葡萄柚中糖基转移酶对槲皮素及其类似物催化机制探讨

研究了葡萄柚中糖基转移酶(FGT)作为催化酶,以UDP-葡萄糖、UDP-N-乙酰-D-氨基葡萄糖、UDP-甘露糖为糖基供体,槲皮素、柚皮素、柚皮苷为糖基受体,研究糖基化合成情况。通过高效液相、质谱及核磁共振氢谱对产物反应进行检测。确定了槲皮素与UDP-葡萄糖、UDP-N-乙酰-D-氨基葡萄糖可以发生糖基化反应,生成的槲皮素糖苷分子量分别为464、505。根据核磁结果鉴定,所得到的产物结构分别为槲皮素-3-O-β-D葡萄糖苷、槲皮素3-O-β-D-N-乙酰-D-氨基葡萄糖苷。以FGT酶为催化酶,对槲皮素进行了糖基化修饰。     

琐琐葡萄化学成分研究

采用大孔树脂、聚酰胺和Sephadex LH-20柱从琐琐葡萄乙醇溶液中分离得到六个化合物,通过波谱学技术及理化手段分别鉴定为齐墩果酸(1)、异槲皮苷(2)、槲皮素-3-O-β-D-葡萄糖醛酸钠盐(3)、槲皮素-3-O-β-D-葡萄糖醛酸乙酯苷(4)、没食子酸(5)、单咖啡酰酒石酸(6)。其中化合物2~6为首次从该植物中分离得到,化合物2~4为首次从该属植物中分离得到。     

核桃分心木化学成分及抗炎活性研究

为研究核桃分心木的化学成分,明确其药效物质基础,采用高速逆流色谱、反相C18柱色谱、制备型高效液相色谱等方法进行分离纯化,通过质谱、核磁共振波谱等多种谱学数据分析鉴定其化学结构。从核桃分心木的乙酸乙酯萃取部位分离纯化得到10个化合物,分别鉴定为:2,3-dihydro-3-hydroxy-2-oxo-1H-indole-3-acetic acid(1)、核桃素D(2)、2-hydroxycinchoninic acid(3)、C-藜芦酰乙二醇(4)、槲皮素-3-O-(6″-没食子酰基)-β-D-半乳糖苷(5)、槲皮素-3-O-(6″-没食子酰基)-β-D-葡萄糖苷(6)、金丝桃苷(7)、异槲皮苷(8)、4,8-二羟基-1-四氢萘醌(9)、槲皮素(10)。化合物3,7和8是首次从核桃分心木中分离得到,化合物1、4和6为首次从该种属植物中分离得到。运用Griess法检测LPS诱导的RAW264. 7细胞中一氧化氮(NO)水平,对各单体化合物的抗炎活性进行初步研究,结果显示化合物10具有较好的抗炎作用。     

红背山麻杆叶的化学成分研究(Ⅱ)——黄酮和苯乙醇苷类化合物

采用80％丙酮提取物的水萃取部位,利用凝胶、MCI、反相碳18、及 Toyopearl Butyl-650C 柱色谱进行分离纯化得到7个黄酮和3个苯乙醇苷类化合物。根据化合物的波谱数据分析鉴定为槲皮素(1)、槲皮苷(2)、异懈皮苷(3)、芦丁(4)、异牡荆素(5)、牡荆素(6)、木犀草素-7-O-α-L-鼠李糖(1→6)-β-D-葡萄糖苷(7)、2-phenethylβ-D-glucoside(8)、icariside D1(9)、2-苯乙基-D-芸香甙(10)。其中化合物1-3、5-6、8-10为首次从本属植物中分离得到。     

贯叶连翘中的新抗真菌黄酮苷

从贯叶连翘 (HypericumperforatumL .)地上部分的 80 %乙醇提取物中分离得到 1个新的黄酮苷和 6个已知黄酮类化合物。采用红外光谱、紫外光谱、质谱和核磁共振光谱等波谱技术测得新黄酮苷的结构为 6″_O_乙酰基槲皮素_3_O_β_D_阿洛糖苷 (1 ,6″_O_acetylquercetin 3_O_β_D_alloside) ,6个已知化合物分别被鉴定为槲皮苷 (quercitrin)、金丝桃苷 (hyperoside)、蓄苷 (avicularin)、芦丁 (rutin)、槲皮素 (quercetin)和山萘酚 (kaemferol)。体外抗真菌活性实验表明 ,新黄酮苷 (1 )、槲皮苷和槲皮素对植物病原菌HelminthosporiumsativumPamelKingetBakke具有一定的抑制作用 ,三者的最小抑制浓度 (MIC)分别为 :2 5、5 0和 5 0 μg/mL。另外 ,1和槲皮苷对禾赤色镰孢霉 (FusariumgraminearumSchw .)的生长也有抑制作用 (MIC值均为 1 0 0 μg/mL)。     

黄芪甲苷Ⅳ对内毒素诱导RAW264.7细胞损伤的保护作用及机制研究

目的:研究黄芪皂苷Ⅳ对LPS诱导的巨噬细胞RAW264.7损伤的保护作用及机制.方法:测定细胞活力判断心肌细胞损伤的程度.TNF-α,IL-1β,IL-6,IL-10的释放以及NF-кB蛋白、Akt和磷酸化Akt表达用以研究作用机制.结果:黄芪皂苷Ⅳ对LPS引起的RAW264.7细胞损伤具有显著的抑制作用,1,3和10μM黄芪皂苷Ⅳ可显著降低LPS诱导的RAW264.7细胞TNF-α,IL-1β和IL-6的生成,促进IL-10的释放.黄芪皂苷Ⅳ剂量依赖性地增加了由LPS刺激而引起的RAW264.7细胞NF-kB蛋白的表达,抑制了LPS所致的p-Akt蛋白表达的升高.结论:黄芪皂苷Ⅳ可通过Akt-NF-kB途径调控促炎因子和抗炎因子表达的失衡,有效发挥对LPS诱导巨噬细胞RAW264.7损伤的保护作用.     

云南透骨草的化学成分研究

从云南透骨草全草95%乙醇提取物中分离得到11个化合物,经波谱分析鉴定为山奈酚3-O-α-L-吡喃鼠李糖苷(1),番石榴苷(2),滇白珠甲苷(3),槲皮苷(4)(,-)-5’-甲氧基异落叶松脂醇9-O-β-D-木糖苷(5),五味子苷(6)(,–)-表儿茶素(7),异槲皮苷(8),金鸡纳素Ia(9),熊果酸(10)和2,5-双-(β-苯乙基)苯酚(11)。其中化合物1、2、9和11为首次从该植物中分得,化合物11是首次报导的天然产物。     

广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7免疫调节作用受体TLR4和TLR2的影响

确定广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7的免疫调节作用受体,探索广叶绣球菌β-D-葡聚糖的免疫调节机制。采用MTT法测定不同浓度广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7增殖活力的影响,筛选出促进巨噬细胞增殖能力最强的浓度。用筛选出的β-D-葡聚糖浓度作用巨噬细胞RAW264.7;TLR4抗体和TLR2抗体分别作用巨噬细胞RAW264.7 1h,再用含有β-D-葡聚糖的细胞培养液培养。收集细胞培养上清和细胞,检测细胞培养上清中NO、IL-6、TNF-α、IFN-β的生成量;提取细胞内总RNA,采用RT-PCR测定巨噬细胞TLR4 mRNA表达量;提取巨噬细胞总蛋白,采用蛋白免疫印迹western blot测定TLR4的蛋白表达。广叶绣球菌β-D-葡聚糖能够促进巨噬细胞RAW264.7增殖,增加NO、IL-6、TNF-α、IFN-β的生成量,提高TLR4 mRNA表达和蛋白表达,差异极显著（P<0.01）。TLR4抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量明显下降,差异极显著（P<0.01）。TLR2抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量下降,但差异不显著。广叶绣球菌β-D-葡聚糖可以通过细胞表面受体TLR4激活信号转导通路,增强下游细胞因子的释放,从而调节巨噬细胞RAW264.7的免疫功能。TLR2可能不是广叶绣球菌β-D-葡聚糖的免疫受体。     

贯叶连翘中的新抗真菌黄酮苷

从贯叶连翘( Hypericum perforatum L.)地上部分的80%乙醇提取物中分离得到1个新的黄酮苷和6个已知黄酮类化合物.采用红外光谱、紫外光谱、质谱和核磁共振光谱等波谱技术测得新黄酮苷的结构为6″-O-乙酰基槲皮素-3-O-β- D -阿洛糖苷(1,6″-O-acetyl quercetin 3-O-β- D -alloside),6个已知化合物分别被鉴定为槲皮苷(quercitrin)、金丝桃苷(hyperoside)、蓄苷(avicularin)、芦丁(rutin)、槲皮素(quercetin)和山萘酚(kaemferol).体外抗真菌活性实验表明,新黄酮苷(1)、槲皮苷和槲皮素对植物病原菌 Helminthosporium sativum Pamel King et Bakke具有一定的抑制作用,三者的最小抑制浓度(MIC)分别为:25、50和50 μg/mL.另外,1和槲皮苷对禾赤色镰孢霉( Fusarium graminearum Schw.)的生长也有抑制作用(MIC值均为100 μg/mL).     

法兰地草莓的芳香类和黄酮类成分

为了解草莓(Fragaria×ananassa Duch.)法兰地品种的化学成分,采用色谱分离方法,从新鲜果实的乙醇提取物中获得19个化合物。通过波谱数据分析鉴定了它们的结构,分别为对羟基苯甲酸(1)、没食子酸乙酯(2)、鞣花酸3-O-α-L-鼠李糖苷(3)、苄基β-D-葡萄糖苷(4)、淫羊藿次苷F2(5)、苄基6-O-α-L-呋喃阿拉伯糖基-β-D-葡萄糖苷(6)、苯乙基6-O-α-L-呋喃阿拉伯糖基-β-D-葡萄糖苷(7)、反式肉桂酰基β-D-葡萄糖苷(8)、顺式肉桂酰基β-D-葡萄糖苷(9)、反式对香豆酰基β-D-葡萄糖苷(10)、顺式对香豆酰基β-D-葡萄糖苷(11)、反式阿魏酰基β-D-葡萄糖苷(12)、山柰酚(13)、反式椴树苷(14)、山柰酚3-O-β-D-葡萄糖醛酸苷(15)、槲皮素(16)、槲皮素3-O-β-D-葡萄糖苷(17)、槲皮素3-O-β-D-葡萄糖醛酸苷(18)和根皮苷(19),化合物1~12为芳香类,其余为黄酮类。所有化合物均为首次从法兰地品种中报道,化合物4~9为首次从草莓中获得。     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

Identification of the potential active components of Abelmoschus manihot in rat blood and kidney tissue by microdialysis combined with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

In this paper, microdialysis combining with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to simultaneously identify components in blood and kidney dialysis after oral administration of Abelmoschus manihot extract. Microdialysis probe was implanted in the jugular vein and the kidney medulla, respectively; microdialysis samples were collected continuously, transferred to microtubes and analyzed by UPLC-Q-TOF/MS. The components in microdialysis samples were separated by an UPLC HSS T3 column and eluted with acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.4 mL/min. The results showed that unbound constituents in blood circulation of the rat include hyperoside, isoquercitrin, quercetin monoglucuronide, quercetin-3'-O-glucoside, quercetin, myricetin, and hibifolin while unbound constituents in kidney are hyperoside, isoquercitrin, quercetin monoglucuronide, which might be the potential active components in vivo. The developed method was simple and reliable, and could be adopted to rapidly screen and identify potential active components contributing to pharmacological effects of TCM and to better clarify its action mechanism.     

The influence of wine polyphenols on reactive oxygen and nitrogen species production by murine macrophages RAW 264.7

The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.     

Microbial Transformation of Quercetin by Bacillus cereus

Biotransformation of quercetin was examined with a number of bacterial cultures. In the presence of a bacterial culture (Bacillus cereus), quercetin was transformed into two crystalline products, identified as protocatechuic acid and quercetin-3-glucoside (isoquercitrin).     

微进化对阿萨希毛孢子菌与小鼠巨噬细胞RAW264.7相互作用的影响

目的研究微进化对阿萨希毛孢子菌(T.asahii)与巨噬细胞RAW264.7相互作用的影响。方法将微进化前后的T.asahii与RAW264.7细胞共培养,检测RAW264.7对两株菌的吞噬和杀伤能力以及两株菌对RAW264.7产生的细胞毒性的差异,同时分析RAW264.7自身细胞因子分泌情况的变化。结果巨噬细胞对原代菌株(TO)的吞噬能力以及杀伤率均明显高于微进化株(TEVO);TO菌株对巨噬细胞的细胞毒性要强于TEVO;当巨噬细胞与菌株按1∶3或1∶6共培养24 h时,与TO共培养的巨噬细胞TNF-α和IL-6的分泌量要高于TEVO组,而按1∶9共培养时TEVO组细胞因子的分泌量却高于TO组。结论微进化后的TEVO菌株与巨噬细胞的相互作用明显弱于原代株TO,这也为TEVO菌株在宿主体内长期共存提供了良好的基础。     

Effect of quercetin conjugates on vascular permeability and expression of adhesion molecules

Quercetin and its glucosides exist in plant foods and are recovered in human plasma as glucuronide and sulfate conjugates. Quercetin and its conjugates show antioxidant activity in model experiments. It remains obscure, however, whether these conjugates retain these biological functions in vivo. We investigated the interaction of quercetin conjugates with lipid bilayers using liposome systems. Less quercetin conjugate was incorporated into liposomes than quercetin aglycone. We also studied the vascular permeability of quercetin-3-glucuronide using cell culture inserts. Incubation of human aortic endothelial cells (HAECs) with IL-1alpha resulted in increased permeability of quercetin-3-glucuronide. Furthermore, quercetin-3-glucuronide showed no suppressive effect on TNF-alpha-induced cell expression of intercellular adhesion molecule-1 (ICAM-1) on HAECs. These observations suggest that circulating conjugates of quercetin pass through the endothelium to reach vascular smooth muscle cells and exert their biological effects in the blood vessels during inflammation followed by deconjugation of the conjugates.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.