【作者】 张海艳；

【导师】 赵天增；

【作者基本信息】 郑州大学 ， 有机化学， 2006， 博士

【摘要】 天然产物化学的目的和方向是寻找活性化合物、活性化合物配伍组合及其合适的生产制作方法，本文就淫羊藿有效部位黄酮醇苷的化学成分、生产工艺和HPLC-NMR指纹图谱以及冬凌草甲素脂质体制作进行了深入研究。一、淫羊蒮应用基础研究淫羊藿为常用中药之一，味辛甘、性温，入肝、肾二经，具有补肾壮阳、祛风除湿之功效。淫羊藿主要含有8位具异戊烯基（及其衍生基团）取代的黄酮醇及其苷类化合物，实验证明其具有提高免疫系统功能、改善心脑血管系统功能、改善生殖系统功能等功效。国内外已在淫羊藿的化学成分、药理作用、药用植物新资源以及质量评价方面作了大量工作，但在淫羊藿有效部位系统工艺以及指纹图谱和化学成分NMR解析等应用基础方面作得工作较少；另外，目前仅对50余种淫羊藿属植物的20种进行了化学成分研究，而且实际上随着产地的不同，同种淫羊藿的次生代谢成分类型也会有较大的变化。因此，有必要继续对淫羊藿进行深入细致的应用基础研究。本论文对丹东产朝鲜淫羊藿有效部位的化学成分进行了研究。利用硅胶柱层析进行分离，根据化合物的光谱数据鉴定其结构。从朝鲜淫羊藿大孔树脂处理物中分离得到了8个化合物，分别鉴定为：3-羟基-2-甲基-4-吡喃酮、淫羊藿次苷Ⅱ、3″′-羰基-2″-β-L-奎诺糖基淫羊藿次苷Ⅱ、朝藿定K、淫羊藿苷、EpimedokoreanosideⅠ、朝藿苷A和3″′-羰基-2″-β-L-奎诺糖基淫羊藿苷，其中，3″′-羰基-2″-β-L-奎诺糖基淫羊藿次苷Ⅱ和3″′-羰基-2″-β-L-奎诺糖基淫羊藿苷为新化合物，其糖基（2″-β-L-奎诺糖基）为新骨架。3-羟基-2-甲基-4-吡喃酮为首次从该植物中分离得到。在此基础上对其特征提取物HPLC-NMR(¹³CNMR，¹HNMR)指纹图谱的建立方法进行了探讨，并推算出了HPLC-¹³CNMR和HPLC-¹H NMR计算公式，已知淫羊藿苷含量（HPLC测定），从定量¹³CNMR或¹HNMR可直接计算出其他主要成分的含量。本论文对日本三菱HP20和SP825大孔吸附树脂进行了优选，最终采用日本三菱HP20大孔吸附树脂和硅胶柱层析分离纯化朝鲜淫羊藿，并优化了HP20大孔吸附树脂和硅胶柱层析分离纯化朝鲜淫羊藿总黄酮的工艺条件及树脂前处理的方法；通过一步硅胶柱层析，可将有效部位中淫羊藿苷的纯度提高一倍以上。通过HP20大孔吸附树脂柱和硅胶柱，可得到不同收率和不同淫羊藿苷含量的系列产品。采用HPLC法测定双指标淫羊藿苷（主要成分）和淫羊藿次苷Ⅱ（较主要成分，保留时间最长）含量，建立了淫羊藿苷相关的HPLC-NMR指纹图谱(其中¹³CNMR定量指纹图谱为国内外首创)，对确保产品质量将发挥重要保障作用。朝鲜淫羊藿中成分极其复杂，但经过分离提纯的有效部位成分较纯，质量指标可控，量效关系明确，疗效稳定。本论文首次研究了光叶淫羊藿全草的化学成分，为阐明其有效成分提供根据。利用硅胶柱层析进行分离，根据化合物的光谱数据鉴定其结构。从光叶淫羊藿根、茎中分离得到了8个化合物，分别鉴定为：（7S，8R，8′R）-3，3′-二甲氧基-4，4′-二羟基-9-乙酰氧基-7，9-环氧木脂素、胡萝卜苷、淫羊藿次苷C、2″-鼠李糖大花淫羊藿苷A、淫羊藿苷、淫羊藿次苷A、朝藿定C和柔藿苷；从光叶淫羊藿叶中分离得到了4个化合物，分别鉴定为：川藿苷A、Thalictoside、Icarside D₂和山奈苷，所有12个化合物均为首次从该植物中分离得到。其中，化合物（7S，8R，8′R）-3，3′-二甲氧基-4，4′-二羟基-9-乙酰氧基-7，9-环氧木脂素纠正了原文献中的结构表达，本文还分别对该化合物和量大的抗HW活性化合物川藿苷A进行了¹H和¹³C MNR检测，通过DEPT和¹H-¹H COSY、HSQC、HMBC、NOESY等2DNMR技术对其所有的¹H和¹³C NMR信号进行了全归属和详细解析。本论文还对从不同种淫羊藿中分离得到的三种黄酮化合物（淫羊藿次苷、淫羊藿苷和朝藿定C）分别进行了¹H和¹³C MNR检测，通过DEPT和¹H-¹H COSY、HSQC、HMBC等2D NMR技术对其所有的¹H和¹³C NMR信号进行了全归属和详细解析；研究了这3种黄酮化合物连接的异同，综合了该类化合物的基本波谱学特征，着重补充了报道较少的糖取代基和糖上羟基的¹HNMR数据，总结了前期文献中未注意到的NMR数据规律，纠正了前期文献中存在的错误。二、冬凌草应用基础研究冬凌草系唇形科香茶菜属植物，味甘苦，性微寒，具清热解毒、消炎止痛、健胃活血及抗肿瘤之功效。冬凌草富含对映-贝壳杉烷类二萜化合物，其中冬凌草甲素为其最主要成分。冬凌草甲素具有较强的抗肿瘤活性，对多种移植性肿瘤有效，临床上用于食管癌、肝癌的治疗取得了一定的疗效。但由于冬凌草甲素水溶性差，生物半衰期短，到目前为止尚未研究出理想的靶向制剂。本文首先采用简单易行的工艺方法，对活性炭处理过乙醇提取物直接上硅胶柱，石油醚-丙酮系统梯度洗脱，再采用分步结晶法，即可得到大量纯度高的冬凌草甲素，大量冬凌草甲素的获得为下一步脂质体的制作打下了良好的基础。我们还在分离冬凌草甲素过程中得到了另外8个化合物，分别鉴定为：β-谷甾醇、齐墩果酸、熊果酸、冬凌草乙素、rabdoternin A、rabdoternin B、wikstroemioidin B和lasiodonin acetonide；其中，lasiodonin acetonide为新的对映-贝壳杉烯型二萜化合物。本文采用了国际上先进的脂质体制备技术和分析方法（依照Hope等发表的挤压法制备大单室脂质体），成功开发出包封率高、稳定性好的长效冬凌草甲素脂质体（包封率98％以上，平均粒径102nm）。长效脂质体既保留了大单室脂质体在血液中的长时效，高载药量，高稳定性等特点，又解决了普通脂质体易从体循环中被肝、脾巨噬细胞迅速清除的缺点，可以使脂质体在血液中保留较长一段时间。药理实验证明长效冬凌草甲素脂质体对实验性小鼠肉瘤S180具有显著的抑制作用。更多还原

【Abstract】 The aim of natural products chemistry is to discover new biologically activecompounds, to combine biologically active compounds and look for suitable productionmethod. In this thesis, we we made a thorough pharmaceutical-chemistry investigationand study on the chemical constituents, production method and HPLC-NMRfingerprints of the flavonol glycoside active fractions of Epimedium, together with thethe production method of oridonin encapsulated in long-circulating liposome.1. the applied basal studies of Epimedium plantsThe aerial parts of Epimedium plants （Berberidaceae）, which have long been usedas a tonic, aphrodisiac and antirhermatic in chinese medicinal medicine under the name"yinyanghuo", have been shown to be rich of flavonoid glycosides with aγ,γ-dimethylallyl group. Much work has benn done on the chemical constituents,pharmacological effects, new medicinal plant resources and quality assessment ofEpimedium plants. But few work has benn done on the systemic production method,active fraction fingerprints and completely NMR assignment of chemical constituents.Moreover, only 20 of the toally 50 species have been studied and the chemicalconstituents of the same speciess may be biggishly changed with the change ofproducing area. So it is necessary to make continuously applied basal study on the theEpimedium plants. Our short-term achievements are established as follows:Two novel flavonol glycosides, 3"’-carbonyl-2"-β-L-quinovosyl icarisideⅡand3’"-carbonyl-2"-β-L-quinovosyl icariin, together with known flavonol glycosides,icarisideⅡ, icariin, epimedin K, epimedo-koreanoside I, caohuoside A, was isolatedfrom the flavonol glycoside active fractions of the aerial parts of Epimedium koreanum（producing area: Dandong, Jilin, China）. The structrue of the first new compound waselucidated as 4’-Methylether, 3-O-[3-one-β-L-quinovosyl-（1→2）-α-L-rhamno-pyranoside], 5,7-dihydroxy-2-（4-hydroxyphenyl）-8-（3-methyl-2-butenyl）-4H-1-benzopyran-4-one. The structrue of the second new compound was elucidated as 4’-Methylether, 3-O-[3-one-β-L-quinovosyl-（1→2）-α-L-rhamnopyranoside], 7-O-D-glucopyranoside, 5,7- dihydroxy-2-（4-hydroxyphenyl）-8-（3-methyl-2-butenyl）-4H-1-benzopyran-4-one.On this basis, we discussed the establishment of HPLC-NMR(¹³CNMR,¹HNMR) fingerprints of active fractions and reckoned （deduced） thecomputing formula of active fractions. Knowing the quantitative content of icariin, wecan directly calcaluted the the quantitative content of other main compounds from thequantitative ¹³CNMR or ¹HNMR fingerprints.After making optimum choice between Diaion HP20 and Sepabeds SP825macroporous absorption resin, we finally selected HP20 macroporous absorption resinand cc （silica gel） to isolate and purificate Epimedium koreanum and optimized thetechnological conditions of the whole isolation and purification process together withthe former treatment method. Using only one extraction solvent （EtOAc: 95％EtOH10:1）, the icariin content in the silica gel active fractions can be heightened to two ormore times than that in the resin active fractions. Through macroporous absorption resinand silica gel, we gained series products of diffient recovery ratios and different icariincontents. Using HPLC methods, the content of the two selected standards, icariin（themain compound） and icarisideⅡ（one of the lesser compounds which had the longestretention time）, was exactly determained. On all this basis, the HPLC-NMR fingerprintsin relation to icariin was establisted, among which the ¹³CNMR quantitative fingerprintswas established for the first time in China.Epimedium sagittatum Maxim. Var. glabratum T. S. Ying is a species with a closerelationship with Epimedium sagittatum which is an important medical species. Itsmedical use has not been officially developed and its chemical constituents has not beenstudied to date. Studies on the chemical constituents of rhizome of this plant resulted inthe isolation of eight known compounds, （7S,8R, 8’R）-3,3’-dimethoxy-4,4’-dihydroxy-9-acetoxy-7,9-exopylignan, daucosterol, epimedoside C, 2"-O-rhamnosyl icariside A,icariin, epimedoside A, epimedin C and rouhuoside. Studies on the chemicalconstituents of leaves of this plant resulted in the isolation of four known compounds,sutchnenoside A, thalictoside, icariside D₂ and kaemferitrin. The stereo-structure of（7S,8R, 8’R）-3,3’-dimethoxy-4,4’-dihydroxy-9-acetoxy-7,9-exopylignan in original literature was wrong and too little NMR data was offered. Sutchuenoside A was anflvone which was found to have inhibitory effects on three different activities of HumanImmunodeficiency Virus-1 Reverse Transcriptase, its sturcture have not completelyassigned. Using a combination of 1D and 2D NMR techniques, their ¹H and ¹³C NMRspectra were completely assigned and analyzed.IcarisideⅡ, icariin and epimedin C were three important flavonoid glycosidesisolated from different Epimedium speices. Using a combination of 1D (¹H, ¹³C NMRand DEPT) and 2D NMR （¹H-¹H COSY, HSQC and HMBC） techniques, their ¹H and¹³C NMR spectra were completely assigned and analyzed. We studied the difference andsimilarity of the linkage of the three flavonoid glycosides, synthesized the basic NMRcharacter of flavonoid glycosides with aγ,γ-dimethylallyl group at 8 position, made upthe ¹HNMR data of sugar groups and the hydroxy of the sugar groups, summarized theNMR character which had not been noticed in earlier literatures and corrected thewrong data existed in earlier literatures.2. the applied basal studies ofRabdosia rubescensRabdosia rubescens, commonly known as "Dong Ling Cao" in China, is a traditionalChinese medicine used as remedies for various diseases. Oridonin, an ent-kauranediterpenoid first isolated from Isodon trichocarpus,is an main active component ofRabdosia rubescens. Oridonin has various pharmacological and physiological effects suchas anti-inflammation, anti-bacteria and anti-tumor effects, and has been used for thetreatment of human cancers, especially esophageal carcinoma and prostate carcinoma.But because of its poor watersolubility, perfect targeted drug delivery system has not beenexcogitated.Firstly, we use a simple method to achieve much oridonin. After decolourized byactive charcoal, the EtOH extraction of dried and powdered leaves was directlysubmitted to cc （silica gel） eluting with increasing proportions of Petroleumether-Me₂CO to stepply give a large number of oridonin crystals. During this process,other 8 compounds,β-sitosterd, Oleanolic acid, ursolic acid, ponicinin, rabdoternin A,rabdoternin B, wikstroemioidin B and lasiodonin acetonide were isolated. In which lasiodonin acetonide was a new compound and its structure was determined as 1α, 6β,7β,11β-tetrahydroxy-cyclic, 1,11-（1-methyl-ethylidene acetal）-7,20-epoxy-Kaur-16-en-15-one by 1D and 2D NMR techniques.Secondly, using an advanced method （proposed by Hope M J, et al.）, oridoninlong-circulating liposome was successfluly developed. The efficiency of oridonin inliposome was more than 98％and the mean particle size of oridonin long-circulatingliposome was 120nm. The results of animal experiments showed that oridoninlong-circulating liposome has higher antitumoral activity and lower toxicity than themasculine standard更多还原