山茱萸种质资源的研究及优良品种的筛选

【作者】 陈随清；

【导师】 冀春茹；

【作者基本信息】 北京中医药大学 ， 中药学， 2003， 博士

【摘要】 山茱萸为山茱萸科（Cornaceae）植物山茱萸（Cornus officinalis Sieb．et Zucc．）除去果核的干燥成熟果肉，具有补益肝肾、涩精固脱之功效；现代研究证明山茱萸具有较好的调节免疫系统功能和显著的降血糖作用。但山茱萸在长期的栽培过程中出现了很多农家栽培品种，这些品种的果实从形状、大小、单果平均重量到产量、干果肉（药材）得率，以及熊果酸、水溶性浸出物、脂溶性浸出物的含量等具有较大的差异，即它们的经济价值和药材质量是明显不同的。为了更好的开发、利用和保护山茱萸的种质资源，作者对其进行了系统的研究。本文分为二部分。第一部分为文献综述。对近20年来山茱萸在品种分类、化学成分、质量控制、药理药效等方面的研究资料进行了研究分析，为本科题的实验研究打下了坚实基础。另外，作者还对RAPD分子标记技术(Random amplified polymorphic DNA，随机扩增多态性DNA)在中药种质资源研究中的应用，以及指纹图谱在中药中的应用及其研究进展进行了资料综述和分析。第二部分为实验研究部分。1．山茱萸的农家栽培品种的分类与鉴定。根据山茱萸果实的不同形状进行了分类，分为椭圆形果型、长圆柱形果型、圆柱形果型、短圆柱形果型、纺锤形果型、长梨形果型、短梨形果型等；并对其不同栽培品种的经济指标进行了评价分析；还对不同栽培品种山茱萸药材的性状、粉末显微特征、果皮表皮细胞的电镜显微特征等进行了研究，为山茱萸栽培品种的分类及鉴定提供了理论依据。2．山茱萸中化学成分的分离与鉴定。为了深入全面的了解山茱萸的化学成分，为建立山茱萸的化学成分指纹图谱奠定基础，本课题对山茱萸的化学成分进行了系统分离。实验采用95%乙醇40℃温浸提取，乙醚、乙酸乙酯、水萃取分部位，各部位经硅胶、Diaion HP-20、Toyopearl HW-40、MCI gel CHP -20P、活性炭等反复柱层析，从中共分离得到17个化合物。经化学检识及核磁共振和质谱等波谱鉴定技术，分别鉴定为：齐墩果酸、β-谷甾醇、熊果酸、胡萝卜苷、苹果酸甲酯、苹果酸丁酯、3-羟基-2，4-二氨基丁酸、β-阿拉伯糖-(1→4)-β-葡萄糖-(1→4)-β-葡萄糖-(1→6)-α-葡萄糖、蔗糖、没食子酸、马钱素、莫诺苷、7-O-乙基莫诺苷、水杨梅素D、特里马素Ⅰ、1-O-galloyl-4，6-O-HHDP-β-D-glucose、1,2,3,4,6-五-O-没食子酰基-β-D-葡萄糖。其中胡萝卜苷、苹果酸甲酯、1-O-galloyl-4，6-O-HHDP-β-D-glucose 、1,2,3,4,6-五-O-没食子酰基-β-D-葡萄糖为第一次从山茱萸中分离得到。3．山茱萸不同栽培品种的品质评价为了全面、客观的评价山茱萸的栽培品种，本课题对不同品种的化学成分含量、药效学作用进行了研究，结合农业经济指标，对山茱萸的不同栽培品种进行了综合评价，筛选出了优良品种。3．1不同栽培品种的化学质量评价 山茱萸中熊果酸的含量测定采用双波长薄层扫描法：硅胶G薄层板，以氯仿:乙醇(20:1)为展开剂，10%硫酸乙醇为显色剂，反射法锯齿扫描，λS=541nm，λR=680nm，回归方程Y=2768.3x-259.66,r=0.999，线性范围：0.62~3.72μg，<WP=5>平均回收率为97.07%,RSD=2.69%；马钱素的含量测定采用RP-HPLC法： Hypersil C18柱（4.5mm×200mm，5μm）；保护柱长1cm，以甲醇—水（30∶70）为流动相，柱温25℃，流速1ml/min，检测波长 231nm，回归方程为：Y=1.01×10-6 X -0.03，r=0.9996，线性范围 0.156—1.560μg平均回收率100.42% ，RSD=1.58%；总多糖的含量测定采用硫酸-苯酚法，测定波长486nm，回归方程Y=1.9189+162.5429x，r=0.9998，平均回收率99.68%，RSD=2.21%；总鞣质的含量、水溶性浸出物和醇溶性浸出物的含量测定采用《中国药典》2000年版一部附录规定的方法。测定结果利用灰色模式识别法进行综合评价。3．2不同栽培品种的药效学评价 药效学实验结果说明，山茱萸水煎液对BALB/C小鼠淋巴细胞转化有一定的抑制作用；对鸡红细胞致敏小鼠溶血素抗体的生成有一定的增强作用；对小鼠网状内皮系统吞噬功能有一定的抑制作用；对四氧嘧啶高血糖小鼠有一定的降血糖作用。利用灰色模式识别法对不同农家品种的药效作用进行了分析评价。通过二甲苯所致小鼠耳廓肿胀法的炎症模型实验证明，山茱萸水煎液的抗炎作用不明显。3．3山茱萸优良品种的筛选 根据化学指标评价和药效学指标评价结果，结合农业经济指标，确定椭圆形、圆柱形、长梨形果型的山茱萸为优良栽培品种。4．山茱萸优良栽培品种的HPLC指纹图谱的研究。采用ODS柱、DAD检测器，对山茱萸3个优良农家栽培品种进行了HPLC指纹图谱的研究。脂溶性部位（FSP）用甲醇-水-磷酸系统，检测波长213nm；水溶性部位（WSP）用甲醇-水梯度洗脱，检测波长231nm，可使山茱萸两个部位分别得到较好的分离。以熊果酸（FSP）和马钱素（WSP）为参照物，椭圆形果型的特征色谱峰是相对保留时间为 0.099 、 0.142 、 0.199 、0.224 、0.940 、 1.000（FSP）、0.251、0.289、0.447、0.500、0.599、0.661、1.000、1.659、2.379（WSP）等15个色谱峰；圆柱形果型是相对保留时间为0.100、0.200、0.941、1.000（FSP）、 0.150、0.248、0.342 0.442、0.490、0.657、1.000?更多还原

【Abstract】 Fructus Corni is the pulp of the ripe fruit of Cornus officinalis Sieb. et Zucc. ( Cornaceae，it is called dogwood fruit in English), which is removed its stones and produced mainly in Henan,Zhejiang and Shanxi provinces. It can supplement the liver and kidney,astringe essence and stem desertion.Pharmacological research on Fructus Corni has showed that it can regulate immune function of mice and decrease blood sugar in diabetic mice.But Cornus officinalis has many cultivated varieties in the main planting areas.The shape,size ,mean weight of their fruit,and dried pulp productivity ,and the contents of Ursolic acid,water-soluble extractives ,ethanol-soluble extractives,and so on,are different from each other.So their economic value and quality of drug are different.To further develop and make better use of the germplasm resources of Cornus officinalis ,systematical research was carried out. This dissertation is composed of two parts. Part 1 A review on the research progresses of Cornus officinalis during the latest 20 years is presented,including the taxonomy of cultivated varieties,chemical constituents,quality control, pharmacological activities,etc.On other hand,the application of RAPD marker technology in medicinal plant germplasm resources and the application of fingerprint in traditional Chinese medicine were reviewed and looked forward.120 references were cited.Part 2 systematical research on Cornus officinalis is reported.1.Taxonomy and identification of cultivated varieties of Cornus officinalis are described.The fruit shape of Cornus officinalis can be divided into seven types , ellipticform, cylindericform, long cylindericform, short cylindericform, long pear-shape, short pear-shape and spindleform. Their economic value is evaluated.The characteristics and microscopical characters of raw drugs from cultivated varieties of are drawn. Scanning Electron Microscope (SEM) examination on surface of fruit coat of Cornus officinalis has been finished.2. 17 compounds were isolated from Et2O-fraction, EtOAc-fraction and water-fraction of alcohol-extracts. On the basis of spectroscopic and chemical evidences, their structures are identified as oleanolic acid , β-sitosterol , ursolic acid , methyl malic acid , butyl malic acid,3-hydroxy-2,4aminobutyric acid,β-ara-(1→4)-β-glu-(1→4)-β-glu-(1→6)-α-glu， daucosterol(β-sitosterol-3-O-β-D-glucoside) , sucrose , gallic acid , loganin , morroniside , 7-O-ethyl-morroniside , Gemin D , TellimagradinⅠ, 1-O-galloyl-4,6-O-HHDP-β-D-glucose , 1,2,3,4,6-pene-gallagyl-β-D-glucose . Among these compounds, daucosterol , methyl malic acid , 1-O-galloyl-4,6-O-HHDP-β-D-glucose, 1,2,3,4,6-pene-galloyl-β-D-glucose, are obtained from Fructus Corni for the first time. 3. The chemical quality and pharmacological activities of different cultivated varieties were initially evaluated. Ursolic acid was determined by daul wavelength TLCS on Silica gel plate ,with using CHCl3-EtOH(20:1) as mobil phase ，10%H2SO4 as developing agent and λS=541nm，λR=680nm.The linear regression equation was Y=2768.3x-259.66 with correlation<WP=7>coefficient r=0.999，the linear range was 0.62～3.72μg. the average recoveries was 97.07%(RSD=2.69%,n=5) Loganin was determined by RP-HPLC.RP-HPLC was performed on SHIMADZU SPD-10A apparatus equipped with a Hypersil C18 column (4.5mm*200mm,5μm,a 1cm protective column was preposed) in an oven at 25℃,and a UV detector adjusted to 231 nm ,a solvent system consisting of MeOH-H2O(30:70,v/v) was used at a flow rate of 1ml?min-1. The linear regression equation was Y=1.01×10-6 X -0.03with correlation coefficient r= 0.9996 ,the linear range was 0.156～1.560μg，the average recoveries was 100.42%(RSD=2.69%,n=5)The content of total polysaccharide was determined by phenol- sulfuric acid method. The detective wavelength is 486 nm. The linearity of this method was quite satisfactory , the linear regression equation was Y=1.9189+162.5429x with correlation coefficient r=0.9998，the average recoveries was 99.68%(RSD=2.21%,n更多还原