柚皮素:新一代免疫调节剂

大量研究表明,植物来源的黄酮类化合物具有广泛的生理活性和药理作用,日常摄取适量的黄酮类化合物能够显著降低许多疾病的发生.柚皮素作为一种自然界分布广泛的黄酮类化合物富含于水果、蔬菜、坚果、咖啡、茶和红酒等日常饮食中,与其他黄酮类化合物相比柚皮素易于胃肠道吸收、生物利用度高且安全剂量大.自2004年起我们对柚皮素调节免疫的分子机制进行了深入系统的研究.本文将重点介绍柚皮素作为一种新型免疫调节剂的研究进展.     

柚皮素合成关键基因表达水平对目标产物积累水平的量化影响

柚皮素是一种天然黄酮类化合物,具有抗炎、抗氧化、抗病毒、预防动脉粥样硬化等多种药理活性,也是其他黄酮类化合物合成的重要前体,具有重要的应用价值。目前,微生物法生产柚皮素等黄酮类化合物由于代谢通路不平衡等原因导致产量较低,在很大程度上限制了其工业应用。文中以一株产柚皮素的酿酒酵母菌株Y-01为研究对象,利用启动子和拷贝数控制柚皮素合成代谢途径关键酶4-香豆酸:CoA连接酶(4CL)、查尔酮合成酶(CHS)和查尔酮异构酶(CHI)编码基因的表达水平,考察这些基因的表达水平对目标产物积累水平的量化影响。结果表明,柚皮素产量与4CL或CHI编码基因的表达量之间关联性较低,而与chs基因的表达量存在显著的正相关性。通过调控chs基因的表达水平,获得一株高产柚皮素的酿酒酵母工程菌株Y-04,产量较出发菌株Y-01提高了4.1倍。研究结果表明,CHS是柚皮素合成过程的关键调控靶点,合理调控CHS表达可以显著促进酿酒酵母积累柚皮素。相关结果为采用代谢工程强化微生物合成柚皮素等重要黄酮类化合物提供了重要的理论参考。     

黄酮类化合物清除羟自由基(HO·)功能及其构效关系研究

本文采用体外实验的方法,系统地研究了八种黄酮类植物化学物槲皮素、异槲皮素、芦丁、橙皮素、橙皮苷、柚皮素、柚皮苷和淫羊藿苷对羟自由基(HO.)的清除作用及构效关系。结果表明,八种黄酮类化合物中,只有槲皮素、芦丁和异槲皮素对HO·有明显的清除作用,在浓度为260μM时其对HO·清除率分别为30.69%±0.06、13.70%±0.04和10.23%±0.03。构效比较结果表明:黄酮类化合物C环C3位羟基和B环3’-4’邻位二羟基是其清除HO.的重要功能基团。本研究为选择有效的能清除HO·的膳食黄酮类化合物用于预防和治疗相关疾病提供了一定实验基础和理论依据。     

关键酶泛素化位点对柚皮素生物合成的影响

黄酮类化合物具有多种生物活性,在食品、药品、化妆品等领域都有重要应用.柚皮素是多种重要黄酮类化合物生物合成的平台化合物.泛素化是蛋白质翻译后修饰的重要一环,参与调控细胞的生命活动.泛素化的蛋白质通过泛素-蛋白酶体系统降解,对维持细胞正常生理活动具有重要意义,对外源蛋白的表达和积累也可能具有显著影响.文中利用荧光双分子互...     

云南栘(木衣)(Docynia delavayi(Franch.)Schneid.)黄酮成分研究

本文首次对采自云南西双版纳的栘(木衣)树皮的主要成分进行了研究,从其乙醚提取物中分离出四种黄酮类化合物,通过NMR、MS和IR等波谱手段,鉴定出它们分别为:白杨素(Chrysin,Ⅰ),柚皮素(Naringenin,Ⅱ),槲皮素(Quercetin,Ⅲ)和广寄生甙(Avicularin,Ⅳ)     

柚皮素酰腙类衍生物的合成、体外抗氧化活性以及细胞毒性研究

柚皮素具有良好的抗氧化活性,但由于其生物利用率低,导致其应用受限。本文合成得到13种柚皮素的酰腙类衍生物,其中12种未被文献报道。采用ABTS、FRAP、DPPH 3种方法测定了合成衍生物与柚皮素的体外抗氧化活性。结果显示13种化合物的抗氧化活性均强于柚皮素,其中8种衍生物的体外抗氧化活性是柚皮素活性的3到6倍。细胞毒性实验中细胞存活率90%时,可认为化合物对细胞无抑制。8种化合物对HEK293细胞的细胞毒性实验结果显示,在0～25μmol/L浓度范围内,衍生物b、g、j、k、l对HEK293细胞存活率大于90%,其中衍生物g、k、l的浓度在0～50μmol/L时,HEK293细胞的存活率仍大于90%,而衍生物j在浓度高达100μmol/L时细胞存活率仍大于90%。研究结果为后期的抗氧化食品添加剂及抗氧化药物筛选提供理论依据。     

合成8二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建

8二甲基异戊烯基柚皮素(8DN)作为生产黄酮类药物淫羊藿苷的重要前体,在医药合成领域具有重大应用潜力。由于其合成路径及相关基因的复杂性,目前主要通过饲喂8DN的直接前体(柚皮素、异黄腐酚等)的方式合成8DN,而在生物体内全合成8DN的研究工作还未见报道。为了实现8DN在酿酒酵母体内的生物全合成,通过组合筛选8DN前体物柚皮素合成所需的多种外源基因(TAL、4CL、CHS、CHI),获得30株柚皮素生产菌,发现不同来源的基因组合使柚皮素产量的具有明显差异(0.37~22.33mg/L)。并且利用Delta位点将较优的基因组合整合至酵母基因组,实现了稳定的柚皮素高产菌株(Sy BE_Sc02050031)构建。在此基础上进一步导入带有苦参来源的异戊烯基转移酶基因(N8DT)多拷贝质粒,实现8DN合成的完整反应过程,8DN的摇瓶发酵产量达到36.7μg/L。另外,通过关键限速酶N8DT的序列优化策略,发现截断定位信号肽序列的N8DT明显提高了从柚皮素到8DN这一关键反应的催化效果,8DN的产量提高到52.6μg/L(144.2%)。首次在酿酒酵母中成功构建高产8DN的生物全合成路径,为在微生物体内合成其他黄酮类天然产物提供了参考,具有重要的指导意义。     

水飞蓟来源黄酮3-羟化酶鉴定及黄杉素发酵优化

黄杉素是一种重要的黄酮类化合物,具有抗炎症、抗氧化等功效,在医药及保健品行业有着广泛的应用。黄酮3-羟化酶 (Flavanone 3-hydroxylase,F3H) 可以催化圣草酚合成黄杉素等3位羟基化的黄酮类化合物。一直以来,由于黄酮3-羟化酶催化效率低,使得利用微生物法合成黄杉素等多种3位羟基化黄酮的产量较低。文中从水飞蓟转录组中鉴定得到一个黄酮3-羟化酶SmF3H。发酵验证结果表明,SmF3H具有黄酮3位羟基化功能,且催化效率显著高于常用的来源于番茄 (Solanum lycopersicum) 的SlF3H。选取6个转录强度不同的启动子优化从黄酮前体柚皮素到黄杉素的合成途径,发现PGAL7启动子起始转录SmF3H时黄杉素合成产量最高,在摇瓶中产量达到695.90 mg/L;在5-L发酵罐上进行发酵优化,以柚皮素为底物发酵得到3.54 g/L黄杉素,是目前可见报道的最高产量。     

柚皮中的有效成分对人子宫颈癌Hela细胞增殖及凋亡的影响

目的探讨柚皮素或柚皮苷对人子宫颈癌Hela细胞增殖及凋亡的影响。方法柚皮素或柚皮苷(0.250、0.125、0.063g/L)作用于人子宫颈癌Hela细胞后,MTT比色法检测Hela细胞增殖活性和半数抑制浓度(half maximal inhibitory concentration,IC50);流式细胞术检测柚皮素对Hela细胞凋亡的影响。结果柚皮素能抑制Hela细胞增殖,且呈剂量和时间依赖性,24、48和72hIC50值分别为0.24、0.11和0.07g/L;柚皮苷无抑制Hela细胞增殖的作用。柚皮素组(0.22g/L、0.11g/L、0.05g/L,作用48h)人子宫颈癌Hela细胞凋亡率高于对照组,差异有统计学意义(P=0.000239681,P=0.00012746,P=5.09328E-05,P0.01)。结论柚皮中黄酮类提取物具有抑制人子宫颈癌Hela细胞增殖作用,其中柚皮素作用明显,部分作用机制与诱导肿瘤细胞凋亡有关。柚皮苷无抗人子宫颈癌作用。     

华中冬青化学成分的研究

从华中冬青（Ｉｌｅｘ ｃｅｎｔｒｏｃｈｉｎｅｎｓｉｓ Ｓ．Ｙ．Ｈｕ）的叶片分离到９ 种黄酮类化合物：１ 种新化合物鉴定为３,５,５,７－四羟基二氢黄酮,命名为华中冬青黄酮（Ⅰ）;８ 种化合物鉴定为柚皮素（Ⅱ）、橙皮素（Ⅲ）、异樱花素（Ⅳ）、枸柑甙（Ⅴ）、橙皮甙（Ⅵ）、洋芹素（Ⅶ）、紫云英甙（Ⅷ）和野漆树甙（Ⅸ）。它们皆首次从该植物分得     

The effect of the skeleton structure of flavanone and flavonoid on interaction with transferrin

Transferrin has been exploited as a potential drug carrier for targeted drug delivery into cancer cells, which express high levels of transferrin receptors. In the present study, we identified specific structural features in flavonoids that were critical for binding to transferrin. Flavanone naringenin and flavonoid apigenin, two flavonoids with characteristic flavonoid core structures were selected for the study of the effects of C2–C3 single bond in the C-ring on transferrin binding. We determined the binding affinities by fluorescence quenching experiments and investigated the binding modes by CD spectra and molecular modeling. Our results demonstrated that naringenin bound transferrin with an affinity almost 100 times higher than that of apigenin attributed to its higher structural flexibility and lower acidity compared with apigenin. Our docking study showed that naringenin had stronger van der Waals interactions with transferrin, which was believed to contribute to its higher binding affinity. We also found that naringenin-binding induced greater increase in the α-helix content in transferrin than apigenin, suggesting that transferrin became more compact upon association with naringenin. Our study demonstrated that naringenin was a ligand for transferrin with good affinity. The results reported herein can facilitate the design and development of drugs that bind transferrin with high affinity.     

Prenylation enhances the biological activity of dietary flavonoids by altering their bioavailability

Flavonoids are distributed across the plant kingdom and have attracted substantial attention owing to their potential benefits for human health. Several studies have demonstrated that flavonoids prenylation enhances various biological activities, suggesting an attractive tool for developing functional foods. This review provides an overview of the current knowledge on how prenylation influences the biological activity and bioavailability of flavonoids. The enhancement effect of prenylation on the biological activities of dietary flavonoids in mammals was demonstrated by comparing the effect of 8-prenyl naringenin (8PN) with that of parent naringenin in the prevention of disuse muscle atrophy in mice. This enhancement results from higher muscular accumulation of 8PN than naringenin. As to bioavailability, despite the lower absorption of 8-prenyl quercetin (8PQ) compared with quercetin, higher 8PQ accumulation was found in the liver and kidney. These data imply that prenylation interferes with the elimination of flavonoids from tissues.     

Regulation of apoptosis by modified naringenin derivatives in human colorectal carcinoma RKO cells

Flavonoids are micronutrients that are widely detected in foods of plant origin and have been ascribed pharmacological properties. Several biological functions of flavonoids have been thus far identified, whereas there currently exists a lack of evidence to support the relationship between the structure-activity relationship and apoptosis-inducing activity. In an attempt to determine the importance of the OH group or substitution of the 5- or 7-carbon in the diphenylpropane skeleton of flavonoids, we selected 14 different flavonoids with different structures, particularly with regard to the 5- or 7-carbon, and found that naringenin treatment caused a slight decrease in the cell viability of the human colorectal carcinoma RKO cells. Next, in order to characterize the effects of specific substitutions of the 7-carbon of naringenin on apoptosis-regulatory activities, and in an attempt to develop anti-proliferative flavonoid derivatives that would be more effective against colon cancer, we originally synthesized several modified naringenin derivatives (MNDs) including 7-O-benzyl naringenin (KUF-1) and 7-O-(m-metoxybenzyl) naringenin (KUF-2). Treatment with KUF-1 or KUF-2 resulted in significant apoptosis-inducing effects concomitant with losses in mitochondrial membrane potential, caspase activation, intracellular ROS production, and sustained ERK activation. Our data show that KUF-1 or KUF-2 regulate the apoptosis of RKO cells via intracellular ROS production coupled with the concomitant activation of the ERK signaling pathway, thereby implying that hydroxylation or substitution at C7 is critical for the apoptosis-inducing activity of flavonoids.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

Effect of complex formation by taxifolin and naringenin with Cu(i) ions on the distribution of the components of complexes in the octanol–water system

The interaction of two structurally close flavanones: taxifolin and naringenin with copper(I) ions and its effect on the distribution of flavonoids and the corresponding ions in a biphasic system octanol–water have been studied. It has been shown that these polyphenols form complexes with copper ions of different stoichiometric ratio depending on the pH of medium (5.4, 7.4, and 9.0). The interaction of the flavonoids with copper ions leads to an increase in the fraction of polyphenols in the water phase at all pH values examined. The fraction of metal ions in octanol in the presence of both taxifolin and naringenin is maximal in the range of neutral pH values. The parameters obtained in the study, such as the partition coefficient and the coefficient of distribution in a biphasic system octanol–water (logP and logD) form the physicochemical basis necessary for the estimation of the bioavailability of flavonoids and the corresponding metal ions upon their combined consumption.     

Suppression of bacterial cell–cell signalling,biofilm formation and type III secretion system by citrus flavonoids

Aim: This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Methods and Results: Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96‐well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi‐quantitative PCR. Differential responses for different flavonoids were observed for different cell–cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell–cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. Conclusion: The results of the study indicate a potential modulation of bacterial cell–cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer‐mediated cell–cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Significance and Impact of the Study: Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell–cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.     

Effect of adding extracted hesperetin, naringenin and pectin on egg cholesterol, serum traits and antioxidant activity in laying hens

In this study three feed additives (hesperetin, naringenin and pectin) for laying hens were investigated on their influence on the egg yolk cholesterol, serum traits and antioxidant activities in hens. Additives were extracted from citrus and grapefruit peels and contained 31.5% crude hesperetin, 39% crude naringenin and 60% galacturonic acid (pectin). Eighty 30-week-old Leghorn laying hens were randomly assigned to four groups and received, for two months, a control diet or diets with 0.05% hesperetin, 0.05% naringenin or 0.5% pectin. All additives reduced the egg yolk cholesterol level significantly. Feeding diets with added flavonoids (hesperetin and naringenin) increased the yolk weight and the ratio of yolk weight/egg weight and the blood serum superoxide dismutase (SOD) activity was elevated. Total antioxidation capacity, the level of thiobarbituric acid-reactive substances and superoxide scavenging capacity in the naringenin group were greater than in the control group. Supplemented flavonoids reduced the serum cholesterol level significantly, while serum triglyceride concentration in the naringenin and pectin groups was reduced. Addition of flavonoids resulted in an enhanced cholesterol level in excreta. The results of this study indicated that intake of hesperetin, naringenin and pectin extracted from citrus and grapefruit peel in laying hens diet, may exhibit positive effects.     

Prevention of Disuse Muscle Atrophy by Dietary Ingestion of 8-Prenylnaringenin in Denervated Mice

Flavonoids have attracted considerable attention in relation to their effects upon health. 8-Prenylnaringenin (8-PN) is found in the common hop (Humulus lupulus) and assumed to be responsible for the health impact of beer consumption. We wanted to clarify the effects of prenylation on the physiological functions of dietary flavonoids by comparing the effects of 8-PN with that of intact naringenin in the prevention of disuse muscle atrophy using a model of denervation in mice. Consumption of 8-PN (but not naringenin) prevented loss of weight in the gastrocnemius muscle further supported by the lack of induction of the protein content of a key ubiquitin ligase involved in muscle atrophy, atrogin-1, and by the activation of Akt phosphorylation. 8-PN content in the gastrocnemius muscle was tenfold higher than that of naringenin. These results suggested that, compared with naringenin, 8-PN was effectively concentrated into skeletal muscle to exert its preventive effects upon disuse muscle atrophy. It is likely that prenylation generates novel functions for 8-PN by enhancing its accumulation into muscle tissue through dietary intake.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.