【作者】 李倩；

【导师】 赵军；

【作者基本信息】 新疆大学 ， 食品科学， 2017， 硕士

【摘要】 叉子圆柏(Juniperus sabina L.)系柏科圆柏属(Sabina mill)植物,是叉子圆柏的枝叶和果实,民间用于风湿免疫性疾病的治疗。本课题以叉子圆柏叶总黄酮有效部位为研究对象,采用单因素试验结合正交设计优化叉子圆柏总黄酮的提取纯化工艺;利用高速逆流色谱分离纯化总黄酮中标识性黄酮单体,并用高效液相、核磁共振等技术鉴定它们的结构;在制备工艺及化学成分研究基础上进行叉子圆柏总黄酮质量标准的研究,并起草了叉子圆柏总黄酮质量标准草案。主要研究结果如下:(1)建立了叉子圆柏叶中3种标识性成分的高效液相含量测定法。色谱条件:Phenomenex Gemini-NX C18色谱柱(250mm×4.6mm,5μm),乙腈(A)-0.2%磷酸水溶液(B)为流动相(梯度洗脱:0 min~2 min~20 min~25 min,19%A~20%A~24%A~19%A),柱温30℃,流速1.0 mL/min,检测波长360 nm。芦丁、异槲皮苷和槲皮苷有效分离,含量测定稳定。不同地区的叉子圆柏叶中3种黄酮苷含量差异较大,可为叉子圆柏叶药效物质的开发提供基础依据。(2)叉子圆柏总黄酮的最佳提取工艺条件:提取溶剂50%乙醇,料液比1:25,回流提取3次,每次提取1.5 h;总黄酮提取得率为7.47%。D101树脂对叉子圆柏总黄酮的吸附和解吸效果较好,其最佳纯化工艺条件为:上样浓度1.2256 mg/m L,上样流速1.0 mL/min,5 BV去离子水除杂,4 BV 50%乙醇洗脱,洗脱流速1.0mL/min。在此条件下,总黄酮纯化回收率为88.36%,纯度为69.96%。三批实验室放大研究结果稳定,说明此提取纯化工艺稳定、可行。(3)应用高速逆流色谱分离叉子圆柏总黄酮,得到3个黄酮类化合物,纯度均大于90%,结果分别为:异槲皮苷(91.89%)、槲皮苷(98.45%)、槲皮素-3-O-(6″-O-乙酰基)-β-D-吡喃葡萄糖苷(95.35%)。该方法快速简便,能够高效分离叉子圆柏中的黄酮类成分,并使纯度达到90%以上。(4)建立了叉子圆柏总黄酮的理化鉴别、薄层色谱鉴别方法;依据《中国药典》2015版初步限定总黄酮中水分不得超过7.5%,炽灼残渣不得过0.9%,重金属不得过百万分之十五,砷盐不得过百万分之零点五。叉子圆柏总黄酮的黄酮含量限度设为不低于50%;采用HPLC测定叉子圆柏总黄酮中标识性成分含量,初步将叉子圆柏总黄酮中3种标识性成分芦丁、异槲皮苷及槲皮苷的含量限度分别设为不小于21.4 mg/g、6.8 mg/g、17.7 mg/g。高温和强光对叉子圆柏总黄酮外观、色泽无明显影响,总黄酮含量和标识性成分含量无明显变化,性质稳定。更多还原

【Abstract】 Juniperus sabina L.belongs to Cupressaceae Juniperus plants,which is its branches,leaves and fruits and is used to treat rheumatic autoimmune disease.With tha study object of total flavonoids from Juniperus sabina L.,this paper studies that the extraction and purification process of total flavonoids from Juniperus sabina L.were optimized by single factor tests and orthogonal experiment design;identification flavonoids were separated from total flavonoids by high-speed counter-current chromatography(HSCCC),and their structures were identified by high performance liquid chromatography(HPLC)and nuclear magnetic resonance(NMR);on the base of the studies of preparation technology and chemical components,the quality standard of total flavonoids from Juniperus sabina L.was researched and drafted out.The conclusions were as follows:(1)The determination method of three characteric ingredients in Juniperus sabina L.leaves was established by HPLC,and chromatographic conditions were showed as follows: Phenomenex Gemini-NX C18 as chromatographic column(250mm×4.6mm,5μm),the acetonitrile-0.2% phosphoric acid as the mobile phase(0 min~2 min~20 min~25 min,19%A~20%A~ 24%A~19%A),the temperature was 30℃,the flow rate was 1.0 m L/min,and detection wavelength was 360 nm.Rutin,isoquercitrin and quercitrin were effectively separated with content determinations of stability.The three kinds of flavonoid glycosides contents from Juniperus sabina L.leaves were different.This method can provide base in order to effective substance development of Juniperus sabina L.(2)The optimum technological conditions for extracting total flavonoids from J.sabina(JSTF)were obtained as follows: ethanol concentration 50%,solid-to-liquid ratio 1:25,extraction time 1.5h and extraction times 3.Under these conditions,the extraction yield of JSTF was 7.47%.The optimal parameters for purified JSTF by D101 macroporous resin were as follows: the sample concentration of 1.2256 mg/m L,the sample flow rate of 1.0 m L/min,5 BV water consumption on removing impurity,eluting at 50% ethanol with 4 BV,desorption rate at 1.0 mL/min.Under these conditions,JSTF had a recovery rate of 88.36 % and a purity of 69.96 %.The experimental results of pilot amplification showed that extraction and purification process was stable and feasible.(3)Three compounds were separated from JSTF by HSCCC including isoquercitrin(91.89%),quercitrin(98.45%),quercetin-3-O-(6″-O-acetyl)-β-Dglucopyranoside(95.35%).Therefore,this method was fast and convient,and can effectively separate flavonoids in Juniperus sabina L.with the purity of 90% above.(4)The quality evaluation method of JSTF has been eatablished by the study of physical and chemical identification,thin-layer chromatography examination and content determination according to Chinese Pharmacopoeia(2015).The quality control of JSTF was expressed as follows: moisture content less than 7.5%,burn residue less than 0.9%,heavy metal less than 15 parts per million and arsenic salt was under 0.5 parts per million;total flavonoid content in JSTF was above 50%;the contents of rutin,isoquercitrin and quercitrin were not less than 21.4 mg/g,6.8 mg/g,17.7 mg/g.The appearance and color of JSTF were not obviously affected by the influence factors experiments(high temperature,high humidity and strong light),at same time the contents of JSTF and identify component has no change and stable character.更多还原