【作者】 贾薇；

【导师】 江滨；

【作者基本信息】 广州中医药大学 ， 中药学， 2009， 博士

【摘要】 随着中药产业的发展,中药的安全性问题日益受到国内外的普遍关注。近些年来,中药重金属问题引起了国际上的高度重视。中药重金属含量超标不仅降低了中药质量、影响了用药的安全性,还制约了中药的出口,因此对中药中重金属进行控制已成为当前急需解决的重要问题之一。解决并控制好中药中重金属的含量,对于提高中药质量、保证用药安全,以及开拓中药的国际市场等均具有重要的意义。对于中药重金属的质控,首先要求建立科学的中药重金属分析方法。按照目前的国内外标准,在中药中通常监控的有害重金属元素主要包括铅（Pb）、镉（Cd）、汞（Hg）、铜（Cu）、砷（As）等。在对中药中重金属元素进行分析时,先要对样品进行适当的前处理,进而采用相应的仪器测定其中重金属元素的含量。目前常用的样品前处理方法主要有湿法消解、干法消解和微波消解法等;常用的测定方法主要有电感耦合等离子体质谱法（ICP-MS）、原子吸收光谱法（AAS）、原子荧光光谱法（AFS）等。中药重金属的质控,还应贯穿于药材的种植等一系列的过程中。中药重金属污染与多方面的因素有关,为了从源头控制中药中重金属的含量,我国开展了中药材的规范化种植研究。对药材而言,其中重金属含量的高低,主要取决于两个方面:一是土壤中重金属元素含量及土壤的性质;二是药材本身对重金属元素的选择和积累能力,而不同种类的药材对不同重金属元素的富集、积累能力是不同的。开展药材及其土壤中重金属元素的相关性研究和药材对重金属元素的吸收富集特征研究对于从源头上控制中药中重金属限量具有十分重要的指导意义,为进一步完善中药材规范化种植基地的土壤环境质量评价标准提供科学依据。砂仁、佛手、巴戟天、穿心莲、广藿香是临床常用药物和南药的主要品种,需求量较大,而且其药用部位不全相同,基体性质各异,具有一定的代表性,因此,本研究选择这五种药材作为研究对象,以铜、砷、镉、铅、汞五种重金属元素为研究指标,先对样品的前处理方法及不同测定方法进行了系统的对比研究,进而在此基础上,建立了一种准确、快速、简便的中药材中重金属分析方法。然后利用所建立的方法对药材样品进行分析,探讨了中药材及其生长土壤中重金属元素的相关性,为揭示中药材重金属污染的来源提供了科学依据,对中药材的规范化种植也具有一定的指导意义。此外,还研究了广东道地药材广藿香对土壤重金属元素的吸收富集特征,为科学管理中药材种植基地土壤环境和生产质量控制提供科学依据。现将主要研究内容和结果概述如下:1.中药材样品前处理方法的研究1.1中药材中铜、砷、镉、铅元素的前处理方法研究分别采用湿法、干法和微波消解法对样品进行前处理,然后利用电感耦合等离子体质谱法（ICP-MS）测定其中铜、砷、镉、铅四种元素的含量。通过对结果进行方差分析,探讨不同前处理方法对四种元素测定结果的影响,以期选出一种适合于中药材中这四种重金属元素分析的最佳前处理方法。在湿法中,对消解体系进行了比较,分别采用硝酸、硝酸-过氧化氢、硝酸-高氯酸对样品进行消解,结果表明,以硝酸-高氯酸作为消解体系时效果最佳;在干法中,对灰化温度进行了考察,分别以400、500、600℃对样品进行灰化处理,结果表明,对于铜和铅,最佳灰化温度为500℃,对于砷和镉,由于在高温下极易损失而使测定结果显著偏低,所以不宜采用该法;在微波消解法中,采用正交试验设计对微波消解条件进行了优化,得到了四种元素的最佳微波消解程序。通过对湿法、干法和微波消解法的对比,得出微波消解法为最佳样品前处理方法。因此,本研究选择微波消解法作为样品前处理方法,通过实验确定的最佳微波消解程序可用于中药材中铜、砷、镉、铅四种元素的前处理。1.2中药材中汞元素的前处理方法研究分别采用湿法和微波消解法对样品进行前处理,然后利用冷原子吸收光谱法（CVAAS）测定其中汞元素的含量。通过对结果进行方差分析,探讨不同前处理方法对汞测定结果的影响,以期选出一种适合于中药材中汞元素的最佳前处理方法。在湿法中,考察了硝酸-高氯酸和五氧化二钒-硝酸-硫酸两种消解体系;在微波消解法中,采用正交试验设计对微波消解条件进行了优化,得到了汞的最佳微波消解程序。通过对不同前处理方法的对比,得出微波消解法为汞元素分析时的最佳前处理方法。因此,本研究选用微波消解法作为样品的前处理方法,通过实验确定的最佳微波消解程序可用于药材中汞元素的前处理。2.中药材中重金属不同测定方法的对比研究2.1 AAS和ICP-MS测定中药材中铜、砷、镉、铅的对比研究样品经微波消解法处理后,分别采用AAS法和ICP-MS法测定其中铜、砷、镉、铅四种元素的含量。通过对测定结果进行统计分析,比较两种方法是否存在差异。配对t检验结果表明,两种测定方法所得结果之间无显著性差异（p＞0.05）。虽然两种测定方法均有较好的准确度,但ICP-MS法比AAS法更加快速、简便,所以本研究首选ICP-MS法作为药材中铜、砷、镉、铅四种重金属元素的测定方法。2.2 CVAAS和AFS测定中药材中汞的对比研究样品经微波消解法处理后,分别采用CVAAS法和AFS法测定其中汞元素的含量。通过对测定结果进行统计分析,比较两种方法是否存在差异。配对t检验结果表明,两种测定方法所得结果之间无显著性差异（P＞0.05）。虽然两种测定方法的准确度均较好,但以CVAAS法的操作更为简便、快速,因此本研究首选CVAAS法作为药材中汞元素的测定方法。3.中药材中重金属分析方法的建立3.1微波消解-ICP-MS法测定中药材中铜、砷、镉、铅方法的建立以微波消解法对样品进行处理,然后采用ICP-MS法测定其中铜、砷、镉、铅四种元素的含量,建立了中药材中这四种有害重金属元素的分析方法。铜、砷、镉、铅四种元素的线性范围分别为0～500μg·L^-1、0～50μg·L^-1、0～10μg·L^-1、0～100μg·L^-1,相关系数r分别为0.9995、0.9997、0.9996、0.9995;检出限（S/N=3）分别为0.013、0.065、0.004、0.010μg·L^-1,定量限（S/N=10）分别为0.045、0.216、0.012、0.033μg·L^-1;方法的重复性分别为1.2%、2.0%、3.5%、1.6%（n=5）;加标回收率分别为103.0%、96.5%、95.8%、98.7%,RSD分别为2.0%、2.7%、3.5%、3.2%（n=6）。结果表明,该法准确、快速、简便,适用于中药材中铜、砷、镉、铅四种重金属元素的含量测定。3.2微波消解-CVAAS法测定中药材中汞方法的建立以微波消解法对样品进行处理,然后采用CVAAS法测定其中汞元素的含量,建立了中药材中有害重金属元素汞的分析方法。汞元素的线性范围为0～6 ng·mL^-1,相关系数r为0.9997;检出限（S/N=3）为0.204 ng·mL^-1,定量限（S/N=10）为0.679ng·mL^-1;方法的重复性为3.0%（n=5）;加标回收率为93.6%,RSD为3.5%（n=6）。结果表明,该法准确、快速、简便,适用于中药材中汞元素的含量测定。4.中药材及其土壤中重金属元素的相关性研究对砂仁、佛手、巴戟天、穿心莲和广藿香五种广东地产药材及其生长土壤中铜、砷、镉、铅、汞五种重金属元素的含量进行测定,然后采用相关分析的方法探讨药材及其土壤中重金属元素的相关关系。相关分析结果表明,砂仁中铜、砷、镉、铅、汞含量与土壤中铜、砷、镉、铅、汞含量相关关系均不显著;佛手中铜含量与土壤中铜含量呈显著的相关关系,而其它四种元素在药材与土壤中的含量均无显著的相关关系;巴戟天中铜含量与土壤中铜含量呈极显著的相关关系,砷、镉、汞含量与土壤中砷、镉、汞含量呈显著的相关关系,而铅与土壤中铅含量相关关系不显著;穿心莲、广藿香中铜含量与土壤中铜含量均呈极显著的相关关系,两种药材中砷、镉含量与土壤中砷、镉含量呈显著的相关关系,而两种药材中铅、汞含量与土壤中铅、汞含量均无显著的相关关系。研究结果说明,不同种类的药材对土壤中重金属元素的吸收具有选择性;不同重金属在植物-土壤系统中的迁移规律是不同的,以土壤中的铜较易被药材吸收积累,砷和镉次之,而铅和汞的迁移性相对较弱。5.广藿香对重金属元素的吸收富集特征研究通过对不同生长时间的广藿香中铜、砷、镉、铅、汞五种重金属元素的含量进行测定,研究重金属在广藿香体内的动态吸收积累特征。结果表明,五种重金属元素在广藿香地上部分中的含量均随着生长时间的延长而增加;在根中,铜和汞的含量随生长时间的延长而增加,但砷和镉的含量却随时间的延长呈下降趋势,而铅在根中的含量随时间变化不明显。分析了广藿香对土壤中重金属元素的富集能力,结果表明,不同部位对各种重金属的富集能力不同,以叶对重金属的富集能力相对较强;由富集系数的大小可以看出,广藿香对铜元素的富集能力相对较强,而对砷、镉、铅、汞四种元素的富集能力相对较弱。本研究还探讨了广藿香中重金属含量与土壤中重金属含量及土壤性质之间的关系,多元回归分析结果表明,广藿香各部位对铜、砷、镉的吸收富集均受土壤性质影响,广藿香根对铅的吸收富集也受土壤性质影响,得到的回归方程可用于根据土壤中重金属含量及土壤性质计算广藿香中该元素的含量;而广藿香各部位对汞的吸收富集与土壤中汞含量和土壤性质的关系不明显,广藿香地上部分对铅的吸收富集与土壤中铅含量和土壤性质的关系也不明显,这说明除了上述因素外,还与其他因素有关,原因有待于进一步研究。更多还原

【Abstract】 With the development of the traditional Chinese medicine,its safety problem is increasingly subjected to a domestic and oversea widespread concern.In recent years,the problem of heavy metals in traditional Chinese medicine aroses a high international recognition.The excess content of heavy metals in traditional Chinese medicine not only influenced its quality and its medication safety,but also restricted the export of traditional Chinese medicine.Therefore,to control the content of heavy metals in traditional Chinese medicine has become one of the important problems which need solving urgently at present. It has an important meaning in ensuring the quality and the medication safety of traditional Chinese medicine,it is also important to expand the international market of traditional Chinese medicine.In order to control the heavy metals in traditional Chinese medicine,we should firstly establish a scientific analytical method of heavy metals in traditional Chinese medicine. According to the current international standard,the harmful heavy metals controlled in traditional Chinese medicine mainly includes lead（Pb）,cadmium（Cd）,mercury（Hg）, copper（Cu）,arsenic（As）,etc..When we analyse the heavy metals in traditional Chinese medicine,we should process the sample properly and then use instrument determine the contents of heavy metal elements.At present,the sample pre-processing methods mainly include wet digestion,dry digestion and microwave digestion and so on;and the determination methods mainly include inductively coupled plasma mass spectrometry （ICP-MS）,atomic absorption spectrometry（AAS） and atomic fluorescence spectrometry （AFS）,etc..To control the heavy metals in traditional Chinese medicine,we should also carry it out in a series of process like herbal planting.Heavy metal contamination in traditional Chinese medicine has something to do with many factors.In order to control the contents of heavy metals in traditional Chinese medicine originally,we put into effect Good Agricultural Practice（GAP） of Chinese medicinal materials.The contents of heavy metals in medicinal materials mainly depends on two factors:one is the contents of heavy metals in the soil in which the medicinal materials grows and the soil property;the other is the accumulation capability of the medicinal materials,and different kinds of medicinal materials have different accumulation capability to different heavy metals.The research on correlation of heavy metals in Chinese medicinal materials and their soils has a very important instruction meaning to control the contents of heavy metals originally,so is the study on absorption and accumulation characteristics of heavy metals in Chinese medicinal materials.And at the same time,they will also provide scientific basis to further perfect soil environment quality evaluation standard of Chinese medicinal materials production base.Fructus Amomi,Fructus Citri Sarcodactylis,Radix Morindae Officinalis,Herba Andrographis and Herba Pogostemonis are commonly used clinical medication and the main breed of south medicines,they are demanded greatly in the market.The medicinal parts of the five Chinese medicinal materials are not same,and their matrix nature are different.Therefore,we choosed the five Chineses medicinal materials as study objects, choosed copper,arsenic,cadmium,lead and mercury as study targets,we studied the sample pre-processing methods systemly,compared the different determination methods, and established the analytical method of heavy metal in Chinese medicinal materials which was accurate,rapid and convenient.Furthermore,we studied the correlation of heavy metals in Chinese medicinal materials and its soils,this study will provide scientific basis to discuss the source of heavy metal in Chinese medicinal materials.We also studied the absorption and accumulation characteristics of heavy metals in Herba Pogostemonis which is one of the famous-region drugs in Guangdong province,this study will provide scientific basis to administer the soil environment quality of the Chinese medicinal materials growth base and to control the production quality.The main contents and results of the study are as follows:1.Study on sample pre-processing method1.1 Study on pre-processing method of copper,arsenic,cadmium and lead in Chinese medicinal materialsWe processed the sample with wet digestion,dry digestion and microwave digestion respectively,and used ICP-MS determine the contents of copper,arsenic,cadmium and lead.Then we analysed the results with analysis of variance in order to discuss the influence of different pre-processing methods.By doing this we wanted to find an optimal pre-processing method for the four heavy metal elements in Chinese medicinal materials.In wet digestion method,we compared the different digestion systems.We digested the samples with nitric acid,nitric acid-hydrogen peroxide and nitric acid-perchloric acid respectively,the results showed that the effect of nitric acid-perchloric acid as the digestion systemwas better.In dry digestion method,we compared different ash temperatures.We processed the samples with the temperatures that were 400℃,500℃and 600℃,the results showed that the optimal ash temperature of copper and lead was 500℃,arsenic and cadmium were not suitable for the dry digestion.In microwave digestion method,we optimized the microwave digestion conditions with the orthogonal experiment and got the optimal microwave digestion process of the four elements.By comparing the three methods, we concluded that the microwave digestion was the best pre-processing method.So we choosed the microwave digestion as the sample pre-processing method in this study,and the optimal microwave digestion process we got in the experiment could be applied for the pre-processing of the four elements in Chinese medicinal materials.1.2 Study on pre-processing method of mercury in Chinese medicinal materialsWe processed the sample with wet digestion and microwave digestion respectively, and used cold-vapor atomic absorption spectrometry（CVAAS） determine the content of mercury.Then we analysed the results with analysis of variance in order to discuss the influence of different pre-processing methods of mercury to the results.By doing this we wanted to find an optimal pre-processing method for mercury in Chinese medicinal materials.In wet digestion method,we choosed nitric acid-perchloric acid and vanadium pentoxide- nitric acid-sulphuric acid respectively as the digestion systems.In microwave digestion method,we optimized the microwave digestion conditions with the orthogonal experiment and got the optimal microwave digestion process of mercury.By comparing the different pre-processing methods,we concluded that the microwave digestion was the optimal pre-processing method of mercury.So we choosed the microwave digestion as the sample pre-processing method in this study,and the optimal microwave digestion process we got in the experiment could be applied for the pre-processing of mercury in Chinese medicinal materials.2.Study on different determination methods of heavy metals in Chinese medicinal materials2.1 Study on determination of copper,arsenic,cadmium and lead in Chinese medicinal materials by comparing AAS with ICP-MSThe samples were processed with microwave digestion,then we used AAS and ICP-MS determine the contents of copper,arsenic,cadmium and lead.We analysed the results in order to compare whether the two methods had difference or not.The paired t-test results showed that the two methods didn’t have significant difference（P>0.05）.The two determination methods were accurate,but ICP-MS was faster and more convenient than AAS.So we choosed ICP-MS firstly as the determination method of the four elements in Chinese medicinal materials in this study.2.2 Study on determination of mercury in Chinese medicinal materials by comparing CVAAS with AFSThe samples were processed with microwave digestion,then we used CVAAS and AFS determine the content of mercury.We analysed the results in order to compare whether the two methods had difference or not.The paired t-test results showed that the two methods didn’t have significant difference（P>0.05）.The two determination methods were accurate,but CVAAS was more convenient and faster.So we choosed CVAAS firstly as the determination method of mercury in Chinese medicinal materials in this study.3.Establishment of analytical method of heavy metals in Chinese medicinal materials3.1 Establishment of determination of copper,arsenic,cadmium and lead in Chinese medicinal materials by microwave digestion- ICP-MSWe processed the samples with microwave digestion,then used ICP-MS determine the contents of copper,arsenic,cadmium and lead,and established the analytical method of the four harmful elements.The linear range of copper,arsenic,cadmium and lead were 0～500μg·L^-1,0～50μg·L^-1,0～10μg·L^-1,0～100μg·L^-1,their correlation coefficient were 0.9995, 0.9997,0.9996,0.9995;their detection limit were 0.013μg·L^-1,0.065μg·L^-1,0.004μg·L^-1, 0.010μg·L^-1,their determination limit were 0.045μg·L^-1,0.216μg·L^-1,0.012μg·L^-1,0.033μg·L^-1;the results of their reproducibility were 1.2%,2.0%,3.5%,1.6%（n=5）;their recovery rates were 103.0%,96.5%,95.8%,98.7%,the values of their RSD were 2.0%, 2.7%,3.5%,3.2%（n=6）.The results showed that the method was accurate,rapid and convenient,it can be applied to the determination of heavy metals in Chinese medicinal materials.3.2 Establishment of determination of mercury in Chinese medicinal materials by microwave digestion-CVAASWe processed the samples with microwave digestion,then used CVAAS determine the contents of mercury,and established the analytical method of mercury.The linear range of mercury was 0～6 ng·mL^-1,the correlation coefficient was 0.9997;the detection limit was 0.204 ng·mL^-1,the determination limit was 0.679 ng·mL^-1;the reproducibility of the method was 3.0%（n=5）;the recovery rate was 93.6%,RSD was 3.5%（n=6）.The results showed that the method was accurate,rapid and convenient,it can be applied to the determination of mercury in Chinese medicinal materials.4.Study on correlation analysis of heavy metals in Chinese medicinal materials and their soilsWe determined the contents of copper,arsenic,cadmium,lead and mercury in Fructus Amomi,Fructus Citri Sarcodactylis,Radix Morindae Officinalis,Herba Andrographis, Herba Pogostemonis and the soils they growed.Then we discussed their correlativity by adopting correlation analysis.The results showed that the contents of copper,arsenic, cadmium,lead and mercury in Fructus Amomi and its soil didn’t have significant correlativity;the content of copper in Fructus Citri Sarcodactylis and its soil had significant correlativity,but the contents of the other four elements in Fructus Citri Sarcodactylis and its soil didn’t have significant correlativity;the contents of copper, arsenic,cadmium and mercury in Radix Morindae Officinalis and its soil had significant correlativity,but the content of lead in Radix Morindae Officinalis and its soil didn’t have significant correlativity;the contents of copper,arsenic,cadmium in Herba Andrographis,Herba Pogostemonis and their soils had significant correlativity,but the contents of lead,mercury in the two medicinal materials and their soils didn’t have significant correlativity.The results of the study revealed that different medicinal materials had selective absorption to heavy metals in the soil.The study also revealed that the transfer character of different heavy metals in plant-soil system was different,the copper in the soil was easily absorbed and accumulated by the medicinal materials,arsenic and cadmium took second place,but lead and mercury in the soil were difficult to be absorbed and accumulated by the medicinal materials.5.Study on the absorption and accumulation characteristics of heavy metals in Herba PogostemonisWe determined the contents of copper,arsenic,cadmium,lead and mercury in Herba Pogostemonis that grew at different time.By doing this we wanted to study the dynamic absorption and accumulation characteristics of heavy metal elements in Herba Pogostemonis.The results showed that the contents of the five heavy metal elements in the aerial parts of Herba Pogostemonis increased along with its growth period;the contents of copper and mercury in the root of Herba Pogostemonis also increased along with its growth period,but the contents of arsenic and cadmium in the root decreased along with growth period,and the content of lead in the root didn’t have distinct change along with time.We studied the accumulation capability of Herba Pogostemonis to heavy metals in soil.The results showed that the accumulation capability of different parts to heavy metals was different,the leaf of Herba Pogostemonis had a relatively big accumulation capability to heavy metals in the soil.By analysing the accumulation coefficients,we found that the accumulation capability of Herba Pogostemonis to copper was relatively bigger,but it had a small accumulation capability to arsenic,cadmium,lead and mercury.We also studied the relation of the contents of heavy metals in Herba Pogostemonis and its soil property.The results of multiple linear regression showed that the absorption and accumulation of copper, arsenic,cadmium in every part of Herba Pogostemonis was influenced by the the soil property,so was lead in its root.The regression equations we got could be applied to calculate the contents of heavy metal elements in Herba Pogostemonis according to the contents of heavy metals in the soil and the soil property.But the absorption and accumulation of mercury in every part of Herba Pogostemonis had nothing to do with the content of mercury in the soil and the soil property,so did lead in its aerial parts.This revealed that there were other influencing factors besides the contents of heavy metals in the soil and the soil property,so the reason need further studying.更多还原