【作者】 王彩红；

【导师】 聂凤禔；

【作者基本信息】 河北医科大学 ， 药物分析学， 2007， 硕士

【摘要】 丹参为唇形科(Labiatae)鼠尾草属(Salvia)植物丹参(Salvia miltiorrhiza Bge.)的根及根茎,此外,在实际应用中还存在一些地方代用品与混淆品,如甘西鼠尾(Salvia przewalskii Maxim),荫生鼠尾(Salvia umbratica Hance),雪见草(Salvia plebeia R. Br)及多种同属植物。近年来,主产地栽培丹参逐步成为药材的主要来源,但是,种子市场基源品种混乱,而且同名异物、同物异名现象严重。几种鼠尾草属植物的种子在外部形态上非常相近,难以凭感官宏观鉴别。为确保丹参药材质量的稳定、可控,在规模化、规范化种植中首先要保证丹参种源纯化、优良。本实验从外观性状、显微特征、理化性质等方面,并利用细胞生物学、分子生物学技术及超微技术对鼠尾草属几种植物的种子进行综合鉴别研究。既有快速、简便、实用、满足基层需要的方法又有新技术与传统中药鉴定技术交叉融合的深层次鉴别研究,也是我省制定中药种子标准前重要的应用基础研究。第一部分性状、显微、理化鉴别目的:应用快速、简便、实用的方法,建立丹参、甘西鼠尾、荫生鼠尾、雪见草种子的外观性状、显微特征、超微特征、理化性质方面的综合鉴别信息指征。方法:1外观性状观察。2种皮脱色表面片制备:(1)脱色:取种皮切成12 mm小块浸入水合氯醛(1:1)溶液中。(2)压片观察。3石蜡横切片制备:(1)取材和固定:种子F.A.A固定液固定一周以上。(2)脱水:依次用浓度为70 %、80 %、90 %、95 %的乙醇溶液、无水乙醇进行脱水,每隔2~3 h调换溶液。(3)透明:二甲苯:无水乙醇(1:1)浸种1~2 h后,转入二甲苯中至种子透明为止。(4)浸蜡:65℃恒温箱中进行,半小时调蜡一次,至种子完全浸透。(5)包埋和切片:石蜡包埋,切片厚度12μm。(6)贴片和融蜡。(7)染色:番红-固绿双重染色。(8)脱水与封藏。4种皮扫描电镜观察:(1)清洗:取种子用50 %乙醇超声清洗5 min。(2)脱水:50 %、60 %、70 %、80 %、90 %、100 %乙醇逐级脱水,每一级脱水两次,每次30 min,其间不断震荡清洗。(3)固定:戊二醛固定30 min。(4)电镜观察:真空喷金后,进行观察。5紫外扫描图谱特征:(1)取过40目筛的种子粗粉各1.0 g用石油醚索氏提取3 h,制备供试液A;挥干石油醚用乙酸乙酯索氏提取3 h,然后挥干乙酸乙酯用甲醇索氏提取3 h,制备供试液B。(2)扫描波长为200~400 nm的紫外吸收图谱。(3)图谱分析。6薄层色谱鉴别:(1)取一定量种子粉末用石油醚索氏提取3 h制备供试液C,挥干石油醚用乙酸乙酯索氏提取3 h,制备供试液D,供点样;(2)点样,展开。(3)显色、观察、照相。7高效液相鉴别:提取:甲醇索氏提取样品中的成分;色谱条件:选用合适的色谱柱,调整流动相的组成、配比、pH、检测波长及流速,使样品中所含成分色谱峰的分离度符合测定要求。结果:1宏观性状:四种种子均为纺锤状,丹参、甘西鼠尾、荫生鼠尾种子稍大,雪见草种子稍小,整体性状相似不易区分。2种皮脱色表面片:四种种子内外表皮细胞的垂周壁形状、增厚程度和后含物方面有一定的区别。3石蜡横切片:四种种子的表面凸起,角质层次级凸起特征、细胞层数和形状上都有明显差别。4扫描电镜观察:四种种子的种脐特征、种脐周围及侧面种皮纹饰区别明显。5紫外扫描图谱:乙酸乙酯和甲醇提取部分的吸收峰数目和吸收峰位置具有差别。6薄层色谱:几种供试液丹参、甘西鼠尾、荫生鼠尾、雪见草种子的薄层色谱具有差别,可用于鉴别。7高效液相色谱:四种种子的色谱峰既有共有峰,又有特征峰,能有效的鉴别。结论:丹参、甘西鼠尾、荫生鼠尾和雪见草的种子在外观性状、显微特征、理化性质存在差异,可用于鉴别。方法快速、简便、实用。第二部分细胞生物学及分子生物学特征研究目的:利用染色体组型分析及种子蛋白质PAGE和SDS-PAGE技术研究药用丹参品种之间及鼠尾草属几种植物种子的综合鉴别信息指征,从细胞水平及分子水平鉴别真伪,并为丹参的品种筛选提供依据。方法:1根尖体细胞染色体标本片制备:(1)预处理:种子25℃下萌发,根尖长0.5~1.0 cm时切取,蒸馏水4℃处理24 h。(2)固定:卡诺固定液(无水乙醇:冰醋酸3:1)固定1.0~1.5 h。(3)解离:95%乙醇:浓盐酸(1:1)解离10~15 min。(4)后低渗:重蒸水低渗,时间8~15 min。(5)染色:改良石炭酸品红液染色。(6)封片:冰冻揭盖片,中性树胶封片。(7)显微照相、计数、测量。2聚丙烯酰胺凝胶电泳(PAGE)图谱特征研究:(1)制备供试品溶液。(2)制备分离胶和浓缩胶。(3)电泳:恒定电压120~180 V。(4)染色和洗脱:考马斯亮蓝(R-250)染色,脱色液洗脱至背景清晰为止。(5)结果分析:绘图并测定特征谱带泳动率。3 SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)图谱特征研究:(1)制备供试品溶液。(2)制备分离胶和浓缩胶。(3)电泳:恒定电压80~120 V。(4)染色和洗脱:考马斯亮蓝蛋白胶快速染色液染色,脱色液洗脱至背景清晰为止。(5)结果分析:绘图并测定蛋白分子量。结果:1根尖体细胞染色体组型特征:丹参染色体总数目为2n=16,染色体总长度26.25μm,平均长度1.64μm,变异倍数为1.72,臂比值变化范围为1.11~1.74;甘西鼠尾染色体总数目为2n=32,染色体总长度42.88μm,平均长度1.34μm,变异倍数为1.60,臂比值变化范围为1.06~1.82。荫生鼠尾染色体总数目为2n=16,染色体总长度24.94μm,平均长度1.56μm,变异倍数为2.03,臂比值变化范围为1.00~2.10;雪见草染色体总数目为2n=16,染色体总长度14.34μm,平均长度0.90μm,变异倍数为1.69,臂比值变化范围为1.00~2.09。2聚丙烯酰胺凝胶电泳(PAGE)图谱特征:丹参、甘西鼠尾、荫生鼠尾、雪见草种子的谱带数目和位置不同。丹参图谱中一级带2条,二级带2条;甘西鼠尾图谱中一级带2条,二级带1条,三级带1条;荫生鼠尾图谱中一级带2条,二级带3条,三级带2条;雪见草图谱中一级带1条,二级带2条,三级带1条。鼠尾草属几种植物种子之间有明显差别。3 SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)图谱特征:丹参、甘西鼠尾、荫生鼠尾、雪见草种子的蛋白分子量不同。丹参图谱中蛋白分子量为24.8、30.4、37.3、71.0、89.7、143.2 KD;甘西鼠尾图谱中蛋白分子量为20.2、22.7、24.8、30.4、37.3、71.0、89.7、143.2 KD;荫生鼠尾图谱中蛋白分子量为20.2、22.7、24.8、27.9、30.4、37.3、71.0、89.7、143.2 KD;雪见草图谱中蛋白分子量为24.8、29.6、34.2、37.3、54.6、79.8、89.7、143.2 KD。鼠尾草属几种植物种子之间有明显差别。结论:应用分子生物学技术进行种子鉴别研究,消除了种子性状特征易受外界环境影响的特点,可以较根本的揭示丹参及其混淆品之间的差异。不但为丹参种源筛选提供依据,也为鼠草属的物种研究提供了一定的方法。更多还原

【Abstract】 Danshen is root and rhizome of Labiatae plants Salvia miltiorrhiza Bge. There are many other kinds of local substitute of Danshen in Hebei, such as Salvia przewalskii Maxim, Salvia umbratica Hance, Salvia plebeia R. Br. At present the medicinal substances source mainly comes from cultivation, but the origin of species is very confusion. It’s necessary to identify seed of Danshen and its adulterants. Since the four kinds of plant belong to the same species, their seeds are similar in exterior morphous characters. We identify them by their Macroscopic Characters, Microscopical Features, Physical-chemical Characters and by molecular biology technology. Through experiments we can prevent planting Adulterants and choose better variety of Danshen. The methods are established. It is not only a basic applying but also a deep study. We can establish standards for identification of Danshen.Part 1 Identification from Macroscopic Characters, Micro scopical Features and Physical-chemical CharactersObjective: To establish identification standards for Salvia miltiorrhiza Bge., Salvia przewalskii Maxim, Salvia umbratica Hance, Salvia plebeia R. Br. from their appearance characters, microscopical features and physical-chemical characters with fast, simple, feasible methods.Methods: 1 Macroscopic identification. 2 Preparation of discolored surface section: (1) Cut samples into 1 mm to 2 mm long and put them into choral hydrate solution. (2) Tabletting and observation. 3 Preparation of the paraffin transverse sections: (1) Selected samples and fix them with F.A.A. fixative liquid. (2) Dehydration: The concentration of ethanol solutions were 70 %, 80 %, 90 %, 95 %, 100 %, and changed solutions at every 2 h to 3 h. (3) Transparention: Used mixture of ethanol and xylene (1:1). Changed solutions at every 2 h to 3 h. (4) Put samples in the constant temperature oven with 65℃and added paraffin every 30 min. (5) Embedment and slice up with sliding microtome, the thickness was 12μm. (6) Pasted the slices of samples and melted paraffin. (7) Dyeing: Use Safranine and Fast Green double dyeing. (8) Dehydrated and sealed the slices with neutral gum. 4 Seed coat observation with scanning electron microscope. (1) Cleaning: Seeds were cleaned with 50 % ethanol and ultrasonic cleaning instrument. (2) Dehydration: The concentration of ethanol solutions were 50 %, 60 %, 70 %, 80 %, 90 %, 100 % and changed solutions at every 30 min. Double dehydration in every grade. (3) Fixation: fix materials with Glutaral in 30 min. (4) Observation. 5 Study on the characters of ultraviolet spectrum: Prepare solutions extracted with petroleum benzene in apparatus, Soxhlet’s for 3 h, then extracted with acetic ether in apparatus, Soxhlet’s for 3 h, last extracted with methanol in apparatus, Soxhlet’s for 3 h. (2) Scanning wavelength was 200~400 nm. (3) Results analysis. 6 Identification by TLC characters: (1) Prepare solutions extracted with petroleum benzene in apparatus, Soxhlet’s for 3 h, then extracted with acetic ether in apparatus, Soxhlet’s for 3 h. (2) Dropped sample solutions on the lamella and spread them out. (3) Coloration, observation and took photos. 7 HPLC: extracted principle from seeds by methanol in apparatus, Soxhlet’s. Chose suitable chromatographic column, adjusted the composition, pH and proportion of mobile phase, flow rate and wavelength to separate the component peaks well.Results: 1 Macroscopic identification: The five kinds of seeds were nealy cambiform, seeds of Salvia miltiorrhiza Bge., Salvia umbratica Hance and Salvia przewlskii Maxim, were a little bigger than Salvia plebeia R. Br.. But there had not obvious differences between the four seeds. 2 Preparation of discolored surface section: There were obvious differences in exterior and interior view of seed coat, cellwall of thickening and content of the four seeds. 3 Preparation of the paraffin transverse sections: The seed had obvious difference in heave, seedcase layers and cutin. 4 Seed coat observation with scanning electron microscope: There were differences in characters of hila, circumjacent area of hila and side surface of four seeds when observed with scanning electron microscope. 5 Ultraviolet scanning spectrums identification: The number and position of absorbing peaks and its absorbability were different in the four kinds of acetic ether and methanol extractive. 6 Identification by the TLC characters: There were differences in the seeds of Salvia miltiorrhiza Bge., Salvia umbratica Hance and Salvia przewlskii Maxim, Salvia plebeia R. Br.by TLC. 7 HPLC: There were owned by both absorption band and characteristic absorption band between the four kinds of seed.Conclusion: From differences of appearance characters, microscopical features, physical-chemical characters and ultrastructure of seeds, we can identify Salvia miltiorrhiza Bge., Salvia przewalskii Maxim, Salvia umbratica Hance and Salvia plebeia R. Br. seeds. The methods were simple, convenient, feasible and accurate .Part 2 Cell biology and Molecular Biology Studies Objective: Using chromosome analysis technology and protein PAGE and SDS-PAGE technology distinguish Danshen and its adulterants.Methods: 1 Preparation of root-tip cell chromosome sections: (1) Seeds were sprouted at 25℃, root-tip with 0.5 cm to 1.0 cm long were cut down and treated in distilled water at 4℃for 24 hours. (2) Fixed the root-tips with Carnoy’s fixative for 1.0~1.5 h. (3) Decomposed the root-tips with 95% ethanol: hydrochloric acid (1:1) in 10~15 min. (4) Dropped the root-tips into double-distilled water 8~15 min. (5) Dyed the root-tips in alkaline Carmine solution. (6) Pressed the root-tips into sections, unclosed the cover at frozen condition and sealed with neutral gum. (7) Took photos with microscope, counted the number and measured the length of chromosome. 2 Study on the polyacrylamide gel electrophoresis (PAGE) features. (1) To prepare sample solutions. (2) To prepare separation gel and concentrating gel. (3) Electrophoresis: constant voltage was 120~180 V. (4) When electrophoresis was over, dyed the gel with coomassie brilliant blue R-250 and decolored the gel to clear background. (5) Result analysis: Took photos and drew pictures of the tapes on gel and measured the move rates of characteristic tapes at the same time. 3 Study on the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) features. (1) To prepare sample solutions. (2) To prepare separation gel and concentrating gel. (3) Electrophoresis: constant voltage was 80~120 V. (4) When electrophoresis was over, fast dyed the albumin gel with coomassie brilliant blue and decolored the gel to clear background. (5) Result analysis: Took photos and drew pictures of the tapes on gel and measured the molecular mass of protein of characteristic tapes at the same time.Results: 1 The differences of root-tip body cell karyotye: Salvia miltiorrhiza Bge. seed’s chromosomal total number was 16, length in all was 26.25μm, average length was 1.64μm, variation coefficient was 1.72; brachi-relative value was 1.11~1.74; Salvia przewalskii Maxim seed’s chromosomal total number was 32, length in all was 42.88μm, average length was 1.34μm, variation coefficient was 1.60; brachi-relative value was 1.06~1.82; Salvia umbratica Hance seed’s chromosomal total number was 16, length in all was 24.94μm, average length was 1.56μm, variation coefficient was 2.03; brachi-relative value was 1.00~2.10; Salvia plebeian R. Br seed’s chromosomal total number was 16, length in all was 14.34μm, average length was 0.90μm, variation coefficient was 1.69; brachi-relative value was 1.00~2.09. 2 PAGE feature: From the photos we can find that Salvia miltiorrhiza Bge. seed had 2 primary tapes and 2 secondary tapes; Salvia przewalskii Maxim seed had 2 primary tapes , 1 secondary tape and 1 thirdly tape; Salvia umbratica Hance seed had 2 primary tapes, 3 secondary tapes and 2 thirdly tapes; Salvia plebeia R. Br seed had 1 primary tape, 2 secondary tapes and 1 thirdly tape. There were different tapes between the four kinds of Danshen seeds which can be used in identification. 3 SDS-PAGE feature: From the photos we can find that the molecular mass of protein of Salvia miltiorrhiza Bge. seed were 24.8, 30.4, 37.3, 71.0, 89.7, 143.2 KD; the molecular mass of protein of Salvia przewalskii Maxim seed were 20.2, 22.7, 24.8, 30.4, 37.3, 71.0, 89.7, 143.2 KD; the molecular mass of protein of Salvia umbratica Hance seed were 20.2, 22.7, 24.8, 27.9, 30.4, 37.3, 71.0, 89.7, 143.2 KD; the molecular mass of protein of Salvia plebeia R. Br seed were 24.8, 29.6, 34.2, 37.3, 54.6, 79.8, 89.7, 143.2 KD. There were different the molecular mass of protein between the four kinds of seeds which can be used in identification.Conclusion: From studying on the root tip body cell chromosome number and karyotype characters, PAGE tape feature and SDS-PAGE tape feature, we can distinguish Danshen and its adulterants from cell and molecular level. It is helpful for study of species of Salvia.更多还原