Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells

Sweet orange (Citrus sinensis L.) peel is a rich resource of flavonoids, especially polymethoxyflavones (PMFs). Citrus flavonoids exert a broad spectrum of biological activity, including antiproliferative and proapoptotic effects in cancer cells. We have recently shown that individual PMFs from orange peel induce Ca(2+)-mediated apoptosis in human breast cancer cells and that hydroxylation of PMFs is critical for enhancing their proapoptotic activity. Here, we report that the fraction of orange peel extract containing a mixture of non-hydroxylated PMFs (75.1%) and hydroxylated PMFs (5.44%) and the fraction containing only hydroxylated PMFs (97.2%) induce apoptosis in those cells as well. Treatment of MCF-7 breast cancer cells with these fractions inhibited growth and induced apoptosis associated with an increase in the basal level of intracellular Ca(2+). Effective concentrations of the hydroxylated PMFs fraction in inhibiting growth, inducing apoptosis, and increasing intracellular Ca(2+) were lower than those of the non-hydroxylated PMFs fraction. Our results strongly imply that bioactive PMFs from orange peel exert proapoptotic activity in human breast cancer cells, which depends on their ability to induce an increase in intracellular Ca(2+ )and thus, activate Ca(2+)-dependent apoptotic proteases.     

Nanoemulsion-based delivery systems for poorly water-soluble bioactive compounds: Influence of formulation parameters on Polymethoxyflavone crystallization

Polymethoxyflavones (PMFs) extracted from citrus peel exhibit potent anti-cancer activity, but are highly hydrophobic molecules with poor solubility in both water and oil at ambient and body temperature, which limits their bioavailability. The possibility of encapsulating PMFs within nanoemulsion-based delivery systems to facilitate their application in nutraceutical and pharmaceutical products was investigated. The influence of oil type (corn oil, MCT, orange oil), emulsifier type (β-lactoglobulin, lyso-lecithin, Tween, and DTAB), and neutral cosolvents (glycerol and ethanol) on the formation and stability of PMF-loaded nanoemulsions was examined. Nanoemulsions (r < 100 nm) could be formed using high pressure homogenization for all emulsifier types, except DTAB. Lipid droplet charge could be altered from highly cationic (DTAB), to near neutral (Tween), to highly anionic (β-lactoglobulin, lyso-lecithin) by varying emulsifier type. PMF crystals formed in all nanoemulsions after preparation, which had a tendency to sediment during storage. The size, morphology, and aggregation of PMF crystals depended on preparation method, emulsifier type, oil type, and cosolvent addition. These results have important implications for the development of delivery systems for bioactive components that have poor oil and water solubility at application temperatures.     

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。     

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.     

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.     

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.     

Minerals, phenolic compounds, and antioxidant capacity of citrus peel extract by hot water

ABSTRACT:  Some dried citrus peels, more familiar as chenpi in China, have been widely used in traditional Chinese medicines from ancient times. This paper reports the efficiency of infusion cooking on extracting minerals and phenolic compounds (flavanone glycosides [FGs], polymethoxylated flavones [PMFs], and phenolic acids), and also antioxidant activity of hot water extract of citrus peels. Peels of 2 citrus varieties, namely, Satsuma mandarin ( C. unshiu Marc.) and Ponkan ( C. poonensis Hort. ex Tanaka), which belong to C. reticulata , were selected. As a result, hot water extraction was efficient in extracting phenolic acids and some minerals. As for citrus flavonoids, narirutin, nobiletin, and tangeretin were easier to extract than hesperidin. The result of antioxidant capacity assays indicated that for citrus peels, hot water extract had almost the same capacity as the methanol extract. We suggested that Ponkan was more suitable as the source of chenpi , since its hot water extract had much higher content of phenolic acids, FGs and PMFs, and higher antioxidant capacity than those of Satsuma mandarin. Generally, to raise the extraction temperature or to prolong the time could not yield higher content of phenolic compounds and stronger antioxidant capacity, though the content of minerals increased to some extent. Furthermore, a 2nd-time extraction seemed necessary since considerable minerals and phenolic compounds could be obtained by doing so. Finally, we suggested that 2 times extraction at 100 °C for 30 min was proper to extract the minerals and phenolic compounds in chenpi .     

HPLC-DAD-FLD同时测定柑橘果汁中12种多甲氧基黄酮和香豆素类物质

建立高效液相色谱-串联二极管阵列检测器和荧光检测器(high performance liquid chromatography-diode array detector-fluorescence detector,HPLC-DAD-FLD)同时检测柑橘果汁中12种多甲氧基黄酮、香豆素及呋喃香豆素类物质的方法。以0.01%磷酸、甲醇、乙腈组成三元流动相进行梯度洗脱,12种物质在30 min内实现基线分离。利用DAD和FLD获得各物质的紫外和荧光光谱信息,将柑橘果汁样品组分的光谱与之比对,并结合色谱保留时间,实现样品成分的定性分析。分别以320 nm和450 nm为紫外和荧光的检测波长,研究该方法的定量性能。结果显示:标准曲线线性关系良好;荧光定量限低至μg/L级,可作为紫外定量的重要补充手段;果汁回收率的紫外和荧光检测值分别为95.2%~104.8%和94.5%~103.5%。该方法对样品定性、定量分析准确,适合柑橘多甲氧基黄酮、香豆素等能发射荧光物质的检测。     

Simultaneous determination of polymethoxyflavones in Citrus species,Kiyomi tangor and Satsuma mandarin,by high performance liquid chromatography

The content of polymethoxyflavones (PMFs) in Kiyomi tangor (Citrus unshiu Marcov. forma miyagawa-wase × C. sinensis) and Satsuma mandarin (C. unshiu Marcov. forma miyagawa-wase) was determined by HPLC/UV. The major PMFs of Kiyomi tangor were determined as 3,6,7,4′-tetramethoxyflavone in the peels (10.1 mg/g) and leaves (9.2 mg/g), and 3-hydroxy-5,6,7,4′-tetramethoxyflavone in the stems (1.5 mg/g). The major PMFs of Satsuma mandarin were determined as 5,6,7,8,4′-pentamethoxyflavone in the peels (2.2 mg/g) and leaves (1.6 mg/g), and 5,6,7,3′,4′-pentamethoxyflavone in the stems (1.4 mg/g). Large amounts of Citrus by-products can ultimately provide a cheap and convenient source of PMFs.     

HPLC–PDA/ESI–MS/MS detection of polymethoxylated flavones in highly degraded citrus juice: a quality control case study

Highly degraded citrus fruit juices collected during a quality control investigation of the Italian Ministry of Agricultural Alimentary and Forestry Policies (MIPAAF) were analyzed for verifying their technological history. The analyzed samples were fermented black fluids stated as degraded first strength juices, in order to justify communitarian aids. Polymethoxylated flavones (PMFs) detection of solid phase extracted (SPE) purified samples provided clean chromatograms and good separation of compounds of interest using high performance liquid chromatography coupled with diode array and mass spectrometry HPLC–PDA/ESI–MS/MS. Since the PMFs concentration is elevate in peel flavedo, it is directly correlated with juice extraction strength. Results showed that all analyzed samples presented a PMFs concentration ranging from two to eight times first strength juices, not respecting the minimum quality requirements of the European regulatory framework.     

Important flavonoids and limonin in selected Thai citrus residues

The distributions of important flavonoids and limonin in selected Thai citrus residues were investigated in this study. The residues of interest were peels of pomelo (Citrus grandis (L.) Osbeck cv. Kao Yai and cv. Kao Nampheung), residues after juice extraction of tangerine (Citrus reticulata Blanco cv. Sainamphueng and cv. Bangmod) and peels and residues after juice extraction of kaffir lime (Citrus hystrix DC). Flavonoids were characterized and quantified in terms of flavanones, i.e., naringin and hesperidin, and polymethoxyflavones (PMFs), i.e., nobiletin, sinensetin and tangeretin. Naringin was a dominant flavanone in polmelo peels. Three PMFs were found in all samples with varying contents. Comparing the samples, residues from tangerine cv. Bangmod exhibited the highest value of each PMFs. Limonin was present in small amounts in pomelo peels and residues after juice extraction of tangerine and kaffir lime. All samples were good sources of dietary fibre, with a total dietary fibre content of more than 60 g/100 g on a dry weight basis with high proportion of soluble dietary fibre.     

生物活性多甲氧基黄酮羧酸和氨基酸衍生物的合成及其结构表征

多甲氧基黄酮(PMFs)是一类存在于柑橘属植物中具有显著抗癌、抗炎和抗氧化等生物活性的天然产物。为了提高多甲氧基黄酮类化合物的水溶性和药用价值,以2种来源丰富且抗癌活性高的多甲氧基黄酮橘皮素和川陈皮素为底物,分别经过氧丙酮氧化得到多甲氧基黄酮醇[3-羟基橘皮素(7)和3-羟基川陈皮素(8)],化合物7和化合物8分别在K₂CO₃和N,N-二甲基甲酰胺条件下与氯乙酸乙酯进行Williamson反应,生成了多甲氧基黄酮羧酸酯类衍生物,再经碱性水解合成了2种未见文献报道的多甲氧基黄酮羧酸衍生物[橘皮素-3-O-乙酸(1)和川陈皮素-3-O-乙酸(2)]。化合物1和化合物2在CH₂Cl₂作溶剂、1-(3-二甲基丙胺)-3-乙基碳二亚胺盐酸盐为缩合剂和4-N,N-二甲氨基吡啶为辅助剂的条件下,分别与2种不同的氨基酸甲酯盐酸盐发生缩合反应,得橘皮素和川陈皮素的氨基酸甲酯衍生物,然后经水解反应得到了4种新型多甲氧基黄酮氨基酸衍生物3～6。对所合成的化合物用¹H NMR、¹³C NMR和MS等方法进行了结构表征。     

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。     

柑橘类黄酮的生物学活性及提高生物利用度技术研究进展

柑橘类黄酮是人类膳食黄酮的主要来源之一,具有抗氧化、抗炎、调节脂质代谢、抗癌和神经保护等多种生物活性,但其存在口服生物利用度较低等问题。本文系统总结了近年来关于柑橘类黄酮发挥各类生物学功效的作用机理,以及包括环糊精包埋、酶处理、脂质体运载体系和乳液运载体系构建等提高生物利用度技术的研究进展,并在此基础上展望柑橘类黄酮的研究方向与应用前景,以期为相关保健品及辅助治疗药物的开发利用提供参考依据。     

体外发酵对柑橘黄酮的生物转化及功能活性研究进展

柑橘黄酮是一类主要存在于柑橘属植物果实外皮中的次生代谢物,具有多种生理功能,可预防和改善与氧化应激、炎症、癌细胞增殖和脂质积累等造成的健康问题。然而,柑橘黄酮普遍存在溶解性不高、稳定性较差、生物利用度低等问题。通过体外发酵技术对柑橘黄酮的特定结构进行修饰,能一定程度上改善其生物利用度并增强功能活性。该文从柑橘黄酮的结构与分类、参与体外发酵的微生物及酶种类、柑橘黄酮的体外转化过程及代谢产物等多个方面详细阐述了生物催化剂对柑橘黄酮的定向结构修饰作用,并总结了生物转化对柑橘黄酮产物抗氧化、抗炎、抑制癌细胞增殖、抗病毒、降脂等多种功能活性的影响,以期为柑橘黄酮的生物转化研究及新型功能食品开发提供理论依据。     

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。     

橙皮苷生物学活性及其改性技术的研究进展

橙皮苷是一种广泛存在于柑橘属水果中的天然黄酮类化合物,具有抗氧化、抗炎、抑菌和抗癌等多种生物活性。已有研究表明,橙皮苷因其水溶性和吸收性较差,导致生物利用度较低,因此国内外学者聚焦于橙皮苷进行物理、化学和生物改性,以增加其溶解性及生物利用度。本文对橙皮苷的生物学活性、改性技术以及改性衍生物的相关内容进行综述,为橙皮苷的高效利用提供理论支撑。