Monodemethylated polymethoxyflavones from sweet orange (Citrus sinensis) peel Inhibit growth of human lung cancer cells by apoptosis

Polymethoxyflavones (PMFs) are almost exclusively found in the Citrus genus, particularly in the peels of sweet orange (Citrus sinensis L. Osbeck) and mandarin (C. reticulate Blanco). We studied the effects of two major PMFs, namely, nobiletin and 3,5,6,7,8,3′,4′‐heptamethoxyflavone (HMF), and two major monodemethylated PMFs, namely 5‐hydroxy‐3,7,8,3′,4′‐pentamethoxyflavone (5HPMF), and 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF), on the growth of human lung cancer H1299, H441, and H460 cells. Monodemethylated PMFs were much more potent in growth inhibition of lung cancer cells than their permethoxylated counterpart PMFs. In H1299 cells, cell cycle analyses further revealed that monodemethylated PMFs caused significant increase in sub‐G0/G1 phase, suggesting possible role of apoptosis in the growth inhibition observed, whereas the permethoxylated counterpart PMFs did not affect cell cycle distribution at same concentrations tested. These results strongly suggested that the phenolic group is essential for the growth inhibitory activity of monodemethylated PMFs. Further studies in H1299 cells demonstrated that monodemethylated PMFs downregulated oncogenic proteins, such as iNOS, COX‐2, Mcl‐1, and K‐ras, as well as induced apoptosis evidenced by activation of caspase‐3 and cleavage of PARP. Our results provide rationale to develop orange peel extract enriched with monodemethylated PMFs into value‐added nutraceutical products for cancer prevention.     

Chemistry and health effects of polymethoxyflavones and hydroxylated polymethoxyflavones

Polymethoxyflavones (PMFs) and hydroxylated polymethoxyflavones exist exclusively in citrus genus, especially in the citrus peels which have been used as herbal medicine for several diseases for thousands of years. In this review, the natural occurrence, isolation and separation of PMFs; synthetic scheme of hydroxylated PMF preparation, especially the reaction mechanism of preparation of 5-hydroxylated PMFs; biotransformation and metabolic fate of PMFs; the preliminary study on PMF bioavailability and the close relationship among solubility, permeability, absorption and oral bioavailability are summarized. The published bioactivity data on anti-inflammatory, anti-cancer and anti-atherogenic properties of PMFs have also been detailed along with a report of new findings of hydroxylated PMFs and their potent biological activities.     

The p53-, Bax- and p21-dependent inhibition of colon cancer cell growth by 5-hydroxy polymethoxyflavones

Scope: Previously, we reported that 5‐hydroxy polymethoxyflavones (5OH‐PMFs) isolated from orange, namely 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone, 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF) and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF), potently induced apoptosis and cell‐cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell‐cycle arrest induced by different 5OH‐PMFs. Methods and results: Annexin V/PI co‐staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53^+/+) cells but not in HCT116 (p53^?/?) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax^+/?) cells, whereas their pro‐apoptotic effects on HCT116 (Bax^?/?) cells were marginal. All three 5OH‐PMFs increased G0/G1 cell population of HCT116 (p53^+/+) cells, and these effects were abolished in HCT116 (p53^?/?) and HCT116 (p21^?/?) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase‐3, cleaved PARP in both HCT116 (p53^+/+) and HCT116 (Bax^+/?) cells and these effects were much weaker in HCT116 (p53^?/?) and HCT116 (Bax^?/?) cells. Conclusion: Our results demonstrated that 5OH‐PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell‐cycle arrest by p53‐, Bax‐ and p21‐dependent mechanism.     

茶枝柑皮提取物中川陈皮素和橘皮素的大鼠肠吸收特性

目的：研究茶枝柑皮提取物中多甲氧基黄酮类成分川陈皮素和橘皮素在大鼠的肠吸收特性。方法：采用大鼠单向肠灌流模型,利用高效液相色谱法分别测定灌流前后经处理的灌流液中川陈皮素和橘皮素的含量变化,计算其肠吸收速率常数Ka和表观吸收系数Kapp,分析茶枝柑皮提取物在大鼠肠吸收的特征。结果：川陈皮素和橘皮素在各肠段吸收有显著差异,同种成分不同肠段间吸收无显著差异,质量浓度和pH值对吸收情况无显著影响。结论：茶枝柑皮提取物中川陈皮素和橘皮素在大鼠各肠段吸收良好,在空肠段吸收最好,其吸收呈简单的被动扩散。     

柑橘中多甲氧基黄酮生物活性及应用研究进展

芸香科柑橘属中的多甲氧基黄酮(PMFs),由于具有抗炎、抗氧化、抗诱变、抗癌等生物活性而备受青睐。而且PMFs的提取使得柑橘果汁加工中的果皮得到充分利用,对其深入研究具有重大的经济效益和社会效益。本文对PMFs的结构及其生物活性,在柑橘分类学研究、柑橘果汁掺假和保健食品中的应用进行了综述。     

14种柑橘皮中多甲氧基黄酮成分分析

建立柑橘皮中9 种多甲氧基黄酮（polymethoxyflavonoids,PMFs）快速定量检测方法,比较不同品种来源柑橘皮中PMFs的含量差异,为寻找高含量PMFs高生物活性柑橘皮的药食两用素材提供参考。采用超声乙醇提取、高效液相色谱方法快速准确检测PMFs,并对14 种柑橘皮、陈皮中9 种特征物质PMFs含量进行比较。结果显示9种PMFs在对照品溶液质量浓度范围内具有良好的线性关系,且准确度和精确度良好。分析发现,不同品种来源柑橘皮中PMFs种类和含量差异很大。江西陈皮、重庆柑橘、广西皇帝柑、广西砂糖橘果皮中PMFs总含量很高,均在7495.19 μg/g以上,其中广西沙糖橘果皮PMFs总含量高达10 412.18 μg/g。该方法简便,稳定性好,可为柑橘皮中PMFs生物活性研究的深入、柑橘皮产品开发和中药原料选材及其质量控制提供科学依据。     

陈皮中黄酮类化合物的抗氧化性

目的研究陈皮中黄酮类化合物的抗氧化性。方法用茜素紫作显色剂,分光光度法测定黄酮类化合物对Co2 H2O2体系所产生的羟基自由基的消除作用。利用连苯三酚自氧化法测定陈皮中黄酮类化合物对超氧阴离子自由基的消除作用。结果陈皮中黄酮类化合物对羟基自由基和超氧阴离子自由基有消除作用。结论陈皮中黄酮类化合物具有良好的抗氧化活性。     

陈皮中黄酮类化合物抗氧化活性的研究

目的:研究陈皮中提取的黄酮类化合物抗氧化活性。方法:测定陈皮提取物中黄酮类化合物的总抗氧化能力,并实验在正常小鼠体内抗氧化作用。结果:显示陈皮提取物黄酮类化合物其总抗氧化能力与提取物浓度有很强的量效关系,线性关系显著,对小鼠体内的MDA(丙二醛)的产生有显著抑制,及显著提高小鼠血浆、组织中SOD(超氧化物岐化酶)、CAT(过氧化氢酶的)活性。结论:表明陈皮提取物中黄酮类化合物具有较强的抗氧化活性。     

纽荷尔脐橙果实发育过程中类黄酮变化与体外抗氧化活性的关系

以纽荷尔脐橙果实为材料,研究了果实发育过程中果皮和果肉总类黄酮含量的变化以及与体外抗氧化活性的关系。结果表明：随着果实发育成熟,果皮中总类黄酮含量、体外总抗氧化活性以及清除H2O2 和O2·能力基本呈下降的趋势,而果肉中各参数指标均呈单峰型变化,其峰值基本出现在9 月中旬。相关性分析表明,果实类黄酮含量与体外总抗氧化活性以及与清除H2O2 和O2·能力均存在显著正相关,其相关系数分别为r=0.9664(p＜ 0.01)、r=0.7723(p ＜ 0.01)和r=0.9144(p ＜ 0.01)。     

Preparation of a polymethoxyflavone-rich fraction (PRF) of Citrus sunki Hort. ex Tanaka and its antiproliferative effects

The peel of Citrus sunki is a rich source of polymethoxyflavones (PMFs). In this study, a PMF-rich fraction (PRF) was obtained from the peels of C. sunki by hot-water extraction and subsequent fractionation with n-hexane. Six PMFs and two demethylated PMFs were isolated by semi-preparative HPLC, and their structures were identified by ¹H NMR and ¹³C NMR. To define the synergistic effects of PRF, the PRF and its eight individual components were assessed for antiproliferative activity using HL-60 cells. The PRF showed greater growth inhibitory activity (IC₅₀ value = 25.14 μg/ml), compared with its individual components. Flow cytometric analysis confirmed that the PRF significantly increased the percentage of sub-G₁ cells (30.1% at 25 μg/ml). Our study provides an efficient method for preparing a PRF for use as a functional food ingredient.     

Thioredoxin system inhibitors as mediators of apoptosis for cancer therapy

The thioredoxin (Trx) system is a major antioxidant system integral to maintaining the intracellular redox state. It contains Trx, a redox active protein, which regulates the activity of various enzymes including those that function to counteract oxidative stress within the cell. Trx can also scavenge reactive oxygen species (ROS) and directly inhibits proapoptotic proteins such as apoptosis signal-regulating kinase 1 (ASK1). The oxidized form of Trx is reduced by thioredoxin reductase (TrxR). The cytoplasm and mitochondria contain equivalent Trx systems and inhibition of either system can lead to activation of apoptotic signaling pathways. There are a number of inhibitors with chemotherapy applications that target either Trx or TrxR to induce apoptosis in cancer cells. Suberoylanilide hydroxamic acid (SAHA) is effective against many cancer cells and functions by up-regulating an endogenous inhibitor of Trx. Other compounds target the selenocysteine-containing active site of TrxR. These include gold compounds, platinum compounds, arsenic trioxide, motexafin gadolinium, nitrous compounds, and various flavonoids. Inhibition of TrxR leads to an accumulation of oxidized Trx resulting in cellular conditions that promote apoptosis. In addition, some compounds also convert TrxR to a ROS generating enzyme. The role of Trx system inhibitors in cancer therapy is discussed in this review.     

Anti-inflammatory effect of the immature peel extract of <Emphasis Type="Italic">Jinkyool</Emphasis> (<Emphasis Type="Italic">Citrus sunki</Emphasis> Hort. ex Tanaka)

The peel of jinkyool (Citrus sunki Hort. ex Tanaka) has been widely used in traditional Asian medicine for treatment of a number of diseases, including indigestion and bronchial asthma. In the present study, we compared the flavonoids content and anti-inflammatory activities of ethanolic peel extracts from both immature and mature fruits. Comparing to the mature peel extract (MPE), the immature peel extract (IPE) contained more abundant flavonoids. IPE more effectively suppressed intracellular reactive oxygen species (ROS) generation (IPE, 67.6±1.2%; MPE, 78.9±2.4% at 300 μg/mL of control), nitric oxide (NO) production, and inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression, as well as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) mRNA expression in lipopolysaccharide-stimulated RAW 264.7 cells. Additionally, IPE reduced c-Jun NH₂-terminal kinase (JNK) and p38 mitogen-activated kinase (p38) activation, suggesting that IPE may exert an anti-inflammatory effect by suppressing pro-inflammatory cytokines that suppress activation of mitogen-activated protein kinases (MAPKs). Taken together, these results indicate that IPE has potential for use as an anti-inflammatory agent.     

Characterization of Polymethoxylated Flavonoids in the Peels of Chinese Wild Mandarin (<Emphasis Type="Italic">Citrus reticulata</Emphasis> Blanco) by UPLC-Q-TOF-MS/MS

China is the most important origin center of the genus Citrus L., especially the wild mandarin (Citrus reticulata Blanco). The aim of this work was to systematically qualitative and quantitative analysis of polymethoxylated flavonoids (PMFs) in seven Chinese wild mandarin citrus using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and UPLC-photodiode array (UPLC-PDA) analyses. This method was validated including the linearity, limit of detection (LOD), limit of quantification (LOQ), precision, repeatability, stability, and recovery. The results demonstrated that UPLC-Q-TOF-MS/MS was a powerful tool to analyze the PMFs in wild citrus peel, and this method was trustable, rapid and sample. Besides, ten PMFs were identified or tentatively characterized as 5,6,7,3′,4′-pentamethoxyflavanone (1), isosinensetin (2), sinensetin (3), 5,7,3′,4′-tetramethoxyflavone (4), 3,5,7,4′-tetramethoxyflavone (5), nobiletin (6), 3,5,6,7,8,3′,4′-heptamethoxyflavone (7), 5,7,4′-trimethoxyflavone (8), 5-Hydroxy-6,7,8,3′,4′-pentamethoxyflavone (9), and tangeretin (10). Among these compounds, 4 was the first time to be identified in citrus. Then the contents of eight PMFs (2, 3, 4, 6, 7, 8, 9, and 10) in these samples were quantified. Compounds 3, 6, and 10 were found to be the prominent PMFs. Cupigoushigan had the highest contents of 3 (2.617 mg/g DW) and 6 (11.111 mg/g DW), and Dakengyeju had the highest content of 10 (5.560 mg/g DW). Our results indicated that wild mandarins are potentially rich source of Citrus PMFs for foods or other utilization.     

Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells

This study investigated the anti-proliferative and apoptotic effect of flavonoids isolated from Korean Citrus aurantium L. using A549 lung cancer cells. Flavonoids potently inhibited of A549 cells in a dose-dependent manner, whereas flavonoids had a weak inhibitory effect on proliferation of WI-38 cells. Flow cytometry and Western blot analysis showed that flavonoids induced cell cycle arrest at the G2/M checkpoint by controlling the proteins expression level of cyclin B1, cdc2, cdc25c and p21^WAF1/CIP1. Also, flavonoids induced apoptosis through the regulation of the expression of caspases, cleaved PARP and Bax/Bcl-xL ratio. The activity of caspase-3 on A549 cells increased in a dose-dependent manner. These results clearly indicated that the anti-cancer effect of flavonoids on A549 cells follows multiple cellular pathways through G2/M arrest and the induction of apoptosis.     

橘皮类黄酮的分离、纯化及结构鉴定研究

柑橘皮内含丰富的类黄酮化合物,具有多种生物学活性,受到医药、食品等行业的关注。本文通过硅胶、聚酰胺及Sephadex HL-20柱对温州蜜桔皮中类黄酮化合物进行反复分离纯化,得到5种类黄酮单体。采用LC-ESI-MS、1H-NMR和13C-NMR及红外光谱等技术对其结构进行鉴定。鉴定结果:化合物A,3,5,6,7,8,3’,4’-七甲氧基黄酮;化合物B,5,6,7,8,4’-五甲氧基黄酮;化合物C,3-羟基-5,6,7,8,3’,4’-六甲氧基黄酮;化合物D,5,6,7,8,3’,4’-六甲氧基黄酮;化合物E,橙皮素芸香糖苷。其中,化合物C是从温州蜜桔皮中首次发现的。     

温州蜜柑皮中氨基酸及脂肪酸的组成分析研究

柑橘是我国大宗水果,皮中富含精油、果胶和类黄酮等生物活性成分,是一种具有较高开发价值的资源。应用氨基酸全自动技术分析温州蜜柑皮中的蛋白质,结果表明蛋白质含量为3.446%(干物质),其中必需氨基酸含量1.239%,比重35.956%,略小于大豆分离蛋白的39.4%;蛋氨酸、胱氨酸为限制性氨基酸。采用索氏法对果皮进行萃取,以GC-MS技术对萃取物中的脂肪酸进行分析鉴定,确定含有11种脂肪酸:6种饱和脂肪酸,占总量的26.06%,主要是十六烷酸19.75%;5种不饱和脂肪酸,占总量的66.20%,主要是9,12-十八碳二烯酸(亚油酸)37.14%和亚麻酸19.06%。     

臭氧对橙皮果脯品质及抗氧化活性的作用

本研究探讨了不同浓度臭氧水前处理对橙皮果脯营养价值和抗氧化能力的影响。经低、中和高三个浓度的臭氧水清洗处理15 min后,新鲜橙皮被制备成橙皮果脯。以未处理组与臭氧水处理组做对比,测定了橙皮果脯的还原性Vc、黄酮、总色差与抗氧化值。结果显示：还原性Vc、黄酮、总色差以及还原力、DPPH?清除率、?OH清除率、O2-?清除率和ABTS+?清除率从高到低均依次为：中浓度（9.10 mg/L）处理组>低浓度（13.78 mg/L）处理组>高浓度（22.23 mg/L）处理组>未处理组。中浓度臭氧水处理组橙皮果脯与未处理组的对比,Vc含量从1.25 mg/g提升到1.40 mg/g,黄酮含量从0.57 mg RE/g提升到0.68 mg RE/g,总色差从34.88下降到28.80。臭氧水处理对橙皮果脯的营养价值和颜色都有显著的保护作用（p<0.05）,对其抗氧化活性也有显著的提高（p<0.05）。其中,橙皮果脯的抗氧化能力与加工过程中橙皮里Vc和黄酮含量变化存在着极显著的正相关。可见,短期的臭氧水处理对橙皮果脯的品质及抗氧化活性均有良好的作用。     

不同晚熟柑橘中酚类物质的含量检测及分析

采用超高效液相色谱(ultra-performance liquid chromatography,UPLC)对9种晚熟柑橘(沃柑、春见、大雅柑、不知火、默科特、红肉脐橙、伦晚脐橙、红翠2号和塔罗科血橙)中的主要酚类物质(13种类黄酮和7种酚酸)进行测定,分析比较不同品种柑橘果皮、果肉和果汁(柑橘原汁)中酚类物质种类和含量的差异。结果表明:9个柑橘品种的果皮、果肉、果汁中,类黄酮均以橙皮苷为主,酚酸以阿魏酸为主,且果皮中多甲氧基黄酮含量丰富。在9个柑橘品种果汁中,春见的橙皮苷、咖啡酸含量最高。果肉中,沃柑中阿魏酸、咖啡酸、芥子酸含量最高,塔罗科血橙的橙皮苷、对香豆酸含量最高。果皮中,默科特的芸香柚皮苷、川皮苷、阿魏酸含量最高。杂柑果皮中咖啡酸、阿魏酸及川皮苷含量高于橙类。晚熟柑橘含有丰富的酚类物质,并呈现显著的多样性。     

橘皮中酚酸的研究进展

酚酸广泛存在于植物中,大量研究表明酚酸具有如抗氧化、抗癌等多种生物活性。橘皮中含有丰富的酚酸,而目前对橘皮酚酸的提取分离等方面的报道很少。本文综述了橘皮酚酸类化合物的理化性质、提取、分离纯化及生物活性的研究新进展。提出了今后橘皮的研究方向和开发前景,为更好地开发利用柑橘提供参考。