【作者】 刘伟；

【导师】 李登武； 王冬梅； 康永祥；

【作者基本信息】 西北农林科技大学 ， 生态学， 2012， 硕士

【摘要】 人类利用药用植物来治病防病已有几千年的历史。当今的处方药有25/%左右来自于药用植物,而药用植物中有效成分大多数为植物次生代谢产物。次生代谢产物(secondary metabolites)是细胞生命活动或植物生长发育正常运行的非必需的小分子化合物,由遗传特性(内因)和植物生长环境(外因)共同调控决定,其产生和分布通常有明显的地域、种属、器官、组织以及生育时期的特异性,并且环境的变化能直接影响次生代谢产物的种类和数量,甚至只有在特定的环境和时间条件下才合成特定的次生代谢产物。因此自然环境的任何一种生态因子都有可能影响次生代谢产物在植物体内的合成和积累。相对于初级代谢产物,次生代谢产物与环境之间有着更强的相关性和对应性,因此明确影响植物次生代谢产物合成和积累的主导环境因子是保证野生药用植物品质和提高药效的关键。但在实际生产中药用植物的质量和产量是互相矛盾的两个方面,要提高次生代谢产物的含量必然要损失一部分产量。因此,本文重点研究主要生态因子对中药材品质的作用和作用大小,揭示主导因子,进而进行人为调控,平衡质量与产量之间的矛盾。金露梅(Potentilla fruticosa)和银露梅(Potentilla glabra)属蔷薇科金露梅属植物,是我国最早应用的中药之一,具有清暑消热,益脑清心,健胃消食,健脾化湿,调经等功能。金(银)露梅是我国高寒地区的一种典型性落叶灌丛,分布于我国新疆、青海、陕西、甘肃、宁夏、内蒙古、四川、山西、黑龙江、吉林等省区。本文选取青海省互助北山、陕西省太白山、陕西省紫柏山、陕西省火地塘、陕西省牛背梁、四川省峨眉山、山西省云顶山、内蒙古自治区贺兰山等8个具有代表性的产地为研究对象,对金(银)露梅的主要有效成分及体外抗氧化活性、产区气候条件、土壤条件进行了分析,旨在探明金(银)露梅有效成分含量及抗氧化活性与生态因子的关系,筛选主导因子,为野生金(银)露梅的保护与利用及可持续经营管理提供理论依据。主要研究结果如下：1.金(银)露梅叶主要次生代谢产物提取工艺的优化在单因素试验基础上,根据Box-Behnken中心组合设计原理,利用响应曲面设计方案优化金(银)露梅叶单宁、总黄酮的提取工艺,经Design-Expert软件分析得金露梅最佳提取工艺为：乙醇浓度为70%、提取温度为80℃、提取时间为2h、提取次数为3次、液料比为20；银露梅最佳提取工艺为：乙醇浓度为90%、提取温度为80℃、提取时间为2h、提取次数为3次、液料比为15。2.两代表性产区金露梅不同部位有效成分含量及抗氧化活性的差异化本试验主要对两个代表性产地——青海省互助北山和陕西省太白山的金露梅不同部位乙醇提取物进行了主要有效成分含量以及抗氧化活性的测定。青海省互助北山金露梅叶中单宁与芦丁含量最高,分别为8.04%和0.46%,而黄酮含量以陕西省太白山金露梅花中最高,达到8.46%。叶子的抗氧化活性是所有部位中最强的,其中以青海省互助北山金露梅叶子为最强,其DPPHIC50值为7.32μg/mL,并且与芦丁标品抗氧化能力(DPPHIC50值为5.79μg/mL)没有显著差异(P>0.05)。结果表明不同产地金露梅不同部位各有效成分含量及抗氧化活性存在显著差异(P<0.05),其中青海省互助北山金露梅叶子有效成分含量及抗氧化活性均较强。造成这种差异的原因可能是：(I)植株自身代谢因素。(II)土壤和气候等生态因子的影响。(III)基因突变。本研究结果可为金露梅叶子能作为天然抗氧化剂和中药使用提供理论依据。3.金露梅叶有效成分及抗氧化活性与生态因子的关系本研究应用相关分析、多元线性逐步回归分析、通径分析研究了生态因子对药用金露梅叶单宁含量、黄酮含量、芦丁含量及抗氧化活性的影响。影响单宁含量的主导因子为速效磷、全钾、年极低温度,其中起直接作用的因子是速效磷,且与单宁含量呈显著负相关关系；影响总黄酮含量的主导因子为土壤pH、年均温,其中起直接作用的因子是pH,且与总黄酮含量呈极显著正相关关系；影响芦丁含量的主导因子为全磷、年日照、无霜期,其中起直接作用的因子是年日照,且与芦丁含量呈显著负相关关系；影响抗氧化活性的主导因子为速效磷、有机质、年均温,其中起直接作用的因子是年均温,且与抗氧化活性呈显著正相关关系。研究结果表明,生态因子是影响药用金露梅叶有效成分含量及抗氧化活性的重要因素,而影响不同有效成分及抗氧化活性能力的主导因子有所不同,但主要集中于速效磷、pH、年日照、年均温。4.银露梅叶有效成分及抗氧化活性与生态因子的关系本研究采用灰色关联度分析了6个不同产地野生银露梅叶中单宁含量、黄酮含量及体外抗氧化活性与生态因子的相关性。研究结果表明不同产地银露梅品质存在显著差异,单宁含量为4.95%-7.74%；黄酮含量为1.07%-3.56%,其中青海互助产银露梅叶中的单宁、黄酮含量均最高,分别达7.74%和3.56%；贺兰山产银露梅叶的抗氧化能力最强,其DPPHIC50值为6.9μg.mL-1；灰色关联度分析显示影响单宁含量的主导因子是年极低温度、速效氮、无霜期、有机质、速效钾、年极高温度；影响黄酮含量的主导因子是年日照时数、年极低温度、pH、年积温、有机质、全氮；影响抗氧化活性的主导因子是年日照、年均降水量、有机质、pH、年极低温度、年极高温度。更多还原

【Abstract】 Human use medicinal plants to treat and prevent disease for thousands of years history.Today, about25%of prescription drugs generate from the medicinal plants.Most of the active ingredients in medicinal plants are plant secondary metabolites. Secondary metabolites regulated commonly by the genetic characteristics (internal) and plant growth environment (external) are the non-essential small molecule compounds in cellular activities or the proper functioning of the plant growing development. Its generation and distribution usually has obvious geographical, species, organ, tissue and reproductive period specificity, while environmental changes can affect the types and quantities of secondary metabolites directly, or synthesize specific secondary metabolites in the particular environment and time conditions only. Therefore, any kind of natural ecological factors in the environment are likely to affect the synthesis and accumulation of secondary metabolites in plants. In contrast with the primary metabolism (metabolites), secondary metabolism (metabolites) has stronger correlation and correspondence with the environment.For this reason, to determine the dominant environmental factors for the synthesis and accumulation of the plant secondary metabolites is the key to guarantee the quality of wild medicinal plants and improve the efficacy of medicine. However, in the actual production, quality and yield of medicinal plant are the two conflicted aspects, since to improve the content of secondary metabolites is bound to lose part of the production. Consequently,This thesis is focus on studying the role and role size of the main ecological factors on quality of Chinese herbal medicine to reveal the dominant factors. Thus, artificially regulating and balancing the contradiction between quality and yield.Potentilla fruticosa, and Potentilla glabra (both in Rosaceae), are one of the earliest applications in traditional Chinese medicine, with heat clearing, heart purifying and brain beneficial, digestion promoting, spleen invigorating and dampness eliminating, menstruation regulating functions, etc. They are typical deciduous shrub in the alpine region in China, located in Xinjiang, Qinghai, Shaanxi, Gansu, Ningxia, Inner Mongolia, Sichuan, Shanxi, Heilongjiang, Jilin Province, etc. The thesis selected eight representational plots, Huzhu Northern Mountain in Qinghai Province Taibai Mountain, Zibai Mountain, Huoditang, and Niubeiliang in Shaanxi Province, Emei Mountain in Sichuan Province, Yunding Mountain in Shanxi Province, Helan Mountain in Inner Mongolia Autonomous Region as the research object, studying the major active ingredients, in vitro antioxidant activity, climatic conditions of the sampled plots, and soil conditions of Potentilla fruticosa and Potentilla glabra, aiming to ascertain the correlation of the active component content and antioxidant activity with all the ecological factors of them, screening the dominant factors, and providing a theoretical basis for the protection, utilization and sustainable management of Potentilla fruticosa and Potentilla glabra.1. Optimization of the main secondary metabolites extraction process of Potentilla fruticosa and Potentilla glabra leavesOn the basis of the single-factor test, according to principle of the Box-Behnken central composite design, using the response surface design to optimize the extraction process of the tannin and total flavonoid of Potentilla fruticosa and Potentilla glabra leaves. According to the analysis of Design-Expert software, the optimized extraction process for Potentilla fruticosa were as follows:ethanol concentration of70%, extraction temperature of80℃, extraction time of2h, extraction times of3, the liquid ratio of20:1. The optimized extraction process for Potentilla glabra were as follows:ethanol concentration of90%, extraction temperature of80℃, extraction time of2h, extraction times of3, the liquid ratio of15:1.2. Differentiation of the effective components and antioxidant activity of Potentilla fruticosa in the two representative plotsThis experiment selects two typical plots—Potentilla fruticosa from Huzhu Northern Mountain in Qinghai Province and Taibai Mountain in Shaanxi Province, measuring the effective components content and antioxidant activity of the ethanol extracts of different parts of them. Tannin and rutin content of Potentilla fruticosa leaves from Huzhu Northern Mountain in Qinghai Province are the highest, reaching8.04%and0.46%, respectively. Flavonoid content of Potentilla fruticosa flowers from Taibai Mountain in Shaanxi Province is the highest, reaching8.46%.It is proven that leaves has the strongest antioxidant activity. Potentilla fruticosa leaves from Huzhu Northern Mountain in Qinghai Province have the strongest antioxidant activity among them, with the DPPHIC50value at7.32μg/ml. While compared with the antioxidant capacity of rutin standard product(DPPHIC50=5.79μg/ml), it can be seen that there is no significant differences between them(P<0.05). The results of this study show that significant differences (P<0.05) between the effective components content and antioxidant activity of different parts of Potentilla fruticosa in different plots. the effiective components content and antioxidant capacity of Potentilla fruticosa leaves from Huzhu Northern Mountain in Qinghai Province is the highest among them. The reason for this difference may be:(Ⅰ) Plant’s own metabolic factors.(Ⅱ) Ecological factors such as soil and climate.(Ⅲ) Genetic mutations. This result is able to provide the theoretical basis for using Potentilla fruticosa leaves as natural antioxidants and traditional Chinese medicine.3. Correlation of the active components and antioxidant activity of Potentilla fruticosa leaves with ecological factorsIn this study, the impact of ecological factors on tannin, flavonoid, rutin content and antioxidant activity of the Potentilla fruticosa leaves are studied using correlation analysis, multiple linear stepwise regression analysis, and path analysis. Rapidly-available phosphorus, total potassium, and annual lowest temperature are the dominant factors affecting the tannin content, among which rapidly-available phosphorus is a direct effective factor, presenting a significant negative correlation with the tannin content; soil pH and annual average temperature are the dominant factors affecting the total flavonoid content, among which pH is a direct effective factor, presenting a significant positive correlation with the total flavonoid content; total phosphorus, annual sunshine hours and frost-free period are the dominant factors affecting the rutin content of, among which annual sunshine hours is a direct effective factor, presenting a significant negative correlation with the rutin content; rapidly-available phosphorus, organic matter and annual average temperature are the dominant factors affecting the antioxidant activity, among which annual average temperature is a direct effective factor, presenting a significant positive correlation with antioxidant activity. The results show that the ecological factors are principle elements affecting active components content and antioxidant activity of Potentilla fruticosa leaves. Though, the dominant factors affecting various active components and antioxidant activity capacity are different, they are mainly focused in the rapidly-available phosphorus, pH, annual sunshine hours, and annual average temperature.4. Correlation of the active components and antioxidant activity of Potentilla glabra leaves with ecological factorsThe correlation of ecological factors on the tannin, flavonoid content and in vitro antioxidant activity of Potentilla glabra leaves in six different plots are analyzed using the gray relational analysis. The results show that there is significant difference in the quality of Potentilla glabra in different plots, with the tannin content between4.95%～7.74%; the flavonoid content between1.07%～3.56%, among which the tannin and flavonoid content of Potentilla glabra leaves from Huzhu Northern Mountain in Qinghai are the highest, reaching7.74%and3.56%, respectively; Potentilla glabra leaves from Helan Mountain have the strongest antioxidant capacity, with its DPPHIC50value at6.9g·ml-1; in the gray correlation analysis, annual lowest temperature, ammonium nitrogen, frost-free periods, organic matter, rapidly-available potassium, and annual highest temperature are the dominant factors affecting the tannin content; annual sunshine hours, annual lowest temperature, pH, effective accumulated temperature, organic matter, and total nitrogen are the dominant factors affecting the flavonoid content; annual sunshine hours, annual average rainfall, organic matter, pH, annual lowest temperature, and annual highest temperature are the dominant factors affecting the antioxidant activity.更多还原