日本毛连菜和细辛的生药学研究
|
摘要
日本毛连菜(Picris japonica Thunb.)隶属菊科毛连菜属(Picris L.),又名枪刀菜,主要分布在我国河北、山西、吉林、黑龙江等地。日本毛连菜全草入蒙药,具有清热、消肿及止痛作用,主治流感、乳痈。目前,关于日本毛连菜的化学成分研究有一些相关报道,有关生药学的研究报道很少。为了建立日本毛连菜显微鉴定方法,为日本毛连菜的显微鉴定提供依据,本文采用显微鉴定和扫描电镜技术,对日本毛连菜根、茎、叶、果实进行组织鉴定,对全草进行粉末鉴定,并对花粉和果实进行了表面微形态观察。其研究结果如下：
1、日本毛连菜的显微结构。①根横切面,表皮细胞一列,老根外侧有木栓层数列,有节乳汁管散在分布于韧皮部中,髓射线明显。②茎横切面,表皮细胞一列,表皮上有腺毛和非腺毛,内皮层明显。③叶横切面,表皮由1列细胞构成,栅栏组织细胞为1-2列,不通过主脉,主脉维管束1-5个,外韧型,大小相间,略呈半环状排列。④叶表皮形态,上、下表皮均有非腺毛、腺毛和气孔分布,上表皮细胞垂周壁较平直,下表皮细胞垂周壁波状弯曲；气孔为不等式或不定式,副卫细胞多为4-6个。⑤日本毛连菜地上部分被覆的非腺毛的顶端由2个细胞特化为分叉钩状,细胞壁增厚明显,基部由多个纵向伸长的细胞构成；腺毛有两种。⑥花粉粒刺状纹饰,有三个萌发孔。⑦全草粉末观察,多见非腺毛2个分叉的顶端碎片和腺毛多细胞的头部；花粉粒常见,类圆形,外壁有刺状突起,有三个萌发孔：导管多为螺纹导管和具缘纹孔导管；断碎的冠毛较多；乳汁管为有节乳汁管。
2、花粉和果实的表面微形态。花粉粒外壁刺状纹饰基部粗大,刺状突起呈环形排列或数个密集成群；网眼小且深,大小、形状相近；萌发孔三个,具大型瘤状突起,瘤状突起为脑纹状纹饰。果实有5-10余条高起的纵肋和5条较深的纵沟,纵沟贯穿果实两端,纵沟内有较多指状突起。外果皮细胞狭长形、先端呈游离的指状突起,游离部分呈层叠环绕果实排列。果实两顶端侧面观和俯面观特征明显,一端俯面观呈五瓣花形；另一端俯面观呈五角星形,中间有孔,表面有许多长条状细胞紧密排列而成。
上述显微结构和扫描电镜微形态特征的观察,为日本毛连菜的生药鉴定提供了重要的依据。
细辛是常用中药,来源于马兜铃科细辛属Asarum植物北细辛A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.、汉城细辛A. sieboldii Miq. var. seoulense Nakai或华细辛A. sieboldii Miq.干燥的根和根茎,具多种功效,主治风寒感冒、头痛牙痛等病症。华细辛广泛分布于黄河和长江流域,其中,陕西华阴的药材历来皆被认为品质最佳。然而,实际上却缺乏有效的依据。本文通过挥发油含量及其化学成分的分析,对华细辛挥发性成分的地区性差异进行了比较。
细辛具一定毒性,自古已有很多记载,然而迄今为止,对于细辛毒性的表现形式、毒性成分以及致毒机制,仍缺乏深入了解。本文较为系统地比较了细辛常用剂型水煎剂、散剂和复方制剂(麻黄附子细辛汤)对SD大鼠的肾、肝、肺、胃、肠等重要脏器的毒害作用。
研究结果如下：
1、挥发油含量及其化学成分分析。测定了浙江、江西、安徽、山东、湖南、湖北、四川、重庆、陕西等省市25个样点华细辛的叶和根及根茎中挥发油的含量,比较了其挥发性成分绝对含量与组成差异。
①华细辛根及根茎的挥发油含量均值为2.87%,而叶的挥发油含量均值为0.23%。华细辛叶中挥发油含量远低于根及根茎中挥发油含量。
②不同产地间华细辛挥发油含量有显著性差异(P<0.05),其部分成分如α-蒎烯、香桧烯、β-蒎烯、松油烯、桉叶素、γ-蒎烯、4-萜品醇、α-松油醇、3,5-二甲氧基甲苯、黄樟醚、甲基丁香酚、肉豆蔻醚、榄香脂素等含量也存在显著性差异(P<0.05),莰烯、月桂烯、α-水芹烯、对-聚伞花素等成分的含量无显著性差异(P>0.05)。
2、细辛的毒性与其剂型剂量有密切相关性。细辛水煎剂和复方制剂麻黄附子细辛汤在细辛生药量0.625 g·kg-1·d-1(6g／人)和3.125 g·kg-1·d-1(30g／人)的剂量下,各项生化指标与空白对照组比较,均无显著性差异,肾脏组织切片亦无病理形态学变化。麻黄附子细辛汤在细辛生药量增加至6.25 g·kg-1·d-1(60g／人)时,大鼠表现出轻度肾脏损伤。细辛散剂在0.94 g·kg-1·d-1(9g／人)的剂量下,大鼠血清肌酐明显偏高,肾脏和肝脏中度损伤,肺脏轻度损伤；在2.81 g·kg-1·d-1(27g／人)的剂量下,大鼠陆续死亡,肾、肝和肺等脏器重度损伤。可见,在大鼠中,细辛的毒性主要表现在肾、肝、肺等脏器损伤,对肠、胃等脏器无损伤。细辛散剂的毒性显著强于水煎剂和复方制剂麻黄附子细辛汤。
3、华细辛AFLP反应体系的建立。采用改良的CTAB法,建立了适于AFLP分析的华细辛基因组DNA的提取方法。结果表明：采用含2%CTAB、2mol/LNaCl、luLβ-琉基乙醇的DNA提取液,酚／氯仿抽提两次,提取得到的华细辛基因组DNA纯度高,基本无降解,适于AFLP分析要求。经对比研究,华细辛AFLP分析的优化反应体系如下：25 uL酶切体系中加入基因组DNA500ng,EcoRI 10U,Mse I 5 U,37℃酶切3h；12.5 uL连接反应体系中加入Mse I adaptor 25 pmol, EcoR I adaptor 2.5 pmol, T4DNA ligase 175 U,酶切产物10μL,在16℃连接过夜(12h～16h)；预扩增产物稀释150倍进行选择性扩增；8 uL选择性扩增产物加入3 uL Loading buffer,95℃变性5 min后,立即冰上冷却,于6%的聚丙烯酰胺凝胶上电泳,然后进行银染,拍照。
Picris japonica Thunb belongs to family Compositae. In China, it mainly grows in Hebei, Shanxi, Jilin and Heilongjiang provinces. It is often used to herba and could clear heat, detume scence, cure flu and mammary abscess. At present, pharmacognosy study of P. japonica Thunb is very few, and only the chemical composition is covered in several reports. In this paper, its microscopic identification was studied based on the identification and scanning electron microscopic technique. The main contents are as follows:
1. The microstructure of P. japonica Thunb.①Root cross-section has an epidermal cell. The old root lateral layers have several cork layers. The laticifer has scattered in the phloem, and medullary ray is obvious.②Stem cross-section has an epidermal cell. Its inner cortex is obviously. There are glandular hairs and non-glandular hairs on the epidermis of stem.③Leaf cross-section has an epidermal cell, has 1-2 palisade tissue cells and don't through the main vein. There are 1-5 main vein vascular bundles, which are collateral bundles, slightly semi-circular array.④Leaf epidermis-chip:The glandular hairs, non-glandular hairs, and stomas distributed on the upper and lower epidermis. The anticlinal walls of the upper epidermis cells are slightly straight, the anticlinal walls of the lower epidermis cells are wavy bending. Andersen matal for inequality or infinitive, the Deputy Guardian cells 4-6.⑤The aboveground part are coated with non-glandular hairs which like bifurcated hooks, the two top cells bifurcated into special hook, thickened obviously, the basal part constituted by a number of elongated cells; There are two types of glandular hairs.⑥Pollen exine has spinulose ornamentation. There are three apertures.⑦Powders observation, two top bifurcation of the non-glandular hairs and debris of the multicellular glandular hairs are seen. Pollen grains common, oval, outer barbed-like protuberances, there are three apertures; Vessels are mostly spiral vessel and bordered pit vessel. There are broken pappus and laticiferous vessels.
2. The surface micromorphology for pollen and fruit of P. japonica Thunb. Pollen exine has spinulose ornamentation, whose base is thick. The Thorn-like protuberances are ring-shaped arrangement or dense clusters; The mesh is small and deep, both size and shape are similar; Three germination holes with three large tumor-like enations which has brain striate ornamentation. Different numbers of tumor-like protuberances on the different observation surfaces. Exocarp fruits were arranged around the tile sheet, "tile" tip with finger-like protrusions. The fruit has about five to ten longitudinal ribs and five deep cannelures that across the fruit both sides. There are more finger-like protrusions in the deep cannelures. Exocarp cells are long and narrow, the tip of the finger-like protrusions are dissociative, finger-like protrusions arrangement were stacked around the fruit. Overview on the other side overlooking the pentacle was the middle hole, the surface of many cells tightly arranged in long strips, point hole, the point of view of the fruit surface, such as layer upon layer of mushrooms.
The micro structure and micro-morphological characteristics using scanning electron microscopy observation provide an important fundamental basis for the identification of P. japonica Thunb.
Herba Asari is a commonly used traditional Chinese medicine, derived from dried roots and rhizomes of the Aristolochiaceae Asarum. heterotropoides Fr. Schmidt var.mandshuricum (Maxim.) Kitag, A. sieboldii Miq. var. seoulense Nakai and A. sieboldii Miq.. It has many kinds of effects, mainly treats cold, headache, toothache and other diseases. Herba Asari (A. sieboldii Miq.) widely distributes in the Yellow River and the Yangtze River basin, which derived from Shaanxi Huayin were traditionally regarded as the best quality. However, it is lack of the effective basis actually. In this paper, through analysis of the volatile oil content and chemical composition, the regional differences in volatile components of Herba Asari were compared. Many records said that the Herba Asari was poisonous since ancient times. However, until now, the toxicity manifestations, toxic components and the toxicity mechanism of Herba Asari are still a lack of understanding. In this paper, a systematic comparative analysis of the commonly used formulations of water decoction of Herba Asari, powder and compound preparations (Mahuangfuzixixin Tang) on the SD rat kidney, liver, lung, stomach, intestines and other important organs of the toxic effects. The results are as follows:
1. Volatile oil content and chemical composition analysis. Measured volatile oil contents in leaves, roots and rhizome of Herba Asari at 25 sampling points such as Zhejiang, Jiangxi, Anhui, Shandong, Hunan, Hubei, Sichuan, Chongqing, Shaanxi Provinces, compared its absolute content and composition of volatile components.
①The average of volatile oil content in root and rhizome of Herba Asari is 2.87%, but the average of volatile oil content in leaf is 0.23%. Obviously, the volatile oil content in the leaf is far lower than the volatile oil content in the root and the rhizome of Herba Asari.
②Some volatile components of Herba Asari between the different sampling points show significance difference (P<0.05), such asα-pinene, sabinene,β—pinene,α-terpene, eucalyptol,γ-pinene,4-terpine,α-terpineol, 3,5-dimethoxytoluene, safrole, methyleugenol, myristicin, n-pentadecane, elemicin and so on. The other volatile components show non-significance differences (P>0.05), such as camphene, myrcene,α-phellandrene, p-cymine and so on.
2. The toxicity of Herba Asari was closely related to its preparation and dose. At the dose of 0.94 g·kg-1·d-1(9g/person) Herba Asari powders have caused obvious injury in rats, including considerably high CREA figures and pathological changes in tissues of kidney, liver and lung; and at the dose of 2.81 g·kg-1·d-1 (27g/person) the rats were dead one after another and their kidney, liver and lung were seriously injured. In contrast, Herba Asari decoction and Mahuang-Fuzi-Xixin Tang made no harm to the rats after 60 days drug treament at both doses of 0.625 g·kg-1·d-1(6g/person) and 3.125 g·kg-1·d-1 (30g/person), in other words, there was no meaningful difference in the biochemical indices TPU, CREA and BUN from those of the blank group, and no pathological alterations were recognized in the tissue sections of kidney. However, in Mahuang-Fuzi-Xixin Tang group occured slight injury in the rats'kidney when the dose of Herba Asari was added to 6.25 g·kg-1·d-1 (60g/person). It's showed that in rats the toxic effects of Herba Asari were manifested in the injury of kidney, liver and lung, but not in stomach and intestines. The toxicity of Herba Asari powders was remarkably stronger than those of Herba Asari decoction and Mahuang-Fuzi-Xixin Tang.
3. The establishment of AFLP reaction system for Asarum sieholdii Miq. Adopting improved CTAB method to establish the method for extracting Asarum sieholdii Miq's genome DNA which is suitable for AFLP analysis. It is shown that Asarum sieholdii Miq's genome DNA extracted with 2%CTAB,2mol/LNaCl, 1uLβ-Mercaptoethanol is purity and no degradation basically and is suitable for AFLP analysis. AFLP reaction system of Asarum sieholdii Miq was studied initially and the analysis system which is more suitable for AFLP analysis was obtained. Genomic DNA (500 ng) was digested with 10 U EcoR I and 5 U Mse I restriction enzymes at 37℃for 3 h in a volume of 25μl. In ligation reaction (12.5uL) containing 2.5 pmol EcoR I ligation adapters,25 pmol Mse I ligation adapters and 175 units of T4 DNA ligase, the mixture was incubated overnight at 16℃(12h～16h). Pre-amplification products were diluted 150 folds. The selective PCR reaction mixture (20μl) also included EcoR I selective primer (8 pmol), Mse I selective primer (8 pmol), dNTPs (4 nmol), and Taq DNA polymerase (1 U) in 1×PCR buffer. Prior to electrophoresis, the reaction products (8μl) was mixed with formamide loading buffer (3μl) of 98%(w/v) formamide/10 mM EDTA, pH 8.0/0.1% bromophenol blue/0.1% xylene cyanol (w/v). The loading mixture was denatured at 95℃for 5 min, then cooled on ice immediately, and resolved on 6% denaturing polyacrylamide gels. AFLP gels were silver stained and photographed.
1、日本毛连菜的显微结构。①根横切面,表皮细胞一列,老根外侧有木栓层数列,有节乳汁管散在分布于韧皮部中,髓射线明显。②茎横切面,表皮细胞一列,表皮上有腺毛和非腺毛,内皮层明显。③叶横切面,表皮由1列细胞构成,栅栏组织细胞为1-2列,不通过主脉,主脉维管束1-5个,外韧型,大小相间,略呈半环状排列。④叶表皮形态,上、下表皮均有非腺毛、腺毛和气孔分布,上表皮细胞垂周壁较平直,下表皮细胞垂周壁波状弯曲；气孔为不等式或不定式,副卫细胞多为4-6个。⑤日本毛连菜地上部分被覆的非腺毛的顶端由2个细胞特化为分叉钩状,细胞壁增厚明显,基部由多个纵向伸长的细胞构成；腺毛有两种。⑥花粉粒刺状纹饰,有三个萌发孔。⑦全草粉末观察,多见非腺毛2个分叉的顶端碎片和腺毛多细胞的头部；花粉粒常见,类圆形,外壁有刺状突起,有三个萌发孔：导管多为螺纹导管和具缘纹孔导管；断碎的冠毛较多；乳汁管为有节乳汁管。
2、花粉和果实的表面微形态。花粉粒外壁刺状纹饰基部粗大,刺状突起呈环形排列或数个密集成群；网眼小且深,大小、形状相近；萌发孔三个,具大型瘤状突起,瘤状突起为脑纹状纹饰。果实有5-10余条高起的纵肋和5条较深的纵沟,纵沟贯穿果实两端,纵沟内有较多指状突起。外果皮细胞狭长形、先端呈游离的指状突起,游离部分呈层叠环绕果实排列。果实两顶端侧面观和俯面观特征明显,一端俯面观呈五瓣花形；另一端俯面观呈五角星形,中间有孔,表面有许多长条状细胞紧密排列而成。
上述显微结构和扫描电镜微形态特征的观察,为日本毛连菜的生药鉴定提供了重要的依据。
细辛是常用中药,来源于马兜铃科细辛属Asarum植物北细辛A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.、汉城细辛A. sieboldii Miq. var. seoulense Nakai或华细辛A. sieboldii Miq.干燥的根和根茎,具多种功效,主治风寒感冒、头痛牙痛等病症。华细辛广泛分布于黄河和长江流域,其中,陕西华阴的药材历来皆被认为品质最佳。然而,实际上却缺乏有效的依据。本文通过挥发油含量及其化学成分的分析,对华细辛挥发性成分的地区性差异进行了比较。
细辛具一定毒性,自古已有很多记载,然而迄今为止,对于细辛毒性的表现形式、毒性成分以及致毒机制,仍缺乏深入了解。本文较为系统地比较了细辛常用剂型水煎剂、散剂和复方制剂(麻黄附子细辛汤)对SD大鼠的肾、肝、肺、胃、肠等重要脏器的毒害作用。
研究结果如下：
1、挥发油含量及其化学成分分析。测定了浙江、江西、安徽、山东、湖南、湖北、四川、重庆、陕西等省市25个样点华细辛的叶和根及根茎中挥发油的含量,比较了其挥发性成分绝对含量与组成差异。
①华细辛根及根茎的挥发油含量均值为2.87%,而叶的挥发油含量均值为0.23%。华细辛叶中挥发油含量远低于根及根茎中挥发油含量。
②不同产地间华细辛挥发油含量有显著性差异(P<0.05),其部分成分如α-蒎烯、香桧烯、β-蒎烯、松油烯、桉叶素、γ-蒎烯、4-萜品醇、α-松油醇、3,5-二甲氧基甲苯、黄樟醚、甲基丁香酚、肉豆蔻醚、榄香脂素等含量也存在显著性差异(P<0.05),莰烯、月桂烯、α-水芹烯、对-聚伞花素等成分的含量无显著性差异(P>0.05)。
2、细辛的毒性与其剂型剂量有密切相关性。细辛水煎剂和复方制剂麻黄附子细辛汤在细辛生药量0.625 g·kg-1·d-1(6g／人)和3.125 g·kg-1·d-1(30g／人)的剂量下,各项生化指标与空白对照组比较,均无显著性差异,肾脏组织切片亦无病理形态学变化。麻黄附子细辛汤在细辛生药量增加至6.25 g·kg-1·d-1(60g／人)时,大鼠表现出轻度肾脏损伤。细辛散剂在0.94 g·kg-1·d-1(9g／人)的剂量下,大鼠血清肌酐明显偏高,肾脏和肝脏中度损伤,肺脏轻度损伤；在2.81 g·kg-1·d-1(27g／人)的剂量下,大鼠陆续死亡,肾、肝和肺等脏器重度损伤。可见,在大鼠中,细辛的毒性主要表现在肾、肝、肺等脏器损伤,对肠、胃等脏器无损伤。细辛散剂的毒性显著强于水煎剂和复方制剂麻黄附子细辛汤。
3、华细辛AFLP反应体系的建立。采用改良的CTAB法,建立了适于AFLP分析的华细辛基因组DNA的提取方法。结果表明：采用含2%CTAB、2mol/LNaCl、luLβ-琉基乙醇的DNA提取液,酚／氯仿抽提两次,提取得到的华细辛基因组DNA纯度高,基本无降解,适于AFLP分析要求。经对比研究,华细辛AFLP分析的优化反应体系如下：25 uL酶切体系中加入基因组DNA500ng,EcoRI 10U,Mse I 5 U,37℃酶切3h；12.5 uL连接反应体系中加入Mse I adaptor 25 pmol, EcoR I adaptor 2.5 pmol, T4DNA ligase 175 U,酶切产物10μL,在16℃连接过夜(12h～16h)；预扩增产物稀释150倍进行选择性扩增；8 uL选择性扩增产物加入3 uL Loading buffer,95℃变性5 min后,立即冰上冷却,于6%的聚丙烯酰胺凝胶上电泳,然后进行银染,拍照。
Picris japonica Thunb belongs to family Compositae. In China, it mainly grows in Hebei, Shanxi, Jilin and Heilongjiang provinces. It is often used to herba and could clear heat, detume scence, cure flu and mammary abscess. At present, pharmacognosy study of P. japonica Thunb is very few, and only the chemical composition is covered in several reports. In this paper, its microscopic identification was studied based on the identification and scanning electron microscopic technique. The main contents are as follows:
1. The microstructure of P. japonica Thunb.①Root cross-section has an epidermal cell. The old root lateral layers have several cork layers. The laticifer has scattered in the phloem, and medullary ray is obvious.②Stem cross-section has an epidermal cell. Its inner cortex is obviously. There are glandular hairs and non-glandular hairs on the epidermis of stem.③Leaf cross-section has an epidermal cell, has 1-2 palisade tissue cells and don't through the main vein. There are 1-5 main vein vascular bundles, which are collateral bundles, slightly semi-circular array.④Leaf epidermis-chip:The glandular hairs, non-glandular hairs, and stomas distributed on the upper and lower epidermis. The anticlinal walls of the upper epidermis cells are slightly straight, the anticlinal walls of the lower epidermis cells are wavy bending. Andersen matal for inequality or infinitive, the Deputy Guardian cells 4-6.⑤The aboveground part are coated with non-glandular hairs which like bifurcated hooks, the two top cells bifurcated into special hook, thickened obviously, the basal part constituted by a number of elongated cells; There are two types of glandular hairs.⑥Pollen exine has spinulose ornamentation. There are three apertures.⑦Powders observation, two top bifurcation of the non-glandular hairs and debris of the multicellular glandular hairs are seen. Pollen grains common, oval, outer barbed-like protuberances, there are three apertures; Vessels are mostly spiral vessel and bordered pit vessel. There are broken pappus and laticiferous vessels.
2. The surface micromorphology for pollen and fruit of P. japonica Thunb. Pollen exine has spinulose ornamentation, whose base is thick. The Thorn-like protuberances are ring-shaped arrangement or dense clusters; The mesh is small and deep, both size and shape are similar; Three germination holes with three large tumor-like enations which has brain striate ornamentation. Different numbers of tumor-like protuberances on the different observation surfaces. Exocarp fruits were arranged around the tile sheet, "tile" tip with finger-like protrusions. The fruit has about five to ten longitudinal ribs and five deep cannelures that across the fruit both sides. There are more finger-like protrusions in the deep cannelures. Exocarp cells are long and narrow, the tip of the finger-like protrusions are dissociative, finger-like protrusions arrangement were stacked around the fruit. Overview on the other side overlooking the pentacle was the middle hole, the surface of many cells tightly arranged in long strips, point hole, the point of view of the fruit surface, such as layer upon layer of mushrooms.
The micro structure and micro-morphological characteristics using scanning electron microscopy observation provide an important fundamental basis for the identification of P. japonica Thunb.
Herba Asari is a commonly used traditional Chinese medicine, derived from dried roots and rhizomes of the Aristolochiaceae Asarum. heterotropoides Fr. Schmidt var.mandshuricum (Maxim.) Kitag, A. sieboldii Miq. var. seoulense Nakai and A. sieboldii Miq.. It has many kinds of effects, mainly treats cold, headache, toothache and other diseases. Herba Asari (A. sieboldii Miq.) widely distributes in the Yellow River and the Yangtze River basin, which derived from Shaanxi Huayin were traditionally regarded as the best quality. However, it is lack of the effective basis actually. In this paper, through analysis of the volatile oil content and chemical composition, the regional differences in volatile components of Herba Asari were compared. Many records said that the Herba Asari was poisonous since ancient times. However, until now, the toxicity manifestations, toxic components and the toxicity mechanism of Herba Asari are still a lack of understanding. In this paper, a systematic comparative analysis of the commonly used formulations of water decoction of Herba Asari, powder and compound preparations (Mahuangfuzixixin Tang) on the SD rat kidney, liver, lung, stomach, intestines and other important organs of the toxic effects. The results are as follows:
1. Volatile oil content and chemical composition analysis. Measured volatile oil contents in leaves, roots and rhizome of Herba Asari at 25 sampling points such as Zhejiang, Jiangxi, Anhui, Shandong, Hunan, Hubei, Sichuan, Chongqing, Shaanxi Provinces, compared its absolute content and composition of volatile components.
①The average of volatile oil content in root and rhizome of Herba Asari is 2.87%, but the average of volatile oil content in leaf is 0.23%. Obviously, the volatile oil content in the leaf is far lower than the volatile oil content in the root and the rhizome of Herba Asari.
②Some volatile components of Herba Asari between the different sampling points show significance difference (P<0.05), such asα-pinene, sabinene,β—pinene,α-terpene, eucalyptol,γ-pinene,4-terpine,α-terpineol, 3,5-dimethoxytoluene, safrole, methyleugenol, myristicin, n-pentadecane, elemicin and so on. The other volatile components show non-significance differences (P>0.05), such as camphene, myrcene,α-phellandrene, p-cymine and so on.
2. The toxicity of Herba Asari was closely related to its preparation and dose. At the dose of 0.94 g·kg-1·d-1(9g/person) Herba Asari powders have caused obvious injury in rats, including considerably high CREA figures and pathological changes in tissues of kidney, liver and lung; and at the dose of 2.81 g·kg-1·d-1 (27g/person) the rats were dead one after another and their kidney, liver and lung were seriously injured. In contrast, Herba Asari decoction and Mahuang-Fuzi-Xixin Tang made no harm to the rats after 60 days drug treament at both doses of 0.625 g·kg-1·d-1(6g/person) and 3.125 g·kg-1·d-1 (30g/person), in other words, there was no meaningful difference in the biochemical indices TPU, CREA and BUN from those of the blank group, and no pathological alterations were recognized in the tissue sections of kidney. However, in Mahuang-Fuzi-Xixin Tang group occured slight injury in the rats'kidney when the dose of Herba Asari was added to 6.25 g·kg-1·d-1 (60g/person). It's showed that in rats the toxic effects of Herba Asari were manifested in the injury of kidney, liver and lung, but not in stomach and intestines. The toxicity of Herba Asari powders was remarkably stronger than those of Herba Asari decoction and Mahuang-Fuzi-Xixin Tang.
3. The establishment of AFLP reaction system for Asarum sieholdii Miq. Adopting improved CTAB method to establish the method for extracting Asarum sieholdii Miq's genome DNA which is suitable for AFLP analysis. It is shown that Asarum sieholdii Miq's genome DNA extracted with 2%CTAB,2mol/LNaCl, 1uLβ-Mercaptoethanol is purity and no degradation basically and is suitable for AFLP analysis. AFLP reaction system of Asarum sieholdii Miq was studied initially and the analysis system which is more suitable for AFLP analysis was obtained. Genomic DNA (500 ng) was digested with 10 U EcoR I and 5 U Mse I restriction enzymes at 37℃for 3 h in a volume of 25μl. In ligation reaction (12.5uL) containing 2.5 pmol EcoR I ligation adapters,25 pmol Mse I ligation adapters and 175 units of T4 DNA ligase, the mixture was incubated overnight at 16℃(12h～16h). Pre-amplification products were diluted 150 folds. The selective PCR reaction mixture (20μl) also included EcoR I selective primer (8 pmol), Mse I selective primer (8 pmol), dNTPs (4 nmol), and Taq DNA polymerase (1 U) in 1×PCR buffer. Prior to electrophoresis, the reaction products (8μl) was mixed with formamide loading buffer (3μl) of 98%(w/v) formamide/10 mM EDTA, pH 8.0/0.1% bromophenol blue/0.1% xylene cyanol (w/v). The loading mixture was denatured at 95℃for 5 min, then cooled on ice immediately, and resolved on 6% denaturing polyacrylamide gels. AFLP gels were silver stained and photographed.
引文
[1]中国植物志.81(1)：54～55.
[2]Kenji S, Kazuo M, Yuko O, Hideki S and Hiroyuki A. New migrated gammacerane triterpenoids from roots of Picris hieracioides subsp. Japonica[J]. Tetrahedron Letters,1989, 30(49):6873～6874.
[3]Kenji S, Kazuo M, Tokuhide L, Hideki S and Hiroyuki A. Three gammacer-16-ene derivatives, novel triterpenoids isolated from roots of Picris hieracioides subsp[J]. Japonica.Tetrahedron Letters,1989,30(37):4977～4980.
[4]Taketo U, Keiichi N, Toshio M and Akira U. Terpenoid glycosides from Picris hieracioides[J]. Phytochemistry,1990,29(9):2947～2951.
[5]Yukio H, Kazuhiro K. Strict requirement of fluctuating temperatures as a reliable gap signal in Picris hieracioides var. japonica seed germination [J].Plant Ecol,2007,193:147～156.
[6]刘永镇,金慧子,刘玉华,等.116种野生植物中抗癌物质的筛选研究[J].中国中医药科技,1998,5(3)：151～154.
[7]王冰,王飞,刘丽,等.兴安毛连菜染色体的核型分析[J].中药材,2003,26(5)：317～318.
[8]张德山.毛连菜的核型报道[J].烟台大学学报(自然科学与工程版),1998,11(3)：232～244.
[9]乌兰图娅,高娃.蒙药材毛连菜的生药鉴定[J].中国民族医药杂志,2008,4(4)：38～39.
[10]徐国钧.生药学.北京：人民卫生出版社,1987.162.
[11]张贵君,金哲雄.常用中药显微鉴定.北京：化学工业出版社,2005：11～20.
[12]康廷国.中药显微鉴定学.北京：中国中医药出版社,2003：37.
[13]高建平,王彦涵,陈道峰.不同产地华中五味子叶表皮结构和导管分子的解剖学特征及其与环境因子的关系[J].西北植物学报,2003,23(5)：716.
[14]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,917.
[15]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,159.
[1]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,159.
[2]邢绍周,李万波.华细辛本草考证及其原产地的品种调查[J].中药实验研究,1981,4：49～51.
[3]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,495～502.
[4]谢伟,陆满文.细辛挥发油的化学与药理作用[J].宁夏医学杂志,1995,17(2)：121～124.
[5]回瑞华,魏倩,盖泽广.辽细辛挥发油化学成分的研究[J].辽宁大学学报自然科学版,1993,20(2)：87～93.
[6]吴艳蓉,贾凌云,高福坤.不同产地和采收期辽细辛挥发油的含量测定[J].沈阳药科大学学报,2006,25(5)：285～288
[7]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,附录XD：57～58.
[8]张峰,王龙星,罗茜,等.气相色谱质谱分析北细辛根和根茎中的挥发性成分[J].色谱,2002,20(5)：467～490.
[9]王朝虹,闵知大.芍药化学成分及药理研究.中草药通讯[J]1978,(9)：7.
[10]刘国卿,蒋莹.几种中药挥发油的急性毒性及对戊巴比妥钠的协同作用[J].中国药科大学学报,1989,20(1)：57.
[11]周慧秋,于滨,乔蜿红,等.甲基丁香酚药理作用研究[J].中医药学报,2000,(2)：79.
[12]周勇,姚三桃,吴琦,等.细辛挥发油抗真菌作用及其有效成分黄樟醚的研究.中医杂志[J]1981,22(12)：62.
[13]刘兴隆,贾波,黄秀深,等.细辛药理研究概况[J].江苏中医药,2005,26(7)：59.
[14]中国医学科学研究所.中药志[M].北京,人民卫生出版社,1986.339.
[15]张峰,付绍平,徐青,等.细辛GC指纹图谱的初步研究[J].中国中药杂志,2004,29(5)：411～413.
[16]周长征,杨春澍,马长华,等.气相色谱法测定细辛挥发油中甲基丁香酚和黄樟醚的含量[J].中国药学杂志,1999,34(12)：836.
[17]杨大峰,闰南,杨春澍,等.五个不同来源细辛挥发油气相色谱-质谱分析.中国中药杂志,1997,22(7)：496.
[18]李照福,苏颖.细辛使用探析,北京中医药[J].2009,28(4)：298.
[19]林大勇,李斌,曲道炜.“亦药亦毒”论细辛[J].辽宁中医药大学学报,2009,11(6)：18.
[20]周祯祥,陈泽斌,李军,等.细辛毒性的研究述评[J].中医药信息,2003,20(6)：30～31.
[21]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,498～499.
[22]周祯祥,杨伟峰,陈泽斌,等.细辛散剂对小鼠重要脏器的病理学影响[J].中医药学刊,2004,22(5)：885.
[23]蔡青.细辛对大鼠肺组织形态学及动脉血气的影响的研究：[D].湖北：湖北中医学院,2006
[24]李晶晶.细辛长期毒性对SD大鼠肝组织形态学及肝功能的影响：[D].湖北：湖北中医学院2007
[25]贾波,曹兰秀,邓中甲,等.细辛毒性及配伍解毒实验研究[J].江西中医学院学报,2006,18(2)：50～51.
[26]宋娜丽,照日格图,却翎,等.细辛的化学成分和生物活性研究概况[J].中国民族民间医药,2008,(4)：50～51.
[27]冯素萍.对细辛药理学作用及应用中有关问题的讨论[J].中医正骨,1999,11(2)：41.
[28]赵书锋.从“细辛不过钱”谈细辛的合理运用[J].光明中医,2009,24(1)：130～131.
[29]梁璇,孙德国.细辛用量研究述要[J].辽宁中医药大学学报,2008,10(11)：203～204.
[30]董霞,范颖.七情配伍与中药复方毒／效之间的内在联系[J].中华中医药学刊,2008,26(3)：618～619.
[31]张宇燕,杨洁红.附子甘草配伍对乌头碱、甘草酸、甘草苷的动态影响[J].中国药学杂志,2009,44(1)：11～14.
[32]周文斌,刘萍.中药鉴定中DNA分子遗产标记技术的应用与展望[J].中国药物应用与监测,2006,(1)：17～20.
[33]葛淑俊,李广敏,马峙英,等.甘草野生种群遗传多样性的AFLP分析[J].中国农业科学,2009,42(1)：47～54.
[34]Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. AFLP:a new technique for DNA fingerprinting. Nucleic Acids Research, 1995,23:4407～4414.
[35]耿盼盼,杨冬之,王勇波,等.华细辛AFLP反应体系的建立和优化[J],中草药,2009,40(4)：625-629.
[36]冯娜,郭美丽,张汉明.红花cDNA-AFLP反应体系的优化研究[J].中草药.2008,39(8)：1242～1246
[37]汪碧涛.细辛在眼科临床的应用,上海中医药杂志,2004,38(1)：32～33.
[1]中国植物志.81(1)：53.
[2]王文房,邱奉同.临沂师范学院学报.2003,25(6)：73.
[3]昆明师范高等专科学校学报2007,29(4)：84～87.
[4]中国植物志.81(1)：54～56.
[5]F. Xavier Sansl,Josep Escarrel, Jacques Lepart and Franz Hopkins. Positive vs. negative interactions in Picris hieracioides L., a mid-successional species of Mediterranean secondary succession [J].Plant Ecology 2002,162:109～122.
[6]Yukio Honda,Kazuhiro Katoh. Strict requirement of fluctuating temperatures as a reliable gap signal in Picris hieracioides var. japonica seed germination [J]. Plant Ecol 2007 193:147～156
[7]中国植物志.81(1)：53～57.
[8]Anake K, Luis MM. Vieira, Jose M. Cardoso and Werner Herza. A eudesmanolide from Picris spinifera[J]. Phytochemistry.1992,31(10):3635～3636.
[9]M. Milovanovic, K. Picuric-Jovanovic.Terpenoids from Picris echoides.Fitoterapia [J].2005, 76 (5):490～492.
[10]Kenji S, Kazuo M, Yuko O, Hideki S and Hiroyuki A. New migrated gammacerane triterpenoids from roots of Picris hieracioides subsp. Japonica. Tetrahedron Letters [J].1989, 30(49):6873～6874.
[11]Kenji Shiojima, Kazuo Masuda, Tokuhide Lin, Hideki Suzuki and Hiroyuki Ageta.Three gammacer-16-ene derivatives, novel triterpenoids isolated from roots of Picris hieracioides subsp. Japonica. Tetrahedron Letters [J].1989,30(37):4977～4980
[12]Taketo U, Keiichi N, Toshio M and Akira U.Terpenoid glycosides from Picris hieracioides. Phytochemistry [J].1990,29(9):2947～2951
[13]刘永镇,金慧子,刘玉华,等.116种野生植物中抗癌物质的筛选研究(Ⅰ)[J].中国中医药科技,1998,5(3)：151～154.
[14]张志勇,席啸虎,康曌,等.日本毛连菜中多糖的含量测定[J].山西医科大学学报,2009,40(4)：329～331.
[15]王冰,王飞,刘丽,等.兴安毛连菜染色体的核型分析[J].中药材,2003,26(5)：317～318.
[16]张德山.毛连菜的核型报道[J].烟台大学学报(自然科学与工程版),1998,11(3)：232～244.
[17]徐炳声,黄少甫,刘仲苓.考察与研究.上海：上海科技文献出版社,1986,1～117
[18]朱海涛,陈吉炎,陈黎,等.白术的生药学研究[J].时珍国医国药,2006,17(6)：1019～1020
[19]乌兰图娅,高娃.蒙药材毛连菜的生药鉴定[J].中国民族医药杂志,2008,4(4)：38～39.
[20]中国植物志.80(1)：37.
[1]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,159.
[2]丁立威.细辛缺口大后市价攀升[J].特种经济动植物,2007,(2)：28～29.
[3]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,497.
[4]程黎晖.细辛的鉴别及药理作用[J].海峡药学,2008,20(5)：68～70.
[5]朱聪明.北细辛、华细辛驯化栽培技术要点[J].河南中医学院学报,2004,19(110)：37.
[6]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,496.
[7]宋娜丽,照日格图,却翎,等.细辛的化学成分和生物活性研究概况[J].中国民族民间医药,2008,(4)：50-51.
[8]谢伟,陆满文.细辛挥发油的化学与药理作用[J].宁夏医学杂志,1995,17(2)：121-124.
[9]吴艳蓉,贾凌云,高福坤.不同产地和采收期辽细辛挥发油的含量测定[J].沈阳药科大学学报,2006,25(5)：285～288
[10]张峰,王龙星,罗茜,等.气相色谱质谱分析北细辛根和根茎中的挥发性成分[J].色谱,2002,20(5)：467～490
[11]杨厚玲,邱琴,陈婷婷,等.不同方法提取的北细辛挥发油的气质联用成分分析[J].中国药学杂志,2007,42(13).
[12]蔡少青,王玉,陈世忠,等.北细辛非挥发性化学成分的研究[J].北京医科大学学报,1996,28(3)：228～230.
[13]李国信,于艳,田原,等.Rp-HPLC法测定细辛根中马兜铃酸A的含量[J].辽宁中医杂志,2005,32(10)：1081～1081.
[14]高钧,孙爽,李德坤,等.高效液相色谱法测定华细辛不同部位马兜铃酸A含量[J].药物分析杂志,2006,26(3)：401-403.
[15]周长征,杨春澍,王中仁.细辛类药材主要种质资源植物遗传多样性及生物系统学研究[J].中药研究与信息,2000,1：56-60.
[16]盛长城.东北细辛和首尔细辛的鉴别方法及其类缘关系研究：[D].吉林：延边大学,2006
[17]黄璐琦,王敏,周长征,等.RAPD方法在细辛类药材鉴别研究中的问题及其对策.药学学报,1998,33(10)：778～784.
[18]张爱华,雷锋杰,陈长宝,等.两种北方药材的RAPD[J].分析安徽农业科学,2008,36(7)：2672～2673.
[19]刘兴隆,贾波,黄绣深,等.细辛药理研究概况[J].江苏中医药,2005,26(7)：59
[20]张瑶,宋志永,王林丽.细辛的药理作用及临床应用[J].2007,16(14)：62～63
[21]陈超.郑卫红.熊素兵,等.细辛与verapamil镇痛协同作用的实验研究.中国药理学通报2003,19(3)：337.
[22]宋娜丽,照日格图,却翎,等.细辛的化学成分和生物活性研究概况[J].中国民族民间医药,2008,(4)：50-51.
[23]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,498～499.
[24]蔡青,周祯祥,陈泽斌,等.细辛长期毒性对大鼠肺组织形态学及动脉血气的影响,湖北中医杂志,2007,29(4)：3～5.
[25]李晶晶.细辛长期毒性对SD大鼠肝组织形态学及肝功能的影响：[D].湖北：湖北中医学院，2007
[26]陈靓,杨伟峰,周祯祥,等.超剂量细辛对SD大鼠肝、肾功能影响的实验研究[J].2009,5(5)：3-5.
[27]甄彦君,刘芳,孙宏勋,等.超剂量应用细辛对小鼠肝、肾影响的实验观察[J].河北中医药学报,2003,18(1)：29～31.
[28]贾波,曹兰秀,邓中甲,等.细辛毒性及配伍解毒实验研究[J].江西中医学院学报,2006,18(2)：50～51.
[29]汪碧涛.细辛在眼科临床的应用,上海中医药杂志,2004,38(1)：32～33.
[30]张海燕,王正君,朱飞奇.细辛在临床中的应用[J].湖北中医杂志,2003,25(1)：38.
[31]张瑶,宋志永,王林丽.细辛的药理作用及临床应用[J].中国药业,2007,16(14)：62.
[2]Kenji S, Kazuo M, Yuko O, Hideki S and Hiroyuki A. New migrated gammacerane triterpenoids from roots of Picris hieracioides subsp. Japonica[J]. Tetrahedron Letters,1989, 30(49):6873～6874.
[3]Kenji S, Kazuo M, Tokuhide L, Hideki S and Hiroyuki A. Three gammacer-16-ene derivatives, novel triterpenoids isolated from roots of Picris hieracioides subsp[J]. Japonica.Tetrahedron Letters,1989,30(37):4977～4980.
[4]Taketo U, Keiichi N, Toshio M and Akira U. Terpenoid glycosides from Picris hieracioides[J]. Phytochemistry,1990,29(9):2947～2951.
[5]Yukio H, Kazuhiro K. Strict requirement of fluctuating temperatures as a reliable gap signal in Picris hieracioides var. japonica seed germination [J].Plant Ecol,2007,193:147～156.
[6]刘永镇,金慧子,刘玉华,等.116种野生植物中抗癌物质的筛选研究[J].中国中医药科技,1998,5(3)：151～154.
[7]王冰,王飞,刘丽,等.兴安毛连菜染色体的核型分析[J].中药材,2003,26(5)：317～318.
[8]张德山.毛连菜的核型报道[J].烟台大学学报(自然科学与工程版),1998,11(3)：232～244.
[9]乌兰图娅,高娃.蒙药材毛连菜的生药鉴定[J].中国民族医药杂志,2008,4(4)：38～39.
[10]徐国钧.生药学.北京：人民卫生出版社,1987.162.
[11]张贵君,金哲雄.常用中药显微鉴定.北京：化学工业出版社,2005：11～20.
[12]康廷国.中药显微鉴定学.北京：中国中医药出版社,2003：37.
[13]高建平,王彦涵,陈道峰.不同产地华中五味子叶表皮结构和导管分子的解剖学特征及其与环境因子的关系[J].西北植物学报,2003,23(5)：716.
[14]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,917.
[15]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,159.
[1]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,159.
[2]邢绍周,李万波.华细辛本草考证及其原产地的品种调查[J].中药实验研究,1981,4：49～51.
[3]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,495～502.
[4]谢伟,陆满文.细辛挥发油的化学与药理作用[J].宁夏医学杂志,1995,17(2)：121～124.
[5]回瑞华,魏倩,盖泽广.辽细辛挥发油化学成分的研究[J].辽宁大学学报自然科学版,1993,20(2)：87～93.
[6]吴艳蓉,贾凌云,高福坤.不同产地和采收期辽细辛挥发油的含量测定[J].沈阳药科大学学报,2006,25(5)：285～288
[7]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,附录XD：57～58.
[8]张峰,王龙星,罗茜,等.气相色谱质谱分析北细辛根和根茎中的挥发性成分[J].色谱,2002,20(5)：467～490.
[9]王朝虹,闵知大.芍药化学成分及药理研究.中草药通讯[J]1978,(9)：7.
[10]刘国卿,蒋莹.几种中药挥发油的急性毒性及对戊巴比妥钠的协同作用[J].中国药科大学学报,1989,20(1)：57.
[11]周慧秋,于滨,乔蜿红,等.甲基丁香酚药理作用研究[J].中医药学报,2000,(2)：79.
[12]周勇,姚三桃,吴琦,等.细辛挥发油抗真菌作用及其有效成分黄樟醚的研究.中医杂志[J]1981,22(12)：62.
[13]刘兴隆,贾波,黄秀深,等.细辛药理研究概况[J].江苏中医药,2005,26(7)：59.
[14]中国医学科学研究所.中药志[M].北京,人民卫生出版社,1986.339.
[15]张峰,付绍平,徐青,等.细辛GC指纹图谱的初步研究[J].中国中药杂志,2004,29(5)：411～413.
[16]周长征,杨春澍,马长华,等.气相色谱法测定细辛挥发油中甲基丁香酚和黄樟醚的含量[J].中国药学杂志,1999,34(12)：836.
[17]杨大峰,闰南,杨春澍,等.五个不同来源细辛挥发油气相色谱-质谱分析.中国中药杂志,1997,22(7)：496.
[18]李照福,苏颖.细辛使用探析,北京中医药[J].2009,28(4)：298.
[19]林大勇,李斌,曲道炜.“亦药亦毒”论细辛[J].辽宁中医药大学学报,2009,11(6)：18.
[20]周祯祥,陈泽斌,李军,等.细辛毒性的研究述评[J].中医药信息,2003,20(6)：30～31.
[21]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,498～499.
[22]周祯祥,杨伟峰,陈泽斌,等.细辛散剂对小鼠重要脏器的病理学影响[J].中医药学刊,2004,22(5)：885.
[23]蔡青.细辛对大鼠肺组织形态学及动脉血气的影响的研究：[D].湖北：湖北中医学院,2006
[24]李晶晶.细辛长期毒性对SD大鼠肝组织形态学及肝功能的影响：[D].湖北：湖北中医学院2007
[25]贾波,曹兰秀,邓中甲,等.细辛毒性及配伍解毒实验研究[J].江西中医学院学报,2006,18(2)：50～51.
[26]宋娜丽,照日格图,却翎,等.细辛的化学成分和生物活性研究概况[J].中国民族民间医药,2008,(4)：50～51.
[27]冯素萍.对细辛药理学作用及应用中有关问题的讨论[J].中医正骨,1999,11(2)：41.
[28]赵书锋.从“细辛不过钱”谈细辛的合理运用[J].光明中医,2009,24(1)：130～131.
[29]梁璇,孙德国.细辛用量研究述要[J].辽宁中医药大学学报,2008,10(11)：203～204.
[30]董霞,范颖.七情配伍与中药复方毒／效之间的内在联系[J].中华中医药学刊,2008,26(3)：618～619.
[31]张宇燕,杨洁红.附子甘草配伍对乌头碱、甘草酸、甘草苷的动态影响[J].中国药学杂志,2009,44(1)：11～14.
[32]周文斌,刘萍.中药鉴定中DNA分子遗产标记技术的应用与展望[J].中国药物应用与监测,2006,(1)：17～20.
[33]葛淑俊,李广敏,马峙英,等.甘草野生种群遗传多样性的AFLP分析[J].中国农业科学,2009,42(1)：47～54.
[34]Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. AFLP:a new technique for DNA fingerprinting. Nucleic Acids Research, 1995,23:4407～4414.
[35]耿盼盼,杨冬之,王勇波,等.华细辛AFLP反应体系的建立和优化[J],中草药,2009,40(4)：625-629.
[36]冯娜,郭美丽,张汉明.红花cDNA-AFLP反应体系的优化研究[J].中草药.2008,39(8)：1242～1246
[37]汪碧涛.细辛在眼科临床的应用,上海中医药杂志,2004,38(1)：32～33.
[1]中国植物志.81(1)：53.
[2]王文房,邱奉同.临沂师范学院学报.2003,25(6)：73.
[3]昆明师范高等专科学校学报2007,29(4)：84～87.
[4]中国植物志.81(1)：54～56.
[5]F. Xavier Sansl,Josep Escarrel, Jacques Lepart and Franz Hopkins. Positive vs. negative interactions in Picris hieracioides L., a mid-successional species of Mediterranean secondary succession [J].Plant Ecology 2002,162:109～122.
[6]Yukio Honda,Kazuhiro Katoh. Strict requirement of fluctuating temperatures as a reliable gap signal in Picris hieracioides var. japonica seed germination [J]. Plant Ecol 2007 193:147～156
[7]中国植物志.81(1)：53～57.
[8]Anake K, Luis MM. Vieira, Jose M. Cardoso and Werner Herza. A eudesmanolide from Picris spinifera[J]. Phytochemistry.1992,31(10):3635～3636.
[9]M. Milovanovic, K. Picuric-Jovanovic.Terpenoids from Picris echoides.Fitoterapia [J].2005, 76 (5):490～492.
[10]Kenji S, Kazuo M, Yuko O, Hideki S and Hiroyuki A. New migrated gammacerane triterpenoids from roots of Picris hieracioides subsp. Japonica. Tetrahedron Letters [J].1989, 30(49):6873～6874.
[11]Kenji Shiojima, Kazuo Masuda, Tokuhide Lin, Hideki Suzuki and Hiroyuki Ageta.Three gammacer-16-ene derivatives, novel triterpenoids isolated from roots of Picris hieracioides subsp. Japonica. Tetrahedron Letters [J].1989,30(37):4977～4980
[12]Taketo U, Keiichi N, Toshio M and Akira U.Terpenoid glycosides from Picris hieracioides. Phytochemistry [J].1990,29(9):2947～2951
[13]刘永镇,金慧子,刘玉华,等.116种野生植物中抗癌物质的筛选研究(Ⅰ)[J].中国中医药科技,1998,5(3)：151～154.
[14]张志勇,席啸虎,康曌,等.日本毛连菜中多糖的含量测定[J].山西医科大学学报,2009,40(4)：329～331.
[15]王冰,王飞,刘丽,等.兴安毛连菜染色体的核型分析[J].中药材,2003,26(5)：317～318.
[16]张德山.毛连菜的核型报道[J].烟台大学学报(自然科学与工程版),1998,11(3)：232～244.
[17]徐炳声,黄少甫,刘仲苓.考察与研究.上海：上海科技文献出版社,1986,1～117
[18]朱海涛,陈吉炎,陈黎,等.白术的生药学研究[J].时珍国医国药,2006,17(6)：1019～1020
[19]乌兰图娅,高娃.蒙药材毛连菜的生药鉴定[J].中国民族医药杂志,2008,4(4)：38～39.
[20]中国植物志.80(1)：37.
[1]国家药典委员会.中国人民共和国药典,第一部.北京：化学工业出版社,2005,159.
[2]丁立威.细辛缺口大后市价攀升[J].特种经济动植物,2007,(2)：28～29.
[3]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,497.
[4]程黎晖.细辛的鉴别及药理作用[J].海峡药学,2008,20(5)：68～70.
[5]朱聪明.北细辛、华细辛驯化栽培技术要点[J].河南中医学院学报,2004,19(110)：37.
[6]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,496.
[7]宋娜丽,照日格图,却翎,等.细辛的化学成分和生物活性研究概况[J].中国民族民间医药,2008,(4)：50-51.
[8]谢伟,陆满文.细辛挥发油的化学与药理作用[J].宁夏医学杂志,1995,17(2)：121-124.
[9]吴艳蓉,贾凌云,高福坤.不同产地和采收期辽细辛挥发油的含量测定[J].沈阳药科大学学报,2006,25(5)：285～288
[10]张峰,王龙星,罗茜,等.气相色谱质谱分析北细辛根和根茎中的挥发性成分[J].色谱,2002,20(5)：467～490
[11]杨厚玲,邱琴,陈婷婷,等.不同方法提取的北细辛挥发油的气质联用成分分析[J].中国药学杂志,2007,42(13).
[12]蔡少青,王玉,陈世忠,等.北细辛非挥发性化学成分的研究[J].北京医科大学学报,1996,28(3)：228～230.
[13]李国信,于艳,田原,等.Rp-HPLC法测定细辛根中马兜铃酸A的含量[J].辽宁中医杂志,2005,32(10)：1081～1081.
[14]高钧,孙爽,李德坤,等.高效液相色谱法测定华细辛不同部位马兜铃酸A含量[J].药物分析杂志,2006,26(3)：401-403.
[15]周长征,杨春澍,王中仁.细辛类药材主要种质资源植物遗传多样性及生物系统学研究[J].中药研究与信息,2000,1：56-60.
[16]盛长城.东北细辛和首尔细辛的鉴别方法及其类缘关系研究：[D].吉林：延边大学,2006
[17]黄璐琦,王敏,周长征,等.RAPD方法在细辛类药材鉴别研究中的问题及其对策.药学学报,1998,33(10)：778～784.
[18]张爱华,雷锋杰,陈长宝,等.两种北方药材的RAPD[J].分析安徽农业科学,2008,36(7)：2672～2673.
[19]刘兴隆,贾波,黄绣深,等.细辛药理研究概况[J].江苏中医药,2005,26(7)：59
[20]张瑶,宋志永,王林丽.细辛的药理作用及临床应用[J].2007,16(14)：62～63
[21]陈超.郑卫红.熊素兵,等.细辛与verapamil镇痛协同作用的实验研究.中国药理学通报2003,19(3)：337.
[22]宋娜丽,照日格图,却翎,等.细辛的化学成分和生物活性研究概况[J].中国民族民间医药,2008,(4)：50-51.
[23]国家中医药管理局《中华本草》编委会.中华本草.上海：上海科学技术出版社,1999,498～499.
[24]蔡青,周祯祥,陈泽斌,等.细辛长期毒性对大鼠肺组织形态学及动脉血气的影响,湖北中医杂志,2007,29(4)：3～5.
[25]李晶晶.细辛长期毒性对SD大鼠肝组织形态学及肝功能的影响：[D].湖北：湖北中医学院，2007
[26]陈靓,杨伟峰,周祯祥,等.超剂量细辛对SD大鼠肝、肾功能影响的实验研究[J].2009,5(5)：3-5.
[27]甄彦君,刘芳,孙宏勋,等.超剂量应用细辛对小鼠肝、肾影响的实验观察[J].河北中医药学报,2003,18(1)：29～31.
[28]贾波,曹兰秀,邓中甲,等.细辛毒性及配伍解毒实验研究[J].江西中医学院学报,2006,18(2)：50～51.
[29]汪碧涛.细辛在眼科临床的应用,上海中医药杂志,2004,38(1)：32～33.
[30]张海燕,王正君,朱飞奇.细辛在临床中的应用[J].湖北中医杂志,2003,25(1)：38.
[31]张瑶,宋志永,王林丽.细辛的药理作用及临床应用[J].中国药业,2007,16(14)：62.