【作者】 马英姿；

【导师】 王平；

【作者基本信息】 中南林业科技大学 ， 植物学， 2010， 博士

【摘要】 蚬壳花椒Zanthoxylum dissitum Hemsl,中药名为单面针,为芸香科花椒属常绿灌木。根、茎及叶均可入药,主治牙痛、腰痛、妇女月经过多,产后月经不调等症。分布于我国西南、广东、广西、湖南、湖北、陕西等地。近年来,相关中成药厂生产规模日趋扩大,而蚬壳花椒的自然繁殖率低,至使该自然资源日趋贫乏。寻求适宜的繁殖途径,并合理利用蚬壳花椒资源已势在必行。本研究对湘西产蚬壳花椒药材进行了化学成分分析,采用乙醇回流得到乙醇浸膏后,再用氯仿连续回流提取,去除酸、碱性成分后,获得中性亲脂性浸膏。通过硅胶柱层析对浸膏的化学成分进行分离,从中得到了7种化合物单体,并最终采用1H-NMR、13C-NMR、MS等现代光谱学手段对这7种单体进行了结构鉴定,确定化合物分别为：正二十六烷、正十六烷醇、β-谷甾醇、熊果酸、异虎耳草素、佛手内酯、白藓碱。除β-谷甾醇和白藓碱外,其余5个化合物均为首次从该植物中分离得到。其中,熊果酸、异虎耳草素、佛手内酯、白藓碱等都具有较重要的药用价值,该结果为进一步开发和利用蚬壳花椒提供了参考依据。建立了蚬壳花椒中白藓碱含量的HPLC测定方法。分别对蚬壳花椒的1-3年生幼苗的根、茎、成熟的叶及生药内的白藓碱含量进行了检测,结果为1、2年生茎、成熟的叶及叶柄中均未检测出白藓碱；3年生茎、1-3年生根中检测出白藓碱；3年生根中白藓碱的含量是3年生茎内含量的3.89倍,是生药含量的2.19倍。不同pH值溶液胁迫处理蚬壳花椒幼苗后,能促进植株内的白藓碱含量增加,其中pH为2.5的酸性处理,幼苗内白藓碱含量达0.04725μg·g-1,是对照的7.13倍；pH为9.5的碱性处理,白藓碱的含量达0.03200μg·g-1,是对照的4.83倍,但植株在酸性或碱性过强的情况下均不能正常生长。该研究为高效合理利用蚬壳花椒及合理选择栽培土壤提供参考依据。对蚬壳花椒的生态学特性进行了研究。蚬壳花椒主要分布在海拔500～1000m的常绿阔叶林中,喜阳但忌强光直射；喜湿,常生长在沟溪边；原始生境土壤pH值范围为5.7～7.6,有机质含量平均值为9.02%,C/N为16.9～22.89,冬季平均绝对含水量为26.89%。解剖学研究表明,其营养器官构造具双子叶植物的基本特征,根的初生木质部有三原型和四原型；幼茎表面有表皮毛和腺体,茎内含少量分泌囊和结晶；叶上、下表皮细胞差异显著,上表皮细胞较大,少量分泌囊紧接下表皮。生物碱组织化学反应显示,茎中,生物碱主要存在于周皮、次生木质部的射线细胞及髓部细胞内；根中,生物碱大量存在于周皮、次生韧皮部及次生木质部的射线细胞中；叶片中不含生物碱。黄酮类化合物显色反应不明显。为探索蚬壳花椒自然繁殖中的致危因素,对其种子的休眠和萌发特性进行了研究。采用多种方法处理蚬壳花椒的当年成熟的种子,在常规萌芽条件下不能萌发；用TTC法检测种子生活力,活力达93.33%,去除种皮后给予合适萌芽的条件后,种子可以萌发,证明蚬壳花椒种子的胚在当年已发育成熟；种皮的透水性也较好,与休眠关系不大；蚬壳花椒种皮浸出液对极易发芽的十字花科白菜种子萌发有一定的抑制作用,推测休眠的主要原因可能是种皮内含有抑制萌芽的物质。获得种子萌芽率较高的方法为：浓硫酸处理6min,去除种皮,然后用50 mg·L-1的GA3浸泡5min,清洗后置于25℃培养箱内培养,萌芽率可达60%。以带顶芽或腋芽的茎段为材料,研究蚬壳花椒微繁殖技术,实验表明：MS+2.0 mg·L-16-BA+0.05 mg·L-1NAA,为最佳腋芽诱导培养基,诱导率达64%;MS+2.0 mg·L-1ZT+0.2 mg·L-1NAA,为最佳丛芽诱导培养基,诱导率达90%,增殖系数为2.7；MS+6-BA 0.5 mg·L-1+NAA 0.05 mg·L-1+生物素1.0mg·L-1,为最佳增殖培养基,增殖系数可达4.0；1/3 MS+IBA 0.5 mg·L-1+NAA 0.05mg·L-1,为最佳生根培养基,生根率可达78%。为了解不同酸碱环境对幼苗生长的影响,用不同pH值溶液处理蚬壳花椒组培苗,结果显示,组培苗在pH为3～8的范围内,7d内基本能正常生长,而在pH为2、10、11.5的处理下,组培苗明显到受伤害；用酶联免疫法测定IAA、ABA、GA、ZR四种内源激素含量变化,结果显示,四种内源激素对于不同的胁迫处理都呈现出一定的变化反应,pH为6时,IAA含量始终高于其它处理,IAA含量的变化与ABA的含量变化呈相反的趋势；ZR的含量在2-3d时达到高峰,后期pH为6的处理含量最高；GA3含量总体上呈波浪状上升的趋势。以上研究为以后蚬壳花椒的人工栽培,有效成分的提取、规模化繁殖提供技术指导和理论依据。更多还原

【Abstract】 Zanthoxylum dissitum Hemsl is a traditional Chinese medicine plant known as Danmianzhen. It is an evergreen shrub and belong to Rutaceae family, Zanthoxylum genus. The root, stem and foliage are as medicines with function of toothache, back pain, women menorrhagia, irregular menstruation postpartum etc., It is sporadically distributed in Guangdong, Guangxi, Hunan, Hubei, Shaanxi province and other places in China. Recently, the Z.dissitum medicine factories have been expanding the production scale, while the species is in a low growth and reproduction rate, lead to threat of drug manufacturer. To seek for suitable means of propagation, and to expand available resources of drug has become inescapabe.Ethanol extract was obtained from Z. dissitum Hemsl powders, nearly neutral lipophilic compositions were gotten by removing acid and alkaline compositions. The fraction were suffered SiO2 column and the products were identified by 1H-NMR, 13C-NMR and MS. Seven components were obtained, named triacontane, 1-hexadecanol,β-sitosterol, ursolic acid, isopimpinellin, bergapten and dictamnine. Exceptβ-sitosterol and dictamnine, the others were isolated from Z. dissitum for the first time. Several components, for instance, ursolic acid, isopimpinellin, bergapten and dictamnine have significant medicinal values, the consequence provided a reference for utilizing and further development of Z. dissitum.The determination method of dictamnine by HPLC was established. The content of dictamnie in roots, stems of 1～3 years old seedlings, and crude drug of Z. dissitum were detected respectively, the result showed that there was no dictamnine could be detected in 1 and 2 year-old stem, foliage and petioles. But in the 3-year old stems,1 to 3 year-old roots, it could be detected, the content in roots was 3.89 times of stems in 3-year-old plants, and was 2.19 times of the crude drug. One year old seedlings were treated with different pH value solutions, The dictamnine content was increased with different treatments, it could be up to 0.04725μg·g-1 with pH of 2.5 solution treatment, it was 7.13 times of the reference sample; with the treatment of pH of 9.5, the content could be up to 0.03200μg·g-1, it was 4.83 times of the reference sample, but the plants in strong acidic or alkaline soil could not grow normally. The study provided a reference for ultilizing Z. dissitum efficiently and choosing the planting soil reasonablly.Ecological characteristics of Z. dissitum has been studied in this research. The result showed that Z. dissitum was mainly distributed at 500-1000m altitude, in the evergreen broad-leaved forest, it is light tolerant species grows in humid area, and often founded beside ditch stream; The pH value range of pristine habitat of Z. dissitum was 5.7-7.6, with an average of value of 6.78; the average organic content was 9.02%; C/N was in the range of 16.9-22.89, the average is 20.74; the average absolute water content in winter was 26.89%. In the high moisture, organic-rich, weak acidic soil environment, the plants grow better.The anatomical observations on the vegetative organs showed that the vegetative organs possessed the basic characters of the dicotyledon. The root’s primary xylem was triarch or tetrarch, and the secondary xylem was developed; there were some epidermal hairs on the young stem, and a few secretory cavities and crystallizations in the cortex; the upper and lower epidermal cells of the leaves were significantly different, the upper epidermal cells were obviously larger than the lower epidermal cells, few secretory cavities were close to the lower epidermis. Specific chromogenic reagent was applied in study of the histochemistry localization of alkaloids of the plant. The result illustrated in the stem, alkaloids were mainly distributed in the periderm, secondary xylem, and pith; in the root, many alkaloids were deposited in the periderm, secondary phloem and secondary xylem ray cells; foliage didn’t contain alkaloids. Specific chromogenic reagent was also applied in flavonoids, but the color reaction was not obvious, demonstrated the content of flavonoids was in a low level in Z. dissitum.In order to explore the underlying mechanisms relevant to the impediment in the sexual reproduction of Z. dissitum, the characteristics of the dormancy and germination of the seeds were studied. It was found that the germination inhibitors existed in the seed coat, which was believed to be a main impediment to the germination. The seeds viability was detected by TTC method, found that the vitality was up to 93.33%. The permeability of water of the seed coat was regular, though the hardness of the seed coat was not main reason to restrict its germination. When the seed coat was peeled off, it germinated, so the embryo would not induce the seed dormancy. A better method to broken the seeds dormancy was that:processed the seeds in 98% H2SO4 for 6 min., then peeled off the seed coat, dipped them in 50 mgL-1 GA3 for 5 min, finally washed them in running water and cultivated them in incubator at 25℃. The percentage of the seeds germination reached 60%. The micro-propagation technique is an effective instrument for rapid propagation of Z. dissitum. The stems with terminal bud or axillary bud were used as the explants, and were carried out by culturing on MS media with different hormones. The best media for various stages were as fellows:(1) Axially bud induction medium:MS+ 2.0 mg·L-16-BA+0.05 mg·L-1 NAA, the inducement rate was 64.0%; (2) Clump shoot induction medium:MS+2.0 mg·L-1ZT+0.2 mg·L-1 NAA, the inducement rate was 90.0%; (3) Clump shoot proliferation medium:MS+6-BA0.5 mg·L-1+NAA 0.05mg·L-1+Auxin 1.0 mg·L-1; and the multiplication quotiety can reach 4. (4) Rooting medium:1/3MS+IBA0.3mg·L-1+NAA 0.05 mg·L-1, and the rooting rate was 78%. Rooting plantlets transplanted to the medium which was matrix of yellow soil, plant ash and sand, with the proportion of 1:1:1, and disposable plastic cup as the container, sealed with transparent plastic, the transplant survival rate was 95%.In order to reveal the effect of acid and alkali stress in Z. dissitum Hemsl, the plantlets were treated in different pH values of 2,3,4,6,8,10, and 11.5. Four endogenous hormones contents of IAA, ABA, GA and ZR were assayed by ELISA The results illustrated that the plantlets could grow basic normally in the scale of pH value of 3 to 8 within 7 days, and the plantlets were suffered obviously in the treatment pH of 2,10,11.5. The IAA content in the treatment pH of 6 which as the reference sample was always higher than that of other treatment, it showed that other treatments all had different degree inhibition to the growth of the plantlets. Resemble results are found in identical endogenous hormones when treated differently, and the change rule of IAA content was just on the contrary to ABA content; the content of ZR reached peak level after having been treated 2d-3d, and the maximum content was in the treatment of pH of 6; the GA3 content change rule raised upon wave-like.更多还原