【作者】 聂小妮；

【导师】 梁宗锁；

【作者基本信息】 西北农林科技大学 ， 植物学， 2011， 博士

【摘要】 许多植物挥发油都具有抗菌、杀菌和杀虫等活性作用,随着近十多年来人们对绿色环保化学品及农药的广泛关注,植物源化学品,尤其是植物挥发油的研究成为了现代生物及中药学科的热点问题之一。本论文研究了土荆芥挥发油的成分差异及其活性。土荆芥中所含有的挥发油具有抗菌、驱虫、抗癌、祛风除湿、通络止痛等功效。国内外众多学者对其进行了研究,但主要集中于其化学成分、杀虫活性、种子萌发特性等方面。目前研究的主要不足之处有:一、不同学者使用的分离条件及色谱条件各异,缺乏统一的标准,从而导致不同产地土荆芥挥发油成分及其含量的差异较大;二、对土荆芥挥发油的抗菌消炎和抑制农田植物病源菌的研究较少。针对以上不足,本文采用统一的提取分离、分析鉴定条件和方法,建立了土荆芥挥发油的标准指纹图谱,对我国9省12个产区土荆芥的化学成分及含量进行了分析测定,为土荆芥的质量控制提供了基本的科学依据,对于提高中药质量,促进中药现代化具有重要意义。在建立土荆芥挥发油指纹图谱的基础上,还分析了栽培时水肥条件和药材采收期、采收部位、储存时间及挥发油提取时间等因素对土荆芥挥发油的品质的影响,研究了陕西杨凌产土荆芥挥发油对皮肤及器械消毒所要求的15种标准菌株的抗菌作用、对西北地区10种常见植物病原真菌的抑菌作用及对3龄粘虫和淡色库蚊成虫的毒杀活性。以期为充分利用土荆芥植物资源,在天然消毒杀菌产品开发利用、天然医药及农药产品的开发方面提供科学依据。主要结论如下:1.本论文在统一了挥发油提取方法,分析仪器和分析方法的基础上,用我国9省12个产地的12个土荆芥样本,建立了土荆芥挥发油的气相色谱指纹图谱,通过该指纹图谱,标定了17个特征共有峰作为土荆芥挥发油质量鉴别的指标峰,建立该图谱所用的GC色谱分析条件可使土荆芥挥发油中各组分达到较好地分离效果,精密度、重复性、稳定性良好,可以作为土荆芥药材质量评价的一个重要标准和科学依据。2.气质联用分析结果表明我国12个产地的土荆芥挥发油的主要化学成分基本相似,均含有4种主要化学成分:α-松油烯、P-伞花烃、冰片烯和驱蛔素,而不同产地这四种主要成分的含量不同。土荆芥挥发油的主要成分都具有薄荷烷骨架,含量大于1%的成分一般不超过5种,主要成分含量占绝对优势,占挥发油总量的90%以上。3.本论文分析研究了栽培水肥条件,不同采收期、不同采样部位和不同储存时间等因素对土荆芥挥发油的影响,结果表明土荆芥挥发油的品质受这些因素的影响较大。施用Zn、Mn、Fe、Mo、和B等五种微量元素肥料处理土荆芥的出油率均有显著性提高,其中施用Zn肥的处理出油率增幅最大,且挥发油中所含主要化学成分的种类与含量变化不大;不同水分处理中,土壤相对含水量为40%的处理土荆芥出油率最高;土荆芥植株不同部位的出油率差异十分显著,其中种子出油率最高,茎的出油率最低,叶片出油率居中。土荆芥植株的3种部位挥发油的化学成分基本相似,但各物质的含量差异较大。不同生育期土荆芥的出油率变化较大,幼苗期出油率最低,随着土荆芥植株的生长,挥发油在植物体内不断积累,到种子成熟期达到最大值,枯草期出油率大大下降。储存时间对土荆芥挥发油出油率具有一定的影响。其中采收到储存15 d之内,出油率随储存时间的延长而逐渐增大,而储存15 d到30 d之间,出油率随储存时间的延长反而逐渐减小。但总体来看,在储存30 d之内,土荆芥出油率变化不大。总之土荆芥的最佳采收期为种子成熟期;采收最佳部位为带有果穗的地上部分;最佳储存时间为采收后15 d左右;用水蒸气蒸馏法提取挥发油时,蒸馏1 h得到的挥发油质和量达到最优。4.土荆芥挥发油具有广普的抗细菌和抗真菌作用,对临床常见的致病菌有较强的抑制和灭活作用。在供试的皮肤及器械消毒所要求的15株标准菌株中除对绿脓假单胞菌CMCC10211株无抑制作用外,对金黄色葡萄球菌等其它14株标准菌株均有较强的抑制作用。土荆芥的体外抗菌浓度较低,对于不同的菌株,最小抑菌浓度1.95-62.5μL/mL,最小杀菌浓度7.8-250μL/mL。所以土荆芥挥发油具有广普的抗菌效果,是一种利用价值很高的潜在抗菌药源,亟待进一步研究开发。急性毒性试验结果表明:土荆芥挥发油的小鼠灌胃处理中毒剂量较高。小鼠灌胃处理的半数致死剂量LD₅₀是1183μL/kg,推算出土荆芥挥发油对于人的半数致死剂量LD₅₀为9.18 mL/70kg。5.采用生长速率法测定了土荆芥挥发油对4个亚门10种植物病原真菌的抑制效果。结果表明对供试的10种植物病原菌均有一定的抑制效果,其中对小麦纹枯病菌抑制作用最强,0.5 uL/mL浓度下9 d内抑制率均达到100%以上;对番茄早疫病菌、辣椒疫霉病菌有较强的抑制作用,0.5 uL/mL浓度下,7 d内的抑制率分别为88.9%和74.5%,再次为小麦全蚀病菌、黄瓜枯萎病菌和小麦赤霉病菌,同样在0.5 uL/mL浓度下,5 d的抑制率分别为84%、70.6%和53.2%;土荆芥挥发油对番茄灰霉病菌、苹果炭疽病菌和黄瓜炭疽病菌等3种病原菌的抑制作用较差,抑菌率均低于35%。随挥发油浓度增大,抑菌效果增强,同时随培养时间的延长,抑菌能力下降。土荆芥挥发油对植物源真菌具有广普、高效且持久的抑菌活性,具有开发成农药的良好前景。6.土荆芥挥发油对昆虫具有高效、快速的熏蒸杀虫效果。作为熏蒸剂,土荆芥挥发油对鳞翅目害虫粘虫3龄幼虫和双翅目的卫生害虫淡色库蚊成虫的熏蒸有效剂量都小于10 ppm。土荆芥挥发油对粘虫的熏蒸LC₅₀在30 min时,仅为2.11μL/L,75 min时快速下降接近一半,LC₅₀值为1.07μL/L。用LC₉₅剂量处理粘虫,得到挥发油在LC₉₅剂量下对3龄粘虫的致死中时LT₅₀值为20.04 min;触杀活性试验结果表明土荆芥挥发油对粘虫的触杀活性随着处理时间的延长而增强,同时随着挥发油浓度的增加,粘虫的死亡率增大,且致死时间大大缩短;与触杀相比,采用土荆芥挥发油熏蒸防治粘虫,效率更高。土荆芥挥发油对淡色库蚊2 h熏蒸处理的致死中浓LC₅₀=1.5853μL/L。LC₉₅剂量处理所得的击倒中时KT₅₀为8.71 min。具有高效、快速的熏蒸活性。但是只有2 h的趋避时间,驱蚊效率低,刺激皮肤,且对人有较强毒性,不适合作为蚊虫的驱避剂和涂肤液使用。更多还原

【Abstract】 The volatile oil of many plants has the antibioticaction, sterilization and desinsection activities. Due to the wide concern on green chemicals and pesticides in ten years or so, botanical chemicals, especially the research of plant volatile oil has become into one of the hotspots in the subjects of modern biology and Chinese traditional medicine.The volatile oil of Chenopodium ambrosioides L. has anticancer, expelling wind and removing damness, expelling parasite and antibioticaction activities. Many researchers in the world have studied the volatile oil of C. ambrosioides, but most of the studies focus on the chemical constituents, desinsection and seed germination. The main deficiencies is different researcher use different separation and identification conditions, so that the main chemical constituents and their contents are different for different habitats and there is only a few researches about antibiotic action and restraint to botanical pathogen. So this study used the uniform extraction and identification conditions and methods, built the standard fingerprints of C. ambrosioides and analyzed the chemical constituents and their contents of C. ambrosioides for 12 habitats in 9 provinces of China. This research will provide the scientific basis for the quality control of C. ambrosioides and have important effect on the improving the quality of Chinese traditional medicine and its modernization. Based on the fingerprint of C. ambrosioides, this thesis researched the antibioticaction of 15 standard strains for skin and instrument sterilization and the antibacterial activity of 10 common botanical pathogens. All of the results will produce important effect on the development and utilization of C. ambrosioides to manufacture natural sterilizing products, natural medicine and pesticides. The main conclusion as follow:1. Based on the uniform extraction and identification conditions and methods, this research built the standard fingerprints of C. ambrosioides for the 12 habitats in 9 provinces of China. The GC analysis condition of the fingerprint has the advantage of very good separating effect, precision, repeatability and stability; therefore the fingerprints can be used as the standard and scientific evidence for the quality control of C. ambrosioides.2. The chemical constituents of the C. ambrosioides volatile oil in 12 habitats are similar, and they areα-terpinene, P-cymene, ascaridole and bornylene, the content of the chemical constituents is different in different habitats. All of the main chemical constituents have the menthane skeleton and they compose more than 90% of the total volatile oil. The constituents whose relative content is over 1% normally less than 5 kinds3. The quality of C. ambrosioides volatile oil was affected by the water and fertility condition in cultivate, time of sampling, plant parts of sampling, storage time and the extraction time of volatile oil. Among the different water treatments, the highest yield of volatile oil was got by the treatment with soil relative water content 40%. The yields of volatile oil are all obviously increased by the five kinds of micro fertilizer treatments, and the highest one was got by the treatment with more Zn and Mn fertilizers. There are significant differences between the yields of oil from different plant parts, the highest yield was got from the seed, the lowest was got from the stem and the oil yield from the leaf of C. ambrosioides was between the former two parts. The main chemical constituents of volatile oil from the three parts are basically similar, but the contents of them are different. Among different growth period, the lowest volatile oil yield was got in seedling period, and the yield of volatile oil was increasing with the growth of C. ambrosioides, the highest value appeared at Ripe stage, then sharply decrease.Storage time can affect the yield of volatile oil. But yield increased with the extending of storage time within 15 days, and then it decreased from 15 to 30 days. As a whole, the yield doesn’t change very much during 30 days.In a short word, the best collection period for C. ambrosioides is ripe period, the best part to collect is the part aboveground with fruit, the best storage time is 15 days after collection, and the quality and quantity of the volatile oil of C. ambrosioides is best after 1 h extraction with vapor distillation.4. The volatile oil from C. ambrosioides has extensive effect of antibioticaction and sterilization. It has inhibiting effect to the 14 experimental strains except Bacillus aeruginosus among the 15 standard strains for skin and instrument sterilization. The inhibitory concentration of volatile oil from C. ambrosioides in vitro is low, and the lowest value are 1.95-62.5μL /mL and the lowest bactericidal concentration are 7.8-250μL /mL to different strains. Therefore, volatile oil from C. ambrosioides has good inhibiting effect, and it is one of the high-value medicinal herbs to exploit.The result of acute toxicity experiment showed that toxic dose of intragastric administration of volatile oil from C. ambrosioides to small mouse is high, the half-lethal dose (LD₅₀) is 1183μL /kg,this result supposed that the LD₅₀ for human being of volatile oil from C. ambrosioides should be 9.18 mL /70kg.In this thesis, we use the growth rate method identified the inhibition effect of volatile oil from C. ambrosioides to 10 botanical pathogen. The result showed the volatile oil from C. ambrosioides has inhibiting activities to 10 botanical pathogen except Sclerotinia sclerotiorum. The inhibiting acitivity to Rhizoctonia cerealis is strongest among the 10 botanical pathogen, and its inhibiting ratio reached 100% at 0.5 uL/mL in 9 days. The inhibiting activity to Alternaria solani and Phytophthora capsici is stonger than to Gaeumannomyces graminis, Fusarium graminearum and Fusarium oxysporum （scho） f.sp.cucumbrum. The inhibiting ratio of the former two at 0.5 uL/mL in 7 days was 88.9% and 74.5%, respectively. And the volatile oil from C. ambrosioides has no inhibiting activity to Botrytis cirerea, Glomerella cingulata, Colletrichum orbiculare, and Sclerotinia sclerotiorum. The inhibiting rates of them are all under 35%.The inhibiting effect increased with the increasing concentration of volatile oil from C. ambrosioides, meanwhile, the inhibiting capability decreased with the lasting of culture time. The volatile oil from C. ambrosioides has common, high efficient and lasting inhibiting activity to lots of botanical pathogen and has a promising application for pesticide.The volatile oil from C. ambrosioides has fumigation effect on insects, and the effective fumigating dose of Mythimna separata and Culex pipien is less than 10 ppm.The median lethal concentration (LC₅₀) of volatile oil from C. ambrosioides is only 2.11μL/L to M. separata after 30 min fumigation, and it decrease sharply to around half （1.07μL/L） after 75 min fumigation. The LT₅₀ of LC₉₅ dose treatment to M. separata is 20.04 min. The result indicate that the contact toxicity activity of volatile oil to M. separata increase with the contact time lasting, meanwhile, the death rate increase and the lethal time obviously decrease with the increasing of volatile oil concentration. The efficiency of fumigation is higher than that of contact toxicity. The median lethal concentration (LC₅₀) of volatile oil from C. ambrosioides to C. pipien is 1.59μL/L after 2 h fumigation. The median knockdown time (KT₅₀) of LC₉₅ dose treatment to C. pipien is 8.71 min. These result indicated the volatile oil from C. ambrosioides has high speed and efficient of fumigation activity as well, but the avoidance time of it is only 2 h, meanwhile, it is irritating to skin and has toxicity to human being, therefore it is unsuited to using as repellent for mosquito and soothing lotion to human being.更多还原