摘要
生漆(Oriental Lacquer)是人类所知使用最早的优良天然涂料,素有“涂料之王”之誉称,是现代工业、农业、国防、科技、民用的重要原料,在我国应用已有6900多年的历史。生漆涂刷的器物入土千年不朽,它所显示的超耐久性是近代合成涂料无法比拟的。此外,漆花含有丰富的蜜液,是重要的蜜源植物;“漆籽”加工的漆油可供食用和作油漆原料;漆树的叶、花、根、皮、果实、干漆和木心均可入药。而干漆为中国药典所收载,其性辛,温;有毒。归干、脾经,具有破淤血,消积,杀虫功效。用于妇女闭经,瘀血症瘕,虫积腹痛等病症。
漆树(Toxicodendron vernicifluum(stokes)F.A.Barkley)是生漆的原植物,属漆树科(Anacardiaceae)漆树属(Toxicodendron),原产中国,在我国的大部分省(区)均有分布,集中分布在陕西、湖北、四川、重庆、甘肃、贵州和云南等七个省(市),其产漆量也最大。
目前对生漆漆酚类化合物的研究主要集中在其含量和结构的测定及防腐蚀性能等方面,对不同产地及品种的生漆漆酚组成和含量缺乏系统分析和综合评价。不同品种、不同产地、高山大木与低山小木的生漆在外观、质量及使用性能等方面均存在显著差异,而对此目前缺乏快速有效的质量检控方法。在生物学方面尚存在诸如不同漆树类群与品种如何划分,漆树各品种之间的亲缘关系如何等需要进一步研究的问题。
本课题以高效液相色谱(HPLC)和高效液相色谱-电喷雾/离子阱多级质谱法(HPLC-ESI-MS~n)为主要手段系统分析测定了陕西生漆中漆酚类化合物,确定了其主要化学组成,建立了生漆漆酚类化合物HPLC分析方法;在此基础上,比较分析测定了陕西13个漆树不同类群和品种所产生漆及树皮提取物的主要化学成分,建立了生漆和漆树树皮提取物HPLC指纹图谱;应用植物解剖学方法比较研究了以上13个不同漆树样本的树皮结构。通过上述几个方面的研究,以期改进目前生漆质量检测方法,为鉴别不同漆树类群和品种的生漆质量以及区分不同漆树类群和品种提供化学指标。通过以上系统研究,取得主要成果如下:
1.应用高效液相色谱-电喷雾/离子阱多级质谱(HPLC—ESI-MS~n)分析测定生漆中漆酚类化合物。仪器采用Agilent公司1100系列高效液相色谱-电喷雾-离子阱质谱,Agilent ZORBAXC-18柱(150×4.6mm,5μm),流速:0.8ml/min,流动相:甲醇与10mmol/L醋酸胺水溶液体积比82∶18;柱温:35℃;紫外检测器检测波长:225nm,进样量:10μl;ESI电离源,负离子模式,雾化气压力:35.0psi,干燥气流速:7.0L/min,干燥气温度:350℃,扫描质荷比范围:50—1200m/z;根据总离子流图确定了9种漆酚类化合物。该方法不仅可有效、快速分离鉴定生漆中漆酚类化合物,而且能提供化合物结构信息。
2.应用高效液相色谱与电喷雾/离子阱多级质谱联用技术,对制取干漆的生漆中漆酚类化合物进行了分析。仪器采用Finnigan Surveyor-LCQ Advantage MAX(LC/MS),Hypersil ODS2色谱柱(5μm,2.0×150mm),流速:0.2ml/min,流动相:甲醇∶水=88∶12(v/v),室温,紫外检测器检测波长:225nm,进样量:10μL;ESI电离源,负离子模式,干燥气流速:5.0L/min,毛细管温度:350℃,毛细管电压:-47V,电源电压:4.5kV,源电流:80.00uA,扫描质荷比范围:m/z 50—1200;液相色谱从中共分离出11组分,经离子阱质谱分析确定了9种漆酚类化合物,该方法可以快速、准确分析鉴别制取干漆的生漆中漆酚类化合物。
3.建立了生漆和漆树树皮提取物的HPLC指纹图谱。HPLC法分析条件为:DIKMA C_(18)色谱柱(5μm,4.6mm×250mm),流速:0.8ml/min,紫外检测波长为225nm,柱温:35℃,流动相:甲醇:水(v/v)=83:17,分析时间130min,进样量:10μl。经对陕西13个不同漆树的生漆样本和树皮提取物样本进行分析测定,结果显示,生漆和树皮石油醚提取物所建立的指纹图谱分别标定了7个共有峰,树皮甲醇提取物建立的指纹图谱标定了5个共有峰。通过方法学验证,精密度、稳定性、重现性试验RSD均小于5%;采用中药色谱指纹图谱相似度评价系统研究版(2004A)对所得指纹图谱进行相似度分析,其生漆指纹图谱的相似度均大于0.990,表明13个不同漆树样本的生漆质量基本相同;树皮甲醇提取物指纹图谱平均相似度为0.974,表明13个漆树不同样本的树皮提取物组成份基本相同,属同一物种;而树皮石油醚提取物指纹图谱的相似度值较低,平均值为0.799,表明13个不同漆树的树皮石油醚提取物化学成分之间存在差异。
4.漆树茎干的树皮是采割生漆的主要部位。应用常规石蜡切片法,番红-固绿双层染色观察了13个不同漆树样本的树皮结构,结果显示其基本结构相同,均由次生韧皮部、周皮及其外的死树皮组成。次生韧皮部由轴向系统和径向系统构成,轴向系统由筛管、伴胞、韧皮部薄壁组织细胞、石细胞和乳汁道组成;径向系统即为射线。经对石蜡切片观察统计分析,13个不同漆树样本的树皮中乳汁道腔的直径及单位面积内乳汁道的数量和面积等存在一定差异。
5.采用Statistica统计软件依据13个不同漆树样本的生漆漆酚含量、树皮提取物含量以及单位面积中乳汁道数量和面积等指标进行了聚类分析,结果显示:通过生漆漆酚含量和树皮提取物含量分析的样本间亲缘关系结果较相似,解剖学指标分析的样本间亲缘关系结果与前两者的结果存在一定差异。通过以上三方面分析均得出亲缘关系较近的样本有大红袍和金州红,陇县大木和太白大木。生漆样品和树皮甲醇溶提取物色谱各峰含量分析中共同得出亲缘关系较近的样本有,大红袍和金州红,黄茸高八尺、火焰子和茄棵头,陇县大木、南郑大木和太白大木。
Oriental lacquer which is wellknown used as the earliest excellent natural coatings, called as the king of paint,is one of the most important raw materials in mordern industries,angriculture,national defence,technology and civil use,having been used for more than 6900 years.The relic painted by oriental lacquer can keep their state for thousands of years,and it is shown that the durabilities of the synthesis paint can not reach those of oriental lacquer.Besides Oriental Lacquer,the flowers of lacquer tree not only contain abundant sweet liquid but also are important nectar source plant.The oil which is squeezed by fructification,can be edible and used as material of paint.The leaves,flowers, roots,skin,fruits,resina toxicodendri and heartwood of lacquer tree can all be used as medicine.Resina toxicodendri registered in the pharmacopoeia of the nation,has the character istics of symplectic.warm.poisonous.meridian,and breaking congestion. eliminauion integrable.disinsection,used for the treatment of amenorrhea.congestion. bollyache of integrable of insect.
Toxicodendron vernicifluum(Stokes)F.A.Barkley originated in China and distributed in most provinces or region of China,such as Shaanxi,Hubei,Sichuan,Chongqing,Gansu, Guizhou and Yunnan,The maximal yield of Oriental Lacquer distributed in the seven provinces above.
At present,although many studies on constituents in extracts of oriental Lacquer focus on the mensuration of their content and structure and the research of their antiseptic performances,little attention has been paid to system analysis and comprehensive estimate on the component and content of oriental Lacquer from different origins and species. Moreover,oriental Lacquer from different origins,varieties,altitude is greatly different in the aspects of appearance,quality,performance,being lack of some efficient ways of quality control of them.There is some problem further to research into biology,such as to differentiate varieties,confirm on relative of in Lacquer tree.
Based on high performance liquid chromatography(HPLC)and high perfor- mance liquid chromatogrammphy-electrospray ionization trap mass spectrometry(HPLC--ESI-MS~n),constituents and main chemical compound have been analysed in Oriental Lacquer,and besides,way that authenticated the constituents of urushiol have been detected by HPLC.On this basis,the main chemical components of urushi and lacquer tree bark extract were analysed from different regions of Shaanxi and 13 varieties,the established extracts HPLC fingerprint of Oriental Lacquer and lacquer tree bark. Application of plant anatomy methods comparative have researched on different lacquer tree bark structure above 13 samples.Through the several aspects of the study,with a view to improving the quality of Oriental Lacquer current detection method to identify different regions of the Oriental Lacquer quality and lacquer tree varieties provide different chemical indicators.The main results acquired through these systematic studies as follows:
1.Application of high performance liquid chromatography(HPLC)and high performance liquid chromatogrammphy-electrospray ionization trap mass spectrometry(HPLC-ESI-MS~n) analyse and determine the urushiol compounds in the Oriental Lacquer.Instrument model of HPLC-ESI-MS~n is 1100 series of Agilent Co.Ltd..The characters were as follows: Agilent ZORBAX C-18 columns(150×4.6 mm,5μn),flow rate:0.8 ml/min;mobile phase: the volume ratio between methanol and ammonmium acetate of aqueos solution:82:18, column temprature:35℃;uv detection at 225 nm;volume of injecting sample,10μL;ESI ionization source,negative ion mode,the press of evaporative gas:35.0 psi,the flow rate and temprature of drying gas:7.0 L/min,350℃;the range of scanning the electronic charge-mass ratio:50~1200m/z.According to the ion flow diagram of HPLC-ESI-MS~n identified nine kinds of urushiol compounds.This method not only can be separate and identify Oriental Lacquer urushiol compounds as effective and rapid but also provide information of compounds structural.
2.Application of HPLC and HPLC-ESI-MS~n connected technology,urushi constituents which processed resina toxicodendri is analysed.Finnigan Surveyor-LCQ Advantage MAX (LC/MS)has been used.The characters were as follows:Hypersil ODS2 chromatography column(5μm,2.0×150 mm),the flow rate was 0.2 ml/min,detecting wavelength was at 225 nm and the mobile phase was that methanol compared with water is equal to 88:12(v/v),the injection volume was 10μl;ESI ionization source,negative ion mode,the press of evaporative gas:35.0 psi,the flow rate of drying gas:5.0 L/min,capillary temprture:350℃,capillary voltage:-47 V;power voltage:4.5 KV;source current:80.00μA,the range of scanning the electronic charge-mass ratio:50~1200 m/z.According to separating 11 components by liquid chromatography and identifying nine kinds of urushiol constituents by ionization trap mass spectrometry,urushiol constituents of oriental Lacquer which processed resina toxicodendri can be efficiently and fast separated in this way.
3.The HPLC fingerprints of Oriental Lacquer and the extraction of lacquer tree bark were established.The main components of them were determined by HPLC-ESI-MS~n.Analysis by HPLC conditions were as follows:DIKMA C_(18)column(5μm,4.6 mm×250 mm)was used,the flow rate was 0.8 ml/min,detecting wavelength was at 225 nm and column temperature was at 35℃,Mobile phase:methanol and water ratio of 83:17(v/v),time for analysis was 130 min,the injection volume was 10 ul.The 13 raw lacquer samples and lacquer tree bark extract samples of Shaanxi Province were analyzed by HPLC.The fingerprint constructed by Oriental Lacquer and the chemical constituents from the petroleum ether extract of lacquer tree bark showed seven common peaks,and that of the chemical constituents from the methanol extract of lacquer tree bark showed five ones. Validating with methodology,RSD of precision,stability,reappearance were all less than 3%.Through similarity evaluation software(2004A)published by committee of codex, analyzing similarity of the fingerprints,whose similarity was all more than 0.990,it is revealed that the quality of 13 kinds of sample of lacquer was roughly the same,the average similarity of the chemical constituents from the methanol extract of lacquer tree bark was 0.974,it is revealed that the quality of 13 kinds of sample of the extraction of lacquer tree bark was roughly the same,belong to the same species,and that of the chemical constituents from the methanol extract of lacquer tree bark was low,whose average was 0.799,we can acquire that the chemical constituent of 13 kinds of the petroleum ether extract of lacquer tree bark present some diversity.
4.Lacquer tree bark from the stem which is made up of secondary phloem,periderm and dead bark around it is main part cutting urushi..The secondary phloem consists of the axial system and the radial system,the axial system consists of sieve vessels,companion cells, parenchyma cells,stone cells and laticiferous canals;the radial system is ray.The bark structure of 13 lacquer tree samples which using safranine and fast green double dyeing were observed by the paraffin section method.The results show that the similar in structure. According to the statistical analysis the amount and area of laticiferous canals in per unit area and diameter of laticiferous canal existed certain difference in 13 kinds of samples of lacqer tree bark.
5.Through the cluster analysis for indexes about urushiol content in Oriental lacquer of 13 kinds of samples of lacqer tree,that of the extraction of lacquer tree bark,amount and area laticiferous canals in per Unit Area by Statistica which is statistical software,the result show:the relationship between the first two samples analysed in chemical index was roughly the same,while that of analysed in anatomy existed certain difference.According to the result analysed from three aspects above,the samples whose relationship were close were:Da Hongpao and Jin Zhouhong,Longxian Da Mu and Taibai Da Mu,and those analysed from contents of chromatographic peaks of Oriental Lacquer sample and the chemical constituents from the methanol extract of lacquer tree bark were:Da Hongpao and Jin Zhouhong;Huang Ronggaobachi,Huo Yanzi and Qie Ketou;Longxian Da Mu, Nanzheng Da Mu and Tai Bai Da Mu.
引文
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