地龙种质资源与品质评价研究
摘要
地龙为钜蚓科动物参环毛蚓Pheretima aspergillum(E Perrier)、通俗环毛蚓Pheretima vulgaris(Chen)、威廉环毛蚓Pheretima guillelmi(Michaelsen)或栉盲环毛蚓Pheretima pectinifera(Michaelsen)的干燥体。前一种习称“广地龙”,后三种习称“沪地龙”,为常用动物类中药,具有清热定惊,通络平喘,利尿的功效。现代研究表明,地龙具有降压、抗血栓、抗心律失常、抗癌、抗溃疡、解热镇痛、镇静、平喘、抗菌等多种药理作用,几乎涉及人体各个系统,因此近年来地龙的需求量与日俱增。
长期以来地龙类药用动物处于自然生长状态,药材来源多为野生采集,人工养殖尚未较大规模开展,地龙药材的产量和质量易受采集方法、环境以及自然气候影响而不定。且由于地龙类药用原动物品种较多,尚缺乏真实有效的鉴别方法,经常出现误采误收的现象,导致各地市场上的广地龙药材品种混杂,价格变化较大,药材品质优劣参差不齐等问题,极大地影响了地龙的生产与发展。
为了确保地龙资源的可持续发展与利用,我们在广东省惠州市博罗县建立了地龙(参环毛蚓)规范化养殖基地,并在此基础上对现有地龙的资源种类、分布进行调查,研究了其生物学特性、遗传多样性和地理背景系统,建立药材综合品质评价指标。研究的结果概括如下:
一、文献综述
查阅了大量与本研究相关的国内外文献资料,系统的总结了前人的研究成果与存在的问题,针对地龙药材资源与鉴定、药材质量控制与评价、安全性研究等方面存在的薄弱环节提出了本论文的研究内容与目标。
二、地龙类药用动物的种质资源调查
对地龙药材进行了药源调查和分类学研究,结果表明:地龙全国均有分布,广东、广西为“广地龙”药材主要产区;上海、浙江、江苏为“沪地龙”主要产区;其余各地习用品种为“土地龙”。全国地龙主要商品来源为“广地龙”。我国地龙药材的原动物主要有14种和变种,分别隶属于钜蚓科、正蚓科的3个属。
三、地龙类药用动物的物种鉴别
通过实地捕捉或委托采集的方式收集到国内地龙主要产区的16个不同地点的蚯蚓样品共351条,利用经典动物分类学方法对所收集的样本进行鉴别,发现广东省主要分布着参环毛蚓Pheretima.aspergillum(E Perrier),其中也少量分布有白颈环毛蚓P.californica(Kinberg)和壮伟环毛蚓P.robusta(E.Perrier);广西地区除了分布有参环毛蚓aspergillum(E Perrier)外,还分布有数量均比较多的通俗环毛蚓P.vulgaris(Chen)和白颈环毛蚓P.californica(Kinberg),以及少量的威廉环毛蚓P.guillelmi(Michaelsen)和壮伟环毛蚓P.robusta(E.Perrier);湖北省主要为背暗异唇蚓Allolbophora caliginoa erapezoides(Ant.Duges),赤子爱胜蚓Eiseniafoetida(Savigny),通俗环毛蚓P.vulgaris(Chen)以及少量的威廉环毛蚓P.guillelmi(Michaelsen);福建省则分布着较多的壮伟环毛蚓P.robusta(E.Perrier)。进一步证实了广东与广西为广地龙(参环毛蚓)的主要产区。
比较了样品不同处理保存方法的优缺点,建议用于性状鉴别的标本,采用福尔马林保存;用于理化分析样品可直接晒干保存;用于分子生物学实验,需提取基因组DNA的原动物样品,应迅速定种后用少量70%乙醇或者生理盐水浸泡,密封在试管中,—20℃冻存。
使用体视镜对样品的体表特征以及内部构造的观察,并与相关标准文献进行比较的性状鉴别方法,基本都可以准确的对样品进行鉴别定种,获得相关的种质资源分布状况。根据研究的结果,为便于快速准确的对药用蚯蚓进行鉴别分类,我们编制了14种地龙原动物的分种检索表和分种检索图。
本研究结果表明,采用传统分类学指标,可较方便地对形态特征保存完好地蚯蚓样品进行鉴别分类。
四、地龙类药用动物的ISSR分子鉴定研究
利用ISSR分子标记技术对地龙不同种质资源进行了DNA水平的分子鉴定研究。
以筛选到的8个ISSR引物对供试的7个样品进行ISSR-PCR扩增,共扩增出137条带,其中多态性条带有127条,占92.7%;利用NTSYSpc-2.10e软件对ISSR表型数据矩阵进行聚类分析,用SimQual程序求Nei-Li相似系数矩阵,探讨其亲缘关系。种质间相似系数最小为0.43;最大的为0.66,说明地龙种质的遗传多样性较为丰富。
聚类分析结果表明,供试材料在相似系数为0.56处分为4个类群,第Ⅲ类群和第Ⅳ类群中各种质之间的亲缘关系较近,而第Ⅰ类群与第Ⅱ类群亲缘关系较远,同属蚯蚓内个体间的亲缘关系较近,不同属间个体的遗传距离较远。这与它们在形态鉴别分类上的结果相一致。
五、地龙药材的多组分的含量测定以及HPLC指纹图谱研究
(一)HPLC法测定地龙中尿嘧啶、次黄嘌呤、尿苷、肌苷的含量
本研究测定了不同产地地龙药材中尿嘧啶、次黄嘌呤、尿苷、肌苷的含量,建立了广地龙质量控制的客观指标。方法:用0.9%生理盐水超声提取地龙药材,采用反相高效液相色谱法,以0.01mol/L磷酸二氢钾溶液和50%甲醇为流动相,梯度洗脱,检测波长254nm,柱温27℃,进样量10μl,可同时测定尿嘧啶、次黄嘌呤、尿苷、肌苷等4种成分的含量。
结果:尿嘧啶的线性范围为0.0075-0.24μg(r=0.9993),平均回收率99.82%,RSD为2.34%;次黄嘌呤的线性范围0.0250-0.8μg(r=0.9992),平均回收率101.93%,RSD为1.45%;尿苷的线性范围0.0125-0.4μg(r=0.9992),平均回收率97.53%,RSD为1.73%;肌苷的线性范围0.0500-1.6μg(r=0.9991),平均回收率102.06%,RSD为2.44%。该方法重现性,回收率较好,可用于广地龙尿嘧啶等4种成分的含量测定。
(二)地龙水溶性成分的指纹图谱研究
建立了地龙水溶性成分的高效液相色谱指纹图谱,旨在反映地龙内在化学信息,全面评价地龙的质量。采用高效液相色谱法,梯度洗脱,对17份地龙样品测定,以主要的11个共有峰的相对保留时间和相对峰面积作为地龙指纹图谱的相关技术参数,得出的11个主要共有峰的相对保留时间重现性好,相对峰面积有明显差别,对获取的指纹图谱数据进行相似度分析,建立了地龙的指纹图谱,可用于地龙的鉴别和质量的评价。
运用SPSS分析软件对所采集图谱数据进行了聚类分析,采用离差平方和法,利用欧氏距离作为样品的测度。17个样品基本分为四大类。从聚类结果来看,广东、广西产地的地龙药材,亲缘关系较近,所含化学成分及其比值比较相似;海南产地药材比较相似;而沪地龙的亲缘关系和化学成分则与它们明显区分开来。这也印证了不同产地,不同品种的地龙药材所含的化学成分存在一定的差异。故在生产中建议固定品种和产地。
六、地龙中重金属及有害元素的检测
采用紫外分光光度法测定了地龙中重金属的含量,分析波长215nm。标准曲线的回归方程为:y=0.7231x+0.043,相关系数R~2=0.9968,结果表明检测质量分数在0～50μg/g范围内具有良好的线性关系。平均加样回收率为99.97%。该法简便、快速、重现性好,能在一次操作中测出中药中大部分重金属的含量。适用地龙药材实际生产中重金属的检测。对9批地龙药材测定结果表明,各样品中重金属元素含量差异较大,且大都超出《中华人民共和国药典》2005版一部附录ⅨE规定的应不超过百万分之三十。地龙药材中的重金属超标问题值得我们关注。
七、生态环境与地龙药材品质的相关性研究
(一)地龙生态环境因子的考察与分析
对道地药材广地龙主要产区的分布和环境情况进行了调查,并采集了广地龙产区以及非道地药材产区11个土壤样本,拟通过对广地龙主要产区的生态环境的调查和生长土壤(水分、DH值、重金属等)的分析,以考察与地龙质量相关的环境因子,了解其产地适宜性,为广地龙的规范化养殖提供理论上的支持。
地龙产区的环境因子考察结果表明:广东、广西、福建、海南四个省份地处中国东部沿海,气候、地势条件优越,适合广地龙生长发育。
产区土壤样品测试分析结果表明:含水量为25%左右的土壤较适合参环毛蚓生存。其生长发育的最适pH值为6.8～7.6。广地龙(参环毛蚓)能够在砷(4.929-16.000mg/kg)、镉(0.999-8.604mg/kg)、铬(26.830-110.168mg/kg)、铜(44.900-349.356mg/kg)、镍(94.400-480.088mg/kg)、铅(15.084-114.715mg/kg)、锌(11.163-601.859mg/kg)含量范围的土壤中生存,对土壤重金属的耐受性与其他蚓种相似。地龙产区工业发展与药材的质量优劣有重要关联。工业发展向环境中排放的重金属越多,对该产区的地龙药材的质量下降值影响越大。
(二)地龙中重金属含量与环境的相关性研究
1.广地龙对重金属富集动力学特性研究
本实验应用半静态条件下的双箱动力学模型,将不同浓度重金属条件下养殖过的参环毛蚓经浓硝酸—过氧化氢低温消解处理后测定重金属含量,探讨参环毛蚓对土壤中Cd、Hg、Pb三种重金属离子的生物富集规律。结果:地龙在Pb高、中、低浓度的富集速率分别为k_1=1.232,K_2=1.325,K_3=1.058;对Cd的高、中、低浓度富集速率分别为k_1=0.1792,K_2=0.1753,K_3=0.1382;对Hg的富集呈现出波动性。结论:广地龙从土壤中富集Pb的能力明显高于Cd,且随着时间的推移而增大;对Hg的富集作用是先快速吸收,积累到一定浓度时,再快速排出,而后再度吸收。
2.重金属对参环毛蚓金属硫蛋白wMT诱导机制研究
金属硫蛋白(Metallothionein,MT)是一类存在于生物体内的低分子量、富含半胱氨酸(Cys)、能被金属诱导表达的金属结合蛋白。蚯蚓体内因存在特殊的金属硫蛋白(wMT),对土壤中的重金属有富集作用,导致体内重金属元素含量较高。
本实验通过对参环毛蚓进行不同重金属诱导30天后,提取wMT蛋白,采用银饱和分析法,结合原子吸收ASS对蚯蚓体内wMT的含量进行测定。以探讨重金属诱导参环毛蚓wMT的有关机制。初步分析得出参环毛蚓体内wMT含量受到不同重金属的诱导影响,大小依次为:Cu＞Cd＞Zn＞Pb;且总的看来内脏中含有的wMT含量高于肌肉中,这也表明wMT主要在蚯蚓的内脏中表达。
八、地龙药材的质量标准研究
在大量实验性研究和检测的基础上,对《中华人民共和国药典》(一部,2005年版)有关“地龙”的药材质量标准作了部分修订。增加了地龙药材的含量测定项,选择次黄嘌呤和肌苷作为地龙药材含量测定的指标,规定本品按干燥品计算,含次黄嘌呤不得少于0.2 mg/g;含肌苷不得少于0.3 mg/g。另对性状、炮制等项进行了补充。产地加工方法中添加“除去两头所带泥土”;性状项增加了“除前后两端外,腹部已经剖开,内脏除去或偶有残留”的描述。炮制项建议添加“除去杂质和体内泥沙”。
本研究建立了地龙药材的含量测定方法,完善了质量标准,为地龙药材的较全面的质量控制提供依据。
九、结论与展望
本论文对地龙种质的多样性进行了较为系统的研究,这为种质资源的进一步利用提供了参考。
研究建立了地龙药材多组分含量测定方法和HPLC的指纹图谱,以及地龙药材重金属含量的紫外分光光度测定法,完善了地龙药材的质量指标体系,为药材的质量控制提供了依据。
对地龙药材质量与生态环境因子的关系进行了研究,为改善地龙种质和药材质量提供了借鉴。
其中地龙重金属富集以及WMT蛋白诱导机制等工作还可做进一步的探讨和研究。
Dilong(Earthworm)is the dried body of Pheretima aspergillwn(E.Perrier), Pheretima vulgaris Chen,Pheretima guillelmi(Michaelsen)or Pheretima pectinifera Michaelsen(Fam.Megascolecidae).The former is known as "Guang Dilong",the latter three are known as "Hu Dilong".Dilong is an animal drug in common use,which has functions of clearing away heat,tranquilizing the mind,antiasthmatic and diuretic effects.
Modern research also indicates that earthworm has many kinds of pharmacological effects such as descending blood pressure,anti-thrombus,anti-arrhythmia,anticancer, anti-ulcer,allaying fever and analgesia,sedation,antiasthmatic,antibacterial and so on, involving almost all the body systems.Therefore the demand for earthworm grew day by day in recent years.
Since long ago the earthworm grows in the nature condition,the raw materials for medicine use have been mainly collected in wild,and the artificial cultivation of Dilong has not yet large-scaly developed.Being affected by the gathering method,environment and weather,the raw material output and quality of Dilong was unstable.Because there are many kinds of earthworms,but no effective species identification method,the earthworm was often collected by mistake.Therefore the medicine varieties in the markets were in a mess,the price of Dilong has changed greatly and its' quality was unstable.As a result the production and the development of earthworm has been affected enormously.
To ensure the sustainable development and the use of the earthworm resources,we established the earthworm(Pheretima aspergillum)GAP base in Huizhou of Guangdong province,carried out the study of investigation of Dilong' germplasm resources,the biology characteristic,heredity multiplicity and its geography background system.A quality evaluating system of have been established.The research summary is as follows:
1.Literature Review
After consulting a large amount of related data both in China and abroad,we systematically summarized the research status of Dilong.In view of the weak links and problems among the previous studies on germplasm resources,identification,quality control and estimation,and safety research of Dilong,we raised the study content and the object.
2.Investigaition of Dilong' germplasm resources
We investigated the medicine source and studied the taxonomy of earthworm,and found that there are earthworms distributed all over the country."Guang Dilong" is mainly produced in Guangdong province and Guangxi province;"Hu Dilong" is mainly produced in Shanghai,Zhejiang province and Jiangsu province;and there is "Tu Dilong" used habitually in other regions.Dilong in the market over the country are mainly "Guang Dilong".There are totally 14 kinds of earthworms used as Dilong in China,belonging to 3 genera of Megascolecidae and Lumbricidae.
3.Species identification of Dilong' medical animal resources
We collected 351 earthworm samples from 16 different places in the domestic earthworm production areas,and identified the samples by classical systematic zoology method.We found that a larger part of samples in Guangdong province were Pheretima aspergillum(E.Perrier),and a small part of which were Pheretima californica(Kinberg) and P.robusto;There were not only Pheretima aspergillum(E.Perrier)in Guangxi province,but also quite many Pheretima.vulgaris(Chen.)and Pheretima californica,as well as few P.guillelmi(Michaelsen)and P.robusto;The samples in Hubei province were mainly Allolbophora caliginoa erapezoides(Ant.Duges),Eisenia foetida and P.vulgaris (Chen.),as well as few P.guillelmi(Michaelsen);There were many P.robusto,in Fujian province.This finding further confirmed that Guangdong and Guangxi were the main production area of Guang Dilong.
After comparing several different earthworm processing and preservation methods, we suggested that if the samples were used for the character distinction,it should be processed and preserved in formalin;samples used for physics and chemistry analysis could be seasoned directly;samples used in the molecular biology experiment must be kept in 70%ethyl alcohol or 0.9%of NaCl solution,-20℃.
Using the stereoscope to observe the sample body surface characteristic and the internal structure,and comparing with the reference,we could identify the species accurately.According to the result,we established a retrieval table of 14 kinds of earthworms used for medicinal purposes,and the table could be helpful in the species identification of Dilong.
This finding indicated that by using the classical systematic zoology method,we could identify the earthworm with good characteristic preservation.
4.Molecular Identification of Dilong' germplasm resources by ISSR
ISSR was applied to identify the DNA varieties of Dilong for the first time.
The ISSR amplification of 7 materials by 8 ISSR premiers screened out resulted in 137 electrophoresis strips with 127 strips(92.7%)polymorphic.The NTSYSpc-2.10e software was applied to cluster analysis to the ISSR phenotype data matrix.The minimal value of similarity coefficient among Dilong' germplasm resources was 0.43,while the maximal was 0.66,which demonstrated greater genetic variance and DNA polymorphism of Dilong' germplasm resources.
The cluster analysis result indicated that the materials were classified to 4 groups in the similarity factor 0.56.The 3rd and the 4th class groups presented closer genetic relationship,while the genetic relationship between 1~(st)and 2~(nd)groups was far.The genetic relationship of individuals in the same genera was closer than those in different genera.The result was the same with the finding by using the classical systematic zoology method.
5.Determination of multi chemical constituents and the HPLC fingerprinting of Dilong
(1)Determination of uracil,hypoxanthine,uridine and inoine in Dilong by HPLC
We established an accurate and stable method for the determination of uracil, hypoxanthine,uridine and inoine in Dilong from different places as to efficaciously control the quality of medicinal material.
Method:The samples were extracted with 0.9%NaCl solution by ultrasonic and determined by RP-HPLC method,0.01mol/L KH_2PO_4 solution and 50%MeOH as flowing,gradient elution,examination wave length was 254nm,column temperature was 27℃,10ul sample was injected.
Result:The calibration curve of uracil was linear in the quality range of 0.0075-0.24/μg(r=0.9993),the average recovery was 99.82%,RSD was 2.34%; hypoxanthine was 0.0250-0.8000μg(r=0.9992),average recovery was 101.93%,RSD was 1.45%;uridine was 0.0125-0.4000/μg(r=0.9992),average recovery was 97.53%,RSD was 1.73%;inoine was 0.0500-1.6000μg(r=0.9991),average recovery was 102.06%,RSD was 2.44%.This method reproduction quality,the returns-ratio is good,may use in the broad earthworm uracil and so on 4 ingredients the content determination.The method displayed good reproducibility and high recovery,and it provides an effective way for the study of Dilong.
(2)HPLC fingerprints of 0.9%NaCl solution extracts from Dilong
We established the HPLC fingerprints of 0.9%NaCl solution extracts from Dilong to evaluate its quality.Gradient elution HPLC was used to analyze 0.9%NaCl solution extracts from Dilong,and the data of their fingerprints were analyzed by similarity.17 samples of Dilong from different places were analyzed to test the HPLC fingerprint.The relative retention time and relative area of 11 main common peaks were obtained as correlation technical parameters of Dilong.The repeatability of the relative retention times was good for the main common peaks and the relative peak areas had significant differences.From the above results,we suggested that the HPLC fingerprinting could be used to evaluate the quality and identification of Dilong.
The HPLC fingerprint data of Dilong were cluster analyzed using the SPSS software. 17 samples were classified to 4 groups basically.
The cluster analysis result indicated that the chemical contents of Dilong from Guangdong and Guangxi province were quite similar,which presented closer genetic relationship;the samples in Hainan province were similar;while Hu Dilong was obviously different from them.
This result also verified that Dilong from different places varied form their chemical composition.So we suggest that the species and origin must be fixed in the processing.
6.Determination and analysis of heavy metals and hazardous elements in Dilong
We established an UV method to determine the content of heavy metals in Dilong and supply the basis for standardizing the content limits.The determination wave length was 215nm.The regression equation was:y=0.7231x+0.043,correlation coefficient R~2=0.9968.
The linear range of the calibration curve was 0～50μg/g,and the average recovery was 99.97%.The method was simple,rapid,reliable and had good reproducibility,and could be used to determine the content of most heavy metals in Dilong.
Due to a great distinction between 9 batches of Dilong samples in the heavy metal element content,which mostly beyond the limit of not surpass 30/1000000(Chinese Pharmacopoeia,Volume 1,2005),we should pay attention to the heavy metal problem in Dilong.
7.Study on the correlation between ecological environmental and quality of Dilong
(1)Investigation and analyzing on the ecological environment factors of Dilong
We investigated the distribution and environment of Dilong,and collected 11 soil samples,planning to inspected Dilong' habitat suitability and the main environment factors concerned with the quality by analyzing on the ecological environment factors and the growth soil(moisture content,pH value,heavy metal and so on).The results would provide as a technical support for the standardized cultivation of Dilong.
The investigation on the ecological environment factors of Dilong' habitats indicated that Guangdong Province,Guangxi Province,Fujian Province and Hainan Province situating at the east China coast,their' climate,topography condition were superior and suitable for P.aspergillum to grow.
P.aspergillum could grow better in the soil with 25%water content.The appropriate pH value was 6.8-7.6.P.aspergillum' tolerance to heavy metal was similarly to the other earthworms,and it could survive in the soil with As(4.929-16.000mg/kg), Cd(0.999-8.604mg/kg),Cr(26.830-110.168mg/kg),Cu(44.900-349.356mg/kg),Ni (94.400-480.088mg/kg),Pb(15.084-114.715mg/kg),and the Zn(11.163-601.859mg/kg). Industrial development in Dilong habitats had close relationship with the quality of Dilong. The more heavy metals emissioned to the environment due to industrial development,the worse Dilong' quality would be.
(2)Study on the correlation between ecological environmental and heavy metal content in Dilong
①The heavy metal concentration dynamics characteristic of P.aspergillum
The double box dynamics model under half static condition was applied in this experiment.P.aspergillum were raised in soil with different heavy metal conditions firstly, been digested with HNO_3-H_2O_2 at low temperature,then the heavy metal in the P. aspergillum been determined.According to the results,we could study the dynamics characteristic of P.aspergillum to the Cd,Hg,and Pb in the soil.
Results:The biology concentration ratio of earthworm P.aspergillum in Pb high, middle and low concentration was k_1=1.232,K_2=1.325,K_3=1.058 respectively;In Cd high, middle and low concentration the ratio was k_1=0.1792,K_2=0.1753,K_3=0.1382 respectively; In Hg,the ratio was undulatory.
Conclusion:The concentration ability of P.aspergillum in Pb was higher than in Cd obviously,and increases with the lapse of time.Concentration characteristic in Hg was absorbed quickly at first,discharged quickly when accumulated to certain density,and then absorbed gain.
②Study on the heavy metal inducing mechanism of P.aspergillum wMT by
MT(Metallothionein)is a low-molecular-weight protein in the organism.It is rich in cysteinyl residues and contains no aromatic amino acids.MT shows high affinity for bivalent heavy metals.Because the earthworm contains a special MT(wMT),it could concentrate heavy metals in the soil,and heavy metal content in its vivo is much high.
P.aspergillum were raised in soil with different heavy metal conditions for 30 days. The wMT in P.aspergillum was extracted and determined by the silver saturation method combined with ASS.
According to the results,we could study the induction mechanism of P.aspergillum wMT to the heavy metals in the soil.
Results:The P.aspergillum wMT content could be induced by different heavy metal, the induce ability was in turn:Cu＞Cd＞Zn＞Pb;And generally speaking wMT content in the internal organs was higher than in the muscle,this result also indicated that wMT mainly expressed in earthworm's internal organs.
8.Study on quality standard of Dilong
Based on the experimental and analytical study in this paper,some substantial modifications have been made to Chinese Pharmacopoeia(Volume 1,2005)concerning the quality of Dilong.Determination item of Dilong have been added,choosing hypoxanthine and inoine as determination target,the minimal limited quantity of hypoxanthine in the dry drug should be no less than 0.2 mg/g;Inoine should be no less than 0.3 mg/g.
In addition,some revision have been made in the character and processing items.The words "to clean the soil in the two ends" have been added in the processing method.The words "the abdomen has already been cut open except two ends,the internal organs have been cleaned or occasionally remain" have been added in the character description.The words "to clean the impurity and soil in the body" have been added in the preparation.
This study has established the determination method for Dilong,and modified the quality standard.The modifications would promote the quality control of Dilong.
9.Conclusion and further study
A systematic research has been made on polymorphism of Dilong' germplasm resources,which will provide the reference for the further use of Dilong.
We have established a HPLC fingerprinting and determination method,as well as an UV method to determine the content of heavy metals in Dilong.This study has modified the quality standard to promote the quality control of Dilong.
Based on the investigation and analyzing of the ecological environment factors of Dilong,the research will serve as a reference to promote the qualiy of Dilong.
Insufficient research has been made on the induction mechanism of P.aspergillum wMT by heavy metal,and it's worth making the further research in the future.
长期以来地龙类药用动物处于自然生长状态,药材来源多为野生采集,人工养殖尚未较大规模开展,地龙药材的产量和质量易受采集方法、环境以及自然气候影响而不定。且由于地龙类药用原动物品种较多,尚缺乏真实有效的鉴别方法,经常出现误采误收的现象,导致各地市场上的广地龙药材品种混杂,价格变化较大,药材品质优劣参差不齐等问题,极大地影响了地龙的生产与发展。
为了确保地龙资源的可持续发展与利用,我们在广东省惠州市博罗县建立了地龙(参环毛蚓)规范化养殖基地,并在此基础上对现有地龙的资源种类、分布进行调查,研究了其生物学特性、遗传多样性和地理背景系统,建立药材综合品质评价指标。研究的结果概括如下:
一、文献综述
查阅了大量与本研究相关的国内外文献资料,系统的总结了前人的研究成果与存在的问题,针对地龙药材资源与鉴定、药材质量控制与评价、安全性研究等方面存在的薄弱环节提出了本论文的研究内容与目标。
二、地龙类药用动物的种质资源调查
对地龙药材进行了药源调查和分类学研究,结果表明:地龙全国均有分布,广东、广西为“广地龙”药材主要产区;上海、浙江、江苏为“沪地龙”主要产区;其余各地习用品种为“土地龙”。全国地龙主要商品来源为“广地龙”。我国地龙药材的原动物主要有14种和变种,分别隶属于钜蚓科、正蚓科的3个属。
三、地龙类药用动物的物种鉴别
通过实地捕捉或委托采集的方式收集到国内地龙主要产区的16个不同地点的蚯蚓样品共351条,利用经典动物分类学方法对所收集的样本进行鉴别,发现广东省主要分布着参环毛蚓Pheretima.aspergillum(E Perrier),其中也少量分布有白颈环毛蚓P.californica(Kinberg)和壮伟环毛蚓P.robusta(E.Perrier);广西地区除了分布有参环毛蚓aspergillum(E Perrier)外,还分布有数量均比较多的通俗环毛蚓P.vulgaris(Chen)和白颈环毛蚓P.californica(Kinberg),以及少量的威廉环毛蚓P.guillelmi(Michaelsen)和壮伟环毛蚓P.robusta(E.Perrier);湖北省主要为背暗异唇蚓Allolbophora caliginoa erapezoides(Ant.Duges),赤子爱胜蚓Eiseniafoetida(Savigny),通俗环毛蚓P.vulgaris(Chen)以及少量的威廉环毛蚓P.guillelmi(Michaelsen);福建省则分布着较多的壮伟环毛蚓P.robusta(E.Perrier)。进一步证实了广东与广西为广地龙(参环毛蚓)的主要产区。
比较了样品不同处理保存方法的优缺点,建议用于性状鉴别的标本,采用福尔马林保存;用于理化分析样品可直接晒干保存;用于分子生物学实验,需提取基因组DNA的原动物样品,应迅速定种后用少量70%乙醇或者生理盐水浸泡,密封在试管中,—20℃冻存。
使用体视镜对样品的体表特征以及内部构造的观察,并与相关标准文献进行比较的性状鉴别方法,基本都可以准确的对样品进行鉴别定种,获得相关的种质资源分布状况。根据研究的结果,为便于快速准确的对药用蚯蚓进行鉴别分类,我们编制了14种地龙原动物的分种检索表和分种检索图。
本研究结果表明,采用传统分类学指标,可较方便地对形态特征保存完好地蚯蚓样品进行鉴别分类。
四、地龙类药用动物的ISSR分子鉴定研究
利用ISSR分子标记技术对地龙不同种质资源进行了DNA水平的分子鉴定研究。
以筛选到的8个ISSR引物对供试的7个样品进行ISSR-PCR扩增,共扩增出137条带,其中多态性条带有127条,占92.7%;利用NTSYSpc-2.10e软件对ISSR表型数据矩阵进行聚类分析,用SimQual程序求Nei-Li相似系数矩阵,探讨其亲缘关系。种质间相似系数最小为0.43;最大的为0.66,说明地龙种质的遗传多样性较为丰富。
聚类分析结果表明,供试材料在相似系数为0.56处分为4个类群,第Ⅲ类群和第Ⅳ类群中各种质之间的亲缘关系较近,而第Ⅰ类群与第Ⅱ类群亲缘关系较远,同属蚯蚓内个体间的亲缘关系较近,不同属间个体的遗传距离较远。这与它们在形态鉴别分类上的结果相一致。
五、地龙药材的多组分的含量测定以及HPLC指纹图谱研究
(一)HPLC法测定地龙中尿嘧啶、次黄嘌呤、尿苷、肌苷的含量
本研究测定了不同产地地龙药材中尿嘧啶、次黄嘌呤、尿苷、肌苷的含量,建立了广地龙质量控制的客观指标。方法:用0.9%生理盐水超声提取地龙药材,采用反相高效液相色谱法,以0.01mol/L磷酸二氢钾溶液和50%甲醇为流动相,梯度洗脱,检测波长254nm,柱温27℃,进样量10μl,可同时测定尿嘧啶、次黄嘌呤、尿苷、肌苷等4种成分的含量。
结果:尿嘧啶的线性范围为0.0075-0.24μg(r=0.9993),平均回收率99.82%,RSD为2.34%;次黄嘌呤的线性范围0.0250-0.8μg(r=0.9992),平均回收率101.93%,RSD为1.45%;尿苷的线性范围0.0125-0.4μg(r=0.9992),平均回收率97.53%,RSD为1.73%;肌苷的线性范围0.0500-1.6μg(r=0.9991),平均回收率102.06%,RSD为2.44%。该方法重现性,回收率较好,可用于广地龙尿嘧啶等4种成分的含量测定。
(二)地龙水溶性成分的指纹图谱研究
建立了地龙水溶性成分的高效液相色谱指纹图谱,旨在反映地龙内在化学信息,全面评价地龙的质量。采用高效液相色谱法,梯度洗脱,对17份地龙样品测定,以主要的11个共有峰的相对保留时间和相对峰面积作为地龙指纹图谱的相关技术参数,得出的11个主要共有峰的相对保留时间重现性好,相对峰面积有明显差别,对获取的指纹图谱数据进行相似度分析,建立了地龙的指纹图谱,可用于地龙的鉴别和质量的评价。
运用SPSS分析软件对所采集图谱数据进行了聚类分析,采用离差平方和法,利用欧氏距离作为样品的测度。17个样品基本分为四大类。从聚类结果来看,广东、广西产地的地龙药材,亲缘关系较近,所含化学成分及其比值比较相似;海南产地药材比较相似;而沪地龙的亲缘关系和化学成分则与它们明显区分开来。这也印证了不同产地,不同品种的地龙药材所含的化学成分存在一定的差异。故在生产中建议固定品种和产地。
六、地龙中重金属及有害元素的检测
采用紫外分光光度法测定了地龙中重金属的含量,分析波长215nm。标准曲线的回归方程为:y=0.7231x+0.043,相关系数R~2=0.9968,结果表明检测质量分数在0～50μg/g范围内具有良好的线性关系。平均加样回收率为99.97%。该法简便、快速、重现性好,能在一次操作中测出中药中大部分重金属的含量。适用地龙药材实际生产中重金属的检测。对9批地龙药材测定结果表明,各样品中重金属元素含量差异较大,且大都超出《中华人民共和国药典》2005版一部附录ⅨE规定的应不超过百万分之三十。地龙药材中的重金属超标问题值得我们关注。
七、生态环境与地龙药材品质的相关性研究
(一)地龙生态环境因子的考察与分析
对道地药材广地龙主要产区的分布和环境情况进行了调查,并采集了广地龙产区以及非道地药材产区11个土壤样本,拟通过对广地龙主要产区的生态环境的调查和生长土壤(水分、DH值、重金属等)的分析,以考察与地龙质量相关的环境因子,了解其产地适宜性,为广地龙的规范化养殖提供理论上的支持。
地龙产区的环境因子考察结果表明:广东、广西、福建、海南四个省份地处中国东部沿海,气候、地势条件优越,适合广地龙生长发育。
产区土壤样品测试分析结果表明:含水量为25%左右的土壤较适合参环毛蚓生存。其生长发育的最适pH值为6.8～7.6。广地龙(参环毛蚓)能够在砷(4.929-16.000mg/kg)、镉(0.999-8.604mg/kg)、铬(26.830-110.168mg/kg)、铜(44.900-349.356mg/kg)、镍(94.400-480.088mg/kg)、铅(15.084-114.715mg/kg)、锌(11.163-601.859mg/kg)含量范围的土壤中生存,对土壤重金属的耐受性与其他蚓种相似。地龙产区工业发展与药材的质量优劣有重要关联。工业发展向环境中排放的重金属越多,对该产区的地龙药材的质量下降值影响越大。
(二)地龙中重金属含量与环境的相关性研究
1.广地龙对重金属富集动力学特性研究
本实验应用半静态条件下的双箱动力学模型,将不同浓度重金属条件下养殖过的参环毛蚓经浓硝酸—过氧化氢低温消解处理后测定重金属含量,探讨参环毛蚓对土壤中Cd、Hg、Pb三种重金属离子的生物富集规律。结果:地龙在Pb高、中、低浓度的富集速率分别为k_1=1.232,K_2=1.325,K_3=1.058;对Cd的高、中、低浓度富集速率分别为k_1=0.1792,K_2=0.1753,K_3=0.1382;对Hg的富集呈现出波动性。结论:广地龙从土壤中富集Pb的能力明显高于Cd,且随着时间的推移而增大;对Hg的富集作用是先快速吸收,积累到一定浓度时,再快速排出,而后再度吸收。
2.重金属对参环毛蚓金属硫蛋白wMT诱导机制研究
金属硫蛋白(Metallothionein,MT)是一类存在于生物体内的低分子量、富含半胱氨酸(Cys)、能被金属诱导表达的金属结合蛋白。蚯蚓体内因存在特殊的金属硫蛋白(wMT),对土壤中的重金属有富集作用,导致体内重金属元素含量较高。
本实验通过对参环毛蚓进行不同重金属诱导30天后,提取wMT蛋白,采用银饱和分析法,结合原子吸收ASS对蚯蚓体内wMT的含量进行测定。以探讨重金属诱导参环毛蚓wMT的有关机制。初步分析得出参环毛蚓体内wMT含量受到不同重金属的诱导影响,大小依次为:Cu＞Cd＞Zn＞Pb;且总的看来内脏中含有的wMT含量高于肌肉中,这也表明wMT主要在蚯蚓的内脏中表达。
八、地龙药材的质量标准研究
在大量实验性研究和检测的基础上,对《中华人民共和国药典》(一部,2005年版)有关“地龙”的药材质量标准作了部分修订。增加了地龙药材的含量测定项,选择次黄嘌呤和肌苷作为地龙药材含量测定的指标,规定本品按干燥品计算,含次黄嘌呤不得少于0.2 mg/g;含肌苷不得少于0.3 mg/g。另对性状、炮制等项进行了补充。产地加工方法中添加“除去两头所带泥土”;性状项增加了“除前后两端外,腹部已经剖开,内脏除去或偶有残留”的描述。炮制项建议添加“除去杂质和体内泥沙”。
本研究建立了地龙药材的含量测定方法,完善了质量标准,为地龙药材的较全面的质量控制提供依据。
九、结论与展望
本论文对地龙种质的多样性进行了较为系统的研究,这为种质资源的进一步利用提供了参考。
研究建立了地龙药材多组分含量测定方法和HPLC的指纹图谱,以及地龙药材重金属含量的紫外分光光度测定法,完善了地龙药材的质量指标体系,为药材的质量控制提供了依据。
对地龙药材质量与生态环境因子的关系进行了研究,为改善地龙种质和药材质量提供了借鉴。
其中地龙重金属富集以及WMT蛋白诱导机制等工作还可做进一步的探讨和研究。
Dilong(Earthworm)is the dried body of Pheretima aspergillwn(E.Perrier), Pheretima vulgaris Chen,Pheretima guillelmi(Michaelsen)or Pheretima pectinifera Michaelsen(Fam.Megascolecidae).The former is known as "Guang Dilong",the latter three are known as "Hu Dilong".Dilong is an animal drug in common use,which has functions of clearing away heat,tranquilizing the mind,antiasthmatic and diuretic effects.
Modern research also indicates that earthworm has many kinds of pharmacological effects such as descending blood pressure,anti-thrombus,anti-arrhythmia,anticancer, anti-ulcer,allaying fever and analgesia,sedation,antiasthmatic,antibacterial and so on, involving almost all the body systems.Therefore the demand for earthworm grew day by day in recent years.
Since long ago the earthworm grows in the nature condition,the raw materials for medicine use have been mainly collected in wild,and the artificial cultivation of Dilong has not yet large-scaly developed.Being affected by the gathering method,environment and weather,the raw material output and quality of Dilong was unstable.Because there are many kinds of earthworms,but no effective species identification method,the earthworm was often collected by mistake.Therefore the medicine varieties in the markets were in a mess,the price of Dilong has changed greatly and its' quality was unstable.As a result the production and the development of earthworm has been affected enormously.
To ensure the sustainable development and the use of the earthworm resources,we established the earthworm(Pheretima aspergillum)GAP base in Huizhou of Guangdong province,carried out the study of investigation of Dilong' germplasm resources,the biology characteristic,heredity multiplicity and its geography background system.A quality evaluating system of have been established.The research summary is as follows:
1.Literature Review
After consulting a large amount of related data both in China and abroad,we systematically summarized the research status of Dilong.In view of the weak links and problems among the previous studies on germplasm resources,identification,quality control and estimation,and safety research of Dilong,we raised the study content and the object.
2.Investigaition of Dilong' germplasm resources
We investigated the medicine source and studied the taxonomy of earthworm,and found that there are earthworms distributed all over the country."Guang Dilong" is mainly produced in Guangdong province and Guangxi province;"Hu Dilong" is mainly produced in Shanghai,Zhejiang province and Jiangsu province;and there is "Tu Dilong" used habitually in other regions.Dilong in the market over the country are mainly "Guang Dilong".There are totally 14 kinds of earthworms used as Dilong in China,belonging to 3 genera of Megascolecidae and Lumbricidae.
3.Species identification of Dilong' medical animal resources
We collected 351 earthworm samples from 16 different places in the domestic earthworm production areas,and identified the samples by classical systematic zoology method.We found that a larger part of samples in Guangdong province were Pheretima aspergillum(E.Perrier),and a small part of which were Pheretima californica(Kinberg) and P.robusto;There were not only Pheretima aspergillum(E.Perrier)in Guangxi province,but also quite many Pheretima.vulgaris(Chen.)and Pheretima californica,as well as few P.guillelmi(Michaelsen)and P.robusto;The samples in Hubei province were mainly Allolbophora caliginoa erapezoides(Ant.Duges),Eisenia foetida and P.vulgaris (Chen.),as well as few P.guillelmi(Michaelsen);There were many P.robusto,in Fujian province.This finding further confirmed that Guangdong and Guangxi were the main production area of Guang Dilong.
After comparing several different earthworm processing and preservation methods, we suggested that if the samples were used for the character distinction,it should be processed and preserved in formalin;samples used for physics and chemistry analysis could be seasoned directly;samples used in the molecular biology experiment must be kept in 70%ethyl alcohol or 0.9%of NaCl solution,-20℃.
Using the stereoscope to observe the sample body surface characteristic and the internal structure,and comparing with the reference,we could identify the species accurately.According to the result,we established a retrieval table of 14 kinds of earthworms used for medicinal purposes,and the table could be helpful in the species identification of Dilong.
This finding indicated that by using the classical systematic zoology method,we could identify the earthworm with good characteristic preservation.
4.Molecular Identification of Dilong' germplasm resources by ISSR
ISSR was applied to identify the DNA varieties of Dilong for the first time.
The ISSR amplification of 7 materials by 8 ISSR premiers screened out resulted in 137 electrophoresis strips with 127 strips(92.7%)polymorphic.The NTSYSpc-2.10e software was applied to cluster analysis to the ISSR phenotype data matrix.The minimal value of similarity coefficient among Dilong' germplasm resources was 0.43,while the maximal was 0.66,which demonstrated greater genetic variance and DNA polymorphism of Dilong' germplasm resources.
The cluster analysis result indicated that the materials were classified to 4 groups in the similarity factor 0.56.The 3rd and the 4th class groups presented closer genetic relationship,while the genetic relationship between 1~(st)and 2~(nd)groups was far.The genetic relationship of individuals in the same genera was closer than those in different genera.The result was the same with the finding by using the classical systematic zoology method.
5.Determination of multi chemical constituents and the HPLC fingerprinting of Dilong
(1)Determination of uracil,hypoxanthine,uridine and inoine in Dilong by HPLC
We established an accurate and stable method for the determination of uracil, hypoxanthine,uridine and inoine in Dilong from different places as to efficaciously control the quality of medicinal material.
Method:The samples were extracted with 0.9%NaCl solution by ultrasonic and determined by RP-HPLC method,0.01mol/L KH_2PO_4 solution and 50%MeOH as flowing,gradient elution,examination wave length was 254nm,column temperature was 27℃,10ul sample was injected.
Result:The calibration curve of uracil was linear in the quality range of 0.0075-0.24/μg(r=0.9993),the average recovery was 99.82%,RSD was 2.34%; hypoxanthine was 0.0250-0.8000μg(r=0.9992),average recovery was 101.93%,RSD was 1.45%;uridine was 0.0125-0.4000/μg(r=0.9992),average recovery was 97.53%,RSD was 1.73%;inoine was 0.0500-1.6000μg(r=0.9991),average recovery was 102.06%,RSD was 2.44%.This method reproduction quality,the returns-ratio is good,may use in the broad earthworm uracil and so on 4 ingredients the content determination.The method displayed good reproducibility and high recovery,and it provides an effective way for the study of Dilong.
(2)HPLC fingerprints of 0.9%NaCl solution extracts from Dilong
We established the HPLC fingerprints of 0.9%NaCl solution extracts from Dilong to evaluate its quality.Gradient elution HPLC was used to analyze 0.9%NaCl solution extracts from Dilong,and the data of their fingerprints were analyzed by similarity.17 samples of Dilong from different places were analyzed to test the HPLC fingerprint.The relative retention time and relative area of 11 main common peaks were obtained as correlation technical parameters of Dilong.The repeatability of the relative retention times was good for the main common peaks and the relative peak areas had significant differences.From the above results,we suggested that the HPLC fingerprinting could be used to evaluate the quality and identification of Dilong.
The HPLC fingerprint data of Dilong were cluster analyzed using the SPSS software. 17 samples were classified to 4 groups basically.
The cluster analysis result indicated that the chemical contents of Dilong from Guangdong and Guangxi province were quite similar,which presented closer genetic relationship;the samples in Hainan province were similar;while Hu Dilong was obviously different from them.
This result also verified that Dilong from different places varied form their chemical composition.So we suggest that the species and origin must be fixed in the processing.
6.Determination and analysis of heavy metals and hazardous elements in Dilong
We established an UV method to determine the content of heavy metals in Dilong and supply the basis for standardizing the content limits.The determination wave length was 215nm.The regression equation was:y=0.7231x+0.043,correlation coefficient R~2=0.9968.
The linear range of the calibration curve was 0～50μg/g,and the average recovery was 99.97%.The method was simple,rapid,reliable and had good reproducibility,and could be used to determine the content of most heavy metals in Dilong.
Due to a great distinction between 9 batches of Dilong samples in the heavy metal element content,which mostly beyond the limit of not surpass 30/1000000(Chinese Pharmacopoeia,Volume 1,2005),we should pay attention to the heavy metal problem in Dilong.
7.Study on the correlation between ecological environmental and quality of Dilong
(1)Investigation and analyzing on the ecological environment factors of Dilong
We investigated the distribution and environment of Dilong,and collected 11 soil samples,planning to inspected Dilong' habitat suitability and the main environment factors concerned with the quality by analyzing on the ecological environment factors and the growth soil(moisture content,pH value,heavy metal and so on).The results would provide as a technical support for the standardized cultivation of Dilong.
The investigation on the ecological environment factors of Dilong' habitats indicated that Guangdong Province,Guangxi Province,Fujian Province and Hainan Province situating at the east China coast,their' climate,topography condition were superior and suitable for P.aspergillum to grow.
P.aspergillum could grow better in the soil with 25%water content.The appropriate pH value was 6.8-7.6.P.aspergillum' tolerance to heavy metal was similarly to the other earthworms,and it could survive in the soil with As(4.929-16.000mg/kg), Cd(0.999-8.604mg/kg),Cr(26.830-110.168mg/kg),Cu(44.900-349.356mg/kg),Ni (94.400-480.088mg/kg),Pb(15.084-114.715mg/kg),and the Zn(11.163-601.859mg/kg). Industrial development in Dilong habitats had close relationship with the quality of Dilong. The more heavy metals emissioned to the environment due to industrial development,the worse Dilong' quality would be.
(2)Study on the correlation between ecological environmental and heavy metal content in Dilong
①The heavy metal concentration dynamics characteristic of P.aspergillum
The double box dynamics model under half static condition was applied in this experiment.P.aspergillum were raised in soil with different heavy metal conditions firstly, been digested with HNO_3-H_2O_2 at low temperature,then the heavy metal in the P. aspergillum been determined.According to the results,we could study the dynamics characteristic of P.aspergillum to the Cd,Hg,and Pb in the soil.
Results:The biology concentration ratio of earthworm P.aspergillum in Pb high, middle and low concentration was k_1=1.232,K_2=1.325,K_3=1.058 respectively;In Cd high, middle and low concentration the ratio was k_1=0.1792,K_2=0.1753,K_3=0.1382 respectively; In Hg,the ratio was undulatory.
Conclusion:The concentration ability of P.aspergillum in Pb was higher than in Cd obviously,and increases with the lapse of time.Concentration characteristic in Hg was absorbed quickly at first,discharged quickly when accumulated to certain density,and then absorbed gain.
②Study on the heavy metal inducing mechanism of P.aspergillum wMT by
MT(Metallothionein)is a low-molecular-weight protein in the organism.It is rich in cysteinyl residues and contains no aromatic amino acids.MT shows high affinity for bivalent heavy metals.Because the earthworm contains a special MT(wMT),it could concentrate heavy metals in the soil,and heavy metal content in its vivo is much high.
P.aspergillum were raised in soil with different heavy metal conditions for 30 days. The wMT in P.aspergillum was extracted and determined by the silver saturation method combined with ASS.
According to the results,we could study the induction mechanism of P.aspergillum wMT to the heavy metals in the soil.
Results:The P.aspergillum wMT content could be induced by different heavy metal, the induce ability was in turn:Cu＞Cd＞Zn＞Pb;And generally speaking wMT content in the internal organs was higher than in the muscle,this result also indicated that wMT mainly expressed in earthworm's internal organs.
8.Study on quality standard of Dilong
Based on the experimental and analytical study in this paper,some substantial modifications have been made to Chinese Pharmacopoeia(Volume 1,2005)concerning the quality of Dilong.Determination item of Dilong have been added,choosing hypoxanthine and inoine as determination target,the minimal limited quantity of hypoxanthine in the dry drug should be no less than 0.2 mg/g;Inoine should be no less than 0.3 mg/g.
In addition,some revision have been made in the character and processing items.The words "to clean the soil in the two ends" have been added in the processing method.The words "the abdomen has already been cut open except two ends,the internal organs have been cleaned or occasionally remain" have been added in the character description.The words "to clean the impurity and soil in the body" have been added in the preparation.
This study has established the determination method for Dilong,and modified the quality standard.The modifications would promote the quality control of Dilong.
9.Conclusion and further study
A systematic research has been made on polymorphism of Dilong' germplasm resources,which will provide the reference for the further use of Dilong.
We have established a HPLC fingerprinting and determination method,as well as an UV method to determine the content of heavy metals in Dilong.This study has modified the quality standard to promote the quality control of Dilong.
Based on the investigation and analyzing of the ecological environment factors of Dilong,the research will serve as a reference to promote the qualiy of Dilong.
Insufficient research has been made on the induction mechanism of P.aspergillum wMT by heavy metal,and it's worth making the further research in the future.
引文
[1]苏颁,等.图经本草.福州:福建科学出版社,1988:463
[2]唐慎微.重修政和经史证类备用本草.北京:人民卫生出版社,1957:445
[3]冯耀南,刘明,刘俭,等.中药材商品规格质量鉴别.广州:暨南大学出版社,1995:416.
[4]中药志编委会.中药志.北京:人民卫生出版社,1961.
[5]中国药用动物志编委会,中国药用动物志.天津:天津科技出版社,1979:5.
[6]于德江,刘亚杰,迟仁智,等.中药材地龙.中国药学杂志,1993,28(10):594-596
[7]刘寿山.中药研究文献摘要.(1920-1961)北京:科学出版社.1975:668.
[8]陈平,叶卯祥,严宜昌,等.中药地龙的药源调查与商品鉴定.中草药,1997,28(8):492-495
[9]肖小芹.地龙原动物种类及活体鉴别.湖南中医杂志,2002,18(4):50
[10]冯孝义,董芷馨.中药地龙原动物的研究.中药通报,1987,12(10):579-582
[11]陈强,冯孝义,董健华,等.西北药用蚯蚓资源考察.中国中药杂志,1995,20(11):650-651
[12]周天元著.蚯蚓无土高效养殖新技术.天津科学技术出版社.2002,1
[13]陈义等编.中国动物图谱:环节动物.北京:科学出版社:1959,09
[14]徐芹.中国陆栖蚯蚓分类研究史探讨.北京教育学院学报.1999,3:52-57
[15]陈德中,药用蚯蚓.中草药.1980:11(9):420
[16]李振荣,等.动物类生药的鉴别研究-Ⅱ,广地龙的生药鉴别.河北医学院学报.1986:6(3):136
[17]顺庆生,包雪声.沪地龙的原动物鉴定.中药材.1999,(22)1:13-16
[18]冯孝义,中国陆栖蚯蚓各属的分类特征.东北师范大学学报,1985,2:44-47
[19]吴文如,李薇.地龙类药用动物的比较鉴别.现代生物医学进展.2007,7(11):1754
[20]徐国钧主编,中药材粉末显微鉴定.北京:人民卫生出版社,1986:746
[21]李兰燕,王树春,吴云山,等.广地龙的X射线衍射Fourier谱鉴定.中成药,24(5):380-382
[22]王西芳.地龙的理化鉴别.陕西中医学院学报 1996,19(1):42
[23]黄福珍.蚯蚓.北京:农业出版社,1986:9
[24]汪珊,梁仁.中药地龙药理与哮喘气道重建相关性研究.广东药学院学报,2004,20(1):60-62.
[25]肖培根主编,新编中药志.第四卷.北京:化学工业出版社.2002:56-70
[26]易八贤,徐丽姗,曾漪青,等.两种蚯蚓及其不同部位的酶谱研究.药物生物技术.1999,6(3):174-176
[27]武金霞,赵晓瑜.蚯蚓体内生物活性成分的研究.自然杂志,26(1):27-30
[28]王宗伟,梁秀文,范兴亚.蚯蚓生物工程技术研究.中草药,2000,31(5):386-387
[29]徐晋佑,黄兴华,欧佩华,等.气相色谱/质谱法研究华南参环毛蚓的脂肪酸.华南师范大学学报(自然科学版),1991,1:73-76
[30]王光忠,胡迪,陈敬炳,等.地龙类药材化学成分分析.中药材.1998,21(3):133-135
[31]杨得坡,王发松,彭劲甫.参环毛蚓脂类挥发性成分分析.中药材.2000,23(1):31-33
[32]葛勤,张恩娟,刘同华,等.地龙的化学成分及制剂质量研究概况.中国药业,2004,13(3):69-71
[33]刘继红,尚娟,贾斌.蜈蚣全蝎地龙氨基酸含量测定.郑州大学学报(医学版).2005,40(3):483-485
[34]蒋艳红,刘敏彦,李向军,等.地龙中游离氨基酸的含量测定.中国中医药信息杂志.2003,10(2):36-37
[35]闫萍,杨琦,王惠珍,等.改良Lowry法和Bradford法测定蚯蚓提取物中蛋白质含量的比较.山西医科大学学报.2006,37(1):9-11
[36]陈可夫,谭锡军.荆门蚂蟥蚯蚓微量元素含量的测定.荆门职业技术学院学报.2003,18(6):52-53
[37]叶兴福.火焰原子吸收法测定蚯蚓中的微量元素.分析测试技术与仪器.1997,3(2):103-106
[38]赵春桂.蚯蚓微量元素含量测定.中药通报,1998,1(9):38.
[39]卢丽萍,曹骋.不同产地地龙质量分析研究.广东微量元素科学.2004,11(6):50-52
[40]唐婉清,廖彩震,冯艳妮,等.地龙药材及地龙配方颗粒中铅的含量测定.广东药学 2003,13(3):1-2
[41]王光忠,邹阳,陈敬炳,等.薄层扫描法测定地龙类药材中琥珀酸的含量.药物分析杂志.1997,17(4):274-275
[42]张文斌,校合香.地龙不同炮制品中琥珀酸含量的比较.中国药事.2001,15(3):216
[43]方铁铮,杨翠平,苏薇薇.地龙及其注射液指纹特征谱研究.中药材,2002,25(11):813-815
[44]李金屏.HPLC法测定广地龙中次黄嘌呤的含量.中国药品标准.2002,3(5):29-31
[45]刘松青,马文秀,唐先哲,等.高效液相色谱法测定地龙中次黄嘌呤和黄嘌呤的含量.华西药学杂志.1994,9(3):179-181
[46]李伟,文红梅,钟进,等.HPLC测定地龙中次黄嘌呤黄嘌呤尿嘧啶尿苷的含量.中药材.1996,19(12):625-627
[47]胡馨,黄文辉.各中沪产地龙与广东产地龙中次黄嘌呤的测定和比较.中成药.1994,16(1):42-43
[48]Mihara H.et al.Thrombotic Proteases from earthworm.25Eur.Pat.Appl.Ep105,092(C1C12N964),11.apr 1984,JP Aprl.82/173,669,02,Oct.1982
[49]陈学东,田道法,刘红萍,等.地龙煎液对人出血、凝血时问的影响.湖南中学院学报,2003,23(2):10-11.
[50]李永吉.地龙对家兔血栓形成的影响.中医药学报,1998,(2).46.
[51]朱长连,王小阳,张国桢,等.蚯蚓提取物的纤溶和抗凝作用[J].河南医科大学学报,1993,28(1):15.
[52]韩彩芝,余素清,李秀芳,等.蚯蚓提取液纤溶及抗凝作用的实验研究.中国生化药物杂志,1997,18(5):252-254.
[53]刘协,李小宁,沈逵,等.蚯蚓提取物对血栓形成和血小板聚集作用的实验研究.江苏预防医学,1997,(2)6-7.
[54]扬树东.蚯蚓纤溶活性成分的抗凝及溶血栓作用.心肺血管病杂志,1997,16(3):228-230.
[55]何执中,郭怀芳,唐宁,等.蚯蚓纤溶酶的分离纯化及抗栓活性研究.中国生化杂志,2001,2(6):284-286.
[56]殷书梅,储益平,吴鹏.地龙活性提取物的主要药效学实验.中草药.2002,33(10):926-928
[57]金鸣.蚯蚓溶栓作用研究进展.中草药,1994,25(1):30
[58]程牛亮,等.双胸蚓溶栓酶制剂对家兔血浆t-PA与PAl活力的影响.山西医学院学报,1992,23(4):301
[59]谢江碧,贺卫国,翁宁,等.蚯蚓中抗肿瘤蛋白组分的提取分离及其抗肿瘤活性.中国生物化与分子生物学报.2003,19(3):359-366
[60]曾小澜,章碧玉,麦羡霞,等.蚯蚓提取物对多种瘤细胞的作用.山西医学院学报,1995,26(2):81-83.2
[61]Tsunoda S,etal.Anticancer Res,1997,17(5A):3349.
[62]吕金胜,吴畏,孟德胜,等.地龙醇提物抗炎及镇痛作用的研究.中国药师.2003,6(1):16-18
[63]李勇文,李梅花.地龙的研究进展.广西医学.2004,26(5):699-671
[64]张复夏,王西发,裒克平,等.蚯蚓体内杀精物质的实验研究.陕西中医.1996,17(5):234
[65]张复夏,邵道通,等.地龙粉治疗男性不育症30例.陕西中医.1996,17(10):438
[66]张风春,陈云峰,苏颜珍,等.地龙对巨噬细胞免疫活性的增强作用.中国药学杂志.1998,33(9):532
[67]黎莉,甘明,黎祥胜.地龙类药材药理作用比较.中药材.1997.20(7):361-363
[68]陈随清,崔瑛,靖慧军,等.地龙抑菌作用初步观察.中药材.2003,26(5):354-355
[69]龚峻梅,曲宏达,陈素云.地龙及其提取物的药理作用介绍.暨南大学学报(医学版.2000,21(4):133-135
[70]陈可夫,石银珍,万前程,等.蚯蚓素安全性毒理学评价实验.荆门职业技术学院学报,2000,15(6):57-64.
[71]张理平.浅谈地龙的收敛功效.福建中医药.1992,23(3):56
[72]张绍章,田琼,李予蓉,等.蚯蚓提取物对小鼠脾脏抗体形成细胞的影响.第四军医大学学报1993,14(5):350
[73]曾子澜,张祖,碧玉,等.蚯蚓提取物对多种癌细胞的作用.山西医学院学报1995,26(2):81
[74]张连仁.地龙治疗颠狂证.宜春医药.1981,(2):49
[75]李凤海.地龙侧柏叶外敷治疗痄腮.中国民间疗法1998,2:28
[76]鲁永保.地龙液治疗百日咳16例.中西医结合杂志.1990,10(4):231
[77]高树良.浅谈地龙在皮肤科几种疾病中的应用.唐山地区医药.1983,(11):54
[78]史宪莹,王树同.地龙临床运用新解.实用中医内科杂志.1997,11(1):24
[78]秦丙义.水蛭地龙汤治疗脑梗塞297例.实用中医内科杂志.1997,11(4):26
[79]李时珍.本草纲目.下册,北京:人民卫生出版社(校正本),1982.2352
[80]中华人民共和国药典,2005版一部.中国药典委员会编.北京:化学工业出版社.2005:80
[81]李薇,吴文如,肖翔林.地龙规范化生产的研究概况.中草药,2005,36(9):1419-1422
[82]邵鹏柱,曹晖编.中药分子鉴定.上海:复旦大学出版社,2004
[83]张文青,闰永红,林雀跃.分子标记技术在道地药材研究中的应用.湖南中医药大学学报.2007,27:269-271
[84]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeats(SSR)anhorcd polymerase chain reaction amplification[J].Genomics,1994,20:176-183.
[85]Li H S.Chen G Z.Genetic diversity of sonneratia alba in China detected by inter simple sequence repeats(ISSR)analysis.Acta Bot Sin,2004,46(5):137-142.
[86]Ammiraju J S.Dholakia B B,Santra D K,et al.Identification of inter simple sequence repeat(ISSR)markers associated with seed size in wheat[J].Theor Appl Genet.2001,102(6):726-732.
[87]陈祖云,王晓丽,宋聚先.贵州天麻遗传多态性的ISSR初步分析.中华中医药杂志,2007,22(7):436-439.
[88]刘海河,侯喜林,张彦萍.西瓜ISSR-PCR体系的正交优化研究.果树学报,2004,21(6):615 617
[89]侯林,曲若竹,邹向阳,等.中国4个两性生殖卤虫品系的ISSR指纹分析.辽宁师范大学学报(自然科学版).2003,26(2):174-177
[90]刘广绪,包振民,胡景杰,等.栉孔扇贝、华贵栉孔扇贝及其种间杂交子代、种内交配子代的ISSR 分析.中国海洋大学学报.2006,36(1):71-75
[91]陈大鹏,沈怀舜,丁亚平,等.文蛤(Meretrix meretrix)地理种群ISSR分子标记的初步研究.南京师大学报(自然科学版).2004,27(3):74-77
[92]郑芳,吕秀玲,孙红英,等.ISSR标记在河蟹种质检测中的应用.中国水产科学.2007,14(1):46-51
[93]周延清主编.DNA分子标记技术在植物研究中的应用.北京:化学工业出版社,2005
[94]姜文红,张清波.地龙HPLC指纹图谱分析方法的研究.中医药学报.2006,34(6):13-14
[95]王雁,毕开顺.三七HPLC指纹图谱的建立.中国中药杂志.2003,12,28(4):316-320
[96]刘荣霞,周婷婷,毕开顺,等.建立评价当归HPLC指纹图谱分析方法.中国药学杂志.2003,10,38(10):757-760
[97]张克荣,毕开顺.赤芍HPLC指纹图谱的研究.中药材.2003,11,34(11):1048-1051
[98]张丽娟,谷学新,周勇义.中药产品中的重金属元素.首都师范大学学报(自然科学版),2004,25(1):34-37
[99]沈宁光,周玲,朱丽丽,等.紫外分光光度法测定地龙中重金属的含量.广东微量元素科学.2005,12(7):48-51
[100]楼小红,吴巧凤.紫外分光光度法测定白芍中重金属的研究.广东微量元素科学.2004,11(1):38-40.
[101]吴巧凤.紫外分光光度法测定元胡生品与醋制品的重金属含量.中国现代应用药学.2002,19(5):401-403.
[102]施寄材.中药地龙的含砷量.中成药研究.1981.3(5):17.
[103]朱艳,崔秀明,施莉屏.中药道地性研究进展.现代中药研究与实践,2006,20(1):58-61
[104]任德权,索风梅,陈士林.道地药材的产地适宜性研究.中药研究与信息,2005,7(9):4-9
[105]向兰,杨美华,陈虎彪,等.论药材道地性的研究方法.世界科学技术.中药现代化.2000(2):44-46
[106]曾中平,张国城,徐芹.蚯蚓养殖学,武汉:湖北人民出版社,1982,196
[107]梁溥,魏清泉.中国大百科全书.中国大百科全书出版社.2004.9
[108]秦权人,汪宇明.中国大百科全书.中国大百科全书出版社.2004.9
[109]吴郭泉,等;广西国土资源与可持续发展初探.广西地质.1999.(12)
[110]陈德高.中国大百科全书.北京:中国大百科全书出版社.2004.9
[111]赵昭昞.中国大百科全书.北京:中国大百科全书出版社.2004.9
[112]魏清泉.中国大百科全书.北京:中国大百科全书出版社.2004-9
[113]陈永森,王霞斐.中国犬百科全书.北京:中国大百科全书出版社.2004.9
[114]叶学齐,唐文雅.中国大百科全书.北京:中国大百科全书出版社.2004.9
[115]刘盛佳.中国大百科全书.北京:中国大百科全书出版社.2004.9
[116]冯国鸣.土壤PH值的测定.农民致富之友.2000.10
[117]环保行业标准HJ╱T166-2004土壤环境检测技术规范.国家环保总局.2005.04
[118]中华人民共和国国家标准土壤环境质量标准GB 15618-1995.国家环境保护局,国家技术监督局.1995.07.13
[119]闫志民,等.药用动植物种养加工技术-蚯蚓.北京:中国中医药出版社.2000
[120]史瑞和,鲍士旦,秦怀英,等.土壤农化分析.北京:农业出版社,1986,第二版
[121]周启星,孔繁翔,朱琳主编.生态毒理学.北京:科学出版社,2004:307
[122]Hook,R.I.van.Cadmium,lead and zinc distributions between earthworms and soils,potentials for biological accumulation.Bull Buviron Contom Toxicol,1974.12.509-515
[123]Ireland MP.The effect of the earthworm Dendrobaem rubiba on the solubility of lead,zinc,and calcium in heavy metal contaminated soilin Wales.Soil Sci.1975.26.313-318
[124]张友梅,王振中,邢协加,等.土壤污染对蚯蚓的影响.湖南师范大学自然科学学报.1996.9(19):86-90
[125]C.Svendsen,D.J.Spurgeon,P.K.Hankard,et al.A review of lysosomal memberane stability measured by neutral red retention:is it a workable earth biomarker ? Ecotoxicology and Environmental Safety 2004,57(1):20-29
[126]田晓丽,郭军华.金属硫蛋白的研究进展.国外医学药学分册.2005,3(2):119-124
[127]王海英,郭祀远,李琳.蚯蚓的研究与应用.氨基酸和生物资源,2002,24(4):17-19
[128]StUrzenbaum SR,Kille P,Morgan AJ.The identification,cloning and characterization of earthworm metallothionein.FEBS Lett.1998,24;431(3):437-442
[129]John E.Morgan and A.J.Morgan The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site.Environmental Pollution.1998,99(2):167-175
[130]张桂春.金属硫蛋白的功能及应用前景.烟台师范学院学报(自然科学版).2005,1(1):142-145
[131]李婧.金属硫蛋白检测方法的研究进展.现代检验医学杂志.2004,19(3):64-65
[132]张保林,盛湘溶,刘兴军,等.金属硫蛋白测定方法-银饱和分析法.药物生物技术,1996,3(1):31-33.
[133]OECD Guidelines for Testing of Chemicals.No.207:Earthworm,Acute Toxicity Tests[R].Paris:OECD Council,2001.
[1]殷书梅,储益平,吴鹏.地龙活性提取物的主要药效学实验.中草药,2002,33(10):926
[2]朱九群.地龙的临床应用进展.现代中西医结合杂志,2000,9(3):205
[3]张燕红,张庭芳,王采芹,等.蚯蚓钙调素结合蛋白的研究.北京大学学报(自然科学版),1997,33(6):731
[4]于德江,刘亚杰,迟仁智,等.中药材地龙.中国药学杂志,1993,28(10):594-596
[5]陈平,叶卯祥,严宜昌,等.中药地龙的药源调查与商品鉴定.中草药,1997,28(8):492-495
[6]陈强,冯孝义,董健华,等.西北药用蚯蚓资源考察.中国中药杂志,1995,20(11):650-651
[7]周天元著.蚯蚓无土高效养殖新技术.天津科学技术出版社.2002,1
[8]陈平,傅杰,严宜昌.地龙的本草考证.中药材,20(3):158-160
[9]肖小芹.地龙原动物种类及活体鉴别.湖南中医杂志,2002,18(4):50
[10]张贵君等.中药鉴定学.北京:科学出版社,2002,452
[11]李兰燕,王树春,吴云山,等.广地龙的X射线衍射Fourier谱鉴定.中成药.24(5):380-382
[12]于占林,邢利华.地龙人工养殖技术.时珍国药研究,1995,5(4):34-35
[13]徐晋佑,蓝宗辉,曾逊生,等.人工养殖参环毛蚓的实验研究.中药材科技,1983,4:14-18
[14]亢霞生,程新,黄文戈编著.蚯蚓高效养殖技术-特种养殖点金术.广西科学技术出版社,2002,1
[15]刘向辉,戈峰.蚯蚓纤溶酶活性影响因子的研究.中国中药杂志,2002,27(6):423-425
[16]王海英,郭祀远,李琳.蚯蚓的研究与应用.氨基酸和生物资源,2002,24(4):17-19
[17]程能能,衫浦隆夫,福天辉夫,等.地龙体内磷脂的组成和血小板活化因子的生物合成.药学学报,1992,27(12):886-890
[18]张祖珣,何俐明,于培兰,等.蚯蚓毒素及其去除方法的初步研究.中国生化药物杂志,2001,22(3):112-113
[19]刘艳玲,李莉,吴红艳,等.蚯蚓两种采收加工方法的比较.中国药学杂志,2002,37(2):96-98
[20]路英华,金汝成.蚯蚓纤溶酶的提取性质鉴定和溶解血栓的研究.兰州大学学报,1986,(2):295
[21]丰素娟.地龙炮制经验谈.中国现代应用药学杂志,16(5):14-15
[22]刘洪章,刘喜梅.地龙炮制之我见.时珍国医国药,1998,9(5):415
[23]蒋艳红,刘敏彦,刘向军,等.地龙中游离氨基酸的含量测定.中国中医药信息杂志,2003,10(2):36-37
[24]李伟,文红梅,钟进,等.HPLC法测定地龙中次黄嘌呤、黄嘌呤、尿嘧啶、尿苷的含量.中 药材,1996,19(12):625-627
[25]王光忠,陈平,胡迪,等.薄层扫描法测定地龙类药材中琥珀酸的含量.药物分析杂志,1997,17(4):274-275
[26]张娴,彭国平,王振中.地龙中肌苷含量测定.中药材,2003,26(2):103
[27]易八贤,徐丽姗,曾漪青,等.两种蚯蚓及其不同部位的酶谱研究.药物生物技术.1999,6(3):174-176
[28]赵春桂.蚯蚓中微量元素含量测定.中药通报,1998,1(9):38
[29]王文东,张英华,崔健,等.地龙及其制剂的质量控制研究.吉林中医药,1996,5:38-39
[30]方铁铮,杨翠平,苏薇薇.地龙及其注射液指纹特征谱研究.中药材,2002,11(11):813-815
[31]王光忠,胡迪,陈敬炳等.地龙类药材化学成分分析.中药材,1998,21(3):13
[32]胡馨,黄文辉.各种沪产地龙与广东产地龙中次黄嘌呤的测定和比较.中成药,1994,16(1):42-43
[33]施寄材.中药地龙的含砷量.中成药研究,1981,(5):17
[2]唐慎微.重修政和经史证类备用本草.北京:人民卫生出版社,1957:445
[3]冯耀南,刘明,刘俭,等.中药材商品规格质量鉴别.广州:暨南大学出版社,1995:416.
[4]中药志编委会.中药志.北京:人民卫生出版社,1961.
[5]中国药用动物志编委会,中国药用动物志.天津:天津科技出版社,1979:5.
[6]于德江,刘亚杰,迟仁智,等.中药材地龙.中国药学杂志,1993,28(10):594-596
[7]刘寿山.中药研究文献摘要.(1920-1961)北京:科学出版社.1975:668.
[8]陈平,叶卯祥,严宜昌,等.中药地龙的药源调查与商品鉴定.中草药,1997,28(8):492-495
[9]肖小芹.地龙原动物种类及活体鉴别.湖南中医杂志,2002,18(4):50
[10]冯孝义,董芷馨.中药地龙原动物的研究.中药通报,1987,12(10):579-582
[11]陈强,冯孝义,董健华,等.西北药用蚯蚓资源考察.中国中药杂志,1995,20(11):650-651
[12]周天元著.蚯蚓无土高效养殖新技术.天津科学技术出版社.2002,1
[13]陈义等编.中国动物图谱:环节动物.北京:科学出版社:1959,09
[14]徐芹.中国陆栖蚯蚓分类研究史探讨.北京教育学院学报.1999,3:52-57
[15]陈德中,药用蚯蚓.中草药.1980:11(9):420
[16]李振荣,等.动物类生药的鉴别研究-Ⅱ,广地龙的生药鉴别.河北医学院学报.1986:6(3):136
[17]顺庆生,包雪声.沪地龙的原动物鉴定.中药材.1999,(22)1:13-16
[18]冯孝义,中国陆栖蚯蚓各属的分类特征.东北师范大学学报,1985,2:44-47
[19]吴文如,李薇.地龙类药用动物的比较鉴别.现代生物医学进展.2007,7(11):1754
[20]徐国钧主编,中药材粉末显微鉴定.北京:人民卫生出版社,1986:746
[21]李兰燕,王树春,吴云山,等.广地龙的X射线衍射Fourier谱鉴定.中成药,24(5):380-382
[22]王西芳.地龙的理化鉴别.陕西中医学院学报 1996,19(1):42
[23]黄福珍.蚯蚓.北京:农业出版社,1986:9
[24]汪珊,梁仁.中药地龙药理与哮喘气道重建相关性研究.广东药学院学报,2004,20(1):60-62.
[25]肖培根主编,新编中药志.第四卷.北京:化学工业出版社.2002:56-70
[26]易八贤,徐丽姗,曾漪青,等.两种蚯蚓及其不同部位的酶谱研究.药物生物技术.1999,6(3):174-176
[27]武金霞,赵晓瑜.蚯蚓体内生物活性成分的研究.自然杂志,26(1):27-30
[28]王宗伟,梁秀文,范兴亚.蚯蚓生物工程技术研究.中草药,2000,31(5):386-387
[29]徐晋佑,黄兴华,欧佩华,等.气相色谱/质谱法研究华南参环毛蚓的脂肪酸.华南师范大学学报(自然科学版),1991,1:73-76
[30]王光忠,胡迪,陈敬炳,等.地龙类药材化学成分分析.中药材.1998,21(3):133-135
[31]杨得坡,王发松,彭劲甫.参环毛蚓脂类挥发性成分分析.中药材.2000,23(1):31-33
[32]葛勤,张恩娟,刘同华,等.地龙的化学成分及制剂质量研究概况.中国药业,2004,13(3):69-71
[33]刘继红,尚娟,贾斌.蜈蚣全蝎地龙氨基酸含量测定.郑州大学学报(医学版).2005,40(3):483-485
[34]蒋艳红,刘敏彦,李向军,等.地龙中游离氨基酸的含量测定.中国中医药信息杂志.2003,10(2):36-37
[35]闫萍,杨琦,王惠珍,等.改良Lowry法和Bradford法测定蚯蚓提取物中蛋白质含量的比较.山西医科大学学报.2006,37(1):9-11
[36]陈可夫,谭锡军.荆门蚂蟥蚯蚓微量元素含量的测定.荆门职业技术学院学报.2003,18(6):52-53
[37]叶兴福.火焰原子吸收法测定蚯蚓中的微量元素.分析测试技术与仪器.1997,3(2):103-106
[38]赵春桂.蚯蚓微量元素含量测定.中药通报,1998,1(9):38.
[39]卢丽萍,曹骋.不同产地地龙质量分析研究.广东微量元素科学.2004,11(6):50-52
[40]唐婉清,廖彩震,冯艳妮,等.地龙药材及地龙配方颗粒中铅的含量测定.广东药学 2003,13(3):1-2
[41]王光忠,邹阳,陈敬炳,等.薄层扫描法测定地龙类药材中琥珀酸的含量.药物分析杂志.1997,17(4):274-275
[42]张文斌,校合香.地龙不同炮制品中琥珀酸含量的比较.中国药事.2001,15(3):216
[43]方铁铮,杨翠平,苏薇薇.地龙及其注射液指纹特征谱研究.中药材,2002,25(11):813-815
[44]李金屏.HPLC法测定广地龙中次黄嘌呤的含量.中国药品标准.2002,3(5):29-31
[45]刘松青,马文秀,唐先哲,等.高效液相色谱法测定地龙中次黄嘌呤和黄嘌呤的含量.华西药学杂志.1994,9(3):179-181
[46]李伟,文红梅,钟进,等.HPLC测定地龙中次黄嘌呤黄嘌呤尿嘧啶尿苷的含量.中药材.1996,19(12):625-627
[47]胡馨,黄文辉.各中沪产地龙与广东产地龙中次黄嘌呤的测定和比较.中成药.1994,16(1):42-43
[48]Mihara H.et al.Thrombotic Proteases from earthworm.25Eur.Pat.Appl.Ep105,092(C1C12N964),11.apr 1984,JP Aprl.82/173,669,02,Oct.1982
[49]陈学东,田道法,刘红萍,等.地龙煎液对人出血、凝血时问的影响.湖南中学院学报,2003,23(2):10-11.
[50]李永吉.地龙对家兔血栓形成的影响.中医药学报,1998,(2).46.
[51]朱长连,王小阳,张国桢,等.蚯蚓提取物的纤溶和抗凝作用[J].河南医科大学学报,1993,28(1):15.
[52]韩彩芝,余素清,李秀芳,等.蚯蚓提取液纤溶及抗凝作用的实验研究.中国生化药物杂志,1997,18(5):252-254.
[53]刘协,李小宁,沈逵,等.蚯蚓提取物对血栓形成和血小板聚集作用的实验研究.江苏预防医学,1997,(2)6-7.
[54]扬树东.蚯蚓纤溶活性成分的抗凝及溶血栓作用.心肺血管病杂志,1997,16(3):228-230.
[55]何执中,郭怀芳,唐宁,等.蚯蚓纤溶酶的分离纯化及抗栓活性研究.中国生化杂志,2001,2(6):284-286.
[56]殷书梅,储益平,吴鹏.地龙活性提取物的主要药效学实验.中草药.2002,33(10):926-928
[57]金鸣.蚯蚓溶栓作用研究进展.中草药,1994,25(1):30
[58]程牛亮,等.双胸蚓溶栓酶制剂对家兔血浆t-PA与PAl活力的影响.山西医学院学报,1992,23(4):301
[59]谢江碧,贺卫国,翁宁,等.蚯蚓中抗肿瘤蛋白组分的提取分离及其抗肿瘤活性.中国生物化与分子生物学报.2003,19(3):359-366
[60]曾小澜,章碧玉,麦羡霞,等.蚯蚓提取物对多种瘤细胞的作用.山西医学院学报,1995,26(2):81-83.2
[61]Tsunoda S,etal.Anticancer Res,1997,17(5A):3349.
[62]吕金胜,吴畏,孟德胜,等.地龙醇提物抗炎及镇痛作用的研究.中国药师.2003,6(1):16-18
[63]李勇文,李梅花.地龙的研究进展.广西医学.2004,26(5):699-671
[64]张复夏,王西发,裒克平,等.蚯蚓体内杀精物质的实验研究.陕西中医.1996,17(5):234
[65]张复夏,邵道通,等.地龙粉治疗男性不育症30例.陕西中医.1996,17(10):438
[66]张风春,陈云峰,苏颜珍,等.地龙对巨噬细胞免疫活性的增强作用.中国药学杂志.1998,33(9):532
[67]黎莉,甘明,黎祥胜.地龙类药材药理作用比较.中药材.1997.20(7):361-363
[68]陈随清,崔瑛,靖慧军,等.地龙抑菌作用初步观察.中药材.2003,26(5):354-355
[69]龚峻梅,曲宏达,陈素云.地龙及其提取物的药理作用介绍.暨南大学学报(医学版.2000,21(4):133-135
[70]陈可夫,石银珍,万前程,等.蚯蚓素安全性毒理学评价实验.荆门职业技术学院学报,2000,15(6):57-64.
[71]张理平.浅谈地龙的收敛功效.福建中医药.1992,23(3):56
[72]张绍章,田琼,李予蓉,等.蚯蚓提取物对小鼠脾脏抗体形成细胞的影响.第四军医大学学报1993,14(5):350
[73]曾子澜,张祖,碧玉,等.蚯蚓提取物对多种癌细胞的作用.山西医学院学报1995,26(2):81
[74]张连仁.地龙治疗颠狂证.宜春医药.1981,(2):49
[75]李凤海.地龙侧柏叶外敷治疗痄腮.中国民间疗法1998,2:28
[76]鲁永保.地龙液治疗百日咳16例.中西医结合杂志.1990,10(4):231
[77]高树良.浅谈地龙在皮肤科几种疾病中的应用.唐山地区医药.1983,(11):54
[78]史宪莹,王树同.地龙临床运用新解.实用中医内科杂志.1997,11(1):24
[78]秦丙义.水蛭地龙汤治疗脑梗塞297例.实用中医内科杂志.1997,11(4):26
[79]李时珍.本草纲目.下册,北京:人民卫生出版社(校正本),1982.2352
[80]中华人民共和国药典,2005版一部.中国药典委员会编.北京:化学工业出版社.2005:80
[81]李薇,吴文如,肖翔林.地龙规范化生产的研究概况.中草药,2005,36(9):1419-1422
[82]邵鹏柱,曹晖编.中药分子鉴定.上海:复旦大学出版社,2004
[83]张文青,闰永红,林雀跃.分子标记技术在道地药材研究中的应用.湖南中医药大学学报.2007,27:269-271
[84]Zietkiewicz E,Rafalski A,Labuda D.Genome fingerprinting by simple sequence repeats(SSR)anhorcd polymerase chain reaction amplification[J].Genomics,1994,20:176-183.
[85]Li H S.Chen G Z.Genetic diversity of sonneratia alba in China detected by inter simple sequence repeats(ISSR)analysis.Acta Bot Sin,2004,46(5):137-142.
[86]Ammiraju J S.Dholakia B B,Santra D K,et al.Identification of inter simple sequence repeat(ISSR)markers associated with seed size in wheat[J].Theor Appl Genet.2001,102(6):726-732.
[87]陈祖云,王晓丽,宋聚先.贵州天麻遗传多态性的ISSR初步分析.中华中医药杂志,2007,22(7):436-439.
[88]刘海河,侯喜林,张彦萍.西瓜ISSR-PCR体系的正交优化研究.果树学报,2004,21(6):615 617
[89]侯林,曲若竹,邹向阳,等.中国4个两性生殖卤虫品系的ISSR指纹分析.辽宁师范大学学报(自然科学版).2003,26(2):174-177
[90]刘广绪,包振民,胡景杰,等.栉孔扇贝、华贵栉孔扇贝及其种间杂交子代、种内交配子代的ISSR 分析.中国海洋大学学报.2006,36(1):71-75
[91]陈大鹏,沈怀舜,丁亚平,等.文蛤(Meretrix meretrix)地理种群ISSR分子标记的初步研究.南京师大学报(自然科学版).2004,27(3):74-77
[92]郑芳,吕秀玲,孙红英,等.ISSR标记在河蟹种质检测中的应用.中国水产科学.2007,14(1):46-51
[93]周延清主编.DNA分子标记技术在植物研究中的应用.北京:化学工业出版社,2005
[94]姜文红,张清波.地龙HPLC指纹图谱分析方法的研究.中医药学报.2006,34(6):13-14
[95]王雁,毕开顺.三七HPLC指纹图谱的建立.中国中药杂志.2003,12,28(4):316-320
[96]刘荣霞,周婷婷,毕开顺,等.建立评价当归HPLC指纹图谱分析方法.中国药学杂志.2003,10,38(10):757-760
[97]张克荣,毕开顺.赤芍HPLC指纹图谱的研究.中药材.2003,11,34(11):1048-1051
[98]张丽娟,谷学新,周勇义.中药产品中的重金属元素.首都师范大学学报(自然科学版),2004,25(1):34-37
[99]沈宁光,周玲,朱丽丽,等.紫外分光光度法测定地龙中重金属的含量.广东微量元素科学.2005,12(7):48-51
[100]楼小红,吴巧凤.紫外分光光度法测定白芍中重金属的研究.广东微量元素科学.2004,11(1):38-40.
[101]吴巧凤.紫外分光光度法测定元胡生品与醋制品的重金属含量.中国现代应用药学.2002,19(5):401-403.
[102]施寄材.中药地龙的含砷量.中成药研究.1981.3(5):17.
[103]朱艳,崔秀明,施莉屏.中药道地性研究进展.现代中药研究与实践,2006,20(1):58-61
[104]任德权,索风梅,陈士林.道地药材的产地适宜性研究.中药研究与信息,2005,7(9):4-9
[105]向兰,杨美华,陈虎彪,等.论药材道地性的研究方法.世界科学技术.中药现代化.2000(2):44-46
[106]曾中平,张国城,徐芹.蚯蚓养殖学,武汉:湖北人民出版社,1982,196
[107]梁溥,魏清泉.中国大百科全书.中国大百科全书出版社.2004.9
[108]秦权人,汪宇明.中国大百科全书.中国大百科全书出版社.2004.9
[109]吴郭泉,等;广西国土资源与可持续发展初探.广西地质.1999.(12)
[110]陈德高.中国大百科全书.北京:中国大百科全书出版社.2004.9
[111]赵昭昞.中国大百科全书.北京:中国大百科全书出版社.2004.9
[112]魏清泉.中国大百科全书.北京:中国大百科全书出版社.2004-9
[113]陈永森,王霞斐.中国犬百科全书.北京:中国大百科全书出版社.2004.9
[114]叶学齐,唐文雅.中国大百科全书.北京:中国大百科全书出版社.2004.9
[115]刘盛佳.中国大百科全书.北京:中国大百科全书出版社.2004.9
[116]冯国鸣.土壤PH值的测定.农民致富之友.2000.10
[117]环保行业标准HJ╱T166-2004土壤环境检测技术规范.国家环保总局.2005.04
[118]中华人民共和国国家标准土壤环境质量标准GB 15618-1995.国家环境保护局,国家技术监督局.1995.07.13
[119]闫志民,等.药用动植物种养加工技术-蚯蚓.北京:中国中医药出版社.2000
[120]史瑞和,鲍士旦,秦怀英,等.土壤农化分析.北京:农业出版社,1986,第二版
[121]周启星,孔繁翔,朱琳主编.生态毒理学.北京:科学出版社,2004:307
[122]Hook,R.I.van.Cadmium,lead and zinc distributions between earthworms and soils,potentials for biological accumulation.Bull Buviron Contom Toxicol,1974.12.509-515
[123]Ireland MP.The effect of the earthworm Dendrobaem rubiba on the solubility of lead,zinc,and calcium in heavy metal contaminated soilin Wales.Soil Sci.1975.26.313-318
[124]张友梅,王振中,邢协加,等.土壤污染对蚯蚓的影响.湖南师范大学自然科学学报.1996.9(19):86-90
[125]C.Svendsen,D.J.Spurgeon,P.K.Hankard,et al.A review of lysosomal memberane stability measured by neutral red retention:is it a workable earth biomarker ? Ecotoxicology and Environmental Safety 2004,57(1):20-29
[126]田晓丽,郭军华.金属硫蛋白的研究进展.国外医学药学分册.2005,3(2):119-124
[127]王海英,郭祀远,李琳.蚯蚓的研究与应用.氨基酸和生物资源,2002,24(4):17-19
[128]StUrzenbaum SR,Kille P,Morgan AJ.The identification,cloning and characterization of earthworm metallothionein.FEBS Lett.1998,24;431(3):437-442
[129]John E.Morgan and A.J.Morgan The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site.Environmental Pollution.1998,99(2):167-175
[130]张桂春.金属硫蛋白的功能及应用前景.烟台师范学院学报(自然科学版).2005,1(1):142-145
[131]李婧.金属硫蛋白检测方法的研究进展.现代检验医学杂志.2004,19(3):64-65
[132]张保林,盛湘溶,刘兴军,等.金属硫蛋白测定方法-银饱和分析法.药物生物技术,1996,3(1):31-33.
[133]OECD Guidelines for Testing of Chemicals.No.207:Earthworm,Acute Toxicity Tests[R].Paris:OECD Council,2001.
[1]殷书梅,储益平,吴鹏.地龙活性提取物的主要药效学实验.中草药,2002,33(10):926
[2]朱九群.地龙的临床应用进展.现代中西医结合杂志,2000,9(3):205
[3]张燕红,张庭芳,王采芹,等.蚯蚓钙调素结合蛋白的研究.北京大学学报(自然科学版),1997,33(6):731
[4]于德江,刘亚杰,迟仁智,等.中药材地龙.中国药学杂志,1993,28(10):594-596
[5]陈平,叶卯祥,严宜昌,等.中药地龙的药源调查与商品鉴定.中草药,1997,28(8):492-495
[6]陈强,冯孝义,董健华,等.西北药用蚯蚓资源考察.中国中药杂志,1995,20(11):650-651
[7]周天元著.蚯蚓无土高效养殖新技术.天津科学技术出版社.2002,1
[8]陈平,傅杰,严宜昌.地龙的本草考证.中药材,20(3):158-160
[9]肖小芹.地龙原动物种类及活体鉴别.湖南中医杂志,2002,18(4):50
[10]张贵君等.中药鉴定学.北京:科学出版社,2002,452
[11]李兰燕,王树春,吴云山,等.广地龙的X射线衍射Fourier谱鉴定.中成药.24(5):380-382
[12]于占林,邢利华.地龙人工养殖技术.时珍国药研究,1995,5(4):34-35
[13]徐晋佑,蓝宗辉,曾逊生,等.人工养殖参环毛蚓的实验研究.中药材科技,1983,4:14-18
[14]亢霞生,程新,黄文戈编著.蚯蚓高效养殖技术-特种养殖点金术.广西科学技术出版社,2002,1
[15]刘向辉,戈峰.蚯蚓纤溶酶活性影响因子的研究.中国中药杂志,2002,27(6):423-425
[16]王海英,郭祀远,李琳.蚯蚓的研究与应用.氨基酸和生物资源,2002,24(4):17-19
[17]程能能,衫浦隆夫,福天辉夫,等.地龙体内磷脂的组成和血小板活化因子的生物合成.药学学报,1992,27(12):886-890
[18]张祖珣,何俐明,于培兰,等.蚯蚓毒素及其去除方法的初步研究.中国生化药物杂志,2001,22(3):112-113
[19]刘艳玲,李莉,吴红艳,等.蚯蚓两种采收加工方法的比较.中国药学杂志,2002,37(2):96-98
[20]路英华,金汝成.蚯蚓纤溶酶的提取性质鉴定和溶解血栓的研究.兰州大学学报,1986,(2):295
[21]丰素娟.地龙炮制经验谈.中国现代应用药学杂志,16(5):14-15
[22]刘洪章,刘喜梅.地龙炮制之我见.时珍国医国药,1998,9(5):415
[23]蒋艳红,刘敏彦,刘向军,等.地龙中游离氨基酸的含量测定.中国中医药信息杂志,2003,10(2):36-37
[24]李伟,文红梅,钟进,等.HPLC法测定地龙中次黄嘌呤、黄嘌呤、尿嘧啶、尿苷的含量.中 药材,1996,19(12):625-627
[25]王光忠,陈平,胡迪,等.薄层扫描法测定地龙类药材中琥珀酸的含量.药物分析杂志,1997,17(4):274-275
[26]张娴,彭国平,王振中.地龙中肌苷含量测定.中药材,2003,26(2):103
[27]易八贤,徐丽姗,曾漪青,等.两种蚯蚓及其不同部位的酶谱研究.药物生物技术.1999,6(3):174-176
[28]赵春桂.蚯蚓中微量元素含量测定.中药通报,1998,1(9):38
[29]王文东,张英华,崔健,等.地龙及其制剂的质量控制研究.吉林中医药,1996,5:38-39
[30]方铁铮,杨翠平,苏薇薇.地龙及其注射液指纹特征谱研究.中药材,2002,11(11):813-815
[31]王光忠,胡迪,陈敬炳等.地龙类药材化学成分分析.中药材,1998,21(3):13
[32]胡馨,黄文辉.各种沪产地龙与广东产地龙中次黄嘌呤的测定和比较.中成药,1994,16(1):42-43
[33]施寄材.中药地龙的含砷量.中成药研究,1981,(5):17