Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating β-cryptoxanthin in Citrus

Carotenoids in plant foods provide health benefits by functioning as provitamin A.One ofthe vital provitamin A carotenoids,β-cryptoxanthin,is typically plentiful in citrus fruit.However,little is known about the genetic basis of β-cryptoxanthin accumulation in citrus.Here,we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrus and determine the taxonomic profile of b-cryptoxanthin.We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of β-cryptoxanthin accumulation through a genome-wide association study.We identified a causal gene,CitCYP97B,which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined.We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the β-ring of β-cryptoxanthin in a heterologous expression system.In planta experiments provided further evidence that CitCYP97B negatively regulates b-cryptoxanthin content.Using the sequenced Citrus accessions,we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B,thereby altering β-cryptoxanthin accumulation in fruit.Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrus types during citrus evolution.Overall,these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of β-cryptoxanthin-biofortified products.

β-cryptoxanthin and fatty liver disease:new insights

β-cryptoxanthin is a nutritionally important xanthophyll found in orange-fleshed tropical and citrus fruits,including papaya,oranges,and tangerines(1).It is also one of the most commonly detected carotenoids in human tissues(1).Uniquely,β-cryptoxanthin is the only regularly consumed dietary xanthophyll to have an intactβ-ionone ring,thus in addition to functioning as an antioxidant it can also be metabolized to vitamin A.A limited number of past studies have shown a beneficial effect ofβ-cryptoxanthin supplementation in animal models of hepatic steatosis(2).As discussed below,the recent publication by Liu et al.from the group led by Dr.Xiang-Dong Wang has provided new insight into the benefits ofβ-cryptoxanthin supplementation in the context of fatty liver disease(3).

Investigation of <i>β</i>-Cryptoxanthin Fatty Acid Ester Compositions in Citrus Fruits Cultivated in Japan

In this study, a catalog of β-cryptoxanthin (βCX) and its fatty acid esters (βCXFAs) in citrus fruits were constructed. The peel and pulp of citrus fruits from nine Japanese cultivars and one foreign cultivar were surveyed by supercritical fluid chromatography/triple quadrupole mass spectrometry (SFC/QqQMS). In each citrus sample, the major components were βCX and its laurate ester, myristate ester, palmitate ester, and oleate ester. However, the composition ratio of βCXFA varied with the citrus breed and between the pulp and the peel of the fruit in most cultivars. The SFC/QqQMS system could be used to obtain new information about βCXs and carry out further discussion on the feature and tendency of each citrus variety.

Xanthophyll β-cryptoxanthin treatment inhibits hepatic steatosis without altering vitamin A status inβ-carotene 9',10'-oxygenase knockout mice

Background:β-cryptoxanthin(BCX),one of the major carotenoids detected in human circulation,can protect against the development of fatty liver disease.BCX can be metabolized throughβ-carotene-15,15'-oxygenase(BCO1)andβ-carotene-9',10'-oxygenase(BCO2)cleavage pathways to produce both vitamin A and apo-carotenoids,respectively,which are considered important signaling molecules in a variety of biological processes.Recently,we have demonstrated that BCX treatment reduced hepatic steatosis severity and hepatic total cholesterol levels in both wide type and BCO1^(-/-)/BCO2^(-/-)double knock out(KO)mice.Whether the protective effect of BCX is seen in single BCO2^(-/-)KO mice is unclear.Methods:In the present study,male BCO2^(-/-)KO mice at 1 and 5 months of age were assigned to two groups by age and weight-matching as follows:(I)-BCX control diet alone(AIN-93 purified diets);(II)+BCX 10 mg(supplemented with 10 mg of BCX/kg of diet)for 3 months.At 4 and 8 months of age,hepatic steatosis and inflammatory foci were evaluated by histopathology.Retinoids and BCX concentrations in liver tissue were analyzed by high-performance liquid chromatography(HPLC).Hepatic protein expressions of SIRT1,acetylated and total FoxO1,PGC1α,and PPARαwere determined by the Western blot analysis.Real-time PCR for gene expressions(MCAD,SCD1,FAS,TNFα,and IL-1βgene expression relative toβ-actin)was conducted in the liver.Results:Steatosis was detected at 8 months but not at 4 months of age.Moreover,BCX supplementation significantly reduced the severity of steatosis in the livers of BCO2KO mice,which was associated with changes in hepatic SIRT1 acetylation of FOXO1,PGC1αprotein expression and PPARαprotein expression in BCO2^(-/-)KO mice.HPLC analysis showed that hepatic BCX was detected in BCX supplemented groups,but there were no differences in the hepatic levels of retinol and retinyl palmitate(RP)among all groups.Conclusions:The present study provided experimental evidence that BCX intervention can reduce liver steatosis independent of BCO2.

Suppression of Sebum Production and Accumulation by <i>β</i>-Cryptoxanthin Due to the Inhibition of the Expression of Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes

Background: Acne vulgaris is characterized by the enhancement of sebaceous lipogenesis and sebum secretion, and apart from retinoids and some natural products there are few effective antiacne agents that directly suppress sebum production and accumulation in sebaceous glands. Objective: We examined the effects of β-cryptoxanthin (β-CRX), which is a carotenoid pigment most abundant in Citrus unshiu Marcovich (Satsuma mandarin orange) and plays a role as a vitamin A precursor on sebum production and accumulation in hamster sebaceous gland cells (sebocytes). Materials and methods: The regulation of sebum production was examined by the measurement of triacylglycerols (TGs), the major sebum component, and oil red O staining in insulindifferentiated hamster sebocytes. The expression of diacylglycerol acyltransferase-1 (DGAT-1), a rate-limiting enzyme of TG biosynthesis, and perilipin 1 (PLIN1), a lipid storage droplet protein, was analyzed using real-time PCR and Western blotting. Results: Hamster sebocytes constitutively produced TGs during cultivation and the production of TGs was enhanced by insulin treatment. Both constitutive and insulin-enhanced TG productions were dose- and time-dependently inhibited by β-CRX as well as 13-cis retinoic acid. In addition, the gene expression of DGAT-1 was suppressed by β-CRX in the sebocytes. Furthermore, the insulin-en- hanced sebum accumulation as lipid droplets was reduced in the β-CRX-treated cells. Moreover, β-CRX was found to suppress the gene expression and production of PLIN1 in insulin-differentiated hamster sebocytes. Conclusions: These results provide novel evidence that β-CRX is an effective candidate for acne therapy by its ability to exert dual inhibitory actions against DGAT-1-dependent TG production and PLIN1-mediated lipiddroplet formation in hamster sebocytes.

柑橘果实主要类胡萝卜素成分及含量分析

应用HPLC技术分析了我国53个柑橘品种资源的6种类胡萝卜素成分——叶黄素、玉米黄素、β-隐黄质、α-胡萝卜素、β-胡萝卜素和番茄红素的含量。结果表明,柑橘果皮和果肉中均以叶黄素、玉米黄素、β-隐黄质为主,β-胡萝卜素含量较低,α-胡萝卜素极低;参试品种中番茄红素仅在红肉脐橙的果肉中检测到。不论果皮还是果肉均以宽皮柑橘类中类胡萝卜素含量最高,柚类最低,表明宽皮柑橘具有较高营养保健价值。在宽皮柑橘中,果肉以积累β-隐黄质为主,果皮β-隐黄质与叶黄素含量接近。与果肉相比,柑橘果皮的单位鲜重叶黄素、玉米黄素、β-隐黄质的含量分别为果肉的2.5～15倍,是柑橘果实中主要类胡萝卜素库存部位。

黄肉桃果实发育阶段类胡萝卜素的变化

【目的】为了分析黄肉桃果实发育过程中类胡萝卜素的组分及含量与果皮和果肉颜色深浅的关系,【方法】以3种不同类型黄肉桃‘香金蟠’、‘独立’和‘弗雷德里克’为试材,采用紫外分光光度计测定桃果实类胡萝卜素的总含量,并对桃果实不同发育阶段的类胡萝卜素进行定性定量分析。【结果】结果表明,果实成熟时,‘香金蟠’、‘独立’、‘弗雷德里克’果肉颜色存在差异,分别为淡黄、金黄和橙黄。‘香金蟠’果皮的类胡萝卜素总含量随着果皮黄颜色的加深逐渐降低,‘独立’保持平稳,而‘弗雷德里克’则逐渐升高。3种黄肉桃果肉的类胡萝卜素总含量则随着果实的生长发育逐渐升高,且以果肉黄颜色最深的黄肉桃(‘弗雷德里克’)含量最大。黄肉桃的果皮和果肉均含有类胡萝卜素的5个主要成分,即叶黄素、玉米黄素、β-隐黄质、α-胡萝卜素和β-胡萝卜素,且以β-胡萝卜素所占比例最大。果皮中的叶黄素随着果实的生长发育逐渐降低,玉米黄素、β-隐黄质和α-胡萝卜素则逐渐升高,β-胡萝卜素与其果皮中类胡萝卜素总含量的变化趋势一致。果肉中的叶黄素、玉米黄素均与果皮中的变化趋势一致,而β-隐黄质、α-胡萝卜素和β-胡萝卜素存在较大的差异。【结论】β-胡萝卜素是影响桃果肉颜色差异的主要因素之一,其次是β-隐黄质和α-胡萝卜素,且与2者的比值有很大关系,β-隐黄质/α-胡萝卜素的比值越大,黄颜色越深,比值越小,黄颜色越浅。β-隐黄质所体现的颜色偏向于橙黄色,α-胡萝卜素则偏向于淡黄色。

不同类型枇杷果实着色期间果肉类胡萝卜素含量的变化

【目的】分析不同类型枇杷果实着色过程中果肉类胡萝卜素的变化特点,探讨果肉外观颜色差异形成的原因。【方法】以‘大红袍’、‘青种’分别代表红沙和白沙两类不同枇杷品种,自果实开始着色起,分期采样,进行枇杷果肉类胡萝卜素提取、纯化和HPLC分析。【结果】果实进入着色期后,果肉叶黄质含量迅速下降,至果实成熟时下降平缓,而α-胡萝卜素、β-胡萝卜素、β-隐黄质和玉米黄素成分均呈上升趋势,尤其在采收前7～14d上升迅速。果实成熟后,‘大红袍’果肉的类胡萝卜素总量为‘青种’的2倍以上;两品种果肉叶黄素类色素含量接近,但类胡萝卜素含量差异较大,‘大红袍’的胡萝卜素含量是‘青种’的4倍左右。【结论】胡萝卜素积累的多少,尤其是β-胡萝卜素含量的高低是造成‘青种’和‘大红袍’果肉类胡萝卜素总量积累差异的主要原因,也是影响这两个品种果肉色泽深浅的主要因素。

柑橘果皮颜色的形成与类胡萝卜素组分变化的关系

分析了果皮分别为红、橙和黄颜色的柑橘品种“满头红”、“尾张”和“胡柚”果实着色过程中果皮类胡萝卜素组分及含量的变化。结果表明 ,随着果实的发育和成熟 ,果皮叶绿素含量下降 ,α 胡萝卜素、β 胡萝卜素含量逐渐下降直至消失 ,而β 隐黄质、β 柠乌素、玉米黄质等叶黄素成分含量逐渐上升 ,果实逐渐褪绿并呈现其特征色泽。“胡柚”果皮中类胡萝卜素总量及其橙、红色类胡萝卜素成分 β 隐黄质和 β 柠乌素积累少是其颜色淡、呈黄色的主要原因 ;三种果实的颜色差异并非由于果皮类胡萝卜素总量的差异 ,而主要是由于果皮不同类胡萝卜素成分组成比例不同。“满头红”以积累红色的 β 柠乌素为主 ,而“尾张”则以积累橙色的β 隐黄质为主。

南丰蜜橘β-胡萝卜素羟化酶基因的克隆和序列分析

以南丰蜜橘[Citrus reticulata Blanco var.kinokuni(Tanaka)H.H.Hu]基因组DNA为模板,根据已报道的柑橘β-胡萝卜素羟化酶基因保守序列设计4对引物,进行PCR扩增,得到4条长度为470、761、294和991 bp的片段.将这些片段克隆到pMD18-T载体,并进行测序.测序结果拼接成1条2 326 bp的序列.分析发现该序列含6个内含子,7个外显子.内含子两侧有典型的GT-AG保守序列.该序列中预测的编码序列与温州蜜柑、拟南芥等植物的β-胡萝卜素羟化酶基因序列保守性达80%以上,表明该序列确实为β-胡萝卜素羟化酶基因.该基因编码了1个含311个氨基酸的蛋白.将该序列递交到GenBank数据库,序列号为AM408552.

酸浆宿萼中β-隐黄质体外淬灭单线态氧能力评价

目的:从酸浆(Physalis alkengi L.var.franchetii(Mast.)Makino)宿萼中制备β-隐黄质(β-cryptoxanthin),应用微弱发光测量方法对β-隐黄质体外淬灭单线态氧的能力进行评价。方法:用正己烷萃取酸浆宿萼中的β-隐黄质酯后经皂化获得单体,使用C18-HPLC-PDA对制备物进行定性和定量。用次氯酸钠-双氧水反应产生单线态氧,使用微弱发光测量仪检测制备组分淬灭单线态氧的能力。结果:经C18-HPLC-PDA鉴定,制备物中的类胡萝卜素主要为β-隐黄质。微弱发光测量仪的数据显示:β-隐黄质的存在能减少次氯酸钠-双氧水单线态氧体系中产生的最大光子计数,且存在量效关系。0.049～0.49g/L的β-隐黄质可减少50%的最大光子计数。

柿果类胡萝卜素化学成份及其含量研究

对柿果中类胡萝卜素化学成份及其含量的季节性变化以及栽培条件的影响等进行了调查,旨在为柿色素代谢及其功能性成分合成与调控等的深入研究奠定基础。结果如下:柿果实中的色素成份为玉米黄质、β-隐黄质、β-胡萝卜素、叶黄素和番茄红素;果皮中的类胡萝卜素总含量约是果肉中的6-11倍;按类胡萝卜素组成成分的含量由高至低,果皮依次为玉米黄质、β-隐黄质、β-胡萝卜素、叶黄素和番茄红素;而果肉依次为β-隐黄质、玉米黄质和β-胡萝卜素。结果表明,柿果肉主要色素成份为功能性β-隐黄质。温室栽培磨盘柿果中功能性成分β-隐黄质含量是露地的3倍。供试7品种间色素成分与含量有很大区别。

贮藏温度对采后枇杷果实类胡萝卜素含量的影响

以“早钟6号”枇杷（Eriobotrya japonica Lindl.cv.Zaozhong No.6）果实为试材,采用高效液相色谱法（HPLC）对果实类胡萝卜素进行定性和定量分析,探讨贮藏温度对枇杷果实类胡萝卜素积累的影响.结果表明：在果实贮藏1～12d期间,25℃贮藏条件下枇杷果肉叶黄素、β-隐黄质、β-胡萝卜素含量始终高于4℃和8℃贮藏,且在8℃和25℃贮藏条件下果肉中这3种色素含量呈相似的“升-降”变化规律,但3种色素含量峰值出现的时间各不相同.4℃贮藏条件下果肉叶黄素、β-隐黄质含量随贮藏时间的延长逐渐下降而不利于积累,总体上4℃贮藏的果肉叶黄素、β-隐黄质、β-胡萝卜素含量低于25℃和8℃贮藏.可见,贮藏温度越低越不利于果肉类胡萝卜素的积累,不同温度贮藏后期果肉类胡萝卜素积累减少,贮藏温度过低或贮藏时间过长均不利于果肉类胡萝卜素的积累.综合评价不同温度贮藏枇杷果实的商品价值,8℃环境条件下较适宜枇杷果实的长期贮藏.

β-隐黄素纳米乳的制备及其稳定性研究

以D-柠檬烯为油相,采用低能乳化法制备β-隐黄素纳米乳。对比研究了温度、光照对β-隐黄素纳米乳及β-隐黄素油溶液的影响,以及酸、碱、氧化剂和还原剂对β-隐黄素纳米乳的影响。结果发现:所制备的β-隐黄素纳米乳平均粒径为12.14 nm;β-隐黄素纳米乳比β-隐黄素油溶液更耐热、耐光;短时间强酸强碱环境对β-隐黄素纳米乳稳定性影响较小,但长时间的强酸条件会加速β-隐黄素的降解,而强碱条件会使β-隐黄素纳米乳变浑浊;高浓度的氧化剂会使β-隐黄素纳米乳有轻微降解,而还原剂对β-隐黄素纳米乳具有一定的保护作用。因此,β-隐黄素纳米乳比β-隐黄素油溶液具有更好的稳定性,但仍需避免长时间处于强酸强碱环境。

二次正交旋转组合优化微波辅助提取温州蜜柑β-隐黄素工艺

用微波辅助提取温州蜜柑皮β-隐黄素,先通过单因素试验选取影响因素和水平,再采用二次正交旋转组合设计方法研究料液比、微波处理时间、微波功率对β-隐黄素得率的影响。结果表明,各因素对温州蜜柑皮β-隐黄素得率的影响大小依次为微波功率>微波处理时间>料液比;频率分析法得到温州蜜柑皮β-隐黄素最优的提取工艺为料液比1:20、微波时间184s,微波功率357W,在此最佳条件下β-隐黄素得率为0.30‰。

植物β-胡萝卜素羟化酶研究进展

-β胡萝卜素羟化酶是植物类胡萝卜素合成代谢中的关键酶,它催化了植物中β-胡萝卜素经中间产物β-隐黄素合成玉米黄素的过程。β-胡萝卜素羟化酶广泛存在于植物、蓝藻和细菌等生物中,其基因在植物中成对出现,在原核生物中则处于基因簇内。该酶是一种非血红素双铁单加氧酶,分子中有富含组氨酸的模体。多种生物的β-羟化酶基因已被克隆并分别在细菌、蓝藻或植物中表达。玉米黄素能帮助植物抵御胁迫环境,β-隐黄素对癌症等人类疾病有抑制作用。采用基因工程手段改造β-羟化酶基因,将为培养高抗逆性作物和大规模生产β-隐黄素提供新途径。

溶剂和超声波辅助提取温州蜜柑皮β-隐黄素的条件优化

研究了普通溶剂提取和超声波辅助提取方法对温州蜜柑皮β-隐黄素得率的影响,并通过单因素和正交实验确定了超声波辅助提取温州蜜柑皮β-隐黄素的最佳工艺条件:以乙醇作为提取剂,料液比1:17.5,超声时间33min,超声波功率180W,超声温度45℃,超声波浸提次数2次。在此提取条件下β-隐黄素的得率为0.3022‰。

玉米蛋白粉叶黄素酶解辅助提取工艺优化研究

对玉米蛋白粉叶黄素酶解辅助提取工艺进行了研究,采用响应面分析法分析了酶浓度、底物浓度和酶解时间对总叶黄素得率的影响,并建立了相应的预测模型。方差分析的结果表明,酶解时间的二次方项(P<0.025)和底物浓度的二次方项(P<0.005)对提取效率有显著影响,3个因素对提取效率也都有显著影响(P<0.005),并且酶浓度和底物浓度及酶浓度和酶解时间之间存在着交互作用(P<0.01)。考虑到提取效率、成本和可行性,利用约束复合形法对结果进行优化,得到的最佳工艺参数为:酶浓度7 682 U/g、底物浓度8.8%、酶解时间2.2 h。在最佳工艺参数下,总叶黄素的得率为64.65μg/g,与预测结果相符。与常规提取方法比较,叶黄素得率显著提高(P<0.05)。

辣椒中β-隐黄质的分离制备

建立从辣椒中分离制备高纯度β-隐黄质的方法。辣椒粉先经丙酮超声提取、皂化,硅胶柱色谱进行粗分后进一步采用制备液相对β-隐黄质分离制备。通过紫外光、电喷雾电离质谱、核磁共振氢谱和核磁共振碳谱对所分离的β-隐黄质进行结构鉴定,分析型高效液相色谱进行纯度测定。结果表明:每5g辣椒色素浸膏与异丙醇的比例为1:9(g/mL)、加入6.5mL质量浓度为20g/100mL的KOH-甲醇溶液,在室温条件下皂化24h,皂化完全;硅胶柱色谱进行粗分时用石油醚与丙酮作为洗脱溶剂,当体积比为10:1时,β-隐黄质得到较好的富集;制备液相的条件为丙酮-水(95:5,V/V)、流速5mL/min,可制备分离出纯度为99.0%的β-隐黄质单体。该方法简便快捷、重现性好、所得β-隐黄质纯度高,可作为β-隐黄质的分离制备研究。

正交试验法优化从酸浆宿萼中提取β-隐黄质的工艺研究

酸浆(Physalis alkekengi L.var.franchetii)宿萼中含有丰富的β-隐黄质,其具有预防肺癌、骨质疏松症、神经性疼痛以及心脏病等多种生理活性。该篇通过正交试验的方法研究了酸浆宿萼中β-隐黄质的最佳提取方法,研究条件包括提取温度、料液比、萃取时间和萃取次数。实验结果表明,酸浆宿萼中β-隐黄质的含量可达3.54mg/g,在最佳提取条件下,提取率可达92.97%。该方案提供了一种高效、简便的从酸浆宿萼中提取类胡萝卜素,尤其是β-隐黄质的方法。