【作者】 王花红；

【导师】 王喆之； 郭五保；

【作者基本信息】 陕西师范大学 ， 植物学， 2004， 硕士

【摘要】 山茱萸(Macrocarpium officinale Sieb．et Zucc．)是山茱萸科(Cornaceae)山茱萸属(Macrocarpium)植物，具有补肝益肾的功能，为我国传统珍稀药材。现代药理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，具有广阔的市场前景。本课题主要通过对陕西佛坪地区山茱萸种质资源的调查，以HPLC法测定不同产地、不同类型、不同采收期、不同加工方法药材中有效成分含量，并结合指纹图谱研究以期建立山茱萸质量的综合评价方法，为山茱萸规范化种植和药材的可持续发展提供依据。实验的主要结果如下： (1) 通过对陕西佛坪地区山茱萸资源的调查，以果形、果色等生物学特征及百果重、出药率等经济性状为指标，对佛坪野生、半野生及栽培的山茱萸进行类型划分，确定为9个不同的类型，并对各类型的特征进行系统的描述； (2) 以生物学特性、产量、出药率及其有效成分等进行综合分析，确定陕西佛坪地区山茱萸优良栽培类型有大红枣、珍珠红、石磙枣。其平均出药率分别为：21.9％，20.3％，25.3％；平均马钱素含量分别为：1.023％，0.681％，0.938％；平均熊果酸含量分别为0.157％，0.154％，0.165％；平均齐墩果酸含量分别为：0.065％，0.068％，0.069％； (3) 分别建立了山茱萸中有效成分马钱素、熊果酸和齐墩果酸的HPLC含量测定法。其中马钱素测定条件为：甲醇一水(32：68，v／v)，检测波长236nm，流速1ml／min；熊果酸和齐墩果酸测定条件为：甲醇—0.4％磷酸(88：12，v／v)，检测波长210nm，流速1ml／min。方法学考察结果表明重现性好，精密度高，可以作为山茱萸中马钱素、熊果酸和齐墩果酸的检测手段； (4) 以不同类型及不同产地山茱萸为材料，比较测定其中马钱素、熊果酸和齐墩果酸的含量。结果表明不同类型、不同产地样品有效成分含量存在较大差异。测得马钱素、熊果酸、齐墩果酸平均含量分别为0.586％，0.157％，0.060％； (5) 对不同采收时期的山茱萸果实进行跟踪测定，以其中主要有效成分马钱素、熊果酸及齐墩果酸为主要参考指标，确定陕西佛坪山茱萸最适采收期为9月下旬至10月上旬； (6) 对山茱萸产地加工方法进行系统的研究，确定其最适产地加工流程为：采摘→净选→清洗→水煮软化(2倍水量，80℃保温5min)→去核→干燥(60℃，连续干燥36h)； (7)建立山茱英药材薄层色谱鉴别系统:脂溶性成分的条件为环己烷一氯仿一乙酸乙酷(20:5二8)，显色可见7个斑点;醇溶性成分的条件为氯仿一甲醇一水(12:4:l)，显色可见8个斑点; (8)对山茱英的指纹图谱进行了系统的研究，建立了山茱英指纹图谱研究方法:以甲醇超声处理制备样品溶液，采用HPLC法在A(乙睛)一B(0.4%磷酸)梯度洗脱系统:2%一5%A(0一15min)，5%一16%A(15一50min)，16%一26%A (50一75min)，26%一90%A(75一80min)，90%A(80一100min);流量:1.oml/min;柱温:室温;检测波长:21Onm的条件下测定山茱英的指纹图谱。方法学考察表明试验精密度RSD%<3%，方法重现性RSD<3%，样品在24h内稳定; (9)通过测定陕西佛坪16批山茱英样品的指纹图谱，确定了10个共有峰，与对照品比较后鉴定了马钱素、齐墩果酸、熊果酸三个峰，制定了陕西佛坪地区山茱英指纹图谱的技术参数，以其中马钱素为参照峰，计算16批样品共有峰的相对保留时Ibl为:1(0.349士O‘003)，2(0.419士0.004)，3(0.956士0.002)，4(0.979士0 .001)，5(51)(l)，6(1 .024士0.004)，7(1.476士0.004)，8(1489士0.003)，9(52)(2.003士0.006)，10(53)( 2.018士0.007);相对峰面积为:1(0.774士0.327)，2(5 .641士1 .444)，3(1 .259士0.642)，4(0.259士0.087)，5(51)(1 .000)，6(0.742士0 .343)，7(0.668士0.247)，8(1.168士0.566)，9(52)(0.152士0.056)，10(53) (0.409士0.140)。以16批样品相对峰面积的中值建立共有模式，计算各样品的平均相似度为0.978士0.033; (10)通过测定全国各主要产地10批山茱英样品的指纹图谱，确定了10个共有峰，与对照品比较鉴定了马钱素、齐墩果酸、熊果酸三个峰，制定了全国山茱英指纹图谱的技术参数，以其中马钱素为参照峰，计算10批样品共有峰的相对保留时间为:l(0.350士0.003)，2(0418士0.004)，3(0.955士0.003)，4(0.979士0 .002)，5( 51)(1)，6(1 .024士0.004)，7(1.481士0.006)，8(1.495士0.006)，9(52) (2.009士0.010)，10(53)(2，025土0.010);相对峰面积为:l(1.690土1.196)，2 (4 .927士2.008)，3(1 .183士1.685)，4(0.242士0.054)，5(51)(l)，6(0.492士0 .1 53)，7(0.856士0.669)，8(0.768士0.603)，9(52)(0.134士0.040)，10(53) (0.377士0.118)。以10批样品的相对峰面积的中值建立共有模式，计算各样品平均相似度为0.925士0.088。更多还原

【Abstract】 Cornus (Macrocarpium officinale Sieb. et Zucc.) belongs to Cornaceae, and its dry fruits were known as one of the precious herbs in China and had the effects of novishing kidney and liver. Modern pharmaceutical studies showed that cornus could better regulate the immune system and notably lower the blood sugar, which provided it great market potential. This paper mainly studied the resources of cornus in Foping, Shaanxi Province by ways of investigation and determination the main active components content of it in different habitats, different varieties, different picking time and different primary process by high performance liquid chromatography (HPLC). The HPLC fingerprints of cornus were also systematically studied in order to establish a general quality control standard of it, and offer some data to its good agriculture process (GAP) and sustainable development. The main results could be concluded as following:(1) There were 9 varieties that were classified by the biological characters of the fruits and leaf like color and figure, the economic characters like the rate of medicine, the weight of 100 fruits after the investigation of the wild, semi-wild and cultured cornus in the area of Foping, Shaanxi Province. The main characters of each variety were described systematically;(2) All varieties were systematically evaluated by the biological character, the ability of anti-virus, the yields, the rate of medicine and the content of the main active components. The results showed that Dahongzao, Zhenzhuhong, Shigunzao were believed as the good varieties. The main indicatrix of them were follows: the average rate of medicine were 21.9%, 20.3%, 25.3% respectively; the average content of loganin were 1.023%, 0.681%, 0.938% respectively; the average content of ursolic acid were 0.157%, 0.154%, 0.165% respectively; the average content of oleanolic acid were 0.065%, 0.068%, 0.069% respectively;(3) The methods of content determination of loganin, ursolic acid and oleanolic acid in cornus by HPLC were established. Determination conditions of loganin were: Methanol-water (32:68,v/v) detected at 236nm, the flow-rate was l.Oml/min; Conditions of ursolic acid and oleanolic acid were: Methanol-0.4% phosphoric acid (88:12,v/v) detected at 210nm, the flow-rate was l.Oml/min. Methodologicalexperiments showed that the method had high precision and good repeatability and can be used as the detection means of loganin, ursolic acid and oleanolic acid in cornus;(4) The results of comparative determination of loganin, ursolic acid and oleanolic acid in the samples of different varieties and different habitats showed obvious distinction. The average content of loganin was 0.586%, the ursolic acid was 0.157%, and oleanolic acid was 0.060%, respectively;(5) Through continuous determination of the fruits of different picking time, the most appropriate picking time was set down between later September and early October according to the content of loganin, ursolic acid and oleanolic acid;(6) Through systematically studying on the primary process of cornus, an appropriate process flow could be concluded as: picking → selection→watering -cooking for intenerate (2 times volume of water at 80℃ preserving heat for about 5min) →coring→drying ( 60℃for about 36h);(7) A fast identification method of cornus was established by TLC: the condition of chloroform extractive was cyclohexane-chloroform-ethylacetate (20:5:8), seven spots could be detected after spraying; the condition of methanol extractive was chloroform-methanol-water (12:4:1), eight spots could be detected;(8) The HPLC fingerprints of cornus were systematically studied, and the method was described as following: Samples were extracted with methanol in ultrasonic bathing, then analyzed by HPLC performed by gradient mobile system using A(acetonitrile) and B(0.4% phosphoric acid): 2%~5% A(0~15min), 5%~16% A( 15~50min), 16%~ 26% A(50~75min), 26%~90% A(75~80min), 90% A(80~100min); the flow-rate was 1.0ml/min;更多还原