温莪术规范化生产部分关键技术研究
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摘要
莪术为多基源品种,《中国药典》2005年版一部莪术项下规定莪术来源于姜科植物蓬莪术Curcuma phaeocaulis Val.、广西莪术C. Kwangsiensis S. G. Lee et C. F. Liang或温郁金C. wenyujin Y. H. Chen et C. Ling的干燥根茎,商品上分别习称为“文术”、“桂莪术”和“温莪术”。本课题属于浙江省科技厅重大科技项目“温郁金规范化种植与新药开发”课题的一部分。莪术品种来源复杂,主产区有个别品种变异；且栽培品种质退化、抗逆性降低,植株间良莠不齐,品质差异较大；加之缺乏对莪术种质资源收集、整理、保存、保护等相关方面的研究,野生资源己灭绝。根据调查和前期的研究工作,我们发现不同居群的莪术质量差异较大,尤其是桂莪术,质量不如温莪术、文术。因此,本课题就收集到的5省15地16份莪术种质建立了种质资源圃,对不同居群的莪术进行了原植物、药材性状、分子生物学、指纹图谱、化学成分等方面的比较研究,建立了莪术优良种质筛选的综合评价模式,筛选出了4份优良的莪术种质材料；在此基础上进行了温郁金优良株系的筛选研究,筛选出6个优质高产的温郁金株系；同时完成了温莪术GAP部分标准操作规程(适宜采收期、产地初加工以及包装、贮藏)的研究。
1莪术种质资源综合评价研究
1.1种质鉴定研究方面：对收集到的5省15地16份莪术种质进行了原植物、性状、薄层色谱、分子生物学以及指纹图谱研究。植物分类学鉴定结果表明：No. 1-No.9为温郁金,No.10-No.12为蓬莪术；对No.13-No.16做了初步判定,No.13为蓬莪术,No.14、No.15为温郁金,No.16待定。薄层鉴别结果表明：No.14、No.15与温莪术化学成分相似程度高；No.13、No.16与蓬莪术化学成分相似程度高。首次将聚丙烯酰胺凝胶电泳方法运用到莪术ISSR分子标记研究中,分析莪术种质的亲缘关系和遗传多样性,从30个引物中筛选了9个特异性好的引物,多态性百分率达到70%。分子生物学研究结果表明：种间的亲缘关系较远,种内的亲缘关系较近,不同种质的遗传信息丰富。通过UPGMA聚类,16份莪术种质聚为两类,No.1～No.9、No.14、No.15、No.16聚为一类,说明No.14、No.15、No.16与温郁金亲缘关系较近；No.10-No.13聚为另一类,说明No.13与蓬莪术亲缘关系较近；该聚类结果与传统的分类结果吻合。根据上述结果,确定福建收集的2份种质,No.13为蓬莪术,No.15为温郁金；广西收集的1份种质No.14为温郁金；云南收集的1份种质No.16由于分子标记与原植物、薄层等鉴别结果有出入,属于变种还是新种有待进一步分析研究。
最后采用指纹图谱考察莪术引种前后化学成分的相关性,发现引种至四川的温莪术(2年)与原产地的温莪术化学成分非产相近,相关性良好；引种至广西、福建的温莪术(多年)与原产地的温莪术化学成分有一定的差异,多了A峰,与薄层鉴别结果一致,初步推测可能与引种的年限或地域有关。引种至温州、福建的蓬莪术与原产地的蓬莪术化学成分相似,相关性良好。
1.2种质综合评价研究方面：首次对不同莪术种质的主根茎进行了经济价值评价、化学成分评价等方面的综合研究。通过对产量以及浸出物、挥发油、吉马酮、莪术二酮多指标的含量测定,运用组内分组的方差分析以及配对比较的统计学方法比较了两地种质资源圃以及同一种质资源圃不同年份种质的差异,并首次运用加权评分法分别对两地种质资源圃的种质进行综合评价研究,筛选出4份莪术优良种质材料。
2.温郁金优良株系筛选研究
首次采用系统选育法从大田筛选健壮、抗病抗逆性强的植株1000株,根据根茎鲜重对温郁金进行产量筛选,得到692株；通过有效成分的含量分析,并运用加权评分法对温郁金主根茎的产量以及化学成分评价中的有效成分含量进行评分,得到70株;再经过2年的跟踪筛选,最终得到产量、含量高且稳定性好的6个优良株系,分别是1-29、1-181、1-254、2-6、5-24、5-29。
3温莪术GAP部分标准操作规程研究
3.1采收期研究在前期的工作基础上继续定点、定期采样,研究温莪术在不同采收期的产量、生药折干率以及化学成分的含量,并结合历年的数据考察不同采收期对药材质量和产量的影响,确立最佳采收期为J月中旬至K月下旬。
3.2产地初加工研究对传统加工方法中的蒸煮条件、干燥方法、干燥温度等方面进行对比研究,筛选出既能使药材迅速干燥,又能保持外观饱满,同时保证有效成分影响较小的加工方法：蒸30min-40min,置于55℃烘箱内烘干或者晒干。
3.3包装、贮藏研究研究不同包装材料及贮藏年限对温莪术药材质量的影响,优选出最佳包装材料为编织袋,贮藏年限为两年。
上述研究结果为莪术种质资源的综合评价、温郁金良种选育繁育以及温莪术GAP规范化标准操作规程提供了科学的理论依据。
Rhizoma Curcumae is multi-source species,the rhizome of Curcuma phaeocaulis Val.,Curcuma Kwangsiensis S.GLee et C.F.Liang or Curcuma wenyujin Y.H.Chen et C.Ling, root in the family of Zingiberaceae. The goods are known as'Wen zhu','Gui Ezhu'and'Wen Ezhu'. The work of this thesis is part of the Project of Zhejiang Department of Science and Technology: Standardized cultivation of wenyujin and new drug's exploitation. Their complex origin, a variety of individual variation of the main producing areas and cultivars's quality degradation, reduced resistance lead to varies between plants and the quality of different. At equal pace,Rhizoma Curcumae's research lack of germplasm collection, sorting, preservation, protection and other related research, wild resources has been extincted.. According to the survey and pre-research work, we found that the different populations of Rhizoma Curcumae have the different quality, especially the Gui Ezhu, which is not as good as Wen Ezhu and Wen zhu. Therefore,we collected 16 germplasm form 5 provinces and established germplasm resources nursery. The thesis mainly compared the Rhizoma Curcumae from different populations in plant morphology, material character, molecular biology,finger print,.chemical composition,,established the comprehensive evaluation model of the excellent germplasm resources, gaining four excellent germplasm materials. On this basis of Wenyujin screening studies,selected four high-quality high-yielding strains.The thesis has also completed a part of GAP standard operating procedures, including harvesting, processing, packaging and storage.
Identification of germplasm:we studied the plant morphology, material character,molecular biology and finger print of the 16 germplasm form 5 provinces,.Identification of plant classification results show that No.1-No.9 is C. wenyujin, No.10-No.12 is C. phaeocaulis; No.13-No.16 have been made a preliminary determination, No.13, No.16 is C. phaeocaulis. No.14, No.15 is C. wenyujin. TLC Studies shown that:No.14,No.15 are similar in the chemical composition with Wen Ezhu to a high degree; No.13, No.16 are similar in the chemical composition with Peng Ezhu.Polyacrylamide gel electrophoresis method is used in ISSR molecular markers of Ezhu study for the first time, analysis Rhizoma Curcumae related germplasm and genetic diversity, screened 9-specific primers from the 30 primers the percentage of the polymorphism reached 70%. The molecular biology results show that: the genetic relationship between species more distant species within the close genetic relationship of different genetic information-rich germplasm. Through UPGMA clustering,16 germplasm are gathered to two types, No.1 -No.9, No.14, No.15, No.16 are assembled in a group,which means No.14, No.15, No.16 have the close genetic relationship with C. wenyujin; No.10-No.13 are assembled in another group, which means No.13 have the close genetic relationship with C. phaeocaulis;.The classification is similar with the traditional classification results. Based on the above results, we determined the two germplasm collection in Fujian, No.13 is C. phaeocaulis, No.15 is C. wenyujin,guanxi No.14 is C. wenyujin, as a result of molecular markers and histology not identical, yunnan No.16 is need to be further analyzed.
Finally, we use the fingerprints to see about the chemical constituents before and after the introduction of Ezhu and found that Wen Ezhu introduction to Sichuan (2 years) is similar to Wen Ezhu in original producing area in chemical composition and have a good correlation. Introduction to Guangxi, Fujian Wen Ezhu (many years) has some differences in chemical constituents with Wen Ezhu in original producing area, which is isidentical with the TLC results, the initial guess may be related to the length of the introduction and area. Introduction to Wenzhou, Fujian area identical with Peng Ezhu in original producing area in chemical composition, have a good correlation.
Comprehensive Evaluation of germplasm:Evaluation rhizome from different germplasm of Ezhu in the economy value, chemistry element and so on. determine the production,the content of volatile oil, germacrone, curdione, compared germplasm from two nursery and the same nursery in different years by statistical methods. Use weighted scoring method to evaluate the two germplasm nursery for the first time, and finally 4 were selected as germplasm materials.
Screening studies of excellen Strain:The system robust selection was first used to select 1000 plants with haleness and disease resistance from Daejeon,.we got 692 plants according to the fresh weight of roots C. wenyujin. we use the weighted score law to grade the main roots of C. Wenyujin production, as well as evaluate the chemical composition of the active ingredient content, get 70 plants;After 2 years of screening, high stability and has a better quality of six strains were 1-29,1-181,1-254,2-6,5-24,5-29.
GAP standard operating procedures:Work in the pre-positioned to build on the regular sampling to study the Ezhu's production at different growth, pharmacognostic exsiccation rate and chemical composition of dry content, combine with the data over the years to examine the quality and production with different harvest time, determined the suitable harvesting time of C. Wenyujin. Comparison of traditional processing methods of cooking condition,drying methods, drying temperature,we select the best Processing method to make sure quickly exsiccation, to maintain the appearance of full. and to ensure destruction of the active ingredients. Study on how the different packaging materials and storage of temperature affect the quality of drug, selected the best packaging materials and storage time.
The results provide the scientific theory basis for evaluation of germplasm resources, the elite selection and GAP standard operating procedures.
1莪术种质资源综合评价研究
1.1种质鉴定研究方面：对收集到的5省15地16份莪术种质进行了原植物、性状、薄层色谱、分子生物学以及指纹图谱研究。植物分类学鉴定结果表明：No. 1-No.9为温郁金,No.10-No.12为蓬莪术；对No.13-No.16做了初步判定,No.13为蓬莪术,No.14、No.15为温郁金,No.16待定。薄层鉴别结果表明：No.14、No.15与温莪术化学成分相似程度高；No.13、No.16与蓬莪术化学成分相似程度高。首次将聚丙烯酰胺凝胶电泳方法运用到莪术ISSR分子标记研究中,分析莪术种质的亲缘关系和遗传多样性,从30个引物中筛选了9个特异性好的引物,多态性百分率达到70%。分子生物学研究结果表明：种间的亲缘关系较远,种内的亲缘关系较近,不同种质的遗传信息丰富。通过UPGMA聚类,16份莪术种质聚为两类,No.1～No.9、No.14、No.15、No.16聚为一类,说明No.14、No.15、No.16与温郁金亲缘关系较近；No.10-No.13聚为另一类,说明No.13与蓬莪术亲缘关系较近；该聚类结果与传统的分类结果吻合。根据上述结果,确定福建收集的2份种质,No.13为蓬莪术,No.15为温郁金；广西收集的1份种质No.14为温郁金；云南收集的1份种质No.16由于分子标记与原植物、薄层等鉴别结果有出入,属于变种还是新种有待进一步分析研究。
最后采用指纹图谱考察莪术引种前后化学成分的相关性,发现引种至四川的温莪术(2年)与原产地的温莪术化学成分非产相近,相关性良好；引种至广西、福建的温莪术(多年)与原产地的温莪术化学成分有一定的差异,多了A峰,与薄层鉴别结果一致,初步推测可能与引种的年限或地域有关。引种至温州、福建的蓬莪术与原产地的蓬莪术化学成分相似,相关性良好。
1.2种质综合评价研究方面：首次对不同莪术种质的主根茎进行了经济价值评价、化学成分评价等方面的综合研究。通过对产量以及浸出物、挥发油、吉马酮、莪术二酮多指标的含量测定,运用组内分组的方差分析以及配对比较的统计学方法比较了两地种质资源圃以及同一种质资源圃不同年份种质的差异,并首次运用加权评分法分别对两地种质资源圃的种质进行综合评价研究,筛选出4份莪术优良种质材料。
2.温郁金优良株系筛选研究
首次采用系统选育法从大田筛选健壮、抗病抗逆性强的植株1000株,根据根茎鲜重对温郁金进行产量筛选,得到692株；通过有效成分的含量分析,并运用加权评分法对温郁金主根茎的产量以及化学成分评价中的有效成分含量进行评分,得到70株;再经过2年的跟踪筛选,最终得到产量、含量高且稳定性好的6个优良株系,分别是1-29、1-181、1-254、2-6、5-24、5-29。
3温莪术GAP部分标准操作规程研究
3.1采收期研究在前期的工作基础上继续定点、定期采样,研究温莪术在不同采收期的产量、生药折干率以及化学成分的含量,并结合历年的数据考察不同采收期对药材质量和产量的影响,确立最佳采收期为J月中旬至K月下旬。
3.2产地初加工研究对传统加工方法中的蒸煮条件、干燥方法、干燥温度等方面进行对比研究,筛选出既能使药材迅速干燥,又能保持外观饱满,同时保证有效成分影响较小的加工方法：蒸30min-40min,置于55℃烘箱内烘干或者晒干。
3.3包装、贮藏研究研究不同包装材料及贮藏年限对温莪术药材质量的影响,优选出最佳包装材料为编织袋,贮藏年限为两年。
上述研究结果为莪术种质资源的综合评价、温郁金良种选育繁育以及温莪术GAP规范化标准操作规程提供了科学的理论依据。
Rhizoma Curcumae is multi-source species,the rhizome of Curcuma phaeocaulis Val.,Curcuma Kwangsiensis S.GLee et C.F.Liang or Curcuma wenyujin Y.H.Chen et C.Ling, root in the family of Zingiberaceae. The goods are known as'Wen zhu','Gui Ezhu'and'Wen Ezhu'. The work of this thesis is part of the Project of Zhejiang Department of Science and Technology: Standardized cultivation of wenyujin and new drug's exploitation. Their complex origin, a variety of individual variation of the main producing areas and cultivars's quality degradation, reduced resistance lead to varies between plants and the quality of different. At equal pace,Rhizoma Curcumae's research lack of germplasm collection, sorting, preservation, protection and other related research, wild resources has been extincted.. According to the survey and pre-research work, we found that the different populations of Rhizoma Curcumae have the different quality, especially the Gui Ezhu, which is not as good as Wen Ezhu and Wen zhu. Therefore,we collected 16 germplasm form 5 provinces and established germplasm resources nursery. The thesis mainly compared the Rhizoma Curcumae from different populations in plant morphology, material character, molecular biology,finger print,.chemical composition,,established the comprehensive evaluation model of the excellent germplasm resources, gaining four excellent germplasm materials. On this basis of Wenyujin screening studies,selected four high-quality high-yielding strains.The thesis has also completed a part of GAP standard operating procedures, including harvesting, processing, packaging and storage.
Identification of germplasm:we studied the plant morphology, material character,molecular biology and finger print of the 16 germplasm form 5 provinces,.Identification of plant classification results show that No.1-No.9 is C. wenyujin, No.10-No.12 is C. phaeocaulis; No.13-No.16 have been made a preliminary determination, No.13, No.16 is C. phaeocaulis. No.14, No.15 is C. wenyujin. TLC Studies shown that:No.14,No.15 are similar in the chemical composition with Wen Ezhu to a high degree; No.13, No.16 are similar in the chemical composition with Peng Ezhu.Polyacrylamide gel electrophoresis method is used in ISSR molecular markers of Ezhu study for the first time, analysis Rhizoma Curcumae related germplasm and genetic diversity, screened 9-specific primers from the 30 primers the percentage of the polymorphism reached 70%. The molecular biology results show that: the genetic relationship between species more distant species within the close genetic relationship of different genetic information-rich germplasm. Through UPGMA clustering,16 germplasm are gathered to two types, No.1 -No.9, No.14, No.15, No.16 are assembled in a group,which means No.14, No.15, No.16 have the close genetic relationship with C. wenyujin; No.10-No.13 are assembled in another group, which means No.13 have the close genetic relationship with C. phaeocaulis;.The classification is similar with the traditional classification results. Based on the above results, we determined the two germplasm collection in Fujian, No.13 is C. phaeocaulis, No.15 is C. wenyujin,guanxi No.14 is C. wenyujin, as a result of molecular markers and histology not identical, yunnan No.16 is need to be further analyzed.
Finally, we use the fingerprints to see about the chemical constituents before and after the introduction of Ezhu and found that Wen Ezhu introduction to Sichuan (2 years) is similar to Wen Ezhu in original producing area in chemical composition and have a good correlation. Introduction to Guangxi, Fujian Wen Ezhu (many years) has some differences in chemical constituents with Wen Ezhu in original producing area, which is isidentical with the TLC results, the initial guess may be related to the length of the introduction and area. Introduction to Wenzhou, Fujian area identical with Peng Ezhu in original producing area in chemical composition, have a good correlation.
Comprehensive Evaluation of germplasm:Evaluation rhizome from different germplasm of Ezhu in the economy value, chemistry element and so on. determine the production,the content of volatile oil, germacrone, curdione, compared germplasm from two nursery and the same nursery in different years by statistical methods. Use weighted scoring method to evaluate the two germplasm nursery for the first time, and finally 4 were selected as germplasm materials.
Screening studies of excellen Strain:The system robust selection was first used to select 1000 plants with haleness and disease resistance from Daejeon,.we got 692 plants according to the fresh weight of roots C. wenyujin. we use the weighted score law to grade the main roots of C. Wenyujin production, as well as evaluate the chemical composition of the active ingredient content, get 70 plants;After 2 years of screening, high stability and has a better quality of six strains were 1-29,1-181,1-254,2-6,5-24,5-29.
GAP standard operating procedures:Work in the pre-positioned to build on the regular sampling to study the Ezhu's production at different growth, pharmacognostic exsiccation rate and chemical composition of dry content, combine with the data over the years to examine the quality and production with different harvest time, determined the suitable harvesting time of C. Wenyujin. Comparison of traditional processing methods of cooking condition,drying methods, drying temperature,we select the best Processing method to make sure quickly exsiccation, to maintain the appearance of full. and to ensure destruction of the active ingredients. Study on how the different packaging materials and storage of temperature affect the quality of drug, selected the best packaging materials and storage time.
The results provide the scientific theory basis for evaluation of germplasm resources, the elite selection and GAP standard operating procedures.
引文
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[36]谢永忠,夏泉.高效液相色谱法测定莪术中吉马酮的含量.中国药房,2005,16(6)：461-462
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[40]秦叔逵,钱军,王琳.榄香烯治疗肺癌的临床观察与实验研究[J].中国肿瘤临床,1997,24(3)：231-233
[41]胡守友,陈龙帮,王靖华,等.榄香烯对小鼠黑色素瘤细胞的分化诱导作[J].南京大学学报,1998,34(5)：550～553
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[44]宋利琼,张昌菊.莪术油联合干扰素对鼠宫颈癌端粒酶活性和细胞凋亡影响的研究.实用医学进修杂志,2005,33(1)：31-35
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[47]王胜春,李剑峰,刘明义,等.莪术当归延胡索及配伍后的活血行气作用.解放军药学学报,2004,20(5)：331-334
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[49]夏泉,黄赵刚,李绍平等.莪术油抗流感病毒和呼吸道合胞病毒作用的实验研究.中国 药理学通报,2004,20(3)：357～358
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[52]魏兰福,邹百仓,魏睦新.莪术对大鼠胃动力影响的实验研究.湖南中医杂志,2003,19(3)：58-59
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[54]魏兰福,邹百仓,魏睦新.莪术对实验性功能性消化不良大鼠胃排空的影响.南京医科大学学报(自然科学版),2003,23(4)：350-352