【作者】 邓胜国；

【导师】 邓泽元；

【作者基本信息】 南昌大学 ， 食品科学， 2009， 博士

【摘要】 日益严重的化学合成物质对环境的污染和毒性问题,使得人们对植物天然产物的需求越来越关注。天然植物中的活性成分已成为人们研究的主要对象之一。荷叶属睡莲科植物莲（Nelumbo nucifera Gaerth）的叶片,是国家卫生部公布的即是食品又是药品的植物,主要分布于印度、中国、日本及埃及,价廉易得。荷叶味苦、气味清香,具有解暑清热,散瘀止血,降脂减肥及抗氧化作用等功效,是临床用于治疗咯血、鼻衄及尿血的特效药。研究表明荷叶的生物活性和生理功能主要与其含有的功能性成分生物碱及黄酮类化合物相关。目前国内外对荷叶的研究主要集中于生物碱,而对于荷叶黄酮类化合物的研究较少,影响了对荷叶活性成分的利用和开发。本文对荷叶主要功能性成分荷叶黄酮进行了较系统的研究,以期为荷叶功能食品的开发和深加工开辟出一条新途径,为新型药物的研究提供有益的指导,全面提高荷叶的经济利用价值。主要研究内容及所得实验结果如下:1.采用乙醇溶液提取法,通过正交实验优化提取荷叶黄酮的工艺条件。实验表明用60%的乙醇为溶剂来提取荷叶黄酮的最佳条件为75℃,2.0 h,液固比为40:1。荷叶黄酮的含量可达6.63%。2.通过静态吸附实验研究AB-8和H103两种大孔吸附树脂对荷叶黄酮的吸附特性,筛选出适合对荷叶黄酮进行初步分离纯化的树脂,并对其进行了动态吸附特性研究,结果表明AB-8大孔树脂对荷叶黄酮具有良好的吸附和解吸性能,经AB-8树脂吸附富集后,样品总黄酮含量可达36.73%。3.采用HPLC对经AB-8大孔树脂初步纯化后的荷叶黄酮进行分离分析,建立了荷叶黄酮高效分离及测定的最佳色谱分析条件:色谱柱为AgilentODS C18反相柱（250 mm×4.6 mm I.D.,5μm）,流动相A:乙腈,流动相B:超纯水/甲酸（99.9/0.1;v/v）,进样量为10μL,流速为0.8 ml/min,柱温为30℃,检测波长为254 nm,梯度洗脱条件为:0-20min,10%A;10-20min,10-20%A;20-35 min,20-30%A。4采用高速逆流色谱对荷叶黄酮苷单体进行纯化及制备,并通过IR,MS,1DNMR,2DNMR等现代仪器方法对其进行了结构鉴定。得到的四种荷叶黄酮苷单体为金丝桃苷、异槲皮苷、紫云英苷及首次在荷叶中发现的槲皮素-3-O-桑布二糖苷。实验建立了高速逆流色谱对荷叶黄酮苷单体进行纯化的条件:1)采用正己烷:醋酸乙酯:甲醇:水=1:5:1:5（v/v）组成的两相溶剂系统,转速为800rpm/min,柱温30℃,流速为1.8m.L/min及检测波长254 nm的色谱条件可从80 mg的经AB-8大孔树脂初步纯化后的荷叶黄酮中制备得到纯度经HPLC检测达到97.5%的金丝桃苷9.1 mg,95.8%的异槲皮苷4.6 mg,98.3%的紫云英苷3.0mg:2)采用醋酸乙酯:正丁醇:水=4:1:5（v/v）组成的两相溶剂系统,转速为800rpm/min,柱温30℃,流速为1.8m.L/min及检测波长254 nm的色谱条件可从100 mg的经AB-8大孔树脂初步纯化的荷叶黄酮中制备得到纯度经HPLC检测达到98.6%的槲皮素-3-O-桑布二糖苷5.0 mg。5.采用荧光光谱法、紫外光谱法研究在生理条件（pH=7.4）下荷叶黄酮及紫云英苷与牛血清白蛋白的相互作用。结果表明荷叶黄酮及紫云英苷均可与BSA结合并通过静态猝灭作用机制对BSA内源性荧光进行猝灭,在温度为298K时,测得其猝灭速率常数（Kq）分别为5.133×10¹³ L·mol^-1·S^-1,4.31×10¹³ L·mol^-1·S^-1;结合常数（K_d）分别12.97×10⁵ L·mol^-1,2.009×10⁵L·mol^-1,结合位点数（n）分别为1.07,0.943;而在温度为308K时荷叶黄酮与紫云英苷对BSA的猝灭常数分别为3.982×10¹³ L·mol^-1·S^-1,3.72×10¹³L·mol^-1·S^-1;结合常数（K_d）分别9.12×10⁵ L·mol^-1,0.927×10⁵ L·mol^-1,结合位点数（n）分别为1.06,0.893。荷叶黄酮与BSA间的作用力主要表现为疏水作用及氢键,而紫云英苷与BSA间的作用力主要表现为氢键和范德华力。6.在pH=7.4的Tris-HCl的缓冲溶液中,采用紫外及荧光光谱法研究荷叶黄酮及紫云英苷与脱氧核糖核酸之间的相互作用。探讨了离子强度和阴离子猝灭剂KI对荷叶黄酮-DNA体系及紫云英苷-DNA体系荧光强度的影响,同时分别考察了荷叶黄酮、紫云英苷及中性红与DNA结合的竞争性。结果表明DNA通过静态猝灭作用机制猝灭荷叶黄酮及紫云英苷的荧光,并测得其在298K时的结合常数（K_d）分别为8.204×10⁴ L·mol^-1,3.412×10⁴ L·mol^-1,结合位点数（n）分别为1.013,1.007:测得其在308K时与荷叶黄酮及紫云英苷的结合常数（K_d）为6.855×10⁴ L·mol^-1,1.762×10⁴L·mol^-1,结合位点数（n）分别为1.025,0.962。紫云英苷以嵌插模式与DNA进行结合,而荷叶黄酮与DNA之间的相互作用为沟槽及嵌插。7.采用DPPH法、碘量法（NaS₂O₃-I₂）测定和评价荷叶黄酬及四种荷叶黄酮苷（HSCCC分离纯化获得）的抗氧化效果。实验结果表明荷叶黄酮及荷叶黄酮苷均具有良好的清除DPPH自由基的能力,并能有效抑制猪油的氧化。更多还原

【Abstract】 Owning to the increasing serious question about the environmental pollution and toxicity caused by those chemical synthetic compounds, the demand for natural products of plant was paid more attention. The bioactive components of natural plant has been become one of important objects researched by people. The leaves of Nelumbo nuclfera Gaertn （family Nymphaceae） which is named Lotus leaf is a kind of plant proclaimed by national Ministry of Health, could be used in food and medicine field, distributed mainly in India, China, Japan and Egypt, inexpensive and received easily. The leaves of Nelumbo nucifera are bitter and aromatic, which are reported to resolve summer heat syndrome, eliminate stasis to activate blood circulation and stop bleeding, exhibit antihyperlipidemic, antiobesity and antioxidant effects, and has been also used as an effective drugs for hematemesis, epistaxis and hematuria in clinical intervention. The results of previous work showed that all biological activities and physiological functions of Lotus leaf are closely related to these two functional components including alkaloids and flavonoids present in the leaves. At present many studies mainly focus on alkaloids, only few investigations on flavonoids of Lotus leaf have been done, which bring negative effects of utilities and exploitation of active components in Lotus leaf.The flavonoids, the main functional components in Lotus leaf, have been researched in system in this paper, which will be expected to open up a new way for the development and deep processing of Lotus leaf, provide a useful guideline for investigation of new drugs, and enhance the commercial value of Lotus leaf in an all-round way. The main investigations and experimental results in this paper were listed as following, respectively:1. The process conditions for flavonoids from Lotus leaf extracted by alcohol solution were optimized by orthogonal experiment. The results showed that the optimum process conditions for flavonoids from Lotus leaf extracted by 60% ethanol: extracting temperature being 75℃, extracting time being 2.0 h, proption of solvent and material being 40:1. Under the condition the total flavonoids content of Lotus leaf was 6.63%.2. The adsorption characteristics of flavonoids from Lotus leaf on AB-8 and H103 macroporous resin were investigated by static adsorption experiments in order to choose one resin suitable for the preliminary separation and purification of those flavonoids. It was found that AB-8 resin had excellent adsorption and desorption capacity to the flavonoids from Lotus leaf. The dynamic adsorption characteristics of the flavonoids from Lotus leaf on AB-8 resin were also studied. 36.73%. of flavonoids could be obtained in the purified sample by AB-8 macroporous resin.3. Using high performance liquid chromatography （HPLC） method to analyze the flavonoids preliminarily cleaned up by AB-8 macroporous resin, the optimum HPLC condition which could efficiently separated and determined these flavonoids was obtained: immobile phase was reversed phase Agilent ODS C18 column （250 mm×4.6 mm, I.D., 5μm）, mobile phase A was acetonitrile, mobile phase B was Ultra-pure water/formic acid （99.9/0.1; v/v）, sample volume was 10μL, flow rate was 0.8 ml/min, temperature of column was 30℃, wavelength of detection was 254 nm, the linear gradient elution system was: 0～20min, 10% A; 10～20min, 10～20% A; 20～35 min, 20～30% A。4 The preparative separation and purification of flavonoid glycosides from Lotus leaf were studied by high-speed counter-current chromatography （HSCCC）. Four flavonoid glycosides, namely Hyperoside, Isoquercitrin, Astragalin and Quercetin-3-O-sambubioside, were isolated and their chemical structures were identified by IR, ESI-MS, 1D NMR and 2D NMR, and Quercetin-3-O-sambubioside was obtained from Nelumbo nucifera for the first time. The optimum HSCCC condition which could efficiently separated and determined these four flavonoid glycosides of lotus leaves was obtained: （1） A total of 9.1 mg of Hyperoside, 4.6 mg of Isoquercitrinand 3.0 mg of Astragalin from 80 mg of the flavonoids preliminarily cleaned up by AB-8 macroporous resin with the purity of 97.5 %, 95.8% and 98.3% determined by HPLC, respectively in one-step HSCCC separation by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water （1:5:1:5, v/v/v/v）, fixing revolution speed, separation temperature , flow rate and detection wavelength to be 800rpm/min, 30℃, 1.8m.L/min and 254 nm, respectively. （2） A total of 5.0 mg of Quercetin-3-O-sambubioside with the purity of 98.6% determined by HPLC were obtained from 100 mg of the flavonoids preliminarily cleaned up by AB-8 macroporous resin in one-step HSCCC separation with a two-phase-solvent system composed of ethyl acetate-n-butanol-water （4:1:5, v/v）, regulating revolution speed, separation temperature , flow rate and detection wavelength to be 800rpm/min, 30℃, 1.8m.L/min and 254 nm, respectively .5. The interaction between bovine serum albumin （BSA） and flavonoids and Astragalin from Lotus leaf in physicological condition （pH=7.4） was studied by fluorescence spectroscopy and ultraviolet absorption spectroscopy. The results demonstrated that flavonoids from Lotus leaf and astragalin could bind to BSA and quenched the intrinsic fluorescence of BSA through static quenching mechanism. The quenching rate constants of biomoleculer, the binding constants and the number of binding sites between flavonoids from Lotus leaf, astragalin and BSA were Kq = 5.133×10¹³ L·mol^-1·S-land 4.31×10¹³ L·mol^-1·S^-1, K_d = 12.97×10⁵ L·mol^-1 and 2.009×10⁵ L·mol^-1, n = 1.07 and 0.943 at 298 K , respectively. When temperature goes to 308K, these parameters between flavonoids from Lotus leaf, astragalin and BSA turns to Kq = 3.982×10¹³ L·mol-1·S^-1 and 3.72×10¹³ L·mol^-1·S^-1, K_d = 9.12×10⁵ L·mol^-1 and 0.927×10⁵ L·mol-1, n = 1.06 and 0.893, respectively. The interaction between BSA and flavonoids from Lotus leaf was driven mainly by hydrophobic interaction force and hydrogen bonds, but hydrogen bonds and van der Waals forces played a major role in the interaction between BSA and astragalin.6. The interaction between flavonoids and Astragalin from lotus leaf and deoxyribonucleic acid （DNA） in Tris-HCl buffer （pH=7.4） was investigated by the application of fluorescence spectroscopy and ultraviolet absorption spectroscopy, the effects of ionic strength and anion quencher KI on the fluorescence intensity of the system of DNA - flavonoids and DNA-Astragalin were explored, the competitive binding to DNA between flvonoids and Astragalin from lotus leaf and Neutral Red（NR） dye were also studied at the same time. The results demonstrated that flavonoids from Lotus leaf and astragalin could bind to DNA and the formed complex quenched their corresponding intrinsic fluorescence of flavonoids from Lotus leaf and astragalin through static quenching mechanism. The binding constants （K_d） of the reaction of flavonoids and Astragalin from Lotus leaf and DNA were computed to be 8.204×10⁴ L·mol^-1 and 3.412×10⁴ L·mol^-1, and the number of binding sites （n） were counted to be 1.013 and 1.007 between the two flavonoids and DNA at 298 K, respectively. When temperature goes to 308K, these parameters between flavonoids from Lotus leaf, astragalin and BSA turns to K_d = 6.855×10⁴ L·mol^-1 and 1.762×10⁴ L·mol^-1, n=1.025 and 0.962, respectively. When bound to DNA, flavonoids from Lotus leaf and astragalin showed hypochromic effect in the absorption spectra. The results showed that flavonoids from Lotus leaf could combine with DNA in the mode of intercalation, but Astragalin could combine with DNA in the mode of interaction and groove bindings.7. The anti-oxidative effects of flavonoids and flavonoids glycosides from Lotus leaf were measured and evaluated by using DPPH free radical scavenging and NaS₂O₃-I₂ methods, respectively. The results showed all the total flavonoids from Lotus leaves and the four flavonoids glycosides purified by HSCCC had strong capacity for scavenging DPPH free radical, and could effectively inhibit lard’s oxidation.更多还原