37 resultados para Paeonia ludlowii


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牡丹(Paeonia suffruticosa Andr.),芍药科芍药属植物,是我国的传统名花,因花朵硕大、花色丰富、花型齐全而显雍容华贵、富丽端庄,“惟有牡丹真国色,花开时节动京城”,深受人民的喜爱。人们在欣赏牡丹的过程中,一方面为其艳丽多姿而赞叹,同时又为其自然花期较短且集中而遗憾,“弄花一年,看花十日”,因此,如何通过栽培等技术措施使牡丹连续开花,一直是牡丹研究者探索的重要课题。近年,在对牡丹栽培的系统研究中,发现不同牡丹品种的花芽分化类型(数量、梯度)直接影响其开花数量和开花次数。为了有效地利用不同类型的牡丹,选育出更多的丰花、同株可连续开花的品种,服务于牡丹的产业化生产,本试验对4个牡丹品种群的135个品种进行了同枝条芽数的统计分析及花芽分化梯度的划分,阐明了花芽分化类型与促成连续二次开花的关系。初步测定了具有促成连续二次开花习性的牡丹品种‘High Noon’不同芽位腋芽内源激素的含量,揭示了牡丹同株连续二次开花过程中不同芽位腋芽内源激素的生理变化规律,为解决牡丹同株花期短、不能同株连续开花的难题奠定了理论基础。本研究主要结果如下: 1、牡丹花芽分化类型与自然开花的关系 通过对135个牡丹品种花芽分化数量和梯度类型的调查,将同枝条的芽数聚类为少(3-4)、中等(5-7)、多(8-10)三类;将花芽分化梯度划分为小、中、大三种类型。芽数类型和花芽分化梯度类型均与品种群有一定的关系。中原品种群中90%的品种属于芽数少的类型,47%的品种属于分化梯度大的类型;日本品种群中73%的品种属于芽数中等的类型,52%的品种属于分化梯度中的类型;法国品种群和美国品种群品种调查数量较少,但大多数品种属于芽数中等的类型,分别占80%和44%,分化梯度小的品种分别占调查总数的40%和67%。绝大部分品种的腋花芽因在枝条上着生的位置不同而有明显的异质性,上部芽顶端优势强,萌动率和开花率均高,芽数多的品种中、下部芽在春天自然开花季节常处于休眠状态。 2、牡丹花芽分化类型与促成连续二次开花的关系 花芽分化数量的多少与梯度的大小直接影响着开花次数。花芽分化数量少、梯度大的品种不建议直接用于同株促成连续二次开花栽培;花芽分化数量中等或多、梯度小的品种可以实现同株促成连续二次开花,10个试验牡丹品种中的中原牡丹品种‘如花似玉(Ru Hua Si Yu)’和美国牡丹品种‘High Noon’具有同株连续二次开花能力,且二次开花率达75%以上,两次开花品质均优良。 3、‘High Noon’不同芽位腋芽内源激素与促成连续开花的关系 通过测定美国牡丹品种‘High Noon’同枝条不同芽位腋芽萌动前期、后期内源激素含量,结果表明:萌动后期不同芽位腋芽的GA3、IAA、ZRs含量增加,尤其1位芽、2位芽含量增加显著;萌动后期ABA含量则随着芽位自上而下增加显著,可能是导致下部芽继续保持休眠状态的主要原因;萌动后期下部芽的ABA/ZRs配比增幅较高,说明ABA/ZRs配比与芽的生长与休眠关系密切。

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牡丹(Paeonia suffruticosa Andr.)是芍药科芍药属牡丹组植物,花大、色艳、型美、香郁、药用范围广、文化内涵丰富,被尊为“国色天香”、“花中之王”。我国植牡丹、赏牡丹有1600多年的历史,培育了许多价值高的品种,遗憾的是个别黄色、鲜红色、绿色等珍贵品种不断消失,而且现有的一些文化价值与观赏价值极高的古牡丹、著名的牡丹园的老牡丹均出现了衰弱现象,有的已奄奄一息。为了探索传统牡丹品种消失及牡丹衰老的原因,本研究在充分调查、整理国内品种资源的基础上,总结了牡丹品种的传承和消失情况,掌握了古牡丹的资源状况。通过连作土壤栽培试验证明了牡丹连作障碍的存在,并选定了生长在同一区域的5、10、15、20、25、30年生6个株龄的‘洛阳红(Luo Yang Hong)’为研究对象,从形态指标的调查、生理指标的测定、根际土壤及根系浸提液的化感成分分析三个方面着手,确定了牡丹开始衰老的时期;揭示了牡丹衰老机理;总结了古牡丹保留、复壮、养护办法;为牡丹专类园的管理及可持续性发展提供了理论和技术指导。本研究主要结果如下: 1、牡丹品种和古牡丹资源 通过对中国牡丹品种资源的文献查阅和实地考察,系统整理了我国不同时代的品种状况,调查结果表明:宋、元、明、清共有品种1109种,目前仅存143种,品种资源消失十分严重。收集整理了49处古牡丹资料,并据此绘制了我国目前古牡丹分布图;实地调查了17处24株古牡丹,详细介绍了部分品种的来历传说、生长状况和衰亡原因。5个优良传统品种在中国科学院植物研究所牡丹种质资源圃内得以保存,其中潞城古牡丹,植株最大(高2.3 m,冠径5.6 m),且生长旺盛;西溪牡丹属丰花品种,40年植株可开花811朵。 2、牡丹连作障碍 利用牡丹连作根际土壤及其浸出液栽培牡丹,结果表明,播种苗及分株苗的生长均受到了抑制;连作年限越久的处理抑制作用越强;根际土壤浸出液对牡丹生长的影响较大,对前期地下部和后期地上部都有很强的抑制作用,而且还抑制了上胚轴休眠的解除;根际土壤对后期株高和展叶幅的影响较大,对播种苗前期生根影响不大。牡丹存在连作障碍现象,连作障碍是牡丹衰弱的重要原因。 3、牡丹衰老时间及机理 通过对6 个株龄‘洛阳红’的形态、生理指标及根际土壤化感成分的比较得出,牡丹在同一地方大约栽植15 年后开始衰弱,20 年以内应该采取复壮措施。利用气相色谱—质谱联用技术(Gas Chromatograph-Mass Spectrometer, GC-MS)检测到根际土壤及根系浸提液中主要存在:烷烃、烯烃、芳香烃、醇、醚、酚醌、有机酸、醛、酮、酯、苯、胺等12 类有机物。对比分析得出有12 种物质可能是根系分泌物,根际土壤中可能具毒害作用的物质有40 种,栽植牡丹使土壤中增加的物质有24 种。 4、古牡丹复壮技术及应用 栽培基质中添加活性炭、沙子、麦饭石等均能有效的促进牡丹的生长,以活性炭的效果最好。对“汉牡丹”实施了综合复壮措施,效果明显。

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本文以9 个芍药野生种(15 份种质)、104 个品种及2 个牡丹芍药组间杂种的花瓣为材料,利用液质联用技术鉴定了花瓣中的色素成分并探讨了芍药花色形成的化学机制和化学分类法。 结果表明,芍药花中主要含有5 种花青素,即芍药花素-3,5-二葡糖苷( peonidin-3,5-di-O-glucoside , Pn3G5G ); 矢车菊素-3 , 5- 二葡糖苷( cyanidin-3,5-di-O-glucoside , Cy3G5G ); 天竺葵素-3 , 5- 二葡糖苷( pelargonidin-3,5-di-O-glucoside , Pg3G5G ); 芍药花素-3- 葡糖苷(peonidin-3-O-glucoside,Pn3G)和矢车菊素-3-葡糖苷(cyanidin-3-O-glucoside,Cy3G)。此外,3 种微量的花青素首次在芍药中发现:它们分别为芍药花素-3-葡萄糖-5-阿拉伯糖苷(peonidin-3-O-glucoside-5-O-arabinoside,Pn3G5Ara)、矢车菊素-3- 葡萄糖-5- 半乳糖苷( cyanidin-3-O-glucoside-5-O-galactoside ,Cy3G5Gal)和天竺葵素-3-葡萄糖-5-半乳糖苷(pelargonidin-3-O-glucoside-5-Ogalactoside,Pg3G5Gal)。特征花青素Cy3G5Gal 和Pg3G5Gal 仅在新疆芍药(Paeonia anomala L.)及其亚种川赤芍(P. anomala subsp. veitchii(Lynch) D. Y.Hong & K. Y. Pan)中被检测出来,表明它们属于同一个种。Pn3G5Ara 仅存在于欧洲的野生芍药花瓣中,表明中国野生芍药和欧洲芍药的花青素代谢途径不同。 芍药花瓣中主要含有11 种花黄素,均为黄酮醇类物质。包括栎精-3,7 二葡糖苷( quercetin-3,7-di-O-glucoside )、山奈酚-3 , 7 二葡糖苷(kaempferol-3,7-di-O-glucoside)、异鼠李素-3,7 二葡糖苷(isorhamnetin-3,7-di-Oglucoside)、栎精-3-O-(6”-没食子酰基)-葡糖苷 [quercetin-3-O-(6”-O-galloyl)-glucoside] 、栎精-3- 葡糖苷( quercetin-3-O-glucoside )、山奈酚-7- 葡糖苷( kaempferol-7-O-glucoside )、山奈酚-3-O- ( 6”- 没食子酰基) - 葡糖苷[kaempferol-3-O-(6”-O-galloyl)-glucoside]、异鼠李素-3-O-(6”-没食子酰基)-葡糖苷 [isorhamnetin-3-O- ( 6”-O-galloyl ) -glucoside] 、山奈酚-3- 葡糖苷(kaempferol-3-O-glucoside)、异鼠李素-3-葡糖苷(isorhamnetin-3-O-glucoside)和山奈酚-丙二酰葡糖苷(kaempferol-malonyl-glucoside)。此外,查耳酮在黄色的栽培品种‘黄金轮’和牡丹芍药组间杂交种‘伊藤杂种’中首次被检测到。其化学结构为查耳酮-2’-葡糖苷(chalcononaringenin 2’-O-glucoside),它是花瓣表现出黄色的主要色素,它与黄色牡丹野生种‘滇牡丹’(P. delavayi Franchet)花瓣中主要黄色色素成分一致。 通过对所有芍药野生种和栽培品种的色素分析,研究发现花青素是芍药花瓣中主要的色素,其中Pn3G5G 是花瓣中含量最高的花青素苷,其次为Cy3G5G。3G 型糖苷仅在少数品种中检测出来。此外,黄酮醇是芍药花瓣中重要的辅助色素。山奈酚苷是花瓣中含量最高的黄酮醇类,其次是栎精。 多元线性回归分析的结果表明,芍药花色的形成主要与花瓣中Pn3G5G、Cy3G5G 和Pg3G5G 的含量及总花青素量(TA)有关。根据8 种花青素结构与花色组成,将国内的野生种和大部分品种进行了化学分类:所有样本聚成3 大类,聚类后的树状图与其花色、花色素组成数据相一致,直观反映了野生种和栽培品种花色形成的化学背景和表型相似性程度。 芍药成色机理和化学分类的初步研究,对芍药新花色育种具有重要意义:芍药鲜红色花的育种中,育种亲本应具有高的Cy3G 含量、低的辅助色素效应指数。选育深紫色花或紫黑色花的品种,亲本应具有高的Pn3G5G 含量和低的Pg3G5G 含量。

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本学位论文共有5章。第一章报道白芍的化学成分及芍药苷的微生物转化研究成果;第二章报道天山雪莲的化学成分研究;第三章报道两面针的化学成分研究;第四章报道通关藤的化学成分研究成果;第五章概述了花椒属植物中最近十年报道的新化合物及药理研究情况。 在第1章的第一部分报道了白芍(Paeonia lactiflora Pall.)的化学成分。我们采用正、反相硅胶柱层析等各种分离方法,从白芍的干燥根中共分离出14个化合物,其中1个为新化合物,其结构通过波谱分析证实为没食子酰白芍苷,另外还有2个为首次从该植物中分离得到。第二部分报道了芍药苷的微生物转化生产芍药苷代谢素-I的研究,从15株厌氧菌中筛选出10株有转化活性的菌株,其中短乳杆菌Lactobacillus brevis AS1.12的转化活性最好,对其转化条件进行了初步的筛选,确定了相对合理的转化工艺。 在第2章报道了天山雪莲(Saussurea involucrate Kar.et Kir.)全草乙醇提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR等波谱解析,共分离鉴定了28个化合物,结构类型分属于黄酮、倍半萜和木脂素等,其中2个新倍半萜化合物的结构分别表征为6α-羟基云木香酸6-β-D-吡喃葡萄糖苷和11βH-11,13-二氢去氢云木香内酯8α-O-(6′-乙酰)-β-D-吡喃葡萄糖苷。 第3章报道了两面针(Zanthoxylum nitidum (Roxb.)DC.)干燥根的乙醇提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR等波谱解析以及X-射线单晶衍射,共分离鉴定了16个生物碱,结构类型分属于苯并啡啶类、喹啉类和阿朴啡类等,其中2个新苯并啡啶类生物碱的结构分别表征为二聚双氢两面针碱和丙酮基双氢崖定椒碱。 第4章报道了通关藤(Marsdenia tenacissima (Roxb.) Wight et Arn.)水提取物化学成分的分离纯化和结构鉴定。通过正、反相硅胶柱层析等分离纯化和MS、NMR等波谱解析以及X-射线单晶衍射,共分离鉴定了14个化合物,结构类型均属于C21多羟基甾醇,其中4个新化合物tenacigenoside A, tenacigenoside B, tenacigenoside C和tenacigenoside D的结构分别表征为3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-17β-tenacigenin B (62), 3-O-2,6- dideoxy-4-O-methyl-D-lyxo-hexopyranosly-11α-O- methylbutyryl-12β-O-acetyl-tenacigenin B (63), 3-O-6-deoxy-3-O-methyl-β-D- allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-tigloyl-tenacigenin C (64)和3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-2- methylbutyryl-tenacigenin C (65)。 第5章概述了花椒属植物的化学成分及药理活性研究进展。 This dissertation consists of 5 chapters. The first chapter elaborate the phytochemical investigation of Paeonia lactiflora Pall., and microbial transformation of paeoniforin. The second, third and four chapters elaborate the phytochemical investigation of Saussurea involucrate Kar.et Kir., Zanthoxylum nitidum (Roxb.) DC. and Marsdenia tenacissima (Roxb.) Wight et Arn., respectively. Chapter 5 is a review on chemical constituents and bioactivities of Zanthoxylum species. The part one of chapter 1 focus on the isolation and identification of chemical constituents from P. lactiflora. Fourteen compounds were isolated from the roots of P. lactiflora by repeat column chromatography over normal and reversed phase silica gel. Among them, one is a new compound and the structure was suggested as galloyl-albiflorin by spectral evidence. In addition, two compounds were firstly reported in this plant. The part 2 is about microbial transformation of paeoniforin. Chapters 2, 3 and 4 were isolations and identifications of chemical constituents from S. involucrate, Z. nitidum and M. tenacissima, respectively. From the aerial parts of S. involucrate, 28 compounds including 7 flavonoids and 13 sesquiterpenoids were isolated and identified. Among them, 2 new compounds were characterized as 6α-hydroxycostic acid 6-β-D-glucoside and 11βH-11,13-dihydrodehydro- costuslactone 8α-O-(6'-acetyl)-β-D-glucoside, respectively, by means of spectroscopic analysis. Otherwise, 11 ones were firstly reported from this plant. The third chapter is about the phytochemical investigation of Z. nitidum. Sixteen compounds were isolated and identified. Among them, 2 new benzophenanthridine alkaloids were characterized as 8-acetonyldihydrofagaridine and 1,3-bis(8-dihydronitidinyl)-acetone by spectroscopic analysis. The fourth chapter is about the phytochemical investigation of M. tenacissima. Fourteen compounds were isolated and identified. Among them, 4 new compounds, tenacigenosides A~D, were characterized as 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-17β- tenacigenin B, 3-O-2,6-dideoxy-4-O-methyl-D-lyxo-hexopyranosly-11α-O-methyl butyryl-12β-O-acetyl-tenacigenin B, 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl- (1→4)-β-D-oleandropyranosyl-11α-O-tigloyl-tenacigenin C, and 3-O-6-deoxy-3-O- methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O-2-methylbutyryl- tenacigenin C. Chapter 5 is a review on recent progress in bioactive constituents from plants of Zanthoxylum species.

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本学位论文分为四个部分,第一部分报道了用串联质谱快速分析合成药物中的微量杂质成分以及分析中药材中的化学成分。第二部分报道了通过质谱和串联质谱发现并合成新型的PdPincer 催化剂,同时对其活性进行测试。第三部分为串联质谱自动解析软件的设计及应用。第四部分概述了应用在质谱中的各种碎裂方式。 第一部分首先总结了5-溴粉防己碱及其类似物的裂解规律,并以此为根据推测出2 个微量杂质的结构。随后针对无患子(Sapindus mukurossi Gatren.)中的皂苷成分,由ESI-QTOF 得到各个皂苷成份的高分辨质量数据进而得到其分子式,然后利用ESI-IT 电喷雾串联质谱对无患子总皂苷中各皂苷成分的结构进行进一步的鉴定。进而以同样的方式,先通过ESI-QTOF 得到黄山药(Dioscoreapanthaica)总皂苷中各个组分化合物的分子式,然后对已有的薯蓣皂苷标准品做串联质谱分析,以得到该类化合物的裂解规律并给出解析该类化合物的流程图。在此利用计算化学的方法讨论了离子的丰度与裂解活化能之间的关系。然后应用APCI-MS/MS 方法探讨了四对同分异构体和几个已知的化合物,并最后用液质联用对其进行确认,同时还给出了4 个未知化合物的可能结构。 第二部分报道通过质谱和串联质谱发现并合成新型的PdPincer 催化剂,同时对其活性进行测试。钯催化的交联反应是有机合成中C-C 键形成的最有效的方法,且硫脲是一类对空气和水都稳定的化合物,因此我们设计并合成了一系列的硫脲钯催化剂并得到了很好的催化活性。我们在对其中一类环状双硫脲化合物进行质谱实验的时候,在正离子模式下发现了反常的[M.H]+,通过串联质谱进一步确定了它是一种新型的PdPincer 结构。我们将其合成出来并通过X-ray 衍射实验确定了它的结构。同时测定其催化活性并与未形成pincer 的类似物进行比较发现该类化合物具有较宽的底物适用性。 第三部分为串联质谱自动解析软件的设计及应用。通过前面两部分的启示,独立设计开发了AuMass(1.0)。其算法是:先通过查找特殊的碎片离子,中性丢失或碎片离子质量差来确定某类化合物的骨架结构,然后利用该类化合物的自动解析流程来对其周边取代基进行确认。通过它快速地对白芍中的化学成分进行解析,并对未知的化合物进行了推测。为了增加它的解析能力,我又对其它类型的化合物裂解规律进行总结,并给出了自动解析流程。实践证明该软件具有相当好的应用价值。 第四部分综述了应用在质谱上的各类母离子的碎裂技术。这里包括了碰撞诱导裂解(CID)、光诱导碎裂(LID)、电子捕获裂解/电子转移裂解(ECD/ETD)、红外多光子解离(IRMPD)、黑体辐射解离(BIRD)和PQD 裂解技术。 This dissertation consists of four chapters. The first chapter reports the rapidanalysis of trace impurities from synthetical medicine and analysis of the chemicalconstitutents from Chinese herb medicines. The second chapter elaborates the studieson the discorvery and synthesis of new type of Pd Pincer catalyst by using MS andtandem MS together with the testing of its catalyst activity. The third chapter dwellson the designation and development of automatic tandem mass spectrometry analysissoftware. The last chapter presents a review on the dissociation technique of massspectrometry. The first chapter reports the rapid analysis of trace impurities from synthesismedicine and analysis of the chemical constitutents from Chinese herb medicines. The fission mechanism of 5-bromotetrandrine was obtained by analysis of the dissociationpathways of major product, by using which the possible structure of the two traceimpurties was assumed. There are lots of saponins in Sapindus mukurossi. Except forthe good spumescence and decontamination,it possesses the bioactivity of antigenand antitch. First of all, the high resolution mass information was obtained by ESI-QTOF. Hence the possible molecular formulars were acquired too. Then weconducted the further detection of the structures of its saponins by using ESI-ITtechnology. In the same manner, first the molecular formulars of every constituentfrom Dioscorea panthaica in total saponins were obtained by ESI-QTOF, and thenacquired the fission mechanism of this type of compounds by tandem massexperiment on a series of known and available saponins. In the same time, theanalysis flowchart was concluded. Here the relationship between the ion intensity andthe corresponding dissiociation activation energy was studied by computer chemistry.Then the four pairs of isomers were differentiated by APCI-MS/MS, as well as thecharacterization of known and unknown compounds. The assumption was confirmed by HPLC-MS/MS. Among them the possible structures of four unknown saponinswas presented. The second part was discovery and synthesis of a new type of Pd pincer catalystby MS and tandem MS. The coupling reaction catalyzed by Pd is the most effectivemethod in C-C formation in organic synthesis. Apart from that, thiourea is type ofcompounds that are stable to atmosphere and moisture. Hence we designed a series ofPd thiourea catalysts. Some of them show the excellent catalyst activity. The abnormalparent ion [M.H]+ was founded in positive ESI mode when we conduct some massspectrometry experiments on the bicyclical thiourea Pd complex. The structure wasproposed by mass and tandem mass spectrometry. Because it was a new type of pincer,we want to test its catalyst activity. So the Pd pincer was synthesized and the detailstructure was obtained by x-ray experiment. It shows the more fitness in catalysis ofSuzuki reaction by comparison with the analogue. The third chapter dwells on the design and development of automatic tandemmass spectrometry analysis software. Inspired by the former two chapters, theAuMass (version 1.0) was developed. Its algorithm is: first check the diagnostic ion,diagnostic neutral loss or diagnostic ions mass intervals in database to find out whatthe analyst’s skeleton belongs to, then identify the peripheral functional group by thecorresponding analysis flowchart. The chemical constituents of Paeonia lactiflorawere identified rapidly by using AuMass. To increase the analysis ability, the othertypes of compounds from Chinese herbs was concluded. Actually, the software isproven to have the much valuable application. The last chapter presented the review on the some kinds of fission technique ofmass spectrometry. It involves the collision induced dissociation (CID), laser induceddissociation (LID), electron capture dissociation/electron transfer dissociation(ECD/ETD), infrared multiple photons dissiociation, black body irraditiondissociation and PQD fission technique from Finnigan.

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Extending phenological records into the past is essential for the understanding of past ecological change and evaluating the effects of climate change on ecosystems. A growing body of historical phenological information is now available for Europe, North America, and Asia. In East Asia, long-term phenological series are still relatively scarce. This study extracted plant phenological observations from old diaries in the period 1834–1962. A spring phenology index (SPI) for the modern period (1963–2009) was defined as the mean flowering time of three shrubs (first flowering of Amygdalus davidiana and Cercis chinensis, 50% of full flowering of Paeonia suffruticosa) according to the data availability. Applying calibrated transfer functions from the modern period to the historical data, we reconstructed a continuous SPI time series across eastern China from 1834 to 2009. In the recent 30 years, the SPI is 2.1–6.3 days earlier than during any other consecutive 30 year period before 1970. A moving linear trend analysis shows that the advancing trend of SPI over the past three decades reaches upward of 4.1 d/decade, which exceeds all previously observed trends in the past 30 year period. In addition, the SPI series correlates significantly with spring (February to April) temperatures in the study area, with an increase in spring temperature of 1°C inducing an earlier SPI by 3.1 days. These shifts of SPI provide important information regarding regional vegetation-climate relationships, and they are helpful to assess long term of climate change impacts on biophysical systems and biodiversity.

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Sodium paeoniflorin sulfonate 2 was isolated from processed, but not unprocessed, Paeonia lactiflora roots and characterized by mass spectrometry and NMR spectroscopy. A notable and characteristic downfield shift in the H-1 NMR was observed for the hydrogens to the alkoxysulfonate moiety in 2 and in other model compounds. (c) 2005 Elsevier Ltd. All rights reserved.