟乙基)-2,5-哌嗪二酮(9)和(3S,6S)-3-苄基-6-(1-羟丙基)-2,5-哌嗪二酮(10)。化合物4目前只见化学合成报道,为新天然产物。     

拟诺卡氏放线菌YIM90087的代谢产物研究

对拟诺卡氏菌YIM90087的固体发酵提取物进行了化学成分研究,从中分离得到10个化合物。通过波谱数据分析,鉴定其结构分别为:(3S)-3-苄基-2,5-哌嗪二酮(1)、(3S,6S)-3-(2-丁基)-6-(1-羟乙基)-2,5-哌嗪二酮(2)、(3S,6S)-3-(2-丁基)-6-羟甲基-2,5-哌嗪二酮(3)、(4S)-4-苄基-1,3-恶唑-2,5-二酮(4)、6'-羟基-4,2',3',4'-四甲氧基-对三联苯(5)、(3S,6S)-3-(4-羟苄基)-6-异丁基-2,5-哌嗪二酮(6)、(3R,8aS)-3-异丙基-六氢吡咯并[1,2-a]吡嗪-1,4-二酮(7)、(3S,6S)-3-异丁基-6-甲基-2,5-哌嗪二酮(8)、(3S,6S)-3-苄基-6-(1-羟乙基)-2,5-哌嗪二酮(9)和(3S,6S)-3-苄基-6-(1-羟丙基)-2,5-哌嗪二酮(10)。化合物4目前只见化学合成报道,为新天然产物。     

药用植物灯笼草的化学成分研究

从药用植物灯笼草(Clinopvdium polycephalum)中分离鉴定了5个化合物。其中主要成分为乌索酸(ursolic acid,Ⅰ),其余4个分别鉴定为:异樱花素(isosakuranetin.Ⅱ),香蜂草甙(didymin,Ⅲ),6'-十六碳酸酯基-α-菠甾醇-3-O-β-D-葡萄糖甙(6’-Palmityl-α-spinasteryl-3-O-β-D-glucoside,Ⅳ a)和十八碳酸酯基-α-波甾醇-3-O-β-D-葡萄糖甙(6’-stearyl-α-spinasteryl-3-O-β-D-glucoside,Ⅳ b)。上述化合物在该植物中均为首次报道。     

在体单向肠灌流模型研究白芷中三种成分的吸收特性

目的:研究白芷中有效成分欧前胡素、异欧前胡素和花椒毒酚在大鼠小肠各段的吸收特征。方法:采用大鼠在体单向肠灌流模型结合HPLC法同时测定肠灌流液中欧前胡素、异欧前胡素和花椒毒酚含量的变化,考察高、中、低三组剂量下三种成分在十二指肠、空肠和回肠的吸收特性。结果:三种成分随着剂量的增大,吸收速率常数(Ka)和有效渗透系数(Peff)逐渐增大。三种成分相同剂量下各肠段间吸收均无显著性差异,其吸收大小顺序为:欧前胡素异欧前胡素花椒毒酚。结论:白芷三种主要成分在各肠段均吸收良好,吸收大小呈剂量相关性。     

狗脊化学成分及抑制乙酰胆碱酯酶生物活性研究北大核心CSCD

采用多种分离方法从狗脊(Woodwardia japonica)乙醇提取物中分离纯化出7个化合物,经现代波谱分析将它们的结构分别鉴定为3-O-[6’-O-(9Z-二十碳酰)-β-D-葡萄糖酰]-谷甾醇(1)、β-谷甾醇-3-O-α-L-(6’-O-正十六酰基)-葡萄糖苷(2)、β-谷甾醇(3)、狗脊酸(4)、胡萝卜苷(5)、山柰素-3-O-α-L-鼠李糖基-7-O-α-L-鼠李糖苷(6)和山柰素-3-O-α-L-(4-O-乙酰基)-鼠李糖基-7-O-α-L-鼠李糖苷(7)。其中化合物1的结构还未见报道。乙酰胆碱酯酶抑制活性测试表明化合物4具有一定抑制乙酰胆碱酯酶活性。     

白及提取物抗变异链球菌致龋效果的研究

目的研究白及提取物对变异链球菌粘附、生物膜形成及活性的影响,评价其抗龋效果。方法市售白及95%乙醇浸提;纸片法、打孔法测定直接抑菌作用;液体稀释法检测MIC;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用荧光显微镜和激光共聚焦显微镜观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响;运用扫描电镜观察白及药液对变异链球菌生物膜的影响。结果白及提取物具有一定的抑菌作用,MIC为16~62 mg/m L;结晶紫法定量研究生物膜结果显示白及药液作用4 h对变异链球菌的粘附均有抑制作用,抑制率为28.63%~60.08%;作用20 h对生物膜总量抑制率达77.08%;白及药液作用20 h,荧光染色显示生物膜活性明显被抑制,抑制率达62.03%;梯度浓度白及药液分别作用20 h后,激光共聚焦显微镜下观察到随着药液浓度增加,绿色的活菌、团块状结构减少,生物膜形成明显被抑制;扫描电镜下可见药液作用后细菌间粘性物质减少。结论高浓度白及提取液对变异链球菌有直接抑菌作用,亚抑菌浓度能抑制其粘附和生物膜的形成,进而具有抗龋作用。     

Increased zinc absorption but not secretion in the small intestine of metallothionein-null mice

Metallothionein (MT) has been assigned a role in intestinal Zn absorption and secretion. The influence of MT was investigated in isolated segments of the small intestine from mice lacking the expression of MT I and II genes (MT−/−). To measure Zn absorption, washed 10- to 12-cm segments of the proximal and distal small intestine of MT−/− and control MT+/+ mice were filled with ⁶⁵Zn as ZnSO₄ (10 μg/mL), and the amount of ⁶⁵Zn appearing in the external buffer was measured over 4 h. To measure Zn secretion, the same procedure was followed using everted gut segments. The ⁶⁵Zn absorption from the small intestine was significantly greater in MT−/− mice, but only in the absence of albumin. In the proximal small intestine, the inclusion of 2% albumin in the external buffer significantly increased Zn absorption from 6.8% (no albumin) to 13.2% (with albumin) for MT−/−, and from 4.9% (no albumin) to 14.2% (with albumin) for MT+/+. In the distal segment, the respective values, with and without albumin respectively were 9.5% and 15.1% for MT−/− mice and 4.3% and 16.1% for MT+/+ mice. Regarding ⁶⁵Zn secretion, there was no difference between MT+/+ and MT−/− in either segment. However, the rate of secretion was higher in the proximal small intestine for both genotypes. Although it can be demonstrated that MT limits Zn absorption under controlled conditions in vitro, the ability of albumin to overcome this effect emphasizes the importance of circulating ligands in Zn transport.     

Nickel absorption and distribution from rat small intestine In situ

The aim of this work was to study the absorption of nickel chloride in rats by means of the intestinal perfusion in situ technique at nickel concentrations of 1, 5, 10, 25, and 100 mg/L. Active transport and facilitated diffusion seem to play an important role in the intestinal absorption of nickel at concentrations≤10 mg/L. At higher concentrations, the absorption rate would be limited by saturation of the carriers. The distribution of the absorbed nickel was studied by intestinal perfusion of a 10-mg Ni/L solution for 30 or 60 min. Both in concentration and amount, the jejunum showed the higher values of absorbed nickel, followed by the kidneys and liver. When all of the collected organs (brain, heart, liver, lungs, spleen, kidneys, and testicles) and blood, but not the small intestine, are analyzed following a 60-min perfusion, it was found that 1% of the initial concentration had passed through the intestinal barrier.     

白及蔗糖合成酶基因的克隆及表达分析

为揭示白及蔗糖合成酶基因与生长发育的关系,该研究以白及为材料,利用RT-PCR技术同源克隆白及蔗糖合成酶的关键基因SuSy,对SuSy基因的生物学特性及表达特征进行了分析,并利用实时荧光定量PCR检测SuSy基因在不同组织中的表达规律。结果表明:(1)白及SuSy基因长度为2 215 bp,编码737个氨基酸,与铁皮石斛、文心兰和蝴蝶兰的蛋白质氨基酸序列的相似性分别为97%、92%和95%。(2)生物信息学分析表明,SuSy蛋白质序列具有较高的亲水性,与拟南芥SuSy蛋白质氨基酸三级结构一致性为75.2%;系统进化树分析发现,白及SuSy蛋白与铁皮石斛处于同一个分支上。(3) qRT-PCR结果表明,SuSy基因在叶片中的表达量最高,块茎中的表达量最低;成熟叶片的表达量高于未成熟叶片的表达量;数据差异性分析显示,SuSy基因在根、块茎中表达量具有极显著性差异,但在一年生叶和二年生叶中的表达量无显著性差异,幼苗叶和一、二年生叶中表达量具有极显著性差异。由此推测,SuSy基因可能受生长发育的诱导,是调控白及生长发育关键基因。     

A new role for enteric glucagon-37: acute stimulation of glucose absorption in rat small intestine

Glucagon-37 is secreted by intestinal L-cells following carbohydrate uptake. It is known to inhibit gastric acid secretion (hence also named oxyntomodulin) and appears to increase intracellular cyclic AMP concentrations. Since cyclic AMP could enhance intestinal glucose absorption, a possible stimulatory effect of glucagon-37 on glucose transport was examined. Glucagon-37 acutely increased glucose absorption in the isolated, vascularly perfused small intestine and in isolated enterocytes of the rat. In these cells the stimulation by glucagon-37 could be completely blocked by the cAMP antagonist Rp-cAMPS and was therefore mediated by cAMP. The stimulation of intestinal glucose absorption by glucagon-37 appears to be a major new physiological function.     

Dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats

The hypothesis was tested that dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats, which in turn causes a decreased copper absorption. Male rats were fed adequate-copper (5 mg Cu/kg) diets containing either fructose or glucose (709.4 g monosaccharide/kg) for a period of 5 wk. Fructose vs glucose significantly lowered copper concentrations in plasma and the liver, but did not alter hepatic copper mass. Fructose feeding resulted in a significantly lesser intestinal solubility of copper as based on either a smaller soluble fraction of copper in the liquid phase of small intestinal contents or a lower copper concentration in the liquid phase. The latter fructose effect can be explained by the observed fructose-induced increase in volume of liquid phase of intestinal digesta. After administration of a restricted amount of diet extrinsically labeled with⁶⁴Cu, rats fed fructose also had significantly lower soluble⁶⁴Cu fraction in the digesta of the small intestine. Although this study shows that fructose lowered intestinal copper solubility, only a slight reduction of apparent copper absorption was observed. It is suggested that the fructose-induced lowering of copper status in part counteracted the fructose effect on copper absorption at the level of the intestinal lumen.     

Comparative in situ study of the intestinal absorption of aluminum, manganese, nickel, and lead in rats

This comparative study of the intestinal absorption of four toxic metals (aluminum, manganese, nickel, and lead) carried out in rats using the in situ intestinal perfusion technique was able to measure the partition of each metal between the intestine (intestinal retention), the blood circulation, and target tissues after 1 h. The perfused metal solutions were at concentrations likely to occur during oral intoxication. It was found that aluminum (48 and 64 mM), even as a citrate complex, crossed the brush border with difficulty (0.4% of the perfused amount); about 60% of this was retained in the intestine and the remainder was found in target tissues (about 36%). Conversely, lead (4.8–48 μM) penetrated the intestine more easily (about 35% of the perfused amount), was slightly retained (about 12% of the input), and was soon found in the tissues (about 58% of the input) and to a lesser degree in circulation (about 29%). Within the same concentration range, nickel and manganese showed certain similarities, such as a reduced crossing of the brush border proportional to the increase in the concentration perfused (0.17–9.5 mM). There was similar intestinal retention and absorption (about 80% and 20% of the input, respectively). Manganese crossed the brush border more easily and was diffused more rapidly into tissues. Finally, the addition of equimolar amounts of iron (4.7 mM) produced opposite effects on the absorption of the two elements, inhibiting manganese and showing a trend to increase in nickel absorption. This could be the result of competition between Fe²⁺ and Mn²⁺ for the same transcellular transporters and the slight predominance of paracellular mechanism in the event of “Fe²⁺-Ni²⁺” association.     

Small Intestinal Glucose Transport : Proximal-Distal kinetic gradients

Proximal and distal small intestinal segments of the rat were perfused in situ at two different rates with isotonic solutions containing glucose in concentrations ranging from 25 to 600 mg/100 ml. Absorption was measured as glucose disappearance rate from the lumen. Glucose absorption had not previously been studied at intraluminal concentrations above and below blood glucose. Absorption was more rapid from the proximal segment. In both segments absorption was independent of perfusion rate and of whether glucose was analyzed by counting ¹⁴C or by the Somogyi method. The latter finding suggests that of the unidirectional fluxes, flux out of the bowel is much greater than flux into the bowel. In contrast to the findings in previous studies neither segment showed rate-limiting kinetics, and the Michaelis-Menten analysis was not applicable. The form of the curve depicting absorption rate in relation to concentration differed between the two segments. At the higher concentrations absorption rate continued to increase much more rapidly in the proximal than in the distal segment. The observations could not be explained by known mechanisms of glucose transport and illustrate the difficulties of achieving biochemically and physiologically meaningful in vivo studies of intestinal absorption.