朝鲜白头翁的原植物鉴定及关键栽培技术研究
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摘要
朝鲜白头翁Pulsatilla koreana为毛茛科白头翁属多年生草本植物,以根入药,是我国传统的中药材之一。随着对朝鲜白头翁有效成分及药理作用研究的不断深入,市场需求量进一步增加,产业化人工种植势在必行。为了大规模开发利用朝鲜白头翁资源并对其质量提供保障,本论文从形态、显微和HPLC化学图谱三个方面对朝鲜白头翁和其它白头翁属植物进行鉴别,并系统研究了朝鲜白头翁的生长发育特性、栽培技术对朝鲜白头翁产量和品质的影响。主要研究结果如下：
对朝鲜白头翁、白头翁、兴安白头翁和细叶白头翁4种白头翁属植物的形态特征进行观测,结果表明4种白头翁属植物在叶形、末回裂片宽、萼片颜色、萼片长度、萼片开展度、雄蕊长度等形态特征方面有明显差异,种内变异较小,种间差异显著,可以作为形态学鉴定的指标。
朝鲜白头翁、白头翁、兴安白头翁和细叶白头翁的根均由周皮、韧皮部、木质部组成,韧皮部均比较发达,约占横切面半径的1/2,其根部横切面的差异主要表现在木质部。朝鲜白头翁根木质部导管居于中心,略作3束状,各束向外又有分叉,放射状排列,有的导管排列不整齐；白头翁根木质部导管位于中心,呈3个扇面状排列,韧皮纤维常单个散在,切向断续排列成环；兴安白头翁和细叶白头翁根木质部均为非三原型,兴安白头翁导管略呈两束状,放射状排列,而细叶白头翁根木质部导管由中心向外多次分叉作放射状排列,约10～14束,有的向外2次分叉。
4种植物粉末显微特征的不同主要表现在导管类型、石细胞的有无、韧皮纤维的多少、木栓化细胞的形状等方面。从石细胞的有无来看,朝鲜白头翁和白头翁均无石细胞,兴安白头翁和细叶白头翁中有少许石细胞；韧皮纤维以白头翁、兴安白头翁和细叶白头翁中较多,朝鲜白头翁中韧皮纤维少。朝鲜白头翁、白头翁和细叶白头翁的木栓化细胞均为长方形或近长方形,而兴安白头翁木栓化细胞形状不一,有长方形、多角形或长圆形。另外,兴安白头翁的导管相对其它3种较粗,但均以具缘纹孔为主。可以将这些指标作为显微鉴别的指标。
对朝鲜白头翁中总皂苷提取方法的研究表明,乙醇浓度对总皂苷提取影响最大,以乙醇浓度80%,超声时间40min,料液比1：20,超声功率80W,提取1次为最佳提取工艺。
利用HPLC测定白头翁属药用植物的指纹图谱,结果表明不同种白头翁属植物均显示了其共性和各自的指纹特征,确立了10个色谱峰为白头翁属植物的共有峰。根据14份样品的色谱数据,运用系统聚类法将14份样品聚成4类：第Ⅰ、Ⅱ和Ⅳ类分别为朝鲜白头翁,蒙古白头翁和白头翁；第Ⅲ类由兴安白头翁、细叶白头翁组成。白头翁属植物的HPLC指纹图谱可以用于白头翁属物种鉴别、种间亲缘关系和分类研究。
对不同产地和来源的朝鲜白头翁药材进行指纹图谱测定,确立了朝鲜白头翁药材的指纹图谱共有模式,标定了23个共有峰；经相似度计算,15批药材整体相似度较好,说明不同来源朝鲜白头翁药材的化学组成一致性较好。此指纹图谱特征性和专属性强,可用于朝鲜白头翁的质量控制。
对朝鲜白头翁生长发育及其总皂苷积累规律进行研究,结果表明朝鲜白头翁植物的个体发育可以划分为5个时期,即：出苗期、开花期、种子成熟期、果后生长期、枯萎期。朝鲜白头翁根部生长在整个生育时期内表现出“S”型曲线变化规律。野生移栽型、二年生和三年生朝鲜白头翁在不同生长期总皂苷含量以8月中旬最高,单株总皂苷产量最高值出现在8月中旬至9月中旬,9月中旬以后逐渐下降。从药材内在质量和产量两方面综合考虑,生产上可将朝鲜白头翁最佳采收时间安排在8月下旬至9月中旬。
研究朝鲜白头翁种子的基本特征和萌发特性,结果表明朝鲜白头翁种子具有快速吸水的生物学特性,100mg·L-1GA3浸种12h、10%～30%PEG6000浸种12h和40℃条件下80W超声波处理20min3种前处理萌发出苗效果最好,其出苗率可达到48%以上。多元复合微肥和氮磷钾复合肥配合施用明显促进了朝鲜白头翁幼苗地上部和根部的生长,幼苗叶鲜重比CK平均增加163.71%,幼苗根鲜重比CK平均增加83.10%。氮磷钾复合肥的适宜施用量为400～600kg·hm-2,复合微肥的喷施浓度为0.10%,每10～15d喷1次,喷施3-5次。
朝鲜白头翁生长发育、产量形成及总皂苷的积累受氮、磷、钾肥的供应水平所影响。氮、磷、钾各元素及其配比对朝鲜白头翁干物质累积总量和根产量的影响程度依次为氮>磷>钾,对总皂苷含量的影响程度依次为钾>磷>氮。从获得朝鲜白头翁高产优质的角度考虑,N109.44～123.12kg·hm-2, P2O5113.94～128.34kg·hm-2, K2O143.21～160.99kg·hm-2为朝鲜白头翁理想的最佳理论施肥量。
Pulsatilla koreana, a perennial plant belonging to the family of Ranuculaceae, is one of traditonal medicine in China. In recent years, with the ceaseless researches on the chemical constituents and the pharmacological action of Pulsatilla koreana Bercht, the demand of market further increased, and the wild resources cannot meet fundamentally to the needs for that, artificial planting is imperative. The Pulsatilla medicinal plants were distinguished by means of morphological, microscopic and chemical characteristics. And the extraction method of effective components, growth characteristics and the cultivation techniques of Pulsatilla koreana were studied, also established preliminarily the HPLC fingerprints for Pulsatilla koreana so as to provide basis for its quality control. The main results were as follows:
Morphological characteristics of Pulsatilla koreana, Pulsatilla chinensis, Pulsatilla dahurica and Pulsatilla turczaninovii were observed. The results showed that4species had significant differences in leaf shape, lobes, sepals and other morphological characteristics. There were small variation within specie and significant differences between species in corolla length, sepal length and achene length, which can be used as indicators of morphological identification.
The root of4species consists of periderm, phloem, vascular cambium, and xylem. The phloem Was more developed, accounted for about1/2of transverse radius.The differences of root cross-section between4species were mainly in the xylem. The root xylem of P. koreana was slightly3bunch of shape, which radially arranged in the center, each beamed to outside and had bifurcates, and some conduits arranged irregularly. Xylem conduits of P.chinensis were also in the center, which has3beam tube.P.dahurica and P. turczaninovii were not triarch, P.dahurica had2xylem conduits that radiated out, while xylem conduits of P.turczaninovii had about10to14bunches spreading outward from the center, some bifurcated2times.
The differences in powder microscopic characteristics between4species were mainly in the conduit type, presence or absence of stone cells, the number of phloem fibers, the shape of cork cell, and so on. The characteristics can be used to identify4species microscopically. View from the presence or absence of stone cells, P. koreana and P.chinensis had no stone cells, while P. dahurica and P. turczaninovii had a few stone cells. P. chinensis, P. dahurica, and P. turczaninovii had more phloem fibers, while less in P. koreana. The cork cells of P. koreana, P.chinensis and P. turczaninovii were rectangular or near rectangular. However, P. dahurica had several different cork cells, some are rectangular or polygonal or oblong. Furthermore, the vessels of P. dahurica were thicker than other3species, but mainly bordered pits.
The extraction conditions of total saponins from P. koreana were studied. The optimum conditons of ultrasound-assisted extraction was in the combination of80%of alcohol concentration,40min of ultrasonic time,1:20of solid to liquid ratio,80W of ultrasonic power and one time of extraction. Among them, alcohol had most significant effect on the extraction of total saponins.
The HPLC was used to establish the fingerprint of Pulsatilla medicinal plants, and to evaluate and distinguish the different species of Pulsatilla based on the chromatographic data. The different species of Pulsatilla showed different HPLC fingerprints, but common with10peaks. Q cluster analysis of14 accessions indicated that they were divided into four groups:all accessions from P. koreana were classified into Group Ⅰ, P. ambigua in Group Ⅱ, P. dahurica and P. turczaninovii in Group Ⅲ, and P. chinensis in Group Ⅳ, respectively.
The HPLC methods were employed to analyz the fingerprint of different sources of P. koreana. The precision, stability and repeatability of the method was investigated and reached the technical requirements of HPLC fingerprint. The characteristic mode of HPLC fingerprint of P. koreana was established, and23common peaks were identified. The similarity of15lots tested was good, which illustrated the chemical composition of different sources had good consistency. The HPLC fingerprint of P. koreana with high characteristics and specificity can be used for quality control of P. koreana.
The growth characteristics and total saponins accumulation in the root of P.koreana were studied. The ontogeny of P. koreana can be divided into five growth stages:budding stage, blooming stage, maturity stage, growth stage and wilting stage. The distribution rates and growth rates of its organs changed greatly during its growth period, the dry matter accumulative curve could be described as "S" shape. The total saponins content of the biennial, triennial, and perennial P.koreana reached the highest in mid-August, total saponin yield per plant reached the highest during the mid-August to the mid-September, then began to decline. Combining the results from the quality and quantity of medicinal material, the best harvest time of P. koreana is from the late August to the mid-September.
Using the seeds of P. koreana as materials, basic characters, water absorption characteristics, germination rates and seedling emergence rates of seeds treated by GA3, PEG6000and ultrasonicwaves, were studied in the laboratory. The results showed that seeds had the biological characteristic of fast water absorption, and the results also showed that the three pretreatments with GA3concentration of100mg·L-1for12hours,10%～30%PEG6000for12hours, ultrasonicwaves conditions with40℃,80W,20min had optimum effects on seed germination and seedling emergence, under which seeding emergence rates are above48.0%. Compound fertilizers had obvious effects on the seedlings growth. Fresh weight of leaves in all treatments increased by163.71%on average compared to the CK. Fresh weight of seedling root increased by83.10%on average. The appropriate level for NPK fertilizer was the400～600kg·hm-2. The spraying concentration of trace-element fertilizer was0.10%, spraying every10～15d for3to5times.
The results of field experiment conducted based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium showed that the effects on the growth, yield and total saponin contents in P. koreana of different proportional of nitrogen, phosphorus and potassium were quite different. The effect of nitrogen, phosphorus and potassium application on the total dry matter accumulation and root yield of P. koreana was as following:nitrogen> phosphorus> potassium, the effect on the content of total saponins was as following:potassium>phosphorus>nitrogen. To reach the goals of high yield and good quality, the optimum proportion was N109.44～123.12kg·hm-2, P2O5113.94～128.34kg·hm-2, K2O143.21～160.99kg·hm-2.
对朝鲜白头翁、白头翁、兴安白头翁和细叶白头翁4种白头翁属植物的形态特征进行观测,结果表明4种白头翁属植物在叶形、末回裂片宽、萼片颜色、萼片长度、萼片开展度、雄蕊长度等形态特征方面有明显差异,种内变异较小,种间差异显著,可以作为形态学鉴定的指标。
朝鲜白头翁、白头翁、兴安白头翁和细叶白头翁的根均由周皮、韧皮部、木质部组成,韧皮部均比较发达,约占横切面半径的1/2,其根部横切面的差异主要表现在木质部。朝鲜白头翁根木质部导管居于中心,略作3束状,各束向外又有分叉,放射状排列,有的导管排列不整齐；白头翁根木质部导管位于中心,呈3个扇面状排列,韧皮纤维常单个散在,切向断续排列成环；兴安白头翁和细叶白头翁根木质部均为非三原型,兴安白头翁导管略呈两束状,放射状排列,而细叶白头翁根木质部导管由中心向外多次分叉作放射状排列,约10～14束,有的向外2次分叉。
4种植物粉末显微特征的不同主要表现在导管类型、石细胞的有无、韧皮纤维的多少、木栓化细胞的形状等方面。从石细胞的有无来看,朝鲜白头翁和白头翁均无石细胞,兴安白头翁和细叶白头翁中有少许石细胞；韧皮纤维以白头翁、兴安白头翁和细叶白头翁中较多,朝鲜白头翁中韧皮纤维少。朝鲜白头翁、白头翁和细叶白头翁的木栓化细胞均为长方形或近长方形,而兴安白头翁木栓化细胞形状不一,有长方形、多角形或长圆形。另外,兴安白头翁的导管相对其它3种较粗,但均以具缘纹孔为主。可以将这些指标作为显微鉴别的指标。
对朝鲜白头翁中总皂苷提取方法的研究表明,乙醇浓度对总皂苷提取影响最大,以乙醇浓度80%,超声时间40min,料液比1：20,超声功率80W,提取1次为最佳提取工艺。
利用HPLC测定白头翁属药用植物的指纹图谱,结果表明不同种白头翁属植物均显示了其共性和各自的指纹特征,确立了10个色谱峰为白头翁属植物的共有峰。根据14份样品的色谱数据,运用系统聚类法将14份样品聚成4类：第Ⅰ、Ⅱ和Ⅳ类分别为朝鲜白头翁,蒙古白头翁和白头翁；第Ⅲ类由兴安白头翁、细叶白头翁组成。白头翁属植物的HPLC指纹图谱可以用于白头翁属物种鉴别、种间亲缘关系和分类研究。
对不同产地和来源的朝鲜白头翁药材进行指纹图谱测定,确立了朝鲜白头翁药材的指纹图谱共有模式,标定了23个共有峰；经相似度计算,15批药材整体相似度较好,说明不同来源朝鲜白头翁药材的化学组成一致性较好。此指纹图谱特征性和专属性强,可用于朝鲜白头翁的质量控制。
对朝鲜白头翁生长发育及其总皂苷积累规律进行研究,结果表明朝鲜白头翁植物的个体发育可以划分为5个时期,即：出苗期、开花期、种子成熟期、果后生长期、枯萎期。朝鲜白头翁根部生长在整个生育时期内表现出“S”型曲线变化规律。野生移栽型、二年生和三年生朝鲜白头翁在不同生长期总皂苷含量以8月中旬最高,单株总皂苷产量最高值出现在8月中旬至9月中旬,9月中旬以后逐渐下降。从药材内在质量和产量两方面综合考虑,生产上可将朝鲜白头翁最佳采收时间安排在8月下旬至9月中旬。
研究朝鲜白头翁种子的基本特征和萌发特性,结果表明朝鲜白头翁种子具有快速吸水的生物学特性,100mg·L-1GA3浸种12h、10%～30%PEG6000浸种12h和40℃条件下80W超声波处理20min3种前处理萌发出苗效果最好,其出苗率可达到48%以上。多元复合微肥和氮磷钾复合肥配合施用明显促进了朝鲜白头翁幼苗地上部和根部的生长,幼苗叶鲜重比CK平均增加163.71%,幼苗根鲜重比CK平均增加83.10%。氮磷钾复合肥的适宜施用量为400～600kg·hm-2,复合微肥的喷施浓度为0.10%,每10～15d喷1次,喷施3-5次。
朝鲜白头翁生长发育、产量形成及总皂苷的积累受氮、磷、钾肥的供应水平所影响。氮、磷、钾各元素及其配比对朝鲜白头翁干物质累积总量和根产量的影响程度依次为氮>磷>钾,对总皂苷含量的影响程度依次为钾>磷>氮。从获得朝鲜白头翁高产优质的角度考虑,N109.44～123.12kg·hm-2, P2O5113.94～128.34kg·hm-2, K2O143.21～160.99kg·hm-2为朝鲜白头翁理想的最佳理论施肥量。
Pulsatilla koreana, a perennial plant belonging to the family of Ranuculaceae, is one of traditonal medicine in China. In recent years, with the ceaseless researches on the chemical constituents and the pharmacological action of Pulsatilla koreana Bercht, the demand of market further increased, and the wild resources cannot meet fundamentally to the needs for that, artificial planting is imperative. The Pulsatilla medicinal plants were distinguished by means of morphological, microscopic and chemical characteristics. And the extraction method of effective components, growth characteristics and the cultivation techniques of Pulsatilla koreana were studied, also established preliminarily the HPLC fingerprints for Pulsatilla koreana so as to provide basis for its quality control. The main results were as follows:
Morphological characteristics of Pulsatilla koreana, Pulsatilla chinensis, Pulsatilla dahurica and Pulsatilla turczaninovii were observed. The results showed that4species had significant differences in leaf shape, lobes, sepals and other morphological characteristics. There were small variation within specie and significant differences between species in corolla length, sepal length and achene length, which can be used as indicators of morphological identification.
The root of4species consists of periderm, phloem, vascular cambium, and xylem. The phloem Was more developed, accounted for about1/2of transverse radius.The differences of root cross-section between4species were mainly in the xylem. The root xylem of P. koreana was slightly3bunch of shape, which radially arranged in the center, each beamed to outside and had bifurcates, and some conduits arranged irregularly. Xylem conduits of P.chinensis were also in the center, which has3beam tube.P.dahurica and P. turczaninovii were not triarch, P.dahurica had2xylem conduits that radiated out, while xylem conduits of P.turczaninovii had about10to14bunches spreading outward from the center, some bifurcated2times.
The differences in powder microscopic characteristics between4species were mainly in the conduit type, presence or absence of stone cells, the number of phloem fibers, the shape of cork cell, and so on. The characteristics can be used to identify4species microscopically. View from the presence or absence of stone cells, P. koreana and P.chinensis had no stone cells, while P. dahurica and P. turczaninovii had a few stone cells. P. chinensis, P. dahurica, and P. turczaninovii had more phloem fibers, while less in P. koreana. The cork cells of P. koreana, P.chinensis and P. turczaninovii were rectangular or near rectangular. However, P. dahurica had several different cork cells, some are rectangular or polygonal or oblong. Furthermore, the vessels of P. dahurica were thicker than other3species, but mainly bordered pits.
The extraction conditions of total saponins from P. koreana were studied. The optimum conditons of ultrasound-assisted extraction was in the combination of80%of alcohol concentration,40min of ultrasonic time,1:20of solid to liquid ratio,80W of ultrasonic power and one time of extraction. Among them, alcohol had most significant effect on the extraction of total saponins.
The HPLC was used to establish the fingerprint of Pulsatilla medicinal plants, and to evaluate and distinguish the different species of Pulsatilla based on the chromatographic data. The different species of Pulsatilla showed different HPLC fingerprints, but common with10peaks. Q cluster analysis of14 accessions indicated that they were divided into four groups:all accessions from P. koreana were classified into Group Ⅰ, P. ambigua in Group Ⅱ, P. dahurica and P. turczaninovii in Group Ⅲ, and P. chinensis in Group Ⅳ, respectively.
The HPLC methods were employed to analyz the fingerprint of different sources of P. koreana. The precision, stability and repeatability of the method was investigated and reached the technical requirements of HPLC fingerprint. The characteristic mode of HPLC fingerprint of P. koreana was established, and23common peaks were identified. The similarity of15lots tested was good, which illustrated the chemical composition of different sources had good consistency. The HPLC fingerprint of P. koreana with high characteristics and specificity can be used for quality control of P. koreana.
The growth characteristics and total saponins accumulation in the root of P.koreana were studied. The ontogeny of P. koreana can be divided into five growth stages:budding stage, blooming stage, maturity stage, growth stage and wilting stage. The distribution rates and growth rates of its organs changed greatly during its growth period, the dry matter accumulative curve could be described as "S" shape. The total saponins content of the biennial, triennial, and perennial P.koreana reached the highest in mid-August, total saponin yield per plant reached the highest during the mid-August to the mid-September, then began to decline. Combining the results from the quality and quantity of medicinal material, the best harvest time of P. koreana is from the late August to the mid-September.
Using the seeds of P. koreana as materials, basic characters, water absorption characteristics, germination rates and seedling emergence rates of seeds treated by GA3, PEG6000and ultrasonicwaves, were studied in the laboratory. The results showed that seeds had the biological characteristic of fast water absorption, and the results also showed that the three pretreatments with GA3concentration of100mg·L-1for12hours,10%～30%PEG6000for12hours, ultrasonicwaves conditions with40℃,80W,20min had optimum effects on seed germination and seedling emergence, under which seeding emergence rates are above48.0%. Compound fertilizers had obvious effects on the seedlings growth. Fresh weight of leaves in all treatments increased by163.71%on average compared to the CK. Fresh weight of seedling root increased by83.10%on average. The appropriate level for NPK fertilizer was the400～600kg·hm-2. The spraying concentration of trace-element fertilizer was0.10%, spraying every10～15d for3to5times.
The results of field experiment conducted based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium showed that the effects on the growth, yield and total saponin contents in P. koreana of different proportional of nitrogen, phosphorus and potassium were quite different. The effect of nitrogen, phosphorus and potassium application on the total dry matter accumulation and root yield of P. koreana was as following:nitrogen> phosphorus> potassium, the effect on the content of total saponins was as following:potassium>phosphorus>nitrogen. To reach the goals of high yield and good quality, the optimum proportion was N109.44～123.12kg·hm-2, P2O5113.94～128.34kg·hm-2, K2O143.21～160.99kg·hm-2.
引文
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