【作者】 陈黎；

【导师】 吴卫； 郑有良；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 鱼腥草药蔬兼用,根据《中华人民共和国药典》2005版,鱼腥草新鲜地上和地下部分以及干燥花期地上植株均可入药,而鱼腥草幼嫩茎叶则是鱼腥草主要产区人们餐桌上的一道特色蔬菜。其原植物为三白草科蕺菜属植物蕺菜Houttuynia cordata Thunb.。据《中国植物志》和《四川植物志》,蕺菜属植物仅蕺菜Houttuynia cordata Thunb.1种;2001年,祝正银和张士良在峨眉山发现蕺菜属一新种峨眉蕺菜Houttuynia emeiensis Z.Y.Zhuet S.L.Zhang。该种在峨眉、乐山等地俗称白侧耳根,也作蔬菜和鱼腥草药用。四川和重庆地区的鱼腥草资源分布很广,遗传多样性较大。鱼腥草作为药材或蔬菜主要是利用其中的化学成分,而鱼腥草种质的不同会对这些化学成分产生影响。正确有效地评估鱼腥草资源各种化学成分间的差异,有助于更合理地开发利用这些资源。本研究对四川和重庆地区的鱼腥草资源挥发油成分、黄酮类成分、维生素成分的组成和含量以及部分重金属和农药的残留量进行了检测与分析,获得到如主要结果:1.对18份新鲜鱼腥草材料地上和地下部分挥发油化学成分进行气相色谱质谱联用（GC/MS）分析发现,鱼腥草鲜草地上部分的挥发油成分中共鉴定出21种成分,材料间挥发油成分具有很大的遗传差异。首次发现根据鱼腥草地上部分挥发油成分可分为两种化学型:即癸醛型（D型）和月桂烯型（M型）。聚类分析和主成分分析结果表明,鱼腥草挥发油化学型与染色体数目有关,染色体数目少于80的所有材料均为D型,染色体数目大于或等于80的材料均为M型。D型鱼腥草的单萜类和倍半萜成分含量很低,平均仅5.72%和0.16%,其它非萜类直链化合物占整个挥发油组成的76.94%至94.71%。M型鱼腥草的单萜类和倍半萜含量较高,平均分别为36.16%和1.38%,其它直链化合物的浓度范围为37.88%～64.89%。试验结果还表明,鱼腥草挥发油化学型与其地理来源无关。鱼腥草地下部分挥发油中共鉴定出16种化合物,其不同材料地下部分挥发油成分组成及含量间差异均较小。本试验还发现,蕺菜和峨眉蕺菜间挥发油成分差异不显著。2.采用RAPD、RAMP、ISSR和PCR-RFLP分子标记技术分析鱼腥草资源遗传多样性与挥发油化学型间关系。结果发现,在评价鱼腥草挥发油化学型与遗传背景多样性间关系上,ISSR分子标记较RAPD、RAMP和PCR-RFLP标记更有效,其遗传距离聚类分析结果与挥发油化学型分类结果几乎一致。但Mental test检验结果表明,基于本试验所用各种分子标记的遗传距离与挥发油成分的欧几里得距离间相关均未达显著水平。3.通过对鱼腥草挥发油的紫外（UV）吸收光谱分析发现,鱼腥草挥发油在乙酸乙酯溶剂中的紫外吸收曲线的最大吸收波长λmax主要在252 nm和267 nm;23份试样的紫外吸收曲线可分为5种类型,化学型与吸收曲线类型不尽一致。各供试样品在两波长下的吸光度差异显著,23份试样的吸光度可分为3类。相关分析结果表明,挥发油成分中各主要萜类化合物含量与两个吸收峰的吸收值均呈正相关,且大多数达到极显著水平,而各主要不饱和烷烃类化合物含量与两个吸收峰的吸收值均呈显著或极显著的负相关。4.分别使用不同浓度的D型和M型挥发油对赤霉菌（Fusarium graminearum）、炭疽菌（Colletotrichum coccodes）、灰霉菌（Botrytis cinerea）、大肠杆菌（Escherichia coli）和金黄色葡萄球菌（Staphylococcus aureus）的抑菌能力进行比较。结果表明,两种化学型挥发油的抑菌能力差异显著。D型挥发油对供试的各菌种,特别是对真菌的抑制能力明显高于M型挥发油。此外,同一挥发油对不同菌种抑制能力不同。5.采用GC/MS法对生产中鱼腥草主栽品系的鲜草地上部分挥发油化学成分变化进行检测。方差分析结果表明,w01-100和w01-94的挥发油主要化学成分含量在一天中日照时间内无显著变化,以w01-94挥发油主要化学成分含量更稳定,w01-100和w01-94在整个白天内均可采收。此外,鱼腥草地上部分鲜草挥发油的部分主要成分间含量变化相关显著,但不同材料间相同两种成分间的相关性可能不同。6.采用紫外-可见（UV-VIS）吸收光谱法和薄层色谱（TLC）法对提取挥发油后的剩余水溶液中黄酮类成分分析。显色鉴定和紫外-可见光谱扫描结果发现,提取剩余液中的黄酮类成分的种类丰富,主要类群有黄酮类、黄酮醇类、异黄酮类、二氢黄酮类和二氢黄酮醇类。薄层色谱结果表明这些成分中以苷类为主,含有芦丁和金丝桃苷成分。14份鱼腥草材料总黄酮含量差异达到极显著水平,平均含量为1.93 mg/g,以w01-16的总黄酮含量最高,为3.09 mg/g。鱼腥草资源的黄酮类成分种类及总黄酮含量与其染色体数目间均无显著相关。提取鱼腥草挥发油后的水溶液回收再利用价值明显。7.采用高效液相色谱（HPLC）法比较鱼腥草资源间槲皮素含量差异。采用HypersilODS C18（250mm×4.0mm 5μm）色谱柱,流动相乙腈:0.4%磷酸=45:55,调pH=3,流速为1.0ml/min,柱温30℃,紫外检测波长370nm。结果发现,槲皮素含量在22.43 ng～2.24 ug范围内具有良好的线性关系,回归方程为Y=1.261458X-16.1218（r=0.9997）,鱼腥草材料间槲皮素含量差异显著,w01-120中槲皮素含量水平最高,达4.02 mg/g。8.采用HPLC法对鱼腥草幼嫩植株的维生素种类及含量进行测定,脂溶性维生素VA、δ-VE、VD₂、VD₃、α-VE和VK₁采用Hyperclone BDS C₁₈（150 mm×4.6 mm,5μm）色谱柱,以甲醇:乙酸乙酯（95:5）为流动相,275 nm下测定;水溶性维生素VB₁、VB₂、VB₆、烟酸和VC采用Luna C₁₈（2）100（?）（200 mm×4.6 mm,5μm）色谱柱,柱温30℃,以0.005 mol·mL^-1己烷磺酸钠（pH=3.2）:甲醇（7:3）,254 nm下测定。结果发现,鱼腥草幼嫩植株主要含有较多的α-VE和VC、含少量的VD₃、VK₁以及烟酸,而其余6种维生素均未检测到。鱼腥草资源间维生素种类差异很小,但含量差异显著。其地上部分维生素种类和含量比地下部分丰富,即鱼腥草地上部分维生素的营养价值高于其地下部分。9.采用原子荧光（AFS）法同时测定不同鱼腥草资源中砷（As）和汞（Hg）含量。结果发现,在土壤中As的含量水平为11.557mg/kg,Hg的含量为0.387mg/kg的栽培条件下,鱼腥草幼嫩地上部分中As平均含量为0.161 mg/kg,Hg平均含量为0.004 mg/kg,幼嫩地下部分中两者的平均含量分别为0.130 mg/kg和0.0025 mg/kg。鱼腥草资源间对As、Hg积累的差异不显著,其As、Hg的含量低于国标规定限量。10.采用气相色谱（GC）法,按GB/T14551-2003测定666和DDT的含量。鱼腥草幼嫩地上部分中仅小部分材料中检出痕量的δ-666。鱼腥草资源间对666、DDT积累的差异不显著,其666、DDT的含量低于国标规定限量。综上所述,鱼腥草资源间在挥发油、黄酮类、维生素等自身代谢化学成分上差异显著;在重金属含量和农药残留量上差异不显著。其中,鲜草挥发油成分化学型的产生与其染色体数目变异高度相关,其他化学成分间差异与其染色体数目变异关系不明显。蕺菜和峨眉蕺菜在化学成分上并无显著差异。挥发油化学型的不同会导致其抗菌效果的不同,这对以鱼腥草挥发油为原料的药品生产和使用有重要意义。更多还原

【Abstract】 HERBA HOUTTYNIAE,is a kind of traditional Chinese medicine and also a kind of delicious vegetable.According to Chinese Pharmacopoeia（2005）,the fresh aerial and underground parts and the dry aerial parts during flowering period could be used as Chinese medicine.In the meantime,the fresh young stems and leaves are also delicious vegetable in its main spreading regions.HERBA HOUTTYNIAE came from Houttuynia cordata Thunb., which belongs to genus Houttuynia of Saururaceae.H.cordata is the only species in Houttuynia Thunb.according to Florae Reipublicae Popularis Sinicae and Flora Sichuanica. Zhu et al.（2001）classified a new species H,emeiensis Z.Y.Zhu et S.L.Zhang in the genus Houttuynia collected at Emei mountain in Sichuan province.It was called commonly as white Yuxingcao,which was also used as vegetable and HERBA HOUTTYNIAE in Emei and Leshan regions.Houttuynia is widely spread in Sichuan and Chongqing regions of China.There existed plentiful genetic variations among genus Houttuynia germplasm resources.The chemical components of genus Houttuynia is the main reason why people utilized it as a kind of medicinal plants or vegetables.The diversities among Houttuynia germplasm resources would influence on these chemical components.Evaluation correctly and effectively on the variations among the chemical components of Houttuynia resources will help to the exploitation and ulilization of Houttuynia resources.In this paper,the compositions and their contents of the essential oils, flavones,vitamins and the rudimental contents of some heavy metals and pesticides of Houttuynia accessions in Sichuan and Chongqing regions were analyzed.The main results were as follows:1.The essential oil（EO）compositions of 18 fresh Houttuynia accessions from Sichuan and Chongqing in China were analyzed by using GC/MS method.In total,21 components could be identified from the essential oils（EO）of the aerial parts of Houttuynia.High level of chemical polymorphism in the EOs of the aerial parts of Houttuynia was observed.On the basis of GC/MS data of the EOs of the aerial parts,two chemotypes could be identified for the frist time within the investigated Houttuynia accessions as follows:chemotypes decanal（D） and myrcene（M）.The results of cluster analysis（CA）and principal component analysis（PCA） showed that the chemotypes of Houttuynia were correlated with the chromosome numbers. All the accessions classified into chemotype D have the chromosome number less than 80, while chemotype M had the chromosome number equal to or more than 80.The chemotype D showed lower amounts of monoterpenes and sequiterpenes,on average of 5.72%and 0.16%, respectively.The other non-terpenic straight chain components made up 76.94%to 94.71%of the whole EO components.In general,the chemotype M showed higher amounts of monoterpenes（average 36.16%）and sequiterpenes（1.38%）,while the concentration of other straight chain components rang was from 37.88%to 64.89%.There was no significant relationship between the chemotypes and their geographical distributions.Totally,16 compounds were characterized from the essential oils（EO）of the underground parts of Houttuynia.There was no significant chemical difference between the EO of the underground parts of the different Houttuynia accessions.In this study,no significant chemical component difference was detected between the H.cordata and H.emeiensis accessions.2.The relationships between the genetic diversity among Houttuynia germplasm resources and the essential oil chemotypes were analyzed by using RAPD,RAMP,ISSR and PCR-RFLP DNA markers technology.The results showed that the ISSR marker was more effective to evaluate the relationships than RAPD,RAMP and PCR-RFLP markers on the evualation of the relationships between the genetic diversity and the essential oil chemotypes.The cluster results of genetic distances（GDs）based on ISSR marker were almost coincident with the cluster results of euclidean distances based on the essential oils chemical components.However,the results of matrix correlation showed,there were insignificant correlation between EDs based on the essential oils chemical components and GDs based on any kind of DNA markers.3.Through analyzing the UV spectrum of the essential oil of Houttuynia,it was found that theλmax of the essential oil of Houttuynia were at 252 nm and 267 nm in acetic ether solvent. Twenty-three samples could be divided into 5 types,which could not be coincident with the essential oil chemotypes.The differences of the absorbency of these samples at 252 nm and 267 nm were remarkable.Twenty-three samples counld be divided into 3 types based on the results of absorbency.All of the correlations between the contents of terpenes of EOs and the absorbencies of EOs at the two wavelengths were positive,and most of them reached 1% remarkable levlel.But all the correlations between the contents of unsaturated alkane of EOs and the absorbencies were negative,and all of them reached 5%or 1%remarkable levlel.4.The abilities to restrain the growths of Fusarium graminearum,Colletotrichum coccodes, Botrytis cinerea,Escherichia coli and Staphylococcus aureus were compared by using the different concentrations of D chemotype and M type essential oils.The results showed that the difference of the restraining abilities of the two chemotypes was obvious.The restraining abilities of D chemotype essential oil were stronger than that of M chemotype essential oil, especially to restrain the growths of the epiphytes.And the same chemotype EO had different abilities to restraine the different microbial species.5.The essential oil constituents of the fresh aerial parts of two lines were analyzed by GC/MS.The results of variance analysis showed that the essential oil of the two new lines w01-100 and w01-94 did not change significantly during daytime.The contents of the main components of the essential oil of w01-94 were more stable.Lines w01-100 and w01-94 can be harvested during the whole daytime.There were significant correlations between the content changes of some main components of the essential oil of the fresh aerial parts of Houttuynia,but the correlations between the two same components might be different in different materials.6.The flavone components in the residuary water solution after essential oil distillation were analyzed by using UV-VIS spectrum and TLC.The results of displaying colors identifications and UV-VIS spectrum showed that there were abundance flavone components in the residuary water solution,mainly included flavone,flavonol,isoflavone, dihydroflavone and dihydroflavonol.The results of TLC showed that most of these flavone components in the residuary water solution were glycoside components,containing rutin and hyperin.The average content of total flavone of 14 Houttuynia accessions was 1.93 mg/g, the content of w01-16 was the highest,reaching to 3.09 mg/g.The difference of the content of total flavone among the 14 Houttuynia accessions was distinct.There were insignificant correlationships between the types of total flavone components,contents of total flavone and the chromosome numbers of Houttuynia accessions.The recycled values of the residuary water solution were obvious.7.The difference of quercetin content of different Houttuynia accessions was determined by using HPLC on Hypersil ODS C18（250mm×4.0mm 5μm）at 30℃with acetonitrile:0.4% phosphoric acid（45:55）,pH=3 as the mobile phase.Detection wavelength was at 370nm. The flow rate was 1.0mL/min.The calibration curve of quercetin was 22.43ng～2.24ug（r= 0.999 7）.The quercetin content of the accession w01-120 was the highest,arriving at 4.02mg/g.8.The vitamin constituents and content of fresh young Houttuynia accessions were determined by using HPLC methods.The fat-soluble vitamins VA,δ-VE,VD₂,VD₃,α-VE and VK₁ were determined by Hyperclone BDS C₁₈column（150mm×4.6mm 5u）at 25℃with methanol:ethyl acetate（95:5）as the mobile phase at 275nm;the water-soluble vitamins VB₁, VB₂,VB₆,Nicotinic acid and V_C were determined by Luna C₁₈（2）100（?）column（200 mm×4.6mm 5u）at 30℃with 0.005mol/L hexane sulfonic sodium（pH 3.2）:methanol（70:30） as the mobile phase.The flow rate was at 1.0 mL/min.Detection wavelength was set at 254nm to determine the content of VB₁,VB₂,VB₆,Nicotinic acid and V_C of Hauttuynia.The mainly vitamin constituents wereα-VE and VC,less of VD₃、VK₁ and Nicotinic acid in Houttuynia,others 6 vitamins not detected in this research.The vitamin constituents and contents of the aerial parts were more abundant than that of underground parts in Houttuynia. The difference of the vitamin constituents among Houttuynia resource were little,but were notable in the content of their vitamins.The nutrition value of the aerial parts was higher than that of underground parts.9.The remains of As and Hg in the young aerial and underground parts of 10 Houttuynia accessions and the soil cultivated these Houttuynia accessions were determined simultaneously by using AFS.The results showed that the content of As was 11.557mg/kg, Hg was 0.387mg/kg in soil,the mean content of As was 0.161mg/kg and Hg was 0.004mg/kg in the young aerial parts.And in the underground parts,the mean content of As was 0.130mg/kg and Hg was 0.0025mg/kg.The diference of the remains As and Hg among the Houttuynia accessions was insignificant.According to GB4810-94 and GB2762-94,the remains of heavy metals As and Hg were not beyond the standard.10.The remains of 666 and DDT in the young aerial and underground parts of 10 Houttuynia accessions and the soil were determined simultaneously by using GC.The results showed that the remains ofδ-666 in soil was 0.17ng/kg,but the trace remains of the young aerial and underground parts in several accessions were detected.According to GB/T14551-2003,the remains of pesticides 666 and DDT were not beyond the standard.To sum up,the differences of the self-metablism chemical components,such as the essential oils,flavones and vitamins were significant among the Houttuynia germplasm resources.And the variations on the rudimental contents of some heavy metals and pesticides were insignificant.The chemotyoes of EOs of fresh aerial parts of Houttuynia were closely related to the diversity of the chromosome numbers,while other chemical components were not related to the diversity of the chromosome numbers,obviously.No significant chemical component difference was detected between the H.cordata and H.emeiensis accessions.The different ehemotypes of EOs could cause the different effects on restraining the microbial species;and it was important to the drugs production and utlization,which used EOs of Houttuynia as the raw materials.更多还原