摘要
[目的]
贵州铜仁地区产的吴茱萸,颗粒均匀、色泽绿、香气浓烈、味辛辣、品质上乘,饮誉中外。因贵州铜仁地区所产吴茱萸产量大、质量优良,被称为贵州道地药材,也为贵州名药之一；湖南新晃产吴莱萸质量最好,为吴茱萸道地药材。研究中药吴茱萸道地性形成的相关因子,探讨中药吴茱萸道地性形成的机制,构建吴茱萸道地药材产地适宜性分析评价的共性技术体系,结合社会经济条件,为因地制宜地进行生产区域布局和发展优势品种提供技术支撑,促进吴茱萸道地药材的可持续发展。
[方法]
通过网络调查了全国吴茱萸资源；实地考察了全国吴茱萸主要产区并对产区的种植品种、引种来源、蕴含量、经纬度、海拔、土壤类型等进行详细记录,采用五点采样法采集这些产区的根、茎、叶、果实和土壤等样品；利用TCMGIS技术对中药吴茱萸道地产区进行区划分析；用吴茱萸的嫩叶为试验材料,利用ITS、AFLP、SRAP等分子标记技术,对不同产地的吴茱萸进行遗传背景分析；采用平板分离法对湖南新晃、浏阳、湘乡、娄底,贵州铜仁,江西万载和浙江平阳、建德等地的吴茱萸内生真菌进行了分离纯化及比较分析,采用HPLC, LC-MS等分析技术对产吴茱萸碱、吴茱萸碱次碱及结构类似物等成分的微生物进行分析；采用HPLC对吴茱萸碱和吴茱萸次碱的含量进行测定,采用原子吸收法测定中药吴茱萸果实中微量元素的含量；采用滴定法测定土壤呼吸强度、采用最大或然计数法(MPN)进行微生物生理类群(钾细菌、氨化细菌、自生好氧固氮菌、硝化细菌、纤维素分解菌)的测定,采用比色法测定土壤酶脲酶、蔗糖酶、过氧化氢酶和蛋白酶等酶的活性,采用原子吸收法测定土壤微量元素的含量。
[结果]
1.文献查阅和实地考察发现,中药吴茱萸的道地性与纬度(27。左右)和海拔(200-600m)有关,说明纬度和海拔是中药吴茱萸道地性形成的关键因子；通过吴茱萸的生物学特性与产量的关系分析可以看出,吴茱萸品种的选择对吴茱萸的品质有很大的影响；通过TCMGIS的吴茱萸产地适宜性区划分析发现吴茱萸中的石虎品种82%的适宜产地在贵州,贵州作为石虎的道地产区与文献记载的完全吻合。
2.ITS分析结果表明,吴茱萸药材ITS序列多态性较差,序列相似度大,仅部分样品存在少量碱基突变；AFLP分析结果表明,AFLP分子标记技术能很好地区分吴茱萸药材三个来源植物吴茱萸和石虎、疏毛吴茱萸之间的遗传差异；湖南新晃、贵州铜仁等高海拔山区作为中药吴茱萸道地产区,植株遗传背景比较稳定,这可能是生产质量稳定的药材原料的基础；SRAP分析结果表明,吴茱萸遗传背景多样性比较明显,由SRAP对吴茱萸的聚类分析可知,吴茱萸遗传背景有品种的差异,即石虎和疏毛吴茱萸的遗传差异较大,各地区有按同一品种聚类的趋势,同一品种不同产地的遗传背景差异较小,突出表现为贵州铜仁、湖南新晃道地产区的样品聚集较紧密。
3.对不同产地同一品种、同一产地不同品种、同一产地同一品种的不同采收期的吴茱萸碱和吴茱萸次碱的含量检测表明,品种、产地、采收期对中药吴茱萸的道地性都有重要的影响。
4.对不同吴茱萸产区的土壤因子研究表明,土壤Zn、土壤蔗糖酶活、土壤Cu等因子对中药吴茱萸道地性有显著影响。
5.从8个吴茱萸产区共分离到31株内生真菌和37株内生细菌,分别从吴茱萸的叶、茎和果实中分离到不同的内生菌,表现为组织特异性,且在不同产地的内生菌的种类和优势种群存在明显的差异,即具有地域特异性；筛选出产吴茱萸碱和次碱及结构类似物的4株内生真菌和1株内生细菌。
6.吴茱萸挥发油的抑菌试验表明,吴茱萸挥发油的抑菌能力可以作为中药吴茱萸道地性的一个重要的药效指标。
[结论]
中药吴茱萸道地性的形成主要是以下因子共同作用的结果：各产地吴茱萸栽培品种的差异、遗传背景的特异性等内在因素起决定性作用,如品质最好的湖南新晃的中药吴茱萸品种单一,全为石虎,其遗传背景差异小且稳定；环境因子中的温度,海拔和土壤微生物的总量和分布规律以及土壤元素的分布规律等外在因素起关键作用。研究吴茱萸道地性的技术包括：用于遗传背景分析的AFLP和SRAP等分子标记技术,用于道地产地区划的TCMGIS-Ⅱ系统分析法,用于土壤微生物的生理类群分析的最大或然法,用于土壤总微生物的含量测定的酸碱滴定法。
[Objective]
Euodia ruticarpa of Tongren Guizhou is popular at home and abroad because uniform particles, green colour and lustre, strong fragrance, acridity sapor and excellent quality. With high yield and good quality, Tongren Guizhou produces Euodia ruticarpa usually is looked as genuine medicinal materials of Guizhou and one of Guizhou famous medicinal materials, too. The Euodia ruticarpa of Hunan Xinhuang is also genuine medicinal materials for the best quality. In order to establish generic technology system of appraiseing Euodia rutaecarpa of adaptive region, the correlation factors and forming mechanism of Euodia rutaecarpa genuine were researched. Combing with social and economic conditions, the research would not only to provide technical support for producing region composition according to the local conditions and developing advantage varieties, but also to promote the sustainable development of superior medicinal materials of Euodia rutaecarpa.
[Methods]
Euodia rutaecarpa resource was investigated by network. The main producing areas of Euodia rutaecarpa was investigated via site visits. Plant varieties, introduction, yields, latitudes and longitudes, elevation and soil types were recorded in the main producing areas. Euodia rutaecarpa samples including roots, stems, fruits and soil were collected using five sampling method. The regionalization of Euodia rutaecarpa genuine region was analysed using TCMGIS technology. The genetic diversity of Euodia rutaecarpa with fresh leaves was studied using ITS, SRAP, AFLP molecular markers. Using plate method, the Endophytes in Euodia rutaecarpa were separated and purified and compared in Xinhuang, Liuyang, Xiangxiang, Luodi of Hunan, Tongren Guizhou, Wanzhai Jiangxi and Pingyang, Jiangde Zhejiang. Using HPLC, LC-MS technology, the components about evodiamine, rutaecarpine and structural analogues were analyzed. HPLC method was used to detect the content of Euodiamine and Rutaecarpine in Euodia rutaecarpa. The content of the trace element in Euodia rutaecarpa was determined by atom absorption. Respiration of soil microbe was studied by titration analytical procedures; the microbial physiological groups (decomposing Potassium bacterium, aminate bacteria, Azotobacter, nitrifying bacteria, cellulose decomposition bacteria)in soils were tested by most probable number. Soil enzymatic activities(urease, catalase, sucrase, Proteinase)were determined by colorimetry. The content of the trace element in soil was determined by atom absorption.
[Results]
1. The result of literature review and investigation showed that the genuineness of Euodia rutaecarpa was associated with the latitude(about27°) and altitude(200～600m). the latitude and altitude were key factors of forming the genuineness of Euodia rutaecarpa. Variety influenced the genuineness of Euodia rutaecarpa distinctly. Using TCMGIS, suitable growth regions was analysed, the resulted showed that82%genuineness regions of E. Benth.var.oficinalis(Dode)Huang located in Guizhou, witch was Completely consistent with records.
2. The result of ITS analysis showed that ITS sequence polymorphism of Fructus Euodia was poorer, the sequence was similar, only some samples contain few base mutations.The result of AFLP analysis showed that it was easy to distinguish the genetic diversity of three Medicinal Materials source plants Euodia rutaecarpa, Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang and Euodia rutaecarpa(Juss) Benth var. bodinieri (Dode) Huang. Plant genetic background was stable in High altitude mountain areas, which are Euodia rutaecarpa authentic region such as Xinhuang of Hunan, Tongren of Guizhou etc. This would be the foundation production quality stable medicinal material. The result of SRAP analysis showed that Euodia rutaecarpa genetic background diversity was obvious. Using cluster analysis of SRAP, we concluded that there were varieties differences in Euodia rutaecarpa genetic background. The genetic differences were obvious between Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang and Euodia rutaecarpa(Juss) Benth var.bodinieri (Dode) Huang, and there was a trend of same variety to clustering in each region. Obviously, the genetic background of the same variety from different origin, those samples gathered more closely from Euodia rutaecarpa authentic region such as Xinhuang of Hunan, Tongren of Guizhou.
3. Content of Euodiamine and Rutaecarpine in different producing areas of the same variety, the same origin of different varieties, the same variety of different harvest time showed that varieties, origin and harvest time influenced on the genuineness of Euodia rutaecarpa distinctly.
4. The results of soil factor showed that Zn, sucrase, Cu influenced on the genuineness of Euodia rutaecarpa significantly.
5.31endophytic fungi and37endophytic bacteria in Euodia rutaecarpa were separated from8Euodia rutaecarpa region, different specific types of the endophytic fungi and bacteria were separated in different leaves, stems, and fruits, namely with organized specificity. Existing obvious differences in different types of the Endophytes and dominant population, namely with regional specificity.4Euodia rutaecarpa endophytic fungi and1endophytic bacteria were screened which could produce evodiamine, rutaecarpine and structural analogues.
6. The result of bacteriostatic experiment on Euodia rutaecarpa volatile oil showed that the bacteriostatic ability of Euodia rutaecarpa volatile oil could be regarded as an important efficacy index of Euodia rutaecarpa genuineness.
[Conclusion]
Influencing factors of forming genuineness of traditional Chinese medicine Euodia rutaecarpa include internal factors and Extrinsic Factors. Internal factors played decisive role on the formation of genuineness such as the variety difference of Euodia rutaecarpa and the specificity of the genetic background. For example, Euodia rutaecarpa of Xinhuang Hunan was the only variety of Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang, the genetic background was little difference and stable. Extrinsic Factors included temperature, altitude, the amount and distribution law of soil microorganisms, distribution law of trace elements in soil, witch played key role. The following technology used to research genuineness of Euodia rutaecarpa:AFLP and SRAP molecular markers used for genetic background analysis, TCMGIS-Ⅱ system analysis for genuine origin division, most probable number was applied to the analysis of soil microorganism physiological groups, acid-base titration method was applied to the total content determination of soil microorganisms.
引文
[1]谢宗万.论道地药材[J].中医杂志,1990,40(10)：43-46.
[2]魏宝阳,曹亮,李顺祥.内生菌与药用植物的关系及对次生代谢产物的影响[J].中国农学通报,2011,27(19)：83-88.
[3]黄璐琦,郭兰萍,吕冬梅,等.道地药材的属性及研究对策[J].中国中医药信息杂志,2007,(02)：44-46.
[4]任德权,索风梅,陈士林,等.道地药材的产地适宜性研究[J].中药研究与信息,2005,(09)：4-9.
[5]胡世林.现代道地论概要[J].中国中医药信息杂志,1995,2(7)：7-8.
[6]胡世林,廖福龙.中药材道地性与生物多样性[J].中国医药学报,1999,14(5) ：16-17.
[7]张重义,李萍,李会军,等.道地与非道地产区金银花质量的比较[J].中国中药杂志,2007,32(9)：786-789.
[8]肖小河,肖培根,王永炎.基于道地药材和生物效价检测的中药质量控制与评价模式的研究[J].湖南中医药大学学报,2007,8(27)：5-8.
[9]肖小河,陈士林.论道地药材的系统研究[J].四川中草药研究,1991,5(2-3)：15-20.
[10]李隆云,钟国跃,卫莹芳,等.中国中药种质资源的保存与评价研究[J].中国中药杂志,2002,27(9)：641-645.
[11]冯学锋,胡世林,郭宝林,等.黄芩种群遗传多样性初步研究[J].世界科学技术-中药现代化,2002,4(4)：38-43.
[12]陈士林.地道药材与生态型的相关性[J].中草药,1988,19(8)：40.
[13]陈美兰,黄璐琦,欧阳少华,等.植物内生菌对道地药材形成的影响[J].中国中医药信息杂志,2006,13(9)：40-42.
[14]黄璐琦,陈美兰,肖培根.中药材道地性研究的现代生物学基础及模式假说[J].中国中药杂志,2004,29(6)：494-496,610.
[15]肖小河,夏文娟,陈善墉.中国道地药材研究概论[J].中国中药杂志,1995,20(6)：323-326.
[16]江峰.DNA指纹图谱技术及其在中药研究中的应用[J].海峡药学,2005,17(4)：183-185.
[17]Jeyaramraja P R, Pius P K, Raj Kumar R, et al. Soil moisture stress induced alterations in bioconstituents determining tea quality[J]. J Sci Food Agri,2003, 83(12):1187-1191.
[18]Clark R, Menary R. The effect of irrigation and nitrogen on the yield and composition of peppermint oil[J]. Aust J Agric Res,1980,31:489-498.
[19]李霞,王洋,阎秀峰.水分胁迫对黄檗幼苗三种生物碱含量的影响[J].生态学报,2007,27(1)：58-64.
[20]郑青松,刘玲,刘友良,等.盐分和水分胁迫对芦荟幼苗渗透调节和渗调物质积累的影响[J].植物生理与分子生物学学报,2003,29(6)：585-588.
[21]李隆云,肖小河,秦松云,等.道地药材的形成与分化探讨[J].中国中医药科技,1999,6(2)：105-108.
[22]Yao H, Song JY, Ma XY, et al. Identification of Dendrobium species by a candidate DNA barcode sequence:The chloroplast psbA-trnH intergenic region [J]. Planta Med,2009,75(6):667-669.
[23]Song JY, Yao H, Li Y, et al. Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique [J]. J Ethnopharmacol, 2009,124(3):434-439.
[24]Luo K, Chen SL, Chen KL, et al. Application of DNA bar coding to the medicinal plants of the Araceae family [J].Planta Med,2009,75(4):416.
[25]朱英杰,陈士林,姚辉,等.重楼属药用植物DNA条形码鉴定研究[J].药学学报,2010,45(3)：376-382.
[26]Pang X, Chen SL. Using DNA barcodes to identify Rosaceae[J]. Planta Med, 2009,75(4):417.
[27]Gao T, Chen SL. Authentication of the medicinal plants in Fabaceae by DNA barcoding technique [J]. Planta Med,2009,75(4):417.
[28]Wen J, Zimmer Z A. Phylogeny and biogeography of Panax L (the Ginseng Genus, araliaceae):inferences from ITS sequences of nuclear ribosomal RNA[J]. Molec Phylogen Evol,1996,6 (2):167-177.
[29]姬可平,李啸红,李应东,等.应用rRNA基因间隔区碱基测序对中药(大黄)进行鉴定[J].世界科学技术：中药现代化,2002,4(4)：44-47.
[30]姬可平,李应东,张西玲,等rRNA基因间隔区碱基测序对当归进行鉴定的研究[J].中草药,2003,34(1)：66-69.
[31]赵国平,新关稔,石川隆二,等.中日当归属药用植物序列分析ITS[J]中草药,2006,37(7)：1072-1076.
[32]高建平,王彦涵,乔春峰,等.中药南五味子及其混淆品绿叶五味子果实的ITS序列分析[J].中国中药杂志,2003,28(8)：706-710.
[33]姬可平,刘丽莎,张西玲,等.家种及野生秦艽、麻花秦艽的rRNA基因间隔区PCR产物电泳图谱的初步研究[J].中草药,2003,26(1)：11-14.
[34]丁平,方琴.巴戟天与常见混伪品的rDNA ITS序列分析及其分子鉴定[J].中草药,2005,36(6)：908-911.
[35]蒋继宏,孟娜,曹小迎,等.苏皖产大戟属药用植物rDNA的ITS序列分析[J].中草药,2005,36(6)：900-902.
[36]汪红,王强.丹参及鼠尾草属植物的rDNA ITS序列分析[J].中草药,2005,36(9)：105-109.
[37]谢晖,晁志,霍克克,等.9种柴胡属植物的核糖体ITS序列及其在药材鉴定中的应用[J].南方医科大学学报,2006,26(10)：1460-1463.
[38]罗玉明,张卫明,丁小余,等.紫苏属药用植物的rDNA ITS区SNP分子标记与位点特异性PCR鉴别[J].药学学报,2006,41(9)：840-845.
[39]张君毅,郭巧生,吴丽伟,等.我国不同地区半夏rDNA序列分析[J].中国中药杂志,2006,31(21)：1768-1772.
[40]吴玲,卢毅军,史树德,等.中国石蒜属种间亲缘关系ITS序列分析[J].亚热带植物科学,2007,36(1)：31.
[41]蔡侃,孔文刚,夏红剑,等.牛蒡18S核糖体RNA基因分析和分类学研究[J].食品工业,2011,(9)：105-107.
[42]罗洪斌,张健,胡泽华,等.五鹤续断18S rRNA基因序列的分析[J].时珍国医国药,2010,21(8)：2004-2005.
[43]Huang D, Li SX, Cai GX, Yue CH, Wei LJ, Zhang P. Molecular Authentication and Quality Control using a High Performance Liquid Chromatography Technique of Fructus Euodiae[J]. Biol. Pharm Bull.,2008,31(2):312-315.
[44]Bao yang WEI, Liang CAO, Shun-xiang LI,et al. Population structure of Euodia rutaecarpa in China revealed by amplified fragment length polymorphism (AFLP) and sequence-related amplified polymorphism (SRAP)[J]. Journal of Medicinal Plant Research,2011,5(30):6628-6635.
[45]曹亮,魏宝阳,李顺祥SRAP和SCAR分子标记应用于中药材研究进展[J].科技导报,2010,28(12)：104-109.
[46]曹亮,李顺祥,魏宝阳,等.吴茱萸RAPD体系构建及道地性遗传背景研究[J].中草药,2010,41(6)：975-978.
[47]曹亮.吴茱萸遗传多样性分析及适宜产区规划[D].长沙：湖南中医药大学,2011.
[48]魏宝阳,曹亮,李顺祥,等.吴茱萸遗传多样性的SRAP分析[J].中草药,2011,42(12)：2523-2528.
[49]魏宝阳,曹亮,李顺祥,等.吴茱萸遗传多样性的AFLP分析[J].中国中药杂志,2012,37(3)：278-282.
[50]黄璐琦,郭兰萍,华国栋.道地药材属性及研究对策[J].中国中医药信息杂志,2007,14(2)：44-46.
[51]黄新,邱德有,黄璐琦.茉莉酸甲酯对中国红豆杉细胞基因表达的mRNA差异显示研究[J].分子植物育种,2006,4(5)：627-632.
[52]李娟,盛维瑾,刘大为,等.培养条件对HBsAg转基因人参细胞的生长及表达量的影响[J].中国生物制品学杂志,2007,20(1)：40-42.
[53]Mi H L, Jae H J, Jin W S, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squanlene synthesis gene [J]. Plant Cell Physiol, 2004,45(8):976-980.
[54]Harsh P B, Ramarao V, Christopher B L, et al. Molecular and biochemical characterization of an enzyme responsible for formation of hypericinin St. Johne swort (Hypericun perforatum L.)[J]. Bio Chem,2001,278 (34):3213-3216.
[55]崔瑛,纪彬,赵素霞,等.吴茱萸外用功效的探讨[J].中华中医药杂志,2011,26(2)：264-267.
[56]崔晓秋.吴茱萸化学成分研究[D].长春：吉林农业大学,2004.
[57]许海玉,刘莹,张铁军,等.氧化铝柱纯化吴茱萸提取液中总生物碱和柠檬苦素的研究[J].中草药,2010,41(5)：741-744.
[58]龚慕辛,宋亚芳,王智民,等.吴茱萸化学成分的研究[J].中国中药杂 志,2009,34(6)：792-794.
[59]唐元清,冯孝章,黄量.吴茱萸化学成分的研究[J].药学学报,1996,31(2)：151-155.
[60]Xiu Wei Yang, Jie Teng. Chemical constituents of the unr ipe fruits of Euodia rutaecarpa[J]. Journal of Chinese Pharmaceutical sciences,2007(16):20-23.
[61]Chuang W C, Cheng C M, Chang HC, et al. Contents of constituents in mature and immature fruits of Euodia species[J]. Planta Med.1999,65(6):567-571.
[62]Guang Xian Cai, Dan Huang, Shun Xiang Li, et al. Comparative analysis of essential oil components of Euodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang and Euodia rutaecarpa (Juss.) Benth. [J]. Natural Product Research,2011,1-3.
[63]国家药典委员会.中国药典2010版.一部[S].北京：中国医药科技出版社,2010.160.
[64]黄丹,李顺祥,魏宝阳,等.不同产地吴茱萸中吴茱萸碱、吴茱萸次碱含量比较研究[A].药用植物化学与中药资源可持续发展学术研讨会,2009,300-305.
[65]袁清照,张水寒,李顺祥,等.吴茱萸道地药材的高效液相指纹图谱研究[J].中草药,2010,41(9)：1534-1538.
[66]张小,郭兰萍,韦霄,等.广西青蒿种植气候适宜性等级区划研究[J].中国中药杂志,2008,33(15)：1794-1798.
[67]王晓春,宋来萍,张远东.大兴安岭北部樟子松树木生长与气候因子的关系[J].植物生态学报,2011,35(3)：294-302。
[68]郭兰萍,黄璐琦,阎洪,等.基于地理信息系统的苍术道地药材气候生态特征研究[J].中国中药杂志,2005,30(8)：565-569.
[69]吴卫刚,杜家方,孙耀志.山茱萸果实的形成及其温度效应研究[J].中国现代中药,2010,12(11)：12-14.
[70]黎开强,吴卫,郑有良,等.温度对川贝母生理指标、鳞茎产量及总生物碱含量的影响[J].中国中药杂志,2008,33(16)：1948-1951.
[71]郭兰萍,黄璐琦,蒋有绪,等.影响苍术挥发油组分的气候主导因子及气候适宜性区划研究[J].中国中药杂志,2007,32(10)：888-893.
[72]孙年喜,李隆云,钟国跃,等.不同生长期土壤水分处理对黄花蒿生理特性及产量的影响[J].中国中药杂志,2009,34(2)：386-389.
[73]郭巧生,周黎君,龚伟慧,等.不同水分处理对夏枯草果穗产量和品质的影响[J].中国中药杂志,2010,35(14)：1795-1798.
[74]刘大会,郭兰萍,黄璐琦,等.土壤水分含量对丹参幼苗生长及有效成分的影响[J].中国中药杂志,2011,36(3)：321-325.
[75]张亚妮,苏文华,张光飞.土壤水分胁迫对短葶飞蓬有效成分含量的影响[J].中国中药杂志,2009,34(10)：1191-1194.
[76]柯用春,周凌云,徐迎春,等.土壤水分对金银花总绿原酸含量的影响[J].中国中药杂志,2005,30(15)：1201-1202.
[77]徐迎春,张佳宝,蒋其鳌,等.水分胁迫对忍冬生长及金银花质量的影响[J].中药材,2006,29(5)：420-423.
[78]文涛,梁莉,曾 杨,等.不同光照强度对虎杖愈伤组织的影响[J].中国中药杂志,2007,32(13)：1277-1280.
[79]刘珊,邵东清,贾云峰.光照对麻黄生长发育及生物碱产量的影响[J].中药材,1999,22(5)：221-222.
[80]陈顺钦,袁媛,罗毓健,等.光照对黄芩黄酮类活性成分积累及其相关基因表达的影响[J].中国中药杂志,2010,35(6)：682-685.
[81]许翔鸿,王峥涛,余国奠.光照对延胡索生长及生物碱积累影响的初步研究[J].中药材,2004,27(11)：804-805.
[82]王志清,郑培和,逢世峰, 等.光照强度对北细辛生长发育及质量的影响[J].中国中药杂志,201 1,36(12)：1558-1567.
[83]郭巧生,梁迎暖,张重义,等.土壤因子对怀菊质量影响研究[J].中国中药杂志,2008,33(2)：123-128.
[84]李卫建,李先恩.连翘有效成分含量与土壤养分的量化关系研究[J].中国中药杂志,2005,30(20)：1577-1580.
[85]王艳茹,郭巧生,邵清松,等.土壤因子对药用白菊花活性成分含量影响研究[J].中国中药杂志,2010,35(6)：676-681.
[86]张莲婷,叶正良,郭巧生.土壤因子对麦冬活性成分影响研究[J].中国中药杂志,2010,35(11)：1372-1377.
[87]翟娟园,吴卫,廖凯,等.土壤环境对川白芷产量和品质的影响研究[J].中草药,2010,41(6)：984-988.
[88]郑艳.中药材的地道性与根际土壤微生物[J].现代中药研究与实践,2007,21(6),60-63.
[89]北京农业大学植保系植物生态病理教研室.植物根际生态学与根病生物防治进展[M].北京：中国人民大学出版社,1991.
[90]江曙,钱大玮,段金廒,等.植物内生菌与道地药材的相关性研究[J].中草药,2008,39(8)：1268-1272.
[91]王兴红.内生真菌与天然产物研究进展[J].中草药,2007,38(1)：140-143.
[92]国家药典委员会.中国药典(2005版)[M].北京：化学工业出版社,2005.118-119.
[93]李紫微,胡莹,秦松云.重庆地区吴茱萸属药用植物资源的调查研究[J].资源开发与市场,2006,22(5)：471-473.
[94]黄成就.中国植物志,43(2),北京：科学出版社,1997,65.
[95]崔晓秋.吴茱萸化学成分研究[D].长春：吉林农业大学,2004.
[96]崔岚,祝德秋,安富荣.吴茱萸碱药理作用研究进展[J].中国中医药信息杂志,2005,12(6)：108-110.
[97]Kobayashi V, Nakano V, Kizaki M, et al. Capsaicin-like anti-obese activities of Euodiamine from fruits of Euodia rutaecarpa, a vanilloid receptor agonist[J]. Planta Med,2001,67(7):628.
[98]Kobayashi V, Hoshikuma K, Nakano V, et al. The positive inotropic and chronotropic effects of Euodiamine and rutaecarpine, indoloquinazoline alkaloids isolated from the fruits of Euodia rutaecarpa, on the guineapig isolated right atria:possible involvement of vanil loid receptors[J]. Planta Med, 2001,67(3):244.
[99]Hung P H, Lin L C, Wang G J, et al. Inhibitory effect of Euodiamine on aldosterone release by Zona glomerulosa cells in male rats[J]. Chin J Physiol, 2001,44(2):53.
[100]胡长平.辣椒素受体介导吴茱萸次碱的降压和心脏保护作用[D].长沙：中 南大学,2003.
[101]段徐,凌峰.吴茱萸次碱的药理作用[J].中华中医药学刊,2007,25(9)：1857-1859.
[102]Dan Huang, Shun Xiang Li, Guang Xian Cai, et al. Molecular Authentication and Quality Control using a High Performance Liquid Chromatography Technique of Fructus Euodiae[J]. Biol. Pharm Bull.,2008,31(2):312-315.
[103]李顺祥,蔡光先,张平,等.吴茱萸超微饮片ITS序列指纹图谱的研究[J].科技导报,2007,25(5)：42-46.
[104]蔡光先,李顺祥,汪小兵,等.吴茱萸超微饮片的HPLC指纹图谱研究[J].科技导报,2006,24(8)：40-43.
[105]陈士林,魏建和,孙成忠,等.中药材产地适宜性分析地理信息系统的开发及蒙古黄芪产地适宜性研究[J].世界科学技术—中医现代化工,2006,8(3)：47-53.
[106]王瑀,魏建和,陈士林,等.应用TCMGIS-I分析人参的适宜产地[J].亚太传统医药,2006,(6)：73-78.
[107]王继永,赵润怀,孙成忠,等.基于TCMGIS-I的乌拉尔甘草生态适宜区分析[J].中国现代中药,2006,8(8)：4-8,20.
[108]陈士林,周应群,谢彩香,等.基于TCMGIS-I的西洋参生态适宜性分析[J].中国中药杂志,2008,33(7)：741-745.
[109]严辉,段金廒,孙成忠,等.基于TCMGIS的当归生态适宜性研究[J].世界科学技术—中医药现代化,2009,11(3)：416-422.
[110]李桂英,张本刚,孙成忠,等.基于TCMGIS的槲蕨的生态产地适宜性研究[J].世界科学技术,2009,11(2)：278-282.
[111]林青青,吴水生,徐惠龙,等.基于TCMGIS的建泽泻产地适宜性分析[J].福建中医药大学学报,2011,21(1)：33-34.
[112]刘杨楠,窦德强,谢彩香,等.基于TCMGIS的牛蒡子适宜产区研究[J].中国现代中药,2009,11(11)：11-13.
[113]王珍,索凤梅,黄林芳,等.基于TCMGIS的溪黄草生态适宜性研究[J].中国现代中药,2011,13(11)：15-17,51.
[114]王踽,魏建和,陈士林,等.基于TCMGIS-I的暗紫贝母生态气候产地适宜性分析[J].世界科学技术—中医药现代化,2006,8(3)：122-124,117.
[115]索风梅,陈士林,张昭,等.基于TCMGIS-I的黄檗生态适宜性分析[J].中国中药杂志,2008,33(13)：1536-1539.
[116]赵润怀,王继永,孙成忠,等.基于TCMGIS-I的道地药材附子产地适宜性分析[J].中国现代中药,2006,8(7)：4-8.
[117]陈君,谢彩香,陈士林,等.管花肉苁蓉产地适宜性数值分析[J],中国中药杂志,2008,33(5)：496-501.
[118]王瑀,魏建和,陈士林,等.基于GIS的川芎产地适宜性分析[J].中国现代中药,2006,8(6)：7-9.
[119]索风梅,丁万隆,谢彩香,等.蒙古黄芪的生态适宜性数值分析[J].世界科学技术-中医药现代化,2010,12(3)：480-485.
[120]林余霖,黄林芳,李卿,等.八角茴香产地适宜性分析与数值区划[J].世界科学技术—中医药现代化,2010,12(3)：468-472.
[121]王丽芝,谢彩香,陈士林.怀山药的生态适宜性数值区划[J].中药材,2010,33(8)：1222-1225.
[122]黄林芳,段宝忠,丁平,等.藏茵陈生态适宜性分析与区划[J].安徽农业科学,2010,38(11)：5614-5618.
[123]黄培堂.分子克隆实验指南(第三版)(中译版)[M].北京：科学出版社,2002,1039-1045.
[124]Yeh F C, Yang R C, Boyle T B, et al. PopGene Version 3.2:the user-friendly shareware for population genetic analysis[M]. Molecular Biology and Biotechnology Center, University of Albert, Edmonton.1999.
[125]Lewontin R C. The apportionment of human diversity[J]. Evol Biol,1972, 6:381-398.
[126]Nei M. Analysis of gene diversity in subdivided populations[J]. Proc Natl Acad Sci USA,1973,70:3321-3323.
[127]Nei M. Estimation of average heterozygosity and genetic distance from a small number of individuals[J]. Genetics,1978,89:583-590.
[128]Joseph F. PHYLIP (Phylogeny Inference Package) Version 3.57c, Washington, USA,1986.
[129]Song Z Q, Li X F, Wang H G, et al. Genetic diversity and population structure of Salvia miltiorrhiza Bge in China revealed by ISSR and SRAP[J]. Genetica, 2010,138:241-249.
[130]Li X X, Ding X Y, Chu B H, et al. Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP[J]. Genetica,2008,133:159-166.
[131]Ni X W, Huang Y L, Wu L, et al. Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae)revealed by amplified fragment length polymorphism (AFLP)[J]. Genetica,2006,127,177-183.
[132]蔡光先,李顺祥,汪小兵,等.吴茱萸超微饮片的HPLC指纹图谱研究[J].科技导报,2006,24(8)：40-43.
[133]袁清照,张水寒,李顺祥,等.吴茱萸道地药材的高效液相指纹图谱研究[J].中草药,2010,41(9)：1534-1528.
[134]罗君,张丽艳,杨玉琴,等.贵州余庆栽培吴茱萸的质量对比研究[J].时珍国医国药2008,19(12)：3001-3002.
[135]夏玉凤,戴岳,张艳立,等.疏毛吴茱萸中吴茱萸碱和吴茱萸次碱含量的动态变化[J].中国野生植物资源,2006,25(1)：53-55.
[136]宋亚芳,王智民,朱晶晶,等.一测多评法测定吴茱萸中吴茱萸内酯、吴茱萸碱及吴茱萸次碱的含量[J].中国中药杂志,2009,34(21)：2781-2785.
[137]张雪琴,周欣,王道平,等.液相色谱-电喷雾质谱联用研究不同产地吴茱萸中生物碱成分[J].分析化学研究简报,2005,33(2)：241-244.
[138]侯晓虹,于治国,徐赞美,等.34种吴茱萸中吴茱萸碱和吴茱萸次碱的含量测定[J].沈阳药科大学学报,2000,17(5)：334-337.
[139]周迎新,肖鸣,张海道.中药吴茱萸类的质量比较[J].中国中药杂志,1993,18(4)：201-203.
[140]段朝辉,张红梅,朱恩圆.吴茱萸质量标准研究——TLC指纹图谱鉴别及HPLC测定吴茱萸碱和吴茱萸次碱含量[J].上海中医药杂志,2008,42(5)：86-88.
[141]敖茂宏,宋智琴,罗小青,等.贵州野生与栽培小花吴茱萸的质量对比研究[J].江苏农业科学,2010(1)：255-256.
[142]郭月秋,陈代贤,于黎明.商品吴茱萸的真伪质量检定考察报告(2)[J].中药材,2009,32(9)：1376-1378.
[143]张丽丽,张丽艳,杨玉琴,等.不同产地吴茱萸中吴茱萸碱及吴茱萸次碱的含量对比分析[J].贵州医药,2004,28(8)：755-756.
[144]郑艳.中药材的地道性与根际土壤微生物[J].现代中药研究与实践,2007,21(6),60-63.
[145]吴洪生,陈佳宏,刘正柱,等.钾细菌制剂对土壤钾素的影响探讨[J].中国生态农业学报,2003,11(3)：92-94.
[146]关松荫.土壤酶及其研究法[M].北京：农业出版社,1986.
[147]Burns R G, Dick R P. Enzymes in the Environment:Ecology, Activity and Applications[M]. Marcel Dekker, Inc., New York.2001.
[148]靳素英,崔明学,蔺继尚.天津东郊盐碱土微生物分布及土壤酶活性[J].应用生态学报(增刊),1996,(7)：139-141.
[149]李跃林,彭少麟.桉树人工林地土壤酶活性与微量元素含量的关系[J].应用生态学报,2003,14(3)：345-348.
[150]南京农学院.土壤农化分析[M].北京：农业出版社,1980.
[151]周国英,陈小艳,李倩茹.油茶林土壤微生物生态分布及土壤酶活性的研究[J].经济林研究,2001,9(1)：9-12.
[152]杨万勤,钟章成,韩玉萍.缙云山森林土壤酶活性的分布特征季节动态及与四川大头茶的关系研究[J].西南师范大学学报(自然科学版),1999,24(3)：318-324.
[153]黄璐琦,张瑞贤.“道地药材”的生物学探讨[J1.中国药学杂志,1997,32(9)：563-566.
[154]王求.不同土壤对白术性状影响的初级观察.中国中药杂志,1993,18(2)：86.
[155]彭锐,卫莹芳,李隆云,等.川党参元素含量特征及影响因素分析[J].中国中药杂志,2009,34(11)：1335.
[156]李兆星,吴晓红,魏宝阳,等.一株产吴茱萸碱结构类似物的内生真菌的筛选和初步鉴定[J].中南药学,2011,9(2)：117-121.
[157]曹望龙,魏宝阳,曹亮,等.产吴茱萸碱或吴茱萸次碱结构类似物的吴茱萸内生真菌的筛选[J].湖南农业科学,2011,(23)：16-17,24.
[158]Stivers T, Mangione-Smith R, Elliott MN, McDonald L, Heritage J. Studies on the chemical constituents of Euodia rutaecarpa [J]. Fam Pract.2003,5(2):140-147.
[159]宫海明,赵桦.不同产地吴茱萸挥发油成分的GC-MS的分析及与小花吴茱萸的比较[J].西北植物学报,2008,28(3)：595-605.
[160]宫海明.不同产地吴茱萸主要药用有效成分分析研究及吴茱萸与小花吴茱萸的对比研究[D].兰州：甘肃农业大学,2008.
[161]付娟,赵桦.吴茱萸和密楝叶中挥发油成分的气相色谱-质谱分析[J].时珍国医国药,2010,(1)：60-64.
[162]王世永.吴茱萸挥发油的提取分离、鉴定及抗氧化和抗菌活性研究[D].湖北：华中农业大学,2008.
[163]弭向辉.花椒挥发油的提取分离及其抗菌和抗肿瘤作用研究[D].南京：南京师范大学,2004.
[164]谢英蜂.青篙挥发油抗菌活性研究[D].吉林：东北师范大学生命科学院,2007：14-17.
[165]张文彤.SPSS统计分析教程[M].北京：北京希望电子出版社,2002：223-229.