【作者】 陶巧凤；

【导师】 赵昱；

【作者基本信息】 浙江大学 ， 药物分析学， 2010， 博士

【摘要】 天然产物研究已经成为发现创新药物的重要来源之一。本文在本实验室前期工作基础上,对我国云南省罗平产生姜的化学成分进行了进一步研究,并对13种生姜化学成分的抗氧化和细胞毒性等进行了评价;另外,对16种2,3-脱氢水飞蓟宾系列结构修饰物的抗氧化活性等进行了研究。一、生姜中化学成分的鉴定和生物活性评价生姜为姜科植物Zingiber officinale Roscoe的根茎,在全世界作为药用的历史悠久,其在我国中药处方中也经常在列。生姜主要用于治疗关节炎、风湿病、呕吐、十二指肠和胃溃疡、扭伤、肌肉痛、疼痛、喉咙痛、便秘、消化不良、高血压、老年痴呆、发热、感染性疾病和蠕虫病等。生姜的植物化学研究已经鉴定出许多具有生物活性的化合物,如姜辣素、姜二酮类、姜烯酚和二苯基庚烷类等;其中二苯基庚烷类化学成分具有抑制前列腺素和白三烯合成生物的活性,以及抗真菌、抗氧化和预防癌症等生物活性。通过对云南省罗平产生姜的深入植物化学研究,采用色谱技术分离和纯化了14个化合物;应用核磁共振波谱(¹H NMR,¹³CNMR和DEPT)、质谱（EI-MS,ESI-MS & HR-ESI-MS）、红外光谱、元素分析、旋光测定等技术,鉴定了所获得化合物的结构,其中发现3个二苯基庚烷类和1个单萜类新化合物,分别为5-[4-羟基-6-（4-羟基苯乙基）四氢-2H-吡喃-2]-3-甲氧苯基-1,2-二醇（5-[4-hydroxy-6-（4-hydroxyphenethyl）tetrahydro-2H-pyran-2-y1]-3-methoxybenzene-1,2-diol）、一对对映体:（E）-7-羟基-1,7-双（4-羟基苯基）庚烷-5-烯-3S-磺酸钠（Sodium（E）-7-hydroxy-1,7-bis（4-hydroxyphenyl）hept-5-ene-3S-sulfonate）和（E）-7-羟基-1,7-双（4-羟基苯基）庚烷-5-烯-3R-磺酸钠（sodium（E）-7-hydroxy-1,7-bis（4-hydroxyphenyl）hept-5-ene-3R-sulfonate）、羟基桉-10-O-β-D葡萄糖苷（Hydroxycineole-10-O-β-D-glucopyranoside）。应用清除超氧阴离子自由基和抑制肝微粒体中过氧化脂质的形成之体外模型,对我们实验室分离获得的化合物的抗氧化活性进行了评价。为了考察这些化合物在更接近生理条件下的抗氧化特性,应用大鼠的肝脏分离原代培养细胞模型,进一步检测了化合物抑制叔丁基过氧化氢引起氧化损伤的活性。为了评价化合物的抗肿瘤活性,在9种体外培养肿瘤细胞株上,测定了它们的细胞毒性。这9种肿瘤细胞株包括KB细胞,BEL7404细胞,P388D1细胞,K562细胞,HL-60细胞,A549细胞,Hela细胞,CNE细胞,及PC-3细胞。在供试验的化合物中,大部分化合物对吩嗪甲基硫酸盐-NADH系统显示出强效的超氧阴离子自由基的清除能力。在更接近生物体条件的系统,这些化合物在鼠肝微粒体暴露于氧化条件之试验中,显示出对肝脏脂质过氧化,具有强的保护作用。另外,我们还发现这些化合物对原代培养大鼠肝细胞接触氧化损伤具有细胞保护活性。二苯基庚烷类化学成分在上述所有实验中,显示出有效的抗氧化生物活性。另一方面,实验也表明:这些化合物多数对肿瘤细胞不具备明显的细胞毒性。二、脱氢水飞蓟宾衍生物的抗氧化生物活性研究2,3-脱氢水飞蓟宾（2,3-Dehydrosilybin,DHS）,一种天然水飞蓟宾的衍生物,存在于水飞蓟中的浓度相当低。不象水飞蓟宾,DHS的生物学研究非常有限,部分原因是其产率低和水溶性差（即低于水飞蓟宾）。然而,最近的研究报告表明,DHS具有非常强的抗氧化活性,其活性高于水飞蓟宾。因此,我们实验室对DHS进行了一系列结构修饰,本文对所有合成的16个DHS衍生物,以及水飞蓟宾和DHS的药理学活性进行了研究,包括2,2-二苯基-1-苦基肼（DPPH）、O₂^-氧自由基清除试验和抗脂质过氧化抑制试验、对抗过氧化氢诱导PC12细胞毒性的神经保护试验,以期获得更全面的有关DHS衍生物在体外对抗氧化损伤所造成的神经元损伤的有效性数据。我们还采用亚铁离子(Fe²⁺)螯合模型评价DHS类似物的生物学性质,以确定不同模型DHS类似物在AD和PD治疗中的抗氧化活性。研究结果表明,极性强的化合物,如C7-OH和C20-OH双醚以及C7-OH单醚具有脂肪族取代的乙酰胺,具有非常好的抑制脂质过氧化活性和Fe²⁺离子的螯合效价。相反,C7-OH和C20-OH双-烯丙基醚对过氧化氢诱导损伤PC12细胞的保护作用比槲皮素还要强。总体而言,更亲脂的DHS烯烃取代类似物是比乙酰胺取代的衍生物更好的神经保护剂。本研究结果将有助于优化黄酮木脂素类的结构,提高治疗潜力,特别是研发神经退行性疾病治疗药物,如,老年痴呆症（AD）和帕金森氏症（PD）。更多还原

【Abstract】 Analysis and Evaluation of Chemical Ingredients in Zingiber officinale Roscoe and Silybin DerivativesIt has become an important source of innovative medicines to discovery new drugs via natural products research.In this paper,based on preliminary work in our laboratory, the chemical constituents of Ginger from Luoping county of China’s Yunnan Province were further studied,and the antioxidant activities and cell toxicity for 13 chemical constituents from ginger were evaluated.In addition,the antioxidant activities of 16 dehydrosilybin derivatives with structural modification were studied.1.Identification and biological activity evaluation of chemical constituents in Ginger, the rhizomes of Zingiber officinale Roscoe（Zingiberaceae）,has a long history as a medicinal in the world and was also often listed in traditional Chinese medicine prescriptions.Ginger is mainly used for treatment of arthritis,rheumatism,vomiting, duodenal and gastric ulcers,sprains,muscle pain,pain,sore throat,constipation, indigestion,hypertension,senile dementia,fever,infectious diseases and worms diseases.Chemical studies of this plant species have led to the isolation and identification of numerous biologically active compounds such as gingerols,gingerones, shogaols and diarylheptanoids,etc.The diarylheptanoids,exhibiting a variety of biological activities including inhibition of the biosynthesis of prostaglandins and leukotrienes as well as antifungal,antioxidative,and cancer chemopreventive activities. Fourteen compounds were separated and purified by using chromatographic techniques from the MeOH extract of Z.officinale collected in Yunnan Province,Southwestern China.Three new diarylheptanoids,one new monoterpenoid,and other known diarylheptanoids were structurally identified by using NMR(¹H NMR,¹³C NMR and DEPT),MS（EI-MS,ESI-MS＆HR-ESI-MS）,infrared spectrometry,elemental analysis,optical measurement,etc.The new compounds are reported as: 5-[4-hydroxy-6-（4-hydroxyphenethyl）tetrahydro-2H-pyran-2-yl]-3-methoxybenzene-1, 2-diol,sodium（E）-7-hydroxy-1,7-bis（4-hydroxyphenyl）hept-5-ene-3S-sulfonate, sodium（E）-7-hydroxy-1,7-bis（4-hydroxyphenyl）hept-5-ene-3R-sulfonate, hydroxycineole-10-O-β-D-glucopyranoside.The antioxidant activities of the isolated compounds in our laboratory were evaluated by the in vitro models of scavenging superoxide anion radicals and inhibiting the formation of lipid peroxides in liver microsomes.To investigate the antioxidant properties of these compounds under more physiological conditions,the compounds were further tested in a primary culture of hepatocytes isolated from rat liver against the oxidative damage of tert-butyl hydroperoxide.In order to evaluate the anticancer activity,the compounds were tested the cell toxicities on 9 cancer cell lines,including KB cell,BEL7404 cell,P388D1 cell,K562 cell,HL-60 cell,A549 cell,Hela cell,CNE cell,and PC-3 cell.Among the tested compounds,most of compounds exhibited strong superoxide anion radical scavenging activities in a phenazine methosulfate-NADH system.In a more biological system,these compounds were demonstrated to exhibit potent protection against lipid peroxidation in mouse liver microsomes exposed to oxidative conditions.These compounds were subsequently tested on primary cultures of rat hepatocytes exposed to oxidative damage,and definitive cytoprotective actions were found.The diarylheptanoids were found to possess potent antioxidant properties in all the above assays.On the other hand,most of these compounds did not possess any appreciable cytotoxiciy on cancer cells.2.Study on the antioxidant biological activity of dehydrosilybin derivatives2,3-Dehydrosilybin（DHS）,a natural derivative of silybin which presents at very low concentrations in Silybum marianum（L） Gaertn.Not as silybin,DHS’s biological research is very limited,partly because of its low yield and poor water-soluble（ie,less than the silybin）.However,a recent report shows that DHS has a very strong antioxidant activity,its activity is higher than silybin.Therefore,our laboratory conducted a series of structural modification on the DHS.So the pharmacological activity of 16 DHS synthetic derivatives,as well as silybin and DHS has been studied in this thesis.The In vitro models used included 2,2-diphenyl-1-picrylhydrazyl（DPPH） and O₂^- free radical scavenging assays,and an inhibition test against LPO.The neuroprotective evaluation of the compounds against H₂O₂-induced toxicity of PC12 cells were performed in order to obtain more comprehensive information of the DHS derivatives’ in vitro efficacy against neuronal injury caused by oxidative damage.We also use ferrous ions(Fe²⁺) chelating model to evaluate the biological nature of the DHS analogues to determine the different models DHS analogues in the treatment of AD and PD potential antioxidant activity.The study revealed that the more polar compounds, the di-ether at C7-OH and C20-OH as well as the mono-ether at C7-OH which possess aliphatic substituted acetamides,demonstrated excellent inhibition against LPO as well remarkable chelating potency on Fe²⁺ ions.Conversely,the di-allyl ether at C7-OH and C20-OH was more potent in protection of PC12 cells against H₂O₂-induced injury than DHS and quercetin.Overall,the more lipophilic alkenylated DHS analogs were better performing neuroprotective agents than the acetamidated derivatives.The results in this study would be beneficial for optimizing the therapeutic potential of lignoflavonoids,especially in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease.更多还原