薄荷和留兰香资源染色体数目及化学成分研究
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摘要
薄荷属(Mentha L.)植物为多年生或稀为一年生芳香草本,存在大量野生资源。常见的栽培种有薄荷(Mentha haplocalyx Briq.)和留兰香(Mentha spicata L.)等,二者均为用途广泛的中药材,薄荷具疏散风热,清利头目,利咽和透疹等功效;留兰香则止血、消炎、镇痛效果明显。同时,它们也是经济价值较高的蔬菜和香料植物。
挥发油为薄荷属植物主要化学成分和药效成分,维生素和无机元素种类及含量对中药材的药理和药效有重要影响,通常情况下植物染色体数目与化学成分间存在一定关系。本文分别对21份不同来源的薄荷和留兰香植物染色体数目、挥发油主要化学成分及化学型进行了分析,对其不同部位维生素和无机元素种类和含量差异进行了分析,比较了综合农艺性状较好的15份薄荷和留兰香材料中上部叶片维生素和无机元素种类及含量差别,探讨了薄荷和留兰香资源挥发油化学型,以及维生素、无机元素与薄荷和留兰香资源染色体数目间的关系。主要研究结果如下:
1.对收集的21份薄荷和留兰香资源进行染色体计数。结果表明,所采集的18份薄荷材料存在八倍体(2n=8x=96)和六倍体(2n=6x=72),3份留兰香材料则均为四倍体(2n=4x=48),说明薄荷属植物存在丰富的多倍体现象。试验结果还表明,薄荷和留兰香资源染色体数目无明显地理分布特征。
2.采用气相—质谱联用法对21份薄荷和留兰香材料地上部分挥发油进行鉴定。结果发现各材料挥发油成分组成和含量存在较大差异。在挥发油中共鉴定出41种化合物,平均相对百分含量最大的10种成分为薄荷醇、薄荷酮、胡薄荷酮、氧化胡椒烯酮、香芹酮、芳樟醇、氧化胡椒酮、胡椒酮、乙酸薄荷酯和石竹烯。
3.根据挥发油主要化学成分相对百分含量,对21份薄荷和留兰香材料进行系统聚类。结果所有材料可聚为8类,即存在8种不同化学型,包括芳樟醇型、薄荷酮-薄荷醇型、薄荷酮-薄荷醇-胡薄荷酮型、胡薄荷酮型、胡薄荷酮-氧化胡椒酮型、薄荷醇型、香芹酮型和氧化胡椒烯酮型。所有留兰香材料均为香芹酮型,而薄荷材料则分别为其他7种化学型,且薄荷材料的化学型同茎色间存在一定关系。
4.采用HPLC法对4份薄荷材料不同部位维生素种类及含量进行测定,水溶性维生素用Luna C18(2)100A 200 mm×4.6mm×5u,流动相0.005mol/L己烷磺酸钠(用稀磷酸调pH=3.2):甲醇=7:3,流速1.0ml/min,柱温30℃,紫外检测波长254nm;脂溶性维生素用Hyperclone BDS C18 150mm×4.6mm×5u,流动相甲醇:乙酸乙酯=95:5,流速1.0ml/min,柱温25℃,紫外检测波长275nm。结果发现,叶片中水溶性维生素的含量均比脂溶性维生素含量高,且除个别材料外,随着叶片的衰老,其维生素含量逐渐降低。总体以嫩叶中含量普遍较高,茎中各种维生素含量均最低。
5.采用上述HPLC法对15份综合农艺性状表现良好的薄荷和留兰香材料中上部叶片维生素含量进行测定。结果仅检测到V_C、V_(D2)和V_(K1),它们均具有良好的线性关系。V_C、V_(D2)和V_(K1)平均空白回收率分别为99.85%、81.61%和90.27%。所有薄荷材料中,来源四川简阳薄荷的V_C含量最高,达118.41mg╱100g,而V_(D2)和V_(K1)在来源于重庆的两份薄荷材料中,含量相对较高。留兰香中V_C含量普遍低于薄荷中V_C含量,多数紫茎薄荷比青茎薄荷V_C含量高。V_(D2)和V_(K1)在薄荷和留兰香间,以及紫茎和青茎薄荷间差异不明显。
6.采用原子吸收分光光度法和原子荧光光谱法测定薄荷和留兰香资源无机元素含量。结果发现,薄荷中上部叶有益元素含量较高,有害元素含量较低。薄荷和留兰香中Ca和Cu含量均高于土壤中含量,其对Ca和Cu有富集作用。Ca、Mn、Cu、Cr、Pb、As含量在供试材料中存在极显著差异,但各元素含量间相关不显著。薄荷和留兰香资源无机元素含量变化趋势相同。其中,薄荷资源各无机元素含量普遍高于留兰香资源无机元素含量,而青茎和紫茎薄荷无机元素含量差异不显著,野生和栽培薄荷资源无机元素含量间也无显著差异。
7.相关分析结果表明,薄荷中共有挥发油成分与维生素和无机元素含量相关关系显著。Ca含量与柠檬烯含量呈显著正相关,Cu、Cr含量与胡薄荷酮含量呈显著负相关。Zn含量与石竹烯含量呈显著正相关,As含量与石竹烯含量呈极显著负相关,V_C与Hg含量呈显著正相关,V_(K1)与Ca含量呈显著负相关。偏相关分析结果表明,除薄荷醇外,无机元素对其它各挥发油共有成分影响显著。采用逐步多元回归分析发现,无机元素与除薄荷醇外的其余挥发油共有成分线性关系良好。
Genus Mentha L.accessions are perennial or far-between annual herbage,many of them distributed wildly.Mentha haplocalyx Briq.and Mentha spicata L.are two common spieces cultivated in genus Mentha.Both of them are traditional Chinese medicine.M. haplocalyx has the function of diaphoretic,febrifuge,salubrious,while M.spicata has the function of astrictive,antiphlogistic and sedative.Meanwhile,they are also used as delicious vegetable and flavoring.
The essential oils are the main chemical components and effective components of genus Mentha resources.The sorts and contents of vitamins and inorganic elements are important to Chinese traditional medicine.Usually,there are some relationships between the chromsome numbers and chemical components.This study analyzed the chromsome number,the components and chemotype of 21 M.haplocalyx and M.spicata accessions which collected from different areas,detected the difference between the sorts and contents of vitamins and inorganic elements of the different parts of them,determined the sorts and contents of vitamins and inorganic elements of 15 accessions had better agronomy traits. The study dicussed the types of chemotype,and the relationship between the contents of vitamins,inorganic elements and the chromsome number of M.haplocalyxand M.spicata accessions.The main results were as follows:
1.The chromsome numbers of M.haplocalyx and M.spicata were counted.The results showed that there were octploid(2n=8x=96) and hexaploid(2n=8x=96) among M. haplocalyx accessions,and all the M.spicata accessions were teraploid(2n=4x=48).The polyploid was widely existed among genus Mentha resources.And the study also showed that there was no obvious geographic distributing characteristic among the chromsome numbers of M.haplocalyx and M.spicata accession.
2.The essential oil compositions of 21 M.haplocalyx and M.spicata accessions were analyzed by using GC/MS method.It was found that the constinuents and contents of volatile oils in different Mentha resources were various.Totally,forty-one chemical components were identified from the volatile oils of 21 Mentha accessions.The average contents of ten chemical components were higher,including menthol,menthone,pulegone, peritenone oxide,carvone,linalool,piperitone oxide,piperitone,menthyl acetate and caryophylle.
3.Based on the percentage of the chemical components of the essential oils of M. haplocalyx and M.spicata accessions,twenty-one accessions we analyzed by using cluster analysis.The results showed that all the accessions were classified into 8 groups,namely 8 chemotypes,including linalool,menthone-menthol,menthone-menthol-pulegone, pelugone,pelugone- piperitone oxide,menthol,carvone and piperitenone xide.All the M. spicata accessions were chemotype carvone,and M.haplocalyx accession were divided into the other 7 chemotypes.The chemotypes of M.haplocalyx accessions had some relationship with with the color of stems.
4.The vitamin constituents and content of different parts of four M.haplocalyx accessions were determined by using HPLC method.Water-soluble vitamins was determined by HPLC on Luna C18(2) 100(?) 200 mm×4.6mm×5u at 30℃with 0.005 mol/L:Hexane sodium:methanol(7:3),pH = 3.2 as the mobile phase.Detection wavelength was 370nm. The flow rate was 1.0ml/ min.Fat-soluble vitamins was determined by HPLC on Hyperclone BDS C18 150mm×4.6mm×5u at 25℃with methanol:Ethyl acetate(95:5). Detection wavelength was 275nm.The flow rate was 1.0 ml/min.It was found that the contents of water-soluble vitamins were higher than that of fat-soluble vitamins.Except for minority accessions,the contents of vitamins decreased gradually.Totally,the vitamin contents in younger leaves were commonly higher,while that in the stems were the lowest.
5.The contents of vitamins of 15 M.haplocalyx and M.spicata accessions which had better agronomic traits ware determined by using the previous methods.Only V_c,V_(D2) and V_(K1) were determined.The calibration curve of each vitamine was good.The average recovery of V_c,V_(D2) and V_(K1) was 99.85%,81.61%and 90.27%.The V_c content of M. haplocalyx from Jianyang in Sichuan province was the highest,and the V_(D2) and V_(K1) contents of two M.haplocalyx accessions from Chongqing were relatively high.The content of V_c in M.spicata was commonly less than that in M.haplocalyx.The content of V_c of most purple-stem M.haplocalyx accessions were higher than that in green- stem M. haplocalyx accessions.There were no significant difference between the the contents of V_(D2) and V_(K1) of M.haplocalyx and M.spicata,green- and purple-stem M.haplocalyx.
6.The sorts and contents of inorganic elements of Mentha haplocalyx Briq.and Mentha spicata L.resources,were analyzed by using AAS and AFS methods.The result showed the contents of the beneficial elements the middle and upper leaves of M.haplocalyx were relatively higher while the contents of the deleterious elements were lower.The result also showed the contents of Ca and Cu of M.haplocalyx and M.spicata resources were higher than that in the soil.The tested materials had the function of mineral elements dressing for Ca and Cu.The contents of Ca,Mn,Cu,Cr,Pb and As among all the accessions were significantly different.But there was no significant correlationship between the contents of different inorganic elements.The varying tendency of the contents of inorganic elements within M.haplocalyx and M.spicata resources was similar.Of them,the inorganic element contents in M.haplocalyx accessions was commonly higher than that in M.spicata.The contents of inorganic elements between green- and purple-stem M.haplocalyx accessions were insignificant.And there was also no significant difference between the wild and cultivated M.haplocalyx accessions.
7.The results of correlation analysis showed that there were significant relationships among the contents of the mutual essential oils components,vitamins and inorganic elements.The content of Ca had significant positive correlation with linmene,and Cu,Cr had significant negative correlation with pulegone.The content of Zn had significant positive correlation with caryophylle.The content of As had fearfully significant negative correlation with caryophylle.It was found that the contents of inorganic elements had effect on the content of each mutual volatile oil component except for menthol by using partial correlation analysis.Stepwise regression analysis also showed that there were well linear connection between inorganic element and each mutual volatile oil component except for menthol.
挥发油为薄荷属植物主要化学成分和药效成分,维生素和无机元素种类及含量对中药材的药理和药效有重要影响,通常情况下植物染色体数目与化学成分间存在一定关系。本文分别对21份不同来源的薄荷和留兰香植物染色体数目、挥发油主要化学成分及化学型进行了分析,对其不同部位维生素和无机元素种类和含量差异进行了分析,比较了综合农艺性状较好的15份薄荷和留兰香材料中上部叶片维生素和无机元素种类及含量差别,探讨了薄荷和留兰香资源挥发油化学型,以及维生素、无机元素与薄荷和留兰香资源染色体数目间的关系。主要研究结果如下:
1.对收集的21份薄荷和留兰香资源进行染色体计数。结果表明,所采集的18份薄荷材料存在八倍体(2n=8x=96)和六倍体(2n=6x=72),3份留兰香材料则均为四倍体(2n=4x=48),说明薄荷属植物存在丰富的多倍体现象。试验结果还表明,薄荷和留兰香资源染色体数目无明显地理分布特征。
2.采用气相—质谱联用法对21份薄荷和留兰香材料地上部分挥发油进行鉴定。结果发现各材料挥发油成分组成和含量存在较大差异。在挥发油中共鉴定出41种化合物,平均相对百分含量最大的10种成分为薄荷醇、薄荷酮、胡薄荷酮、氧化胡椒烯酮、香芹酮、芳樟醇、氧化胡椒酮、胡椒酮、乙酸薄荷酯和石竹烯。
3.根据挥发油主要化学成分相对百分含量,对21份薄荷和留兰香材料进行系统聚类。结果所有材料可聚为8类,即存在8种不同化学型,包括芳樟醇型、薄荷酮-薄荷醇型、薄荷酮-薄荷醇-胡薄荷酮型、胡薄荷酮型、胡薄荷酮-氧化胡椒酮型、薄荷醇型、香芹酮型和氧化胡椒烯酮型。所有留兰香材料均为香芹酮型,而薄荷材料则分别为其他7种化学型,且薄荷材料的化学型同茎色间存在一定关系。
4.采用HPLC法对4份薄荷材料不同部位维生素种类及含量进行测定,水溶性维生素用Luna C18(2)100A 200 mm×4.6mm×5u,流动相0.005mol/L己烷磺酸钠(用稀磷酸调pH=3.2):甲醇=7:3,流速1.0ml/min,柱温30℃,紫外检测波长254nm;脂溶性维生素用Hyperclone BDS C18 150mm×4.6mm×5u,流动相甲醇:乙酸乙酯=95:5,流速1.0ml/min,柱温25℃,紫外检测波长275nm。结果发现,叶片中水溶性维生素的含量均比脂溶性维生素含量高,且除个别材料外,随着叶片的衰老,其维生素含量逐渐降低。总体以嫩叶中含量普遍较高,茎中各种维生素含量均最低。
5.采用上述HPLC法对15份综合农艺性状表现良好的薄荷和留兰香材料中上部叶片维生素含量进行测定。结果仅检测到V_C、V_(D2)和V_(K1),它们均具有良好的线性关系。V_C、V_(D2)和V_(K1)平均空白回收率分别为99.85%、81.61%和90.27%。所有薄荷材料中,来源四川简阳薄荷的V_C含量最高,达118.41mg╱100g,而V_(D2)和V_(K1)在来源于重庆的两份薄荷材料中,含量相对较高。留兰香中V_C含量普遍低于薄荷中V_C含量,多数紫茎薄荷比青茎薄荷V_C含量高。V_(D2)和V_(K1)在薄荷和留兰香间,以及紫茎和青茎薄荷间差异不明显。
6.采用原子吸收分光光度法和原子荧光光谱法测定薄荷和留兰香资源无机元素含量。结果发现,薄荷中上部叶有益元素含量较高,有害元素含量较低。薄荷和留兰香中Ca和Cu含量均高于土壤中含量,其对Ca和Cu有富集作用。Ca、Mn、Cu、Cr、Pb、As含量在供试材料中存在极显著差异,但各元素含量间相关不显著。薄荷和留兰香资源无机元素含量变化趋势相同。其中,薄荷资源各无机元素含量普遍高于留兰香资源无机元素含量,而青茎和紫茎薄荷无机元素含量差异不显著,野生和栽培薄荷资源无机元素含量间也无显著差异。
7.相关分析结果表明,薄荷中共有挥发油成分与维生素和无机元素含量相关关系显著。Ca含量与柠檬烯含量呈显著正相关,Cu、Cr含量与胡薄荷酮含量呈显著负相关。Zn含量与石竹烯含量呈显著正相关,As含量与石竹烯含量呈极显著负相关,V_C与Hg含量呈显著正相关,V_(K1)与Ca含量呈显著负相关。偏相关分析结果表明,除薄荷醇外,无机元素对其它各挥发油共有成分影响显著。采用逐步多元回归分析发现,无机元素与除薄荷醇外的其余挥发油共有成分线性关系良好。
Genus Mentha L.accessions are perennial or far-between annual herbage,many of them distributed wildly.Mentha haplocalyx Briq.and Mentha spicata L.are two common spieces cultivated in genus Mentha.Both of them are traditional Chinese medicine.M. haplocalyx has the function of diaphoretic,febrifuge,salubrious,while M.spicata has the function of astrictive,antiphlogistic and sedative.Meanwhile,they are also used as delicious vegetable and flavoring.
The essential oils are the main chemical components and effective components of genus Mentha resources.The sorts and contents of vitamins and inorganic elements are important to Chinese traditional medicine.Usually,there are some relationships between the chromsome numbers and chemical components.This study analyzed the chromsome number,the components and chemotype of 21 M.haplocalyx and M.spicata accessions which collected from different areas,detected the difference between the sorts and contents of vitamins and inorganic elements of the different parts of them,determined the sorts and contents of vitamins and inorganic elements of 15 accessions had better agronomy traits. The study dicussed the types of chemotype,and the relationship between the contents of vitamins,inorganic elements and the chromsome number of M.haplocalyxand M.spicata accessions.The main results were as follows:
1.The chromsome numbers of M.haplocalyx and M.spicata were counted.The results showed that there were octploid(2n=8x=96) and hexaploid(2n=8x=96) among M. haplocalyx accessions,and all the M.spicata accessions were teraploid(2n=4x=48).The polyploid was widely existed among genus Mentha resources.And the study also showed that there was no obvious geographic distributing characteristic among the chromsome numbers of M.haplocalyx and M.spicata accession.
2.The essential oil compositions of 21 M.haplocalyx and M.spicata accessions were analyzed by using GC/MS method.It was found that the constinuents and contents of volatile oils in different Mentha resources were various.Totally,forty-one chemical components were identified from the volatile oils of 21 Mentha accessions.The average contents of ten chemical components were higher,including menthol,menthone,pulegone, peritenone oxide,carvone,linalool,piperitone oxide,piperitone,menthyl acetate and caryophylle.
3.Based on the percentage of the chemical components of the essential oils of M. haplocalyx and M.spicata accessions,twenty-one accessions we analyzed by using cluster analysis.The results showed that all the accessions were classified into 8 groups,namely 8 chemotypes,including linalool,menthone-menthol,menthone-menthol-pulegone, pelugone,pelugone- piperitone oxide,menthol,carvone and piperitenone xide.All the M. spicata accessions were chemotype carvone,and M.haplocalyx accession were divided into the other 7 chemotypes.The chemotypes of M.haplocalyx accessions had some relationship with with the color of stems.
4.The vitamin constituents and content of different parts of four M.haplocalyx accessions were determined by using HPLC method.Water-soluble vitamins was determined by HPLC on Luna C18(2) 100(?) 200 mm×4.6mm×5u at 30℃with 0.005 mol/L:Hexane sodium:methanol(7:3),pH = 3.2 as the mobile phase.Detection wavelength was 370nm. The flow rate was 1.0ml/ min.Fat-soluble vitamins was determined by HPLC on Hyperclone BDS C18 150mm×4.6mm×5u at 25℃with methanol:Ethyl acetate(95:5). Detection wavelength was 275nm.The flow rate was 1.0 ml/min.It was found that the contents of water-soluble vitamins were higher than that of fat-soluble vitamins.Except for minority accessions,the contents of vitamins decreased gradually.Totally,the vitamin contents in younger leaves were commonly higher,while that in the stems were the lowest.
5.The contents of vitamins of 15 M.haplocalyx and M.spicata accessions which had better agronomic traits ware determined by using the previous methods.Only V_c,V_(D2) and V_(K1) were determined.The calibration curve of each vitamine was good.The average recovery of V_c,V_(D2) and V_(K1) was 99.85%,81.61%and 90.27%.The V_c content of M. haplocalyx from Jianyang in Sichuan province was the highest,and the V_(D2) and V_(K1) contents of two M.haplocalyx accessions from Chongqing were relatively high.The content of V_c in M.spicata was commonly less than that in M.haplocalyx.The content of V_c of most purple-stem M.haplocalyx accessions were higher than that in green- stem M. haplocalyx accessions.There were no significant difference between the the contents of V_(D2) and V_(K1) of M.haplocalyx and M.spicata,green- and purple-stem M.haplocalyx.
6.The sorts and contents of inorganic elements of Mentha haplocalyx Briq.and Mentha spicata L.resources,were analyzed by using AAS and AFS methods.The result showed the contents of the beneficial elements the middle and upper leaves of M.haplocalyx were relatively higher while the contents of the deleterious elements were lower.The result also showed the contents of Ca and Cu of M.haplocalyx and M.spicata resources were higher than that in the soil.The tested materials had the function of mineral elements dressing for Ca and Cu.The contents of Ca,Mn,Cu,Cr,Pb and As among all the accessions were significantly different.But there was no significant correlationship between the contents of different inorganic elements.The varying tendency of the contents of inorganic elements within M.haplocalyx and M.spicata resources was similar.Of them,the inorganic element contents in M.haplocalyx accessions was commonly higher than that in M.spicata.The contents of inorganic elements between green- and purple-stem M.haplocalyx accessions were insignificant.And there was also no significant difference between the wild and cultivated M.haplocalyx accessions.
7.The results of correlation analysis showed that there were significant relationships among the contents of the mutual essential oils components,vitamins and inorganic elements.The content of Ca had significant positive correlation with linmene,and Cu,Cr had significant negative correlation with pulegone.The content of Zn had significant positive correlation with caryophylle.The content of As had fearfully significant negative correlation with caryophylle.It was found that the contents of inorganic elements had effect on the content of each mutual volatile oil component except for menthol by using partial correlation analysis.Stepwise regression analysis also showed that there were well linear connection between inorganic element and each mutual volatile oil component except for menthol.
引文
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