【作者】 郭鹏；

【导师】 潘超美；

【作者基本信息】 广州中医药大学 ， 中药学， 2019， 硕士

【摘要】 牛大力为蝶形花科植物美丽崖豆藤(Millettia speciosa Champ.)的干燥块根。具有补虚润肺,强筋活络的功效。临床上用于治疗腰肌劳损,风湿性关节炎,肺热,肺虚咳嗽,肺结核,慢性支气管炎,慢性肝炎,遗精,白带等症。目的:本课题旨在对南药牛大力药材的质量要求进行系统研究,为牛大力药材质量标准的建立提供依据。方法:1、运用植物分类学方法、中药鉴定显微方法、薄层色谱法等方法,对不同产地、不同生长年限的牛大力药材进行综合的观察分析。2、按照《中国药典》上的含量指标检测要求,运用烘干法测定了牛大力药材样品的水分;用马弗炉测定牛大力药材样品的总灰分和酸不溶性灰分;用浸泡法和加热回流法测定了牛大力药材样品中热水浸出物、热醇浸出物、冷水浸出物和冷醇浸出物含量。使用电感耦合等离子体质谱(ICP-MS)法对牛大力样品中的五项重金属含量进行测定,并对五项重金属污染指数做计算。3、运用响应面曲线法探究牛大力中总黄酮的最佳提取方法,运用硝酸铝显色法测定不同批次牛大力样品中总黄酮的含量,测定五种不同年份牛大力中总黄酮的含量,以及测定牛大力根、老叶、新叶、枝条和花五个不同部位中的总黄酮含量。运用苯酚-硫酸法测定不同批次牛大力样品中总多糖的含量,测定五种不同年份牛大力中总多糖的含量,以及测定牛大力根、老叶、新叶、枝条和花五个不同部位中总多糖含量。4、采用高效液相色谱法对不同批次牛大力样品中刺桐碱、槲皮素、芒柄花素和高丽槐素进行含量测定,测定五种不同年份牛大力中以上四种成分的含量,以及测定牛大力五个不同部位中以上四种成分的含量。5、采用高效液相色谱法对27批牛大力样品进行HPLC指纹图谱研究,建立了特定提取条件下牛大力的指纹图谱;通过中药指纹图谱相似度评价系统,得出HPLC指纹图谱共有峰,求出27组牛大力样品的相似度。6、使用氨基酸测定仪测定牛大力样品中十六种氨基酸含量;使用电感耦合等离子体质谱(ICP-MS)测定牛大力样品中十种微量元素含量;利用SPSS对氨基酸总量和微量元素总量进行聚类分析,评估牛大力中氨基酸的含量及微量元素含量蕴藏情况。结果:1、获得了牛大力根横切面和粉末显微特征,得到了以芒柄花素和高丽槐素为对照的薄层色谱显色条件,三氯甲烷-乙酸乙酯-乙酸-石油醚(7:3:0.5:1)为展开剂。2、确定牛大力药材的含水量,建议含水量不超过11.0%。确定牛大力药材的总灰分含量,建议总灰分含量不超过6.50%。确定牛大力药材的酸不溶性灰分含量,建议酸不溶性灰分含量不超过0.55%。确定牛大力药材四种浸出物含量范围,建议热水浸出物含量不低于11.0%,热醇浸出物含量不低于4.50%,冷水浸出物含量不低于,7.50%,冷醇浸出物含量不低于2.00%。确定牛大力药材五项重金属引起的综合污染指数范围。3、总黄酮最佳提取条件为,温度为40°C,乙醇浓度为60%,物料比为1:25,提取时间为30min。总黄酮含量在0.042%～0.551%之间。五种年份中3年生牛大力总黄酮含量最高。老叶的总黄酮含量最高,为2.508%。牛大力中总多糖含量的最佳提取条件为,温度为55℃,提取时间为30min,料液比为1:20。牛大力药材的总多糖含量在3.13%～8.64%之间。3年生牛大力总多糖含量最高。花的总多糖含量最高,为6.88%。4、牛大力药材中刺桐碱的含量在0.081g/kg到0.358g/kg之间。牛大力中槲皮素的含量在0.232mg/kg到31.037mg/kg之间。牛大力中芒柄花素的含量在1.612mg/kg到263.785mg/kg之间。牛大力中高丽槐素的含量在0.379mg/kg到246.97mg/kg之间。五种年份中2年生牛大力刺桐碱含量最高,为0.358g/kg。3年生牛大力槲皮素、芒柄花素和高丽槐素的含量最高,分别为17.830mg/kg、37.247mg/kg、87.715mg/kg。枝条的槲皮素含量最高,为17.384mg/kg,花朵的芒柄花素含量最高,为5.475mg/kg。而高丽槐素只有根茎和新叶中有,新叶中含量还高于根茎。而刺桐碱在老叶、新叶、枝条和花朵中不存在,只存在于根茎中。5、27批牛大力样品种,有21个样品的相似度在9.0以上,6个没有达到9.0;有共有峰22组,其中4组有对照品可以识别。6、氨基酸聚类分析结果为:Ⅰ组为S4、S17、S10、S27、S30、S13、S19、S15、S20、S29。Ⅱ组为 S6、S28、S9、S14。Ⅲ组为 S5、S7、S22、S1、S11、S24、S23、S18、S21、S16、S8、S12、S2、S3、S25、S26。微量元素聚类分析结果为:Ⅰ组为S3、S7、S23、S2、S5、S1。Ⅱ 组为 S27。Ⅲ组为 S17、S19、S16、S18、S20、S29、S6。Ⅳ组为 S4、S26、S10、S11、S9、S25、S8、S12、S15、S24、S14、S28、S13、S21、S22、S30。结论:1、牛大力根的横切面及显微构造特征可以作为牛大力药材的主要鉴别依据。2、牛大力药材的水分、总灰分、酸不溶性灰分、浸出物和重金属可以作为其质量评判优劣的主要参考依据。3、以芦丁为对照品,使用硝酸铝显色法测定特定提取条件下牛大力总黄酮的方法是可行的,且牛大力总黄酮可以作为牛大力质量优劣的评价指标;以葡萄糖为对照品,使用苯酚-硫酸法测定特定提取条件下用特定处理方法得到的牛大力总多糖的方法是可行的,且牛大力总多糖可以作为牛大力质量优劣的评价指标。4、刺桐碱、槲皮素、高丽槐素和芒柄花素等指标成分可以作为牛大力药材的质量优劣的评价指标。5、此种提取条件下牛大力HPLC指纹图谱的相似度较为良好。6、对牛大力氨基酸和微量元素的聚类分析表明牛大力中的氨基酸总量随生长年限的增加而增加,且外观性状越好,氨基酸总量越多。更多还原

【Abstract】 Millettia speciosa Champ.is a plant of the Millettia genus belonging to the Leguminosae family.It has the effect of mending the lungs and activating the strong gluten.Mainly used for lumbar muscle strain,rheumatoid arthritis,and cure lung fever,lung deficiency cough,tuberculosis,chronic bronchitis,chronic hepatitis,spermatozoa,Leucorrhea and so on.Objective:The purpose of this project is to systematically study the quality standards of the southern drug Millettia speciosa,and to formulate a strong quality evaluation method for cattle.Methods:1、The base source,characters,microscopic characteristics and TLC identification of Millettia speciosa were carried out in-depth and meticulous identification of raw medicines.2、According to a series of requirements in the Chinese pharmacopoeia,the water content,total ash content,acid insoluble ash and four kinds of leaching objects of different batches of Millettia speciosa were measured.The content of five heavy metals was measured in the above different batches of Millettia speciosa samples,and five heavy metal pollution indices were calculated.In addition,more than five years of Millettia speciosa detection items,as well as the comparison of Millettia speciosa five different parts of the above detection items.3、The optimum extraction method of total flavonoids from Millettia speciosa was investigated by using the RSM,and the content of total flavonoids in different batches of Millettia speciosa samples was determined by the chromogenic method,and the content of total flavonoids in five different years was determined.and compare the total flavonoids content in five different parts of Millettia speciosa.The content of total polysaccharide in different batches of Millettia speciosa samples was determined by phenol-sulfuric acid method,and the content of total polysaccharide in five different years of Millettia speciosa.and compare the total polysaccharide content in five different parts of Millettia speciosa.4、The content of Paulownia,Quercetin,Fermononetin and Maackiain in different batches of Millettia speciosa samples was determined by HPLC,and the content of four kinds of ingredients in five different years of Millettia speciosa was determined.And the content of more than four components in five different parts of Millettia speciosa were determined by comparison.5、The fingerprint of 27 batches of Millettia speciosa was studied,and the fingerprint of Millettia speciosa was established under specific extraction conditions.Through fingerprint,22 groups of common peaks were obtained,and the similarity of 27 groups of cattle in large samples was calculated.6、The content of 16 amino acids and 10 kinds of trace elements in different batches of Millettia speciosa samples were determined,and the corresponding results were analyzed and studied.At the same time,the content of 16 amino acids and trace elements in five different years were determined,the content of amino acids and the content of trace elements in five different parts of Millettia speciosa were determined,and the content of amino acids and the content of trace elements in Millettia speciosa were evaluated by SPSS analysis of the above experimental data.Results:1、The basis for the identification of Millettia speciosa pharmacognosy was determined.2、To determine the water content of Millettia speciosa medicinal materials,it is recommended that the water content does not exceed 11%.To determine the total ash content of Millettia speciosa medicinal herbs,it is suggested that the total ash content should not exceed 6.5%.To determine the acid insoluble ash content of Millettia speciosa medicinal materials,it is suggested that the content of acid insoluble ash should not exceed 0.55%.To determine the content range of four kinds of leaching objects of Millettia speciosa medicinal materials,it is suggested that the content of hot water leaching is not less than 11%,the content of hot alcohol leaching is not less than 4.5%,the content of cold water leaching is not less than 7.5%,and the content of cold alcohol leaching is not less than 2%.To determine the range of comprehensive pollution index caused by five heavy metals of Millettia speciosa medicinal materials.3、The optimum extraction condition of total flavonoids was 40℃,the ethanol concentration was 60%,the material ratio was 1:25,and the extraction time was 30min.The total flavonoids content is between 0.042%～0.551%.The total flavonoids content of 3 years of raw cattle was the highest.The total flavonoids content of the old leaves was the highest,at 2.508%.The optimum extraction condition of total polysaccharide content in Millettia speciosa is that the temperature is 5℃,the extraction time is 30min,and the material liquid ratio is 1:20.The total polysaccharide content of Millettia speciosa medicinal herbs is between 3.13%～8.64%.The total polysaccharide content of 3 years of raw cattle was the highest.The total polysaccharide content of flowers was the highest,at 6.88%.4、The content of paulownia in Millettia speciosa medicinal is between 0.081g/kg and 0.358g/kg.The content of quercetin in Millettia speciosa medicinal is between 0.232mg/kg and 31.037mg/kg.The content of Fermononet in Millettia speciosa medicinal is between 1.612mg/kg and 263.785mg/kg.The content of Maackiain in Millettia speciosa medicinal is between 0.379mg/kg and 246.97mg/kg.The content of 2-year-old Millettia speciosa paulownia was the highest,0.358g/kg.The contents of quercetin,fermononetin and maackiain were the highest in 3 years,respectively,17.830mg/kg,37.247mg/kg and 87.715mg/kg.The content of quercetin in the branches was the highest,17.384mg/kg,and the flowers had the highest content of the fermononetin,which was 5.475mg/kg.and maackiain only in the rhizome and new leaves,the content of the new leaves is higher than the rhizome.The paulownia does not exist in the old leaves,new leaves,branches and flowers,but only in the rhizome.5、27 batches of Millettia speciosa sample similarity in more than 9.6、The results of amino acid clustering analysis were as:The Ⅰ group was S4,S17,S10,S27,S30,S13,S19,S15,S20,S29.The Ⅱ group is S6,S28,S9,S14.The Ⅲ group is S5,S7,S22,S1,S11,S24,S23,S18,S21,S16,S8,S12,S2,S3,S25,S26.The results of trace element clustering analysis are as:Ⅰ group is S3,S7,S23,S2,S5,S1.The Ⅱ group is S27.Ⅲ group is S17,S19,S16,S18,S20,S29,S6.The Ⅳ group is S4,S26,S10,S11,S9,S25,S8,S12,S15,S24,S14,S28,S13,S21,S22,S30.Conclusion:1、The transverse and microscopic tectonic features of Millettia speciosa roots can be used as the main identification basis for Millettia speciosa medicinal materials.2、The moisture,total ash,acid insoluble ash,leaching and heavy metals of Millettia speciosa medicinal materials can be used as the main reference basis for their quality evaluation..3、Taking rutin as the control product,it is feasible to use aluminum nitrate coloring method to determine the total flavonoids of cattle under specific extraction conditions,and the total flavonoids of cattle can be used as the evaluation index of Millettia speciosa quality,and glucose is the control product,It is feasible to use phenol-sulfuric acid method to determine the total polysaccharide of Millettia speciosa obtained by specific treatment method under specific extraction conditions,and the total polysaccharide of Millettia speciosa can be used as an evaluation index of the quality of it.4、The content range of paulownia,quercetin,fermononetin and maackiain can be used as evaluation indexes for the quality of Millettia speciosa medicinal materials.5、Under this extraction condition,the similarity degree of Millettia speciosa HPLC fingerprint is good.6、The clustering analysis of Millettia speciosa amino acids and trace elements shows that the total amount of amino acids in Millettia speciosa increases with the increase of growth years,and the better the appearance traits,the more amino acids.更多还原