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【基本知识】
1.天然药物鉴定的依据 《中华人民共和国药品管理法》规定：“药品必须符合国家药品标准，中药材饮片按照国家药品标准炮制，国家药品没有规定的，必须按照省、自治区、直辖市人民政府药品监督管理部门制定的炮制规范炮制。国务院药品监督管理部门颁布的《中华人民共和国药典》和药品标准为国家药品标准。”因此天然药物鉴定工作的依据是国家及各级药品标准。
2.天然药物真实性鉴定方法 天然药物的真实性鉴定（authenticity identif ication）即基原鉴定（original identif ication），是根据该天然药物的性状、显微、理化等鉴别特征，鉴定原植（动）物基原学名，其主要包括性状鉴定(macroscopic identif ication)、显微鉴定(microscopic identif ication)、理化鉴定（physical-chemical identif ication）和DNA分子遗传标记技术（DNA molecular genetic marker technologies）等。本实验涉及前3种方法，也是天然药物真实性鉴定的主要方法。
（1） 天然药物性状鉴定 性状鉴定是通过眼观、手摸、鼻闻、口尝等比较简便的鉴定方法，通过观察天然药物的形状、大小、颜色、表面、质地、横切面、气、味、水试和火试来鉴定天然药物的真假优劣的方法。
（2） 天然药物显微鉴定 显微鉴定是利用显微技术观察天然药物的细胞、内含物和颗粒物质的形状特征及性质来达到鉴定的目的。对天然药物组织构造和粉末特征进行分析鉴定，以确定其真伪、纯度、品质的一种鉴别方法。显微鉴定不但可以用于植物、动物和矿物药类天然药物的鉴定，也可用于以其为原材料投药所制成的中成药制剂的鉴定。
（3） 天然药物理化鉴定 理化鉴定是利用物理和化学方法，对天然药物中所含主要化学成分、指标性成分或有效成分进行定性分析的方法。主要的理化鉴别方法是色谱法，如纸色谱、薄层色谱、柱色谱、气相色谱、高效液相色谱等。其中薄层色谱法是较常用的方法，其原理是将适当的吸附剂或载体涂布于玻璃板、塑料或铝片上，使成一均匀薄层，点样、展开，使供试品所含成分分离，所得色谱图与适宜的对照物（对照品或对照药材）按同法在同一薄层板上所得的色谱图做对比，用以进行天然药物某些化学成分有无的鉴别、检查或含量测定的方法。
【目的要求】
1.掌握天然药物性状鉴别技术和方法。
2.掌握天然药物及其制剂显微鉴别技术和方法。
3.掌握天然药物及其制剂薄层色谱鉴别技术和方法。
4.通过上述鉴别技术和方法的综合运用，正确判断几种未知天然药物的真伪优劣，选择正品优质药材进行后续实验。
【仪器试剂】
仪器：生物显微镜、镊子、载玻片、盖玻片、酒精灯、单（双）面刀片、紫外分析灯（254nm和365nm）、硅胶G板、硅胶GF254板、层析缸、天平、锥形瓶、超声振荡仪、量筒、点样毛细管等。
试剂：水合氯醛试液、蒸馏水、稀甘油、甲醇、乙酸乙酯、氯仿。
【实验材料】 待鉴定天然药物及对应药材粉末各5份，分别为研究用正品、混乱品种、劣质不合格正品、伪品1和伪品2；该原料药制剂；该原料药制剂对应的质量检验对照品和对照药材。以葛根为例，5份未知药材分别为葛根（豆科植物野葛 Pueraria lobata (Willd.)Ohwi的干燥根）、粉葛（混乱品种：豆科植物甘葛藤 Pueraria thomsonii Benth.的干燥根）、劣质葛根、未知生药1（伪品）、未知生药2（伪品）；葛根制剂愈风宁心片；葛根素对照品、葛根对照药材。
【实验内容】
1.葛根及粉葛的性状鉴定 观察并记录（文字记录及数码采集图像）药材性状特征，鉴定其是否符合2015年版《中华人民共和国药典》相关项下标准。
（1）葛根的性状鉴定 完整的块根呈圆柱形。表面褐色，具纵皱纹，可见皮孔及须根痕。质坚实。饮片呈纵切的长方形厚片或小方块，长5～35cm，厚0.5～1cm。外皮淡棕色，有纵皱纹，粗糙。切面黄白色，纹理不明显。质韧，纤维性强。气微，味微甜。（图3—1）
图3—1 葛根药材图
（2）粉葛的性状鉴定 呈圆柱形、类纺锤形或半圆柱形，长12～15cm，直径4～8cm；有的为纵切或斜切的厚片，大小不一。表面黄白色或淡棕色，未去外皮的呈灰棕色。体重，质硬，富粉性，横切面可见由纤维形成的浅棕色同心性环纹，纵切面可见由纤维形成的数条纵纹。气微，味微甜。（图3—2）
图3—2 粉葛药材图
2.葛根、粉葛和愈风宁心片的显微鉴定 观察并记录（文字记录及数码采集图像）原料药粉末显微特征，鉴定其是否符合2015年版《中华人民共和国药典》相关项下标准，鉴定其制剂愈风宁心片是否符合该药品质量标准。
（1）葛根的显微特征 粉末淡棕色。淀粉粒单粒球形，直径3～37μm，脐点点状、裂缝状或星状；复粒由2～10分粒组成。纤维多成束，壁厚，木化，周围细胞大多含草酸钙方晶，形成晶纤维，含晶细胞壁木化增厚。石细胞少见，类圆形或多角形，直径38～70μm。具缘纹孔导管较大，具缘纹孔六角形或椭圆形，排列极为紧密。（图3—3）
（2）粉葛的显微特征 粉末黄白色。淀粉粒甚多，单粒少见，圆球形，直径8～15μm，脐点隐约可见；复粒多，由2～20分粒组成。纤维多成束，壁厚，木化，周围细胞大多含草酸钙方晶，形成晶纤维，含晶细胞壁木化增厚。石细胞少见，类圆形或多角形，直径25～43μm。具缘纹孔导管较大，具缘纹孔圆形，排列极为紧密。（图3—4）
图3—3 葛根粉末显微鉴别图
1.淀粉粒 2.晶纤维
图3—4 粉葛粉末显微鉴别图
1.淀粉粒 2.晶纤维
3.葛根、粉葛及其制剂的TLC鉴别
（1）葛根素对照品溶液、天然药物原料药及其制剂供试品溶液的制备 ①对照品溶液制备：取葛根素对照品，加甲醇制成每1mL含1mg的溶液，作为对照品溶液；另取葛根对照药材0.8g，同法制成的对照药材溶液。②药材供试品溶液制备：天然药物原料药（5个样品）0.1g加甲醇10mL，超声处理20分钟，将上清液滤出，药液置蒸发皿中水浴上蒸干，残渣加甲醇1mL使溶解，作为供试品溶液。③制剂供试品制备：取愈风宁心片2片，除去包衣，研细，加醋酸乙酯20mL，超声处理20分钟，滤过，滤液蒸干，残渣加甲醇1mL使溶解，作为供试品溶液。
（2）点样 照2015年版《中华人民共和国药典》四部薄层色谱法(通则0502)试验，吸取上述8种溶液各10 μl，分别点于同一硅胶G薄层板（或硅胶GF254薄层板）上，使成条状。
（3）展开 以三氯甲烷—甲醇—水(7:2.5:0.25)为展开剂，展开，取出，晾干。
（4）结果观察 置紫外光灯(365nm和254nm)下检视。供试品色谱中，在与对照药材色谱和对照品色谱相应的位置上，显相同颜色的荧光条斑或暗斑。记录（数码照相）实验结果。葛根素对照品、葛根对照药材及样品的薄层色谱见图（图3—5）。
图3—5 葛根薄层色谱图
S.葛根素 2.葛根对照药材 3—5.葛根市售药材
【结论及分析】 根据上述实验结果分析，首先确定正品葛根，再选出符合《中华人民共和国药典》的优质葛根进行后续实验。
【教学方式】 教师讲授和演示及学生操作。
【注意事项】 实验记录要求：药材粉末显微鉴别特征显微拍照并测量相应长度、直径、厚度等，天然药物原料药材、TLC结果要求数码拍照并记录实验结果。
【思考题】
1.天然药物的性状鉴定、显微鉴定和TLC鉴定3种鉴定方法各有什么优点？什么情况下适用？
2.粉末进行显微鉴定制片时如何选择透化剂？
【知识链接】 葛根和粉葛的全项检测参考2015年版《中华人民共和国药典》收载的该品种质量标准。
Drug Administration Law of the People’s Republic of China regulates:“Drugs must comply with the national drug standards; Slices of natural medicines should be processed in accordance with the national standards,in the absence of provisions in national standards,must be processed according to the processing specif ications promulgated by the pharmaceutical supervisory and administrative department of provinces,autonomous regions and municipalities directly under the central government. Pharmacopoeia of the People’s Republic of China and drug standards promulgated by the pharmaceutical supervisory and administrative department under the state council are the national drug standards.” Hence identif ication of natural medicines should be based on the national drug standards at all levels.
Authenticity identif ication,also known as original identif ication of natural medicines,is to identify the scientif ic name of the original plant (or animal) based on the macroscopic,microscopic,physicochemical characters of natural medicines,which includes the use of macroscopic identif ication,microscopic identif ication,physiochemical identif ication,and DNA molecular genetic marker technologies.This experiment involves in the first three methods,which are also the principal methods for authenticity identif ication of natural medicines.
1.1.2.1 Macroscopic identification of natural medicines Macroscopic identif ication is a method for the identif ication of authenticity and superiority/inferiority of natural medicines simply through observing,touching,smelling and tasting,which includes observing the shape,size,color,surface features,texture,and transverse section,smelling odor,tasting,and conducting water test and fire test (to observe certain characteristics such as the variation of color,sinking or floating in water,‘bursting voice’ and the color of the flame on burning the natural medicines).
1.1.2.2 Microscopic identification of natural medicines Microscopic identif ication is a method for the identif ication of natural medicines by observing the shape and properties of cell,cell contents and particles contained in natural medicines with the application of microtechnique.To be specif ic,it is a method for the identif ication of the authenticity,purity and quality of natural medicines by analyzing the tissue structures and powder features.Besides the identif ication of natural medicines from categories of plant,animal and mineral,microscopic identif ication can be applied for the identif ication of Chinese patent preparations which are made of natural medicines.
1.1.2.3 Physiochemical identification of natural medicines Physiochemical identif ication is a general term for physical and chemical methods applied in the qualitative analysis of main constituents,marker compounds or active components contained in natural medicines.Chromatography in particular,can be classif ied into paper chromatography (PC),thin-layer chromatography (TLC),column chromatography (CC),gas chromatography (GC),high performance liquid chromatography (HPLC), etc .Among them,thin layer chromatography (TLC)is the most commonly used method.Thin-layer chromatography (TLC) is a separation technique in which test solutions are deposited in a uniform thin layer on a support (plate) and developed by the mobile phase in a chromatographic chamber to separate the components of the substance being examined.The chromatogram thus obtained is then compared with that obtained in the same way of the suitable reference solutions.
1.2.1 To master the basic techniques and operational method of macroscopic identif ication of natural medicines.
1.2.2 To master the basic techniques and operational method of microscopic identif ication of natural medicines and their preparations.
1.2.3 To master the basic techniques and operational procedure of the thin-layer chromatography for natural medicines and their preparations.
1.2.4 To correctly identify the authenticity and quality of several unknown natural medicines and select natural medicines of high quality for the subsequent experiments by comprehensive application of identif ication techniques and methods mentioned above.
Instruments:Optical microscope,tweezers,glass slide,coverslips,alcohol lamp,singleside/double-side blades,UV lamp (254nm and 365nm),silica gel G plate,silica GF 254 plate,chromatography cylinder,balance,conical flask,ultrasonic vibration machine,measuring cylinder,capillary tubes.
Reagents:chloral hydrate solution,distilled water,diluted glycerol,methanol,ethyl acetate,chloroform.
Natural medicines and their corresponding powders five each including authentic natural medicine,confused natural medicine,sub-standard natural medicine,fake samples 1 and 2 respectively; Preparations of natural medicines under test; the corresponding reference substances and the reference drugs for quality control.Taking Puerariae Lobatae Radix for an example,samples are superior Puerariae Lobatae Radix (Kudzuvine Root is the dried root of Pueraria lobata (Willd.) Ohwi (Fam.Leguminosae)),Puerariae Thomsonii Radix (Thomoson Kudzuvine Root is the dried root of Pueraria thomsonii Benth.(Fam.Leguminosae)),sub-standard Puerariae Lobatae Radix,unknown fake samples 1 and 2; Yufeng Ningxin Tablets; Puerarin (reference substance),Puerariae Lobatae Radix (reference drug).
Observe and record (written records and digital images) macroscopic characteristics of natural medicines,and identify whether they conform to those recorded in the 2015 edition of Chinese Pharmacopoeia .
1.5.1.1 Macroscopic identif ication of Puerariae Lobatae Radix The intact tuberous root is cylindrical,having brown outer bark,longitudinal wrinkles,skin pore,fibrous scars and solid texture.It is often cut longitudinally into rectangular thick slices or small square pieces,5—35cm long and 0.5—1cm thick.The outer bark is pale brown and rough with longitudinal wrinkles.The cut surface is yellowish-white with indistinct striations.It has pliable texture,which is strongly fibrous,slight odor and slight sweat taste.(Figure.3—1)
1.5.1.2 Macroscopic identif ication of Puerariae Thomsonii Radix It has cylindrical,sub-fusiform or semi-cylindrical shape,which is 12—15cm in length and 4—8cm in diameter.It can be cut either longitudinally or obliquely into thick slices with varied sizes.The external surface is yellowish-white or pale brown,or grayish-brown when unpeeled.It is heavy in mass,hard in texture and starchy with pale brown fiber-formed concentric rings on the transverse section and several fiber-formed longitudinal striations on the longitudinal section.It has slight odor and slight sweat taste.(Figure.3—2)
Figure.3—1 Picture
Figure.3—2 Picture
Observe and record (written records and digital images) microscopic characteristics of natural medicines and their preparation (Yufeng Ningxin Tablet),and identify whether they conform to those recorded in the 2015 edition of Chinese Pharmacopoeia .
1.5.2.1 Microscopic identif ication of Puerariae Lobatae Radix Powder is pale brown.The single starch granules is spheroidal shaped,3—37μm in diameter,and hilum can be pointed,cleft or stellate.Compound granule is made from 2—10 single granules.Fibers are mostly in bundles,walls are thickened and lignif ied,parenchymatous cells surrounded often contain prisms of calcium oxalate which forms crystal fibers,and walls containing crystal fibers are lignif ied and thickened.Stone cells are infrequently visible,either semi-rounded or polygonal shaped,with 38—70μm in diameter.Bordered pitted vessels relatively larger,either in the shape of elliptical,and arranged in a very dense way (Figure.3—3).
1.5.2.2 Microscopic identif ication of Puerariae Thomsonii Radix Powder is yellowishwhite.Starch granules are abundant while single granule is rarely seen.It is in the shape of spheroid with 8—15μm in diameter and indistinct hilum point.Compound granules are abundant,which have 2—20 components.Fibers are mostly in bundles,walls are thickened and lignif ied,parenchymatous cell surrounded often contain prisms of calcium oxalate which forms crystal fibers,and walls containing crystal fibers are lignif ied and thickened.Stone cells are infrequently visible,either semi-rounded or polygonal shaped,with 25—43μm in diameter.Bordered pitted vessels are relatively large with round shape and dense arrangement(Figure.3—4).
Figure.3—3 Powder
1.tarch granules 2
Figure.3—4 Powder
1.Starch granules
1.5.3.1 Preparation of reference solution,raw material solution of the natural medicine and its preparation solution
1.5.3.1.1 Preparation of reference solution Dissolve Puerarin CRS in methanol to produce a solution containing 1mg permL as the reference solution.Weigh 0.8g Puerariae Lobatae Radix reference drug and operate as in the above method,then produce the reference drug solution.
1.5.3.1.2 Preparation of sample solution Place 0.1g powder of the authentic natural medicine (Puerariae Lobatae Radix),a confused natural medicine,an inferior unqualif ied quality natural medicine,and false samples 1 and 2 and dissolve in 10mL of methanol in a conical flask respectively.Dissolve the sample under ultrasonic treatment for 20 minutes,and then filter off.After the filtration,evaporate the filtrate to dryness and dissolve the product in 1mL of methanol as sample solutions.
1.5.3.1.3 Preparation of preparation solution Dissolve the powder of Yufeng Ningxin Tablets in 20mL of ethyl acetate.Treat the solution with the same method above.
1.5.3.2 Spotting Carry out the method for thin layer chromatography (General Requiremennts 0502) regulated in the 2015 edition of Chinese Pharmacopoeia ,using silica gel G(or silica GF 254 ) mixed with sodium carboxymethylcellulose as the coating substance,take 10 μl of each of the eight solutions and spot in a strip separately on a thin layer plate.
1.5.3.3 Developing Develop the plate under the mobile phase (chloroform:methanol :distilled water= 7:2.5:0.25).Take out the thin-layer plate and dry it.
1.5.3.4 Observation Observe the plate at 365nm and 254nm respectively,under ultraviolet light.The fluorescent or dark strip in the chromatogram obtained with the test solution corresponds in position and color to the fluorescent or dark strip in the chromatogram obtained with the reference drug solution and the reference solution.Record the data and take photos.Puerarin,Puerariae Lobatae Radix reference drug,and samples TLC chromatogramis seen in the picture （Figure.3—5）.
First,identify the authentic Puerariae Lobatae Radix and then choose the superior sample which conforms to the Chinese Pharmacopoeia .
Teachers:Lectures ＆ demonstration.
Students:Hands-on practice ＆ operation.
Figure.3—5 TLC chromatogram
S.Puerarin 2.Puerariae
Experiment record requirements:Take microscopic photos of microscopic identif ication characteristics of natural medicines and record corresponding length,diameter,thickness and so on.Take pictures of macroscopic identif ication of natural medicines and the TLC chromatogram.
1.9.1 What are the advantages of macroscopic identif ication,microscopic identif ication and TLC identif ication respectively? When to apply them?
1.9.2 How to choose transparent agent when natural medicine powder tries to make transparent in microscopic identif ication?
For the entire test of Puerariae Lobatae Radix and Puerariae Thomsonii,please refer to quality monographs of the two species recorded in 2015 edition of The Chinese Pharmacopoeia .
Glossary
1.macroscopic identif ication 性状鉴别
2.Puerariae Lobatae Radix 野葛
3.Puerariae Thomsonii Radix 粉葛
4.authenticity 真实性
5.texture 质地
6.fibrous 纤维性的
7.starchy 粉性的
8.concentric ring 同心环
9.odor 气味
10.starch granule 淀粉粒
11.fibre 纤维
12.stone cells 石细胞
13.bordered pitted vessel 具缘纹孔导管
14.filtration 过滤
15.evaporate 蒸发
16.ultrasonic 超声的
【基本知识】
1.提取分离概述 天然药物中化学成分的组成一般比较复杂，往往有效成分和其他杂质共存，各成分的含量差别也比较大。
提取工艺应根据被提取有效成分的主要理化性质并考虑各种提取分离技术的原理和特点进行选定，使所需要的成分能充分地得到提取。
纯化工艺是采用适当方法最大限度除去无效成分，保留有效成分。分离纯化技术有利于提高产品有效成分的含量，确保天然药物的质量、疗效和稳定性。
2.提取方法 提取就是用适当的溶剂将待研究的化学成分从药材组织中抽提出来的过程。在进行提取时，尽量使需要的成分和不需要的成分分开，使杂质不被提取出来，或在处理过程中尽可能除去，去粗取精。
提取可在室温下进行，也可以加热。一般常温提取杂质较少，而加热提取效率较高。在不了解有效成分性质之前，一般采用温和的条件，不宜使用酸碱溶液，以免有效成分被破坏。
常用的经典提取方法包括溶剂提取法、水蒸气蒸馏法和超临界流体提取法等。
（1）溶剂提取法 溶剂提取法是实际工作中应用最普遍的方法，常见的提取溶剂包括水、亲水性有机溶剂和亲脂性有机溶剂。选择溶剂的要点是根据相似相溶的原理，最大限度地提取所需化学成分；溶剂应以容易回收、安全低毒、价格低廉为原则。
水为极性最大的常用提取溶剂。水提取可使用煎煮、浸泡和渗漉3种方式，也可用酸水或碱水提取。碱性、酸性成分或苷类等大极性成分，如小檗碱、甘草酸、芸香苷等，较易溶于水，可选择水作为提取溶剂。但是使用水提取时，提取液中包含糖类、氨基酸、蛋白质、无机盐类等较多水溶性杂质，较难过滤，不利于进一步分离；此外，如不能及时处理，其中糖类、蛋白质等营养物质易霉变，要注意防腐。因此，有些化合物虽能溶于水，但为了使杂质尽量少带出来，也常用有机溶剂提取。
有机溶剂提取常采用回流提取、连续回流提取（索氏提取法）、浸渍和渗漉等方式。也可采用几种极性不同的有机溶剂，按照极性由低到高依次分步提取，根据各成分在不同极性溶剂中溶解度的差异进行提取和分离。乙醇、甲醇是最常用的有机溶剂，能与水按任意比例混合，且能和大多数亲脂性有机溶剂混合，渗入药材细胞能力较强，能溶解大多数中药成分。一般来说，甲醇比乙醇的提取效果好，但毒性比乙醇大，故甲醇多在实验室研究中应用，而乙醇更适用于工业化生产。甲醇和乙醇的浓度要根据被提取物质的性质而定。
（2）水蒸气蒸馏法 水蒸气蒸馏法只用于提取能随水蒸气蒸馏，而不被破坏的难溶于水的成分。这类成分有挥发性，在100℃时有一定蒸气压，当水沸腾时，该类成分一并随水蒸气带出，再用油水分离器或有机溶剂萃取法，将这类成分自馏出液中分离。水蒸气蒸馏可以用来提取某些挥发油和挥发性成分，如麻黄碱就可以用水蒸气蒸馏法从麻黄中直接蒸馏出来。
（3）超临界流体提取法 超临界流体提取法是天然药物有效成分提取的新技术。超临界流体是介于液体和气体之间的流体，同时具有液体和气体的双重特性，它的扩散系数和黏度接近气体，而分子密度却大大增加，溶解性能类似液体，因此可以用来提取天然药物有效成分。最常用的流体物质是二氧化碳，因为它具有临界条件好，无毒、安全、无污染等优点。
3.有效成分富集方法 天然药物的提取液或浓缩后得到的提取物通常仍是比较复杂的混合物，需要进一步富集才能得到有效部位或者相应的单体。天然药物化学成分的富集是根据提取物中各成分之间物理或化学性质的差异，运用一定的方法使各成分彼此分开，获得有效部位或者相应的单体的过程。
天然药物化学成分的富集方法很多，原理通常是根据天然药物中各化学成分在不同溶剂中的溶解度、分配系数，以及在不同色谱填料中的吸附性、解离程度等性质上的差异进行分离。下面介绍天然药物有效成分富集的一些常用方法。
（1）萃取 通常所指的萃取，即液—液分配萃取，是利用混合物中的各成分在两种互不相溶的溶剂中分配系数的不同而达到分离的目的。当提取溶剂选用的是水或乙醇水溶液时，提取后浓缩成适当浓度的水溶液，选择合适的有机溶剂与其萃取。若所需成分是脂溶性，可用有机溶剂如石油醚、氯仿或乙醚；若所需成分是亲水性物质，可用弱亲水性溶剂如乙酸乙酯、正丁醇等进行萃取。
（2）沉淀法 沉淀法是在提取液中加入某种溶剂或试剂产生沉淀，以获得有效成分或除去杂质的方法。工业上常用的沉淀法主要包括溶剂沉淀法和酸碱沉淀法。
溶剂沉淀法是在提取液中，加入某种溶剂，使某些成分沉淀出来的分离方法。例如在水提取液中，加入一定量的乙醇，使含醇量达到一定比例，则难溶于乙醇的成分如淀粉、树胶、黏液质、蛋白质等杂质从溶液中沉淀出来，经过滤除去沉淀，即可达到有效成分与这些杂质相分离的目的。这便是中药制剂中通用的“水提醇沉法”的基本原理。
酸碱沉淀法是利用酸性成分在碱水中成盐而溶解，在酸水中游离而沉淀，而碱性成分则在酸水中成盐而溶解，在碱水中游离而沉淀的性质，来进行分离的一种分离方法。如游离生物碱一般难溶于水，遇酸生成盐而溶于水，过滤除去水不溶性杂质，滤液再加碱碱化，则重新生成游离的生物碱，从水溶液中析出而与水溶性杂质相分离。
（3）色谱分离法 色谱法是天然药物化学成分分离的最常用方法，利用混合物中各种成分对固定相和流动相亲和作用的差异而使之相互分离。通过选用不同分离原理、不同操作方式、不同色谱材料或将各种色谱组合应用，达到对各类型化学成分的富集和精制。该法具有分离效能高、快速简便等特点。随着近年来色谱技术的发展，色谱法也逐步向仪器化、自动化、高速化及与其他仪器的联用方向发展，成为天然药物化学成分分离最有效、应用范围最广、使用最多的手段。
目前大孔吸附树脂吸附法是工业上最常用的有效部位富集方法之一。大孔吸附树脂是一种具有大孔结构的有机高分子共聚体。以大孔吸附树脂为吸附剂，利用其对不同成分的选择性吸附和筛选作用，通过选用适宜的吸附和解吸条件可以分离纯化某一个或某一类化合物。目前国内市售大孔吸附树脂约有几十种，每一种都有不同的适用范围。
【目的要求】
1.掌握天然药物有效成分常用的提取、纯化方法。
2.熟悉水提醇沉、大孔树脂富集有效成分的原理与方法。
3.比较水提、水提醇沉、大孔树脂等提取纯化工艺对浸膏得率，有效成分含量及其提取率的影响。
【仪器试剂】 旋转蒸发仪、鼓风干燥箱、水浴锅、煎煮锅、玻璃层析柱、纱布、量筒、容量瓶、蒸发皿、移液管、洗耳球、乙醇、甲醇、蒸馏水。
【实验材料】 通过实验一选择正品药材，以此药材作为有效成分提取纯化的原料。本实验以豆科植物野葛 Pueraria lobata (Willd.) Ohwi的干燥根为研究实例；选用AB—8大孔吸附树脂为富集有效成分的填料。
【实验内容】
1.水煎提取工艺 本部分实验采用水煎提取法提取葛根总黄酮。
取葛根（野葛）饮片80g，置煎煮锅中，加10倍量水（800mL），室温浸泡半小时。大火煮沸然后小火保持微沸状态1小时，纱布过滤提取液，药渣用8倍量水（640mL）再煎煮半小时，纱布过滤提取液。合并两次提取的滤液，混合均匀，测量总体积并记录。精密量取0.5mL置10mL容量瓶中，以30%乙醇定容（测试样品1），以备测定水提物总黄酮的含量；其余水煎液常压浓缩至1g生药/mL（80mL），另精密量取5.0mL（相当于5g生药量）的药液于已经记录重量的蒸发皿中，水浴蒸干，烘干，称重，计算水煎法浸膏得率。
水煎法提取葛根总黄酮流程图如图3—6：
图3—6 水煎法流程图
2.醇沉纯化工艺 其余水煎浓缩液（75mL）加2倍量95%乙醇（150mL），搅拌，4℃放置约1个小时，沉淀。减压抽滤，测量滤液体积，量取0.5mL置10mL容量瓶中，以30%乙醇定容（测试样品2），以备测定水提醇沉纯化后样品总黄酮的含量；减压回收剩余滤液，浓缩至2.5g生药/mL（30mL）。另精密量取2mL相当于5g生药量的药液于已经记录重量的蒸发皿中，水浴蒸干，烘干，称重，计算水提醇沉纯化法所得浸膏得率。剩余样品，14mL供药效试验（药理样品A）、14mL供大孔树脂试验。
水提醇沉法富集葛根总黄酮流程图如图3—7。
图3—7 醇沉纯化工艺流程图
3.大孔树脂纯化工艺 取90mL已经预处理好的大孔树脂AB—8吸附剂，装柱，上样，上样量14mL（相当于35g生药）葛根提取液，吸附平衡半小时。先以去离子水洗脱除去水溶性杂质，至检测无多糖反应（Molish反应）为止。再以70%乙醇洗脱，收集洗脱液，至无黄酮络合反应（FeCl 3 反应）。测量70%乙醇洗脱液的总体积。量取0.5mL置10mL容量瓶中，以备测定大孔树脂纯化后总黄酮含量（测试样品3）。剩余洗脱液减压浓缩至上样体积（14mL，相对于2.5g生药/mL），精密量取2mL（相当于5g生药量）的药液于已经记录重量的蒸发皿中，水浴蒸干，烘干，称重，计算大孔树脂纯化法浸膏得率。剩余样品（12mL）供药效试验（药理样品B）。
大孔树脂法富集葛根总黄酮流程图如图3—8：
图3—8 大孔树脂纯化工艺流程图
【实验结果及分析】
表3—1 样品中黄酮含量测试
表3—2 不同提取纯化工艺结果的比较
【教学方式】 教师讲授和演示及学生操作。
【注意事项】
1.注意本实验共计有3个样品需要取样干燥并称重。
2.注意本实验共计有3个样品需要测定总黄酮含量。
3.注意本实验需要制备2个样品供药理实验使用。
【思考题】
1.常用的提取纯化方法有哪些？
2.葛根中的主要化学成分有哪些？
3.本实验是否可以使用乙醇提取法？
【知识链接】 色谱法指导原则参考2015年版《中华人民共和国药典》四部通则0500色谱法。
Bioactive constituents,which could be responsible for particular biological activities,with some exception,occur in low amount in plants.These compounds may be unstable and be present as part of a complex mixture.
To suff iciently extract the targeted components,a procedure should be developed according to the main physicochemical properties of the components and various technical principles and characteristics.
Purif ication is a process that separates components required in an extract from other compounds as much as possible.
The primary goal of the extraction is to concentrate and separate the product from the biomass.The extraction process may be conducted at room temperature or through heating.Generally,extracts obtained at a lower temperature contain fewer impurities,whereas higher extraction eff iciency can be reached at a higher temperature.Regarding unknown constituents,processing under soft conditions free from acid and alkali can effectively avoid decomposition of the constituents.
The following subsections present methods frequently used for extraction:
2.1.2.1 Solvent extraction The most common method adopted for obtaining a plant extract is solvent extraction.Solvent extraction is based on the “like dissolves like” principle(i.e.,a nonpolar solvent extracts nonpolar compounds,and a polar solvent extracts polar compounds).
The simplest form of solvent extraction is water extraction,which comprises of three processes:decoction,immersion,and percolation.Specif ic methods can be employed to isolate a specif ic class of compound,such as alkaloids and glycosides,from a plant source.For instance,to extract alkaloids present as free bases,the plant material can be extracted using dilute acids.However,water extraction may produce more substances than expected ( e.g. ,inorganic salts,proteins,and starches),which will hinder further separation.Therefore,some compounds are extracted using organic solvents to produce fewer unwanted components.
Organic solvent extraction often involves ref lux extraction,Soxhlet extraction,immersion,and percolation.Several solvents of increasing polarity may also be used.The main advantage of incorporating immersion and percolation in cold solvent extraction is that neither the plant material nor the solvent is heated,resulting in low compound degradation.However,cold solvent extraction does not produce as high a yield as that produced when the solvent is heated.The plant material can be heated under ref lux.A sophisticated form of ref lux is Soxhlet extraction.Because ethanol has a satisfactory dissolving capacity,strong penetration behavior,and low risk of toxicity,ethanol-water in various ratios is widely used,depending on the properties of the desired material.
2.1.2.2 Steam distillation Volatile oil and some volatile components can be extracted mainly using steam distillation.For example,ephedrine is separated directly from Ephedra sinica by applying this method.In a laboratory,steam is generated and passed into a flask containing the plant material.The heat releases oil from the material.The oil vaporizes and gathers on the condenser.The cooled oil can then be collected.The process can be scaled up for industrial extraction of large amounts of plant materials.
2.1.2.3 Supercritical fluid extraction Recently,commercial procedures have been developed using supercritical carbon dioxide (CO 2 ) as a mild solvent.This process is based on a liquid attaining a temperature and pressure at which the liquid does not evaporate but remains in a fluid state,a supercritical fluid.At this point,no difference in density between the liquid and gaseous forms of the substance exists.The advantage of CO 2 is that it is nonf lammable and suitable for extracting compounds that have a low thermal stability and nonpolar compounds.The disadvantage is that the apparatus required for the process is more complex and expensive than that required for steam distillation.
All separation processes involve dividing a mixture into numerous discrete fractions.These fractions may be obvious,physically discrete divisions,such as the two phases of a liquid–liquid extraction,or the contiguous eluate from a chromatographic column that is artif icially divided by the extractor into fractions.
Typically,the initial extraction is then separated into fractions.The following subsections detail methods frequently used during fractionation:
2.1.3.1 Fractionation using solvents The most frequently used extraction is liquid–liquid distribution.There are different distribution coeff icients of compounds in two solvents that are insoluble in each other and can separate themselves.When a water or alcohol system is used as the extraction solution,the extract is concentrated and the solution is extracted using suitable organic solvents.When the desired materials are hydrophobic,the options for organic solvents are petroleum ether,chloroform,or diethyl ether.When the materials are hydrophilic,ethyl acetate and n-butanol can be used.
2.1.3.2 Precipitation A specif ic class of compounds can be precipitated from concentrated solutions by using reagents or other solvents.For example,an effective method for removing polysaccharides in a water extract is to treat the concentrated extract by increasing the ethanol concentration.This method is called “water extraction and alcohol precipitation” in Chinese Materia Medica preparation.
When the desired substance is an acid or alkali,acid or alkali must be added to the extracting solvent to change the substance into salt to render it free for extraction.Some substances react with acids or alkalis,and the products have varied solubility.In addition,these properties can be used as long as the reactions are reversible.
2.1.3.3 Chromatography Chromatography involves the distribution of a compound and related analogues between two phases:a moving mobile phase is passed over an immobile stationary phase .Separation is based on the characteristic process in which compounds distribute themselves between these two phases.Thus,as one phase carrying the solute passes over the stationary phase,the solutes are in a constant dynamic equilibrium between the two phases.For any compound,the position of this equilibrium is determined according to the interaction strength of the compound with the stationary phase and the competition for the stationary phase between the compound and mobile phase.The stationary phase may be a solid or liquid,and the fluid mobile phase may be liquid to enable liquid chromatography,which is widely used and appears in several forms.A preliminary fractionation on a column of macroporous resin is the most convenient method.This technique is suited for managing numerous samples.
2.2.1 To understand the extraction and purif ication principles of natural products;
2.2.2 To master the macroporous resin purif ication method;
2.2.3 To compare the differences in yield,recovery,and content of active constituents by applying decoction,water extraction and alcohol precipitation,and macroporous resin purif ication methods.
Rotary evaporator,boiler,water bath,drying cabinet,chromatographic column,volumetric cylinder,volumetric flask,filter cloths,pipette,rubber bulb,ethanol,methanol,and distilled water.
Puerariae Lobatae Radix and AB-8 macroporous resin.
First,80g of cut Puerariae Lobatae Radix is soaked in 800mL of water for 30 min and extracted by boiling for 60 min.The extract is filtered through a piece of cheesecloth,and then the filtrate is reserved.The extracted material is extracted again with 640mL of water for another 30 min in the same process and filtered as described.The combined extract is mixed evenly and the total volume is measured.To detect the content of total flavonoids,0.50mL of the solution is placed into a 10mL volumetric flask ( Sample 1 ) and left to make a certain volum with 30% ethanol.
The volume of the remaining extract is reduced to 80mL (equal to 1g of crude drug permL).Subsequently,5.0mL of the concentrate is placed into an evaporating dish and dried in a water bath.The dried material is weighed to calculate Yield 1 .(Figure.3—6)
Under a stirring condition,150mL of 95% ethanol is added to the remaining 75mL of the aforementioned concentrate and then placed in an ice bath for 60 min.The precipitate is removed by using Buchner filtration,and the total volume is measured.To detect the content of total flavonoids,0.50mL of the solution is placed into a 10mL volumetric flask (Sample 2) and left.The residual solution is condensed to 30mL (equal to 2.5g of crude drug permL) by using a rotary evaporator.Subsequently,2.0mL of the concentrate is placed into an evaporating dish and dried in a water bath.The drying material is weighed to calculate Yield 2.For pharmacological testing and the purif ication process using macroporous resin,14mL and 14mL of the concentrate is reserved ( Sample A ),respectively.(Figure.3—7)
Figure.3—6
Figure.3—7 Purif
First,14mL of the extract (equal to 35g of crude drug) is loaded on top of a short column of AB-8 macroporous resin (90mL) and retained for 30 min.The column is eluted using deionized water until no more polysaccharides can be detected using the Molish reaction followed by 70%ethanol,and this fraction is collected until no more flavonoids can be detected using FeCl 3 .
The total volume of this fraction is measured,and 0.5mL of the aforementioned solution is placed into a 10mL volumetric flask ( Sample 3 ),which is left for detecting the content of total flavonoids.The residual solution is condensed to 14mL by using a rotary evaporator.Subsequently,2mL of the concentrate is placed into an evaporating dish and dried in a water bath.The dried material is weighed to calculate Yield 3 .For pharmacological testing,12mL of the remaining extract is reserved ( Sample B ).(Figure.3—8)
Figure.3—8 Purif ication
Tab.3—1 Quantitative analysis of total flavonoid content
Tab.3—2 Results
Teachers:Lectures ＆ demonstration.
Students:Hands-on practice and operation.
2.8.1 To prepare three samples for calculating yields;
2.8.2 To prepare three samples for analyzing total flavonoid content;
2.8.3 To prepare two samples for pharmacological testing.
2.9.1 What extraction methods are commonly used for natural products?
2.9.2 What are the main chemical constituents of Pueraria lobata ?
2.9.3 Can this experiment be performed using the alcohol-extraction method?
About guidelines for chromatography,please refer to General Requirements 0500,Volume IV,2015 edition Chinese Pharmacopoeia .
Glossary
1.flavonoid 黄酮
2.isof lavonoid 异黄酮
3.process 工艺
4.separate 分离
5.component 成分
6.alkaloid 生物碱
7.terpene 萜类
8.steroid 甾体
9.glycoside 糖苷
10.anthraquinone 蒽醌
11.coumarin 香豆素
12.cellulose 纤维素
13.chlorophyll 叶绿素
14.grease 油脂
15.water-extraction 水提
16.decoct 煎煮
17.immerse 浸泡
18.percolate 渗漉
19.berberine 黄连素
20.glycyrrhizinic acid 甘草酸
21.globulariacitrin 芸香苷
22.alcohol precipitation 醇提
23.organic solvent-extraction 有机溶剂萃取
24.macroporous resin 大孔树脂
25.precipitation 沉淀
26.chromatographic column 色谱柱
【基本知识】 天然药物可以借鉴《中华人民共和国药典》或文献收载的与其相同成分的测定方法，但因品种不同，均要进行方法学考察研究，包括测定方法的线性关系、精密度、重现性、稳定性试验及回收率试验等，主要考察项目的内容和要求如下。
1.方法学考察
（1）测定条件的选择 目的：找出最佳测定条件。
如液相色谱法中固定相和流动相的选择、薄层扫描法层析与扫描条件的选择等。
紫外可见分光光度法或液相色谱法研究过程中，所测总成分以某一单体成分为对照品，进行方法学考察时，应首先考察对照品溶液与供试品溶液最大吸收波长是否一致或相近。
（2）专属性试验 目的：防止假阳性现象。
在色谱法中常用阴性对照法，即以被测成分或药材与除去该成分或该药材的成药做对照，以考察被测成分的斑点（或峰）的位置是否与干扰组分重叠，从而确证测定指标（如吸收度、峰面积）是否仅为被测成分的响应，防止假阳性的干扰。
紫外可见分光光度法中的空白对照液常见的为有机溶剂空白和试剂空白（溶剂加显色剂）。复方制剂应同色谱法一样做阴性对照试验，以确证吸收度仅为被测成分的响应。
（3）线性关系 目的：确定样品浓度与响应值如吸收度、色谱峰面积（或峰高）之间的线性关系。
紫外可见分光光度法须制备相关标准曲线，考察样品浓度与响应值呈线性关系的范围，并获取相应的回归方程，用以确定取样量并计算含量。标准曲线中至少包含5个浓度点，相关系数r值应在0.999以上。
色谱法一般均采用对照品比较法、外标法或内标法测定，并进行线性考察。
（4）稳定性试验 目的：选定最佳的测定时间范围。
用紫外可见分光光度法或薄层扫描法等测定时，应对被测液或薄层色谱斑点的吸收度值稳定性进行考察，即每隔一定时间测定1次，延续3～4小时（12小时），视其是否稳定，以确定适当的测定时间。
液相色谱法中，样品的稳定性应至少测定至12小时，24～48小时更佳，相对标准偏差（RSD）应低于3%。
（5）精密度试验 目的：考察分析方法在不同时间、不同操作人员、不同试验室下，所得结果的重现性和重复性。
1）中间精密度 中间精密度系指在同一试验室，由于试验室内部条件改变，如时间、分析人员、仪器设备、测定结果的精密度。对同一样品，进行多次测定，平行试验至少6次以上，即 n ≥6，计算相对标准偏差（RSD），一般要求低于3%。
2）重复性试验 按拟定的含量测定方法，对同一批样品进行多次测定，要求 n ≥6，低于3%。
同一人短时间内测定多次称重复性，即日内精密度；不同人或实验室测定称重现性。
（6）回收率实验 目的：考察测定方法的准确度。
含量测定方法的建立，多以回收率估计分析的误差和操作过程的损失，以评价方法的可靠性。对单味药材的回收率测定，因不易制备除去被测成分的药材空白对照品而采用加样回收法。
加样回收：即于已知被测成分含量的样品中再精密加入一定量的被测成分纯品，依法测定。用实测值与原样品中含测成分之差，除以加入纯品量计算回收率。
计算公式：
式中， A ：样品所含被测成分量； B ：加入纯品量； C ：实测值。
回收率试验至少需要进行6次试验（ n ＝6），或三组平行试验（ n ＝9），即在同一批样品中加入高、中、低不同纯品量。回收率一般要求在95%～105%，如某些方法操作步骤繁复，可要求略低，但一般应不小于90%。
2.含量测定 目的：得到样品中被分析物含量的准确结果。
未知样品的分析方法需经验证后方可用于含量测定。样品分析可以只进行一次，也可根据需要重复分析测定，则每个批次的样品在分析测定时均需相应的标准曲线。一般来说，质量控制样品的含量测定结果相对标准偏差应不超过5%。
【目的要求】
1.掌握天然药物含量测定方法学研究的内容。
2.以葛根为例，掌握天然药物中指标成分的含量测定方法。
【仪器试剂】 紫外可见分光光度计、电子天平、超声振荡仪，研钵、100mL具塞锥形瓶、量瓶（5mL、10mL、25mL、50mL）、移液管（1mL、2mL、5mL、10mL、25mL），乙醇、蒸馏水。
【实验材料】 药品试剂：葛根（野葛）、粉葛药材、愈风宁心片、葛根素对照品。
【实验内容】 葛根素及总黄酮在紫外区250nm处有强吸收峰，可通过紫外分光光度计在250nm处测定吸光度，以确定其含量。随行空白。
1.样品的制备
（1）药材供试品溶液制备 分别取野葛（6份）和粉葛（2份）药材0.1g，精密称定，置于锥形瓶中，精密加入30%乙醇50mL并称重，超声处理20分钟后重新称重，用30%乙醇补足重量，过滤，滤液即为供试品溶液。
（2）制剂供试品溶液制备 取愈风宁心片2片，除去包衣，捣碎研细，精密称定50mg，同上处理后，取滤液作为供试品溶液。
2.标准曲线的建立 对照品母液制备：配制1mg/mL的葛根素对照品，精密吸取1mL置10mL量瓶中，以30%乙醇稀释并定容。
分别精密量取适量对照品母液，以30%乙醇定容于10mL量瓶，配成一系列浓度梯度的对照品溶液。以30%乙醇为参比溶液，测250nm处的吸收值（以吸光度A表示）。随行空白。以吸光度值为纵坐标Y，葛根素对照品浓度为横坐标X（mg/mL），绘制标准曲线，计算得回归方程，线性相关系数r及线性范围。
表3—3 葛根素标准曲线的建立
3.精密度试验 取一份对照品溶液，测定其吸光度，重复6次，计算总黄酮吸光度的RSD值。
表3—4 葛根素精密度试验结果
4.稳定性试验 取1份样品，每隔一定时间（30分钟）测定一次吸光度，共计测定3小时，计算RSD值。
表3—5 样品稳定性试验结果
5.重复性试验 平行取6份样品，分别测定紫外吸光度，计算总黄酮的平均含量。
表3—6 样品重复性试验结果
6.加样回收率试验 精密称取野葛粉末0.05g，每2份为一组，共3组，于锥形瓶中，根据“5”计算的总黄酮平均含量，分别加入样品中已知总黄酮含量的80%、100%、120%。按（1）法制备供试液，测定样品含量。计算平均回收率及其RSD值。
表3—7 葛根素加样回收率试验结果
7.样品的含量测定 每组样品（葛根、粉葛和愈风宁心片）各平行取2份样品，测定吸光度，计算各组样品中葛根总黄酮的含量（样品浓度应在线性范围之内，如超过线性范围，须稀释一定倍数）。
表3—8 葛根总黄酮含量测定结果
【教学方式】 教师讲授和演示及学生操作。
【注意事项】
1.药材用乙醇溶液超声提取后，应补足重量，防止溶剂挥发所致测定偏差。
2.吸收池的光学面必须清洁干净，不可用手触摸，只能用擦镜纸擦拭。
3.在标准曲线法定量测定实验中，应使样品溶液的被测组分浓度落在标准曲线的线性范围内。
【思考题】 归纳天然药物含量测定方法学研究的内容和要求。
【知识链接】
1.2015年版《中华人民共和国药典》四部通则：药品质量标准分析方法验证指导原则［9101］.中药质量标准分析方法验证指导原则，2015年版《中华人民共和国药典》一部附录ⅪⅩA——Guidelines for validation of analytical procedures on quality standard
2.ICH Q2A.Test on Validation of Analytical Procedures.
3.ICH Q2B.Validation of Analytical Procedures:Methodology.
Quantitative determination of natural medicine can refer to Chinese Pharmacopoeia or literature procedures for the same component.Due to different varieties of the test drug,it is required to conduct method validation,including linearity,precision,reproducibility,stability test and recovery test,etc.Typical validation items which should be considered are listed below:
3.1.1.1 Selection of test condition
Objective:to find out the optimum determination conditions.
For instance,stationary phase and mobile phase need to be optimized in liquid chromatography as well as chromatographic and scanning conditions should be chosen in thin layer chromatography (TLC) method.
Some monomer standards are selected as reference for determination of bulk compositions in ultraviolet and visible (UV-vis) spectrophotometry or liquid chromatography research process.The method validation study,therefore,should firstly examine whether the maximum absorption wavelength of standard solution is consistent with or close to that of the sample solution.
3.1.1.2 Specif ic test
Objective:to prevent false positive results.
In order to inspect whether the tested component spots (or peaks) will be overlapped by interference components,a negative control test is commonly used in chromatography.It is performed by comparing the tested components or herbs to ones that are absent of them.Thereby,the results will conf irm that the response of measurement indicators (such as absorption,peak area) refers only to the tested components,so that false positive interferences will be prevented.
The commonly used blank control solution in UV-vis spectrophotometry is organic solvent or reagent blank (blank solvent adding a different chromogenic agent).Similarly,to ensure that the absorbance response is only obtained from the measured component in the compound preparation,it has to perform the same negative control test as the one used in the chromatographic separation.
3.1.1.3 Linearity
Objective:to determine the linear relationship between the sample concentration and response such as absorbance,and chromatographic peak area (or the peak height) value.
Standard curve solutions should be prepared in order to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample in UV-vis spectrophotometry.Then the sample amount or content will be determined accurately based on the corresponding regression equation.For the establishment of linearity,a minimum of 5 concentrations is recommended as well as the value of correlation coeff icient should be above 0.999.
In chromatographic analysis procedure,generally use the reference comparison method,external and internal standard methods for the determination,and also conduct a linear investigation.
3.1.1.4 Stability test
Objective:to select the best range for measuring time.
The stability test should be performed by investigating the absorbance change of a measured solution or TLC spots when UV-vis spectrophotometry or TLC is applied.The measurement should be carried out at regular intervals to determine a particular time,and it should last for the continuation of 3～4 hours (total of 12 hours).The appropriate time range of the measurement will be determined depending on the stability results.
In Liquid Chromatography， it is necessary to test the sample stability for at least 12 hours（24 to 48 hours will be even better）.The relative standard deviation (RSD) of the measurement results should be below 3%.
3.1.1.5 Precision
Objective:to investigate the reproducibility and repeatability of the analytical method at different time,by different operation personnels or in different laboratories.
● Intermediate precision Intermediate precision expresses within-laboratory variation:different days,different analysts,different equipment, etc .A parallel test should be carried out at least six times ( n ≥6).Generally the RSD of the testing results should be below 3%.
● Repeatability According to the established quantitative method,samples within the same batch were repeatedly measured.The results should also be in accordance with the requirements of n ≥6,RSD＜3%.
Repeatability expresses the precision under the same operating conditions over a short interval of time.It is also termed “intra-assay” precision.Reproducibility expresses the precision between laboratories (collaborative studies,usually applied to standardization of a methodology).
3.1.1.6 Recovery test
Objective:to study the accuracy of the determination method.
The recovery rate can be used to evaluate the reliability of the established determination method by estimating analysis error and loss in the operation process.A sample recovery test is commonly applied to the determination of a single herb due to the lack of blank reference samples,which are not easily prepared by removing the analytes of the test herb.
Recovery:Determine the content of samples which are prepared by adding a certain amount of pure chemical to the known content sample.Calculate the recovery rate by way of dividing the difference of the measured value and the measured component amount in the original sample by a pure chemical spiked amount.
Formula:
In the formula, A :the measured component amount in the original sample; B :the pure chemical spiked amount; C :the measured value.
The recovery test requires at least 6 parallel tests ( n =6),or three groups of parallel tests( n =9),which means three different amounts of pure chemical at high,medium and low level will be added to the same batch samples to obtain the nine test samples.The general requirement of the recovery range is from 95% to 105%.In case that some method and operation steps involved are complicated,we can lower the standard,which should not be less than 90%.
Objective:to obtain the accurate content of the analytes.
The assay of an unknown sample should be performed after the analytical method is validated and accepted.This procedure can be carried out by single determination,or replicate analysis if necessary.The corresponding standard curve of samples in each batch should be generated for analysis.In general,the RSD from the results of the quality control samples should be not more than 5%.
3.2.1 To master the methodological research content of a natural medicine assay.
3.2.2 To master the determination method of marker compounds by taking Puerariae Lobatae Radix as an example.
Ultraviolet-visible spectrophotometer,electronic balance,ultrasonic apparatus,mortar,conical flask with cover (100mL),volumetric flask (5mL,10mL,25mL,50mL),pipette (1mL,2mL,5mL,10mL,25mL),ethanol,distilled water.
Decoction pieces of Puerariae Lobatae Radix,Decoction pieces of Puerariae Thomsonii Radix,Yufeng Ningxin Tablets,Reference substance of puerarin.
Puerarin and total flavonoids have a strong absorption peak at the wavelength of 250nm.The content can be determined by absorbance determination at 250nm using an ultraviolet spectrophotometer with the blank solution of 30% ethanol.
3.5.1.1 Preparation of the herbal test solution Weigh 6 portions of Puerariae Lobatae Radix and 2 portions of Puerariae Thomsonii Radix per 0.1g,and put them into conicial flask.Add 50mL of 30 % ethanol and record the total weight of each flask.Subject them to ultrasonication for 20 min,then complement each weight with 30 % ethanol.After filtration,the filtrate is used as the test sample solution.
3.5.1.2 Preparation of pharmaceutical sample Peel two Yufeng Ningxin Tablets and grind them into a fine powder.Weigh 50mg of powder.Then treat them with the same method mentioned above.
Preparation of the reference solutions:Make puerarin reference solution of 1mg/mL,transfer 1mL of the reference solution precisely to a volumetric flask of 10mL,then add 30% ethanol to the graduation line.Dilute the solution into 5 concentrations with different folds.Determine the absorbance values (A) at 250nm with the blank solution of 30% ethanol.Draw the standard curve with the absorbance value as Y and the concentration as X,calculate the regression equation,correlation coeff icient and linear range.
Tab.3—3 Standard curve of puerarin
Take 1 sample solution,dilute to a suitable concentration,determine the UV absorbance at 250nm continuously for 6 values,and calculate the RSD value of the absorbance.
Tab.3—4 Precision test result of puerarin
Take 1 sample solution,determine the absorbance every 30 min,within 3 hours,and then calculate the RSD value of the absorbance.
Tab.3—5 Stability test result of sample solution
Take 6 sample solutions,determine the absorbance at the wavelength of 250nm.Calculate the average content of the total flavonoids.
Tab.3—6 Repetition test result of sample solution
Weigh 6 portions of herbal powder per 0.05g,adding puerarin reference substance (80%,100%,120% of the flavonoids in the herbs).Prepare the sample solution as procedures in section 3.5.1.1.Determine the absorbance value.
Tab.3—7 Recovery test result of puerarin
Take two sample solutions of Puerariae Lobatae Radix,Puerariae Thomsonii Radix and Yufeng Ningxin Tablet separately,and determine the absorbance values.Calculate the content of total flavonoids.
Tab.3—8 Determination of total flavonoids in different samples
Teachers:Lectures and demonstration
Sstudents:Hands-on practice and operation.
3.7.1 The total weight should be complemented after sample extraction by ethanol solution in order to prevent the measurement deviation caused by solvent evaporation.
3.7.2 Keep the cuvette clean,do not touch the optical surfaces with fingers,cleaning the cells with lens paper.
3.7.3 The concentration of the sample solutions should be within the scope of the standard curve in designing a quantif ication experiment by using standard curve method.
Try to sum up the methodological research content of assay for natural medicine products.
3.9.1 Guidelines for validation of analytical procedures on quality standard of drug or Chinese Materia Medica, Chinese Pharmacopoeia 2015 edition,general rule 〔9101〕 or appendixⅪⅩA.
3.9.2 ICH Q2A.Test on Validation of Analytical Procedures.
3.9.3 ICH Q2B.Validation of Analytical Procedures:Methodology.
Glossary
1.methodology 方法学
2.linearity 线性
3.precision 精密度
4.repetition 重复性
5.stability 稳定性
6.recovery 回收率
7.blank test 空白试验
8.ultraviolet and visible (UV-vis) spectrophotometry 紫外可见分光光度法
9.assay 含量测定
10.standard solution 对照品溶液
11.test solution 供试品溶液
12.sample solution 样品溶液
13.absorption value 吸收值
14.absorbance 吸光度
15.puerarin 葛根素
16.total flavonoids 总黄酮
17.standard curve 标准曲线
18.regression equation 回归方程
19.correlation coeff icient 相关系数
20.linear range 线性范围
21.relative standard deviation (RSD) 相对平均偏差
【基本知识】 中药是天然药物的重要组成部分。本章重点介绍中药新药的药效学研究。
中药新药的药效学研究，应遵循中医药理论，运用现代科学方法，制定具有中医药特点的实验计划。根据中药新药的功效、主治，结合其剂型、给药途径、临床应用，并参考相关文献资料，以拟开发为新药的中药复方或配伍、中药组分、单味中药及其提取物、中药单体成分等为研究对象，根据《中药新药药效学研究指南》的要求，选用或建立与中医“证”或“病”相符或相近似的动物模型和实验方法，制定实验方案。实验设计应严密整合三大要素，严格遵循三大基本原则，并采用正确的统计学方法处理实验结果，研究工作才能对中药新药的有效性做出客观准确的评价。
1.实验设计的三大要素 实验设计的三大要素为处理因素、实验对象和实验效应。
（1）处理因素 处理因素是指研究者根据研究目的欲施加或观察的，能作用于实验对象并引起直接或间接效应的因素，又称实验因素。与处理因素同时存在，能使实验对象产生效应的其他因素称非处理因素，又称干扰因素。完整的实验设计需事先明确其中处理因素及其水平的数量。如研究药物的抗高血压作用，只观察不同药物对血压的影响时，药物是唯一的处理因素，而不同种类药物或药物不同剂量的组别数则是水平数，但是同时血压也受年龄、季节等非处理因素的影响而干扰了处理因素产生的药理效应。因此，在确定处理因素的同时，根据专业知识与实验条件，应尽可能找出对研究结果产生影响的非处理因素，并加以有效控制。最好通过一些预实验，初步筛选处理因素并确定应取哪些水平，以免实验设计过于复杂。
（2）实验对象 即受试对象。不同的药效学实验研究需要选取不同的敏感动物，以解剖、生理、习性及反应性与人类相近的动物为佳。任何新药的研制在进入临床前最终均需得到整体动物模型的验证。实验设计中受试对象的总数称为样本含量，一般需根据特定的设计类型估计出较合适的样本含量，样本过大或过小均有弊端。
（3）实验效应 实验效应是反映处理因素作用强弱的标志，用实验指标来表达。因此应尽量选用灵敏度高、特异性强、精确可靠的客观指标。对一些半客观或主观指标，一定要事先规定读取数值的严格标准，才能准确地分析实验结果，提高可信度。
2.实验设计的三大基本原则 实验设计的三大基本原则是随机、对照、重复。
（1）随机 随机是使每个实验对象在接受处理时，机会均等地分配到各个组别。可采用随机排列表法、随机数字表法等随机方法。随机可减轻主观因素的干扰，尽量保持非处理因素组间均衡可比，减小或避免偏性误差。在实验对象的抽样、分组和实验实施中均应遵循随机化原则。
（2）对照 对照是比较的基础。只有设立了对照，才能消除非处理因素对实验结果的影响，从而把处理因素的效应分离出来。实验组别一般至少分为阴性对照组、阳性对照组和受试实验组。对照应符合“齐同可比”的原则，即除处理因素外，对照组的其他一切条件（尤其是影响实验结果的非处理因素）应与受试实验组完全相同。药效学实验中，阴性对照组以无效药物或受试药物溶媒、赋型剂等作为对照，应产生阴性结果；阳性对照组采用疗效肯定的药物作为对照，应产生阳性结果。进行组间比较时，在确定差异是否具有统计学意义后再下实验结论；受试实验组的药效判定需与阴性对照组进行比较。受试实验组可设多组别，如同种药物不同剂量（等比）、同种中药不同提取物，前者比较可反映药物作用的量效关系及特点，后者比较可了解中药不同提取物的作用差异。
（3）重复 重复包括两方面的内容：良好的重现性和足够的重复数。重现性是指实验结果的重复稳定性，即在同等实验条件下能重复出相同的实验结果。重复数是指样本要达到一定的实验例数或实验次数，抽样的个数才能反映整体。足够的重复数才会取得较高的重现性，为了得到统计学所要求的重现性，必须选择相应的适当的重复数。
3.中药药理研究的一些特殊方法 中药单味药或复方所含的化学成分较为复杂，而且中药药理作用的研究不能脱离中医药理论的指导，因此中药药理研究方法有其特殊性。
（1）中药活性物质和药理活性的筛选方法 分为广筛法和定向筛选法。广筛法是设定明确的实验指标，从大量的药物、有效部位或单体中筛选出具有某种特殊作用的药物。定向筛选法则是根据中药及其复方的功效主治设计药理实验，验证药效并探讨机制。
（2）中药血清药理学方法 将中药或中药复方于动物灌胃一定时间后采集动物血液、分离血清，再用含药血清进行体外实验，称为中药血清药理学方法。此法可防止中药粗制剂本身的理化性质对实验的干扰，可代表药物在体内产生作用的真正有效成分。
（3）病证结合动物模型方法 中药有其特殊的功效主治，因此需建立与临床一致的动物病理模型进行药理实验。病证结合动物模型是指在中医药理论指导下，采用生物学等方法复制出相应中医证候。如将家兔禁水禁食18小时后，以速尿二度利尿脱水，造成“阴津亏虚”状态，再注射大肠杆菌内毒素以致“热盛”，从而制备出温病“阴虚热盛”模型。
4.毒理研究 “安全、有效”是药物必须具备的两大要素。在证明中药新药有效的基础上，还需对其进行毒理学研究，检验中药及其制剂的安全性，阐明其毒性作用的部位和机制以及毒性损伤的程度，以便找出防治毒性损伤的办法和措施，保证人类安全用药。
【目的要求】
1.掌握动物内毒素发热模型的制备方法。
2.比较不同工艺中药提取物对发热模型动物体温的影响。
【仪器试剂】 电子秤、计时器、肛温计、恒温水浴、注射器（2mL、5mL、10mL）、木质兔笼、酒精棉球、凡士林。
【实验材料】
1.动物 新西兰家兔，雄性，普通级，体重2.0±0.2kg。
2.药品 葛根水提液（含葛根生药2.5g/mL）、葛根树脂提取液（含葛根生药2.5g/mL），安乃近注射液（31.25mg/mL），生理盐水，内毒素溶液（250ng/mL），凡士林。
【实验方法】
1.实验分组 取家兔24只，称重、编号，按随机数字表法完全随机分组，共分4组，每组6只。实验组别：阴性对照组，阳性对照组，葛根水提组，葛根树脂组。
2.测基础体温 固定家兔，待家兔安静30分钟后，将家兔臀部抬高，将头端涂有少许凡士林的肛温表（事先将水银柱甩到35℃以下）插入家兔肛门，深度约4cm并保持3分钟，之后取出，读表，记录体温。每30分钟测定一次，共测定3次，取平均值即为基础体温，将所测体温记录于表3—9。基础体温为38.5～39.5℃者作为筛选合格动物；否则弃用，必要时另筛选补充。
3.制备发热模型 各组家兔缓慢耳缘静脉注射内毒素溶液（250ng/mL；注射前于38℃水中温浴）1mL/kg。
4.造模后给药 30分钟后各组分别后肢深部肌注给药2.5mL/kg。阴性对照组：生理盐水；阳性对照组：安乃近注射液；葛根水提组：葛根水提液；葛根树脂组：葛根树脂提取液。
5.测体温 造模后每过30分钟，每只家兔各测肛温一次，至少测6次，记录数据至表3—10。
6.观察症状变化 造模后对比观察家兔有无耸毛、发抖、蜷缩、心率加快、胸腹灼热、眼结膜充血明显等异常反应。
7.统计方法 使用统计软件（如SPSS软件）处理实验数据，采用重复测量的方差分析方法。
【实验结果及分析】
1.原始体温记录
表3—9 原始数据记录表（基础体温测定表）
2.体温变化情况 不同工艺草根提取物对内毒素所致发热家兔体温的影响见表3—10。
表3—10 不同工艺葛根提取物对内毒素所致发热家兔体温的影响（
n
=6
±
s
）
注：温差=造模后体温—基础体温
3.结果分析 根据家兔的肛温变化，分别绘制出以下退热曲线：
（1）时间-体温曲线 X轴为时间（以造模为起点，30分钟为间隔）；Y轴为相应时间点的所测体温（图3—9）。
（2）时间-温差曲线 X轴为时间（以造模为起点，30分钟为间隔）；Y轴为相应时间点的所得温差（图3—10）。
图3—9 时间-体温曲线
图3—10 时间-温差曲线
【教学方式】 教师讲授和演示及学生操作。
【注意事项】
1.以上是规范设计与操作，实际操作应根据学生分组或实验组别增减而调整。
2.若每批次实验学生分为5组；且实验组别按原设计为4组，则每批次可提供家兔15只，其中阴性对照3只，其余组别各4只，随机分配至实验小组和实验组别。
3.基础体温按家兔平静30分钟后，以间隔10分钟的两次肛温测定结果取均值。
4.葛根水提液和葛根树脂提取液为各实验小组前期提取处理后能适用肌注的混合液。
【思考题】
1.关于中药退热作用的研究，是否有其他简便灵敏的实验方法？
2.如何设计合适的实验研究中药不同提取物的药效？
【知识链接】
1.李仪奎.中药药理实验方法学［M］.第2版.上海：上海科学技术出版社，2006.
Ⅰ—4 中药新药的药理学研究
Ⅰ—5 中药的血清药理实验方法
Ⅱ—3 热毒证动物病理模型
2.陈奇.中药药理研究方法学［M］.第3版.北京：人民卫生出版社，2011.
第1篇 第3章 中药药理研究的基本知识
第3篇 中医证候模型中药药理研究方法
3.魏伟.药理实验方法学［M］.第4版.北京：人民卫生出版社，2010.
第三章 药理实验设计
第二十二章 抗炎、解热、镇痛药物的实验方法
Traditional Chinese medicine (TCM) is the important part of natural medicines.The chapter focuses on the pharmacodynamics research of natural medicines.
The pharmacodynamics research of Chinese Materia Medica should follow the TCM theories and adopt modern scientif ic methods to design experiments with TCM characteristics.The design of pharmacodynamics research of Chinese Materia Medica should consider its function,eff icacy,formulation,administration and clinical uses by referring to the relevant documents.Chinese compound formula,fraction,single herb and its extract,and single compound isolated from Chinese Materia Medica may be the research subject.The animal model and the experimental method selected or established should conform or be similar to the “syndrome” or “disease” required by “The Pharmacodynamics Research Guidelines of New Drugs of Chinese Materia Medica”.The experimental design should strictly follow the three elements and the three principles,and use correct statistical methods to evaluate the results.On that basis accurate and reliable conclusions can be drawn.
The three elements of experimental design are studying factor,experimental subject and experimental effect.
4.1.1.1 Studying factor Studying factor is the factor that the researchers want to observe according to the aims of the experiment.The studying factor causes a direct or indirect effect on the experimental subject.It is also known as the experimental factor.Meanwhile,some other factors affecting the experimental result are called non-studying factors,which are also known as interference factors.The experiment designer should know clearly the studying factor and its level number in advance.For example,if a drug is the only studying factor when we study antihypertensive drugs,the level number will be the different kinds or dosages of the drug.Some non-studying factors such as ages and seasons will also affect the result.Therefore,according to the professional knowledge and experimental conditions,it is important to identify as far as possible the non-studying factors,which should be effectively under the control.The best way to avoid the over-complicated experimental design is to select the studying factors and determine levels to be taken by conducting some pre-tests.
4.1.1.2 Experimental Subject Different experiments need to select different sensitive animals whose anatomy,physiology,behavior and reactivity are similar to human beings.Any new drug should be verif ied on the whole animal model before being used in the clinic.The total number of subjects required in the experimental design is called the sample content.Appropriate sample content is estimated according to the specif ic design type.Too big or too small a sample content will be disadvantageous.
4.1.1.3 Experimental Effect The experimental effect ref lects the strength of the studying factor and it is expressed by the experimental index.So some objective indicators of high sensitivity,strong specif icity,accuracy and reliability should be chosen.To some semiobjective or subjective indices,the strict standard of measurement should be made in advance so that the experimental result would be accurately analyzed and its credibility would be improved.
Three fundamental principles of experimental design are randomization,control and repetition.
4.1.2.1 Randomization Randomization means that each experiment object is at a fair chance of getting a different treatment (dosing,analysis,grouping,sampling, etc ).Random number table sampling and random arrange table sampling can be used.Randomization can reduce disturbances originated from subjective factors,reduce or prevent def lective error.Subject sampling,grouping and implementation of the experiment should always follow the principle of randomization.
4.1.2.2 Control Control is the fundamental of comparison.Control is set up to eliminate the inf luence of non-studying factor,so that the effect of studying factor would be obvious.There are at least three different types of control groups:negative control group,positive control group and experiment group.Comparison against a control group should adhere to the principle of“uniform and comparable”.Apart from the studying factor,the conditions of the control group(especially those non-studying factors affecting the result of the experiment) should be the same as those of the comparison group,in order to be comparable.
In a pharmacological experiment,the negative control group refers to a control group which does not hold the studying factor (drug administration) and gives a negative result.Positive control group is a control group which receives a well-recognized medicinal treatment and gives positive results.The experiment group is a group used to conf irm the therapeutic effect of a drug compared with a negative control group.Researchers can establish more than one experiment group.such as groups in different doses with geometric arrangement of the same medicine or groups with various active components from the same Chinese Materia Medica.Different doses are established to determine a dose-response relationship.Various active components are compared together to observe the difference of their effects.
4.1.2.3 Repetition Repetition covers two aspects,that is repeatability and repetition number.Repeatability means the repetition stability of the results in the same experimental condition.The repetition number means that the number of samples or times of experiments can ref lect the overall only by achieving the minimal number acquired.Considerable repeatability results in suff icient repetition number.In order to achieve considerable repeatability for statistical purposes,it is important to choose relevant and adequate repetition number.
The chemical composition of a single herb or a compound formula is complex.Besides,Chinese pharmacological research can not be performed without the guide of TCM theory.Therefore the method of Chinese pharmacological research has its particularity.
4.1.3.1 The screening method of active substances of Chinese Materia Medica and pharmacological activities It is divided into extensive screening method and directional screening method.The extensive screening method is to screen out drugs which have some special functions from a large number of drugs,and effective components or monomers by setting the def inite index.The directional screening method is to design a pharmacological experiment based on the effect of Chinese Material Medica and its compound formula for the eff icacy verif ication and mechanism exploration.
4.1.3.2 Serum pharmacological method of Chinese Materia Medica Animal blood is collected after a period of time since Chinese Materia Medica or its compound formula is given by gavage.The serum separated from blood is used in an in-vitro experiment.The method could prevent the interference of crude extracts of Chinese Materia Medica to the experiment.Therefore,it represents the true in-vivo effective components of the drug.
4.1.3.3 Disease-syndrome-combined animal model method Chinese Materia Medica has its special function and eff icacy.Therefore the animal model should be established in accordance with the disease and syndrome in the clinic.The animal model of a disease and syndrome refers to using biological methods to create the corresponding syndrome of TCM under the guidance of TCM theory.For instance,after rabbits have fasted for 18 hours,furosemide is used to increase the severity of diuresis and dehydration,which will result in “Yin def iciency and liquid depletion” state.Then E.coli endotoxin injected will cause “exuberant heat”,thus the model of “Yin def iciency with exuberant heat” is prepared.
“Safety and eff iciency” are two major elements of new drugs.Hence,on the basis that the new Chinese Materia Medica is proven to be effective,it is also necessary to undertake toxicological studies,which can detect the toxicity of Chinese Materia Medica and its preparation,clarify the location and mechanism that the toxicity acts on,and indicate the damage extent of the toxicity,so as to help people find out the measures to prevent and control the toxic injury,to ensure the safe use of drugs in humans.
4.2.1 To master the establishment of endotoxin-induced rabbit fever model.
4.2.2 To investigate the antipyretic effects of different extracts from Chinese medicine on endotoxin-induced fever in rabbits.
Electronic scales,timers,rectal thermometers,syringes (2mL,5mL,10mL),wooden rabbit hutches,alcohol cotton ball, etc .
Male New Zealand Rabbits,general level,weight 2.0 ± 0.2kg.
Water extract from Pueraria Lobatae Radix (Contains 2.5g/mL crude drug of Pueraria Lobatae Radix),Resin extract from Pueraria Lobatae Radix (Contains 2.5g/mL crude drug of Pueraria Lobatae Radix),Analgin injection (31.25mg/mL),Normal saline (NS),Endotoxin solution (250ng/mL),Vaseline.
24 male New Zealand Rabbits are randomly divided into four groups:Negative control group,Positive control group,Water extract group and Resin extract group.
Fix the rabbit and keep it calm,elevate its hip 30 minutes later,insert the rectal thermometer coated with vaseline into its anus about 4cm and maintain for 3 minutes (keep the mercury column below 35 ℃ ahead),then read and record the temperature.Measure the temperature three times every 10 minutes.The average temperature is calculated as the BBT value and recorded in table 3—9.
Every rabbit is slowly injected in the ear vein with endotoxin solution of 1.0mL/kg body weight (250ng/mL,bathed in 38℃ warm water before injection).
After treatment with endotoxin for 30 minutes,the rabbits of each group were given an intramuscular injection with the drug dosage of 2.5mL/kg body weight.Negative control group:NS; Positive control group:Analgin injection; Pueraria Lobatae Radix water extract group:Pueraria Lobatae Radix water extracts; Pueraria Lobatae Radix resin extract group:Pueraria Lobatae Radix resin extracts.
The rectal temperature is measured every 30 minutes for six time periods (30,60,90,120,150,180 minute point) and the data are recorded.
After administration,the symptoms of rabbits are observed,such as hair shrugging,trembling,crouching,increase in heart rate,abdomen-thorax scorching or conjunctive congestion.
Results are expressed as mean ±S.E.M.and all the data are analyzed by statistical software(such as SPSS).MANOVA of repeated measuring is used for the temperature variations.
Tab.3—9 The original data record（BBT）
Tab.3—10 Antipyretic effects of different
PLR
extract on endotoxin-induced fever in rabbits model（
n
=6；
±
s
）
Temperature variation
According to the changes of rectal temperature,draw the antipyretic curves of each group.
4.6.3.1 Time-Temperature Curve The X axis is the time (Endotoxin injection as the starting point,30 minutes interval),the Y axis is the rectal temperature on the corresponding time point.(Figure.3—9)
4.6.3.2 Time-Temperature variation Curve The X axis is the time (Endotoxin injection as the starting point,30 minutes interval),the Y axis is the temperature variation on the corresponding time point.(Figure.3—10)
Figure.3—9
Figure.3—10
Teachers:Lectures and demonstration
Students:Hands-on practice and operation
Above is the specif ic design and operation.The actual operation should be adjusted to the division of student groups or experimental groups.
In each round,students are divided into five experimental groups.Animals are randomly divided into four groups.In each stage,there are 15 rabbits,including 3 rabbits in the negative control group,4 rabbits in the other groups,randomly divided into experimental groups.
The basal body temperatures of rabbits are the average of two measurements in 10 minutes intervals after keeping the rabbits calm for 30 min.
The water extract from PLR and the resin extract from PLR are the mixture,which were extracted from the previous experiments and processed so as to be eligible for intramuscular injection.
4.9.1 Are there any other simple and sensitive experiments for the research on the antipyretic effects of Chinese Materia Medica?
4.9.2 How would you design proper experiments to explore the effects of different extracts from Chinese Materia Medica?
4.10.1 Li,Yikui. Pharmacological Experimental Methodology of Chinese Materia Medica (2 nd Edition) Shanghai:Shanghai Press of Science ＆ Technology,2006
Ⅰ—4 Pharmacological research of Chinese materia medica
Ⅰ—5 Serum pharmacological method of Chinese materia medica
Ⅱ—3 The animal model of heat toxin syndrome
4.10.2 Chen,Qi. Pharmacological Research Methodology of Chinese Materia Medica (3 rd Edition) Beijing:People’s Medical Publishing House,2011
Chapter 3,Section 1 The basic pharmacological knowledge of Chinese material medica research
Section 3 The method of pharmacological research on TCM syndrome model
4.10.3 Wei Wei. Experimental Methodology of Pharmacology (4 th Edition) Beijing:People’s Medical Publishing House,2010
Chapter 3 The design of pharmacological experiment
Chapter 22 The experimental methods of anti-inf lammatory,antipyretic and analgesic drugs
Glossary
1.endotoxin 内毒素
2.antipyretic 退热的，退烧的
3.Analgin 安乃近（一种解热镇痛抗炎药）
4.Vaseline 凡士林
5.Basal Body Temperature (BBT) 基础体温
6.rectal 直肠的
7.anus 肛门
8.intramuscular 肌肉的，肌肉内的
9.duplication 复制
10.symptom 症状，征兆
11.shrug 耸肩
12.crouch 屈膝，蜷伏
13.scorch 灼热
14.conjunctive 结膜的
15.congestion 充血
16.multivariate analysis of variance (MANOVA) 多因素方差分析
17.least signif icant difference (LSD) 最小显著差法
18.pairwise 成对，配对
【基本知识】
1.滴丸 滴丸，系指饮片经适宜的方法提取、纯化后与适宜的基质加热熔融混匀，滴入不相混溶的冷凝介质中制成的球形或类球形制剂。该剂型的主要特点包括：①生产设备简单，操作容易，生产车间无粉尘，有利于劳动保护。②工艺条件易控制，质量稳定，剂量准确，可提高药物稳定性。③起效迅速，生物利用度高。④由于其载药量小，相应含药量低，服药剂量大。⑤液体药物可制成固体滴丸，便于服用和运输。⑥可制成内服、外用、速释、缓释、控释或局部治疗等多种类型的滴丸剂。
2.滴丸的制备过程 滴丸制备的主要过程：将主药溶解、混悬在已选择好的加热熔融的基质中，保持恒定的温度（80～100℃），经过一定大小管径的滴头，等速滴入冷却液中，凝固形成的丸粒徐徐沉于器底，或浮于冷却液的表面，取出，洗去冷却液，干燥，即成。
图3—11 滴丸制备的基本流程
【目的要求】
1.掌握制备滴丸的基本工艺过程。
2.熟悉滴丸制备过程中的工艺影响因素。
3.了解滴丸机的基本组成及其相应功能。
【仪器试剂】 滴丸试验机、紫外分光光度计、电子天平、崩解仪、恒温水浴锅。
【实验材料】 葛根总黄酮提取物，葛根素对照品（纯度≥98%）；PEG4000，二甲基硅油，30%乙醇。
【实验内容】
1.滴丸的制备
（1）葛根总黄酮提取物与PEG4000熔融液的制备 取葛根总黄酮提取物适量，加入适量无水乙醇，充分溶解后，加入PEG4000熔融液（药物:基质=1:5）中，搅拌混合均匀，直至乙醇挥发尽为止，继续静置于80℃水浴中保温30分钟，待气泡除尽，备用。
（2）滴丸机的参数设定 打开滴丸机油箱，加入二甲基硅油；打开压缩空气开关；打开滴丸机总电源，并设置相关参数：油浴温度80℃；药液温度80℃，管口温度50℃，制冷温度10℃。
（3）滴丸的制备 将上述除尽气泡的葛根总黄酮—PEG4000混匀熔融液转入滴丸机的贮液筒内，在保温70～80℃的条件下，控制滴速20滴/分钟，滴距约为6cm，开始滴制；在滴丸机出料口收集滴丸，沥净，并用滤纸除去丸上的冷凝液，干燥，即得。
2.滴丸的质量评价
（1）外观检查 应呈球状，大小均匀，色泽一致，无粘连现象。
（2）重量差异的测定 取供试品20丸，精密称定总重量，求得平均丸重后，再分别精密称定每丸的重量，每丸重量与平均丸重相比较。结果应符合相关规定（表3—11）。
表3—11 重量差异限度规定
（3）溶散时限检查 按照2015年版《中华人民共和国药典》四部通则0921“崩解时限检查法”项下之规定。
使用崩解仪，不锈钢丝网的筛孔内径为0.42mm。取供试品6粒，按“崩解时限检查法”不加挡板进行检查，应在30分钟内全部溶散。如有1粒不能完全溶散，应另取6粒复试，均应符合规定。
（4）滴丸中总黄酮的含量测定 供试品溶液的制备：取滴丸20丸，研细，取粉末适量，精密称定，置于100mL具塞三角烧瓶中，精密加入30%乙醇50mL，密塞，称重。超声处理20分钟，放冷，再称定重量，用30%乙醇补足减失的重量，摇匀，滤过，取续滤液作为供试品溶液。
对照品溶液的制备：取葛根素对照品适量，精密称定，置于10mL容量瓶中，以30%乙醇溶解，并定容至刻度线，配制得葛根素对照品溶液。
测定法：采用紫外分光光度法，于λ=250nm处分别测定对照品溶液和供试品溶液的吸光度A，并用外标法计算样品中总黄酮的含量（%）。
【实验结果及分析】
1.描述制成的滴丸的外观情况。
2.记录每丸的重量，计算平均丸重，并计算重量差异（表3—12）。
表3—12 重量差异测定结果
续表
20丸总重
3.记录制成滴丸的溶散时限（表3—13）。
表3—13 滴丸的溶散时限测定结果
4.记录紫外分光光度法测定制成的滴丸中葛根总黄酮的含量（表3—14）。
表3—14 滴丸中总黄酮的含量测定结果
【教学方式】 教师讲授和演示、多媒体课件和学生操作。
【注意事项】
1.在熔融药液和基质时应混匀，且将熔融液内的气泡除尽，才可使滴丸呈高度分散且表面光滑。
2.保温油浴可控制贮液筒内熔融液的黏度，应以顺利滴出为度，滴速可用阀门控制。
3.冷凝液的高度、滴距以及冷凝液的温度，均可影响滴丸的外形、粘连程度以及拖尾等，应以圆整为度。
4.制备滴丸时，药液温度应不低于80℃，否则在滴口易凝固而不易滴下。
【思考题】
1.制备滴丸时，应考虑的关键工艺参数有哪些？
2.影响滴丸成型、形状与重量的因素有哪些？在实际操作中应如何控制？
3.在滴丸剂的质量评价中，主要包括哪些项目？
【知识链接】 滴丸剂的一般质量评价项目，参考2015年版《中华人民共和国药典》附录ⅠK项下相关内容。
Dripping pills are prepared by putting the melted mixture of the purif ied and concentrated extract of crude drugs and appropriate bases into an immiscible cooling liquid and let it congeal to a spherical or sphere-like form.The main characteristics of dripping pills are simple production equipment and easy operation,it is produced by uncomplicated equipment and operation within a dust-free production workshop favorable for labor protection,easy control of process conditions with constant quality,accurate dosage and increased stability,quick action,high biological availability,relatively large dosage due to its low loading capacity,solidif ication of liquid medicine into solid dripping pills,which is convenient to administration and transportation,and formation of multitypes of dripping pills for oral administration or externally application,with sustained or controlled release,or just for local application.
The main process for preparing dripping pills is as follows.Take the main drug,which is dissolved or suspended fully,into a heat melting base.Maintain the mixture at a constant temperature of 80—100℃,drip it to the cooling media constantly by a circular dripper.After the solidif ied pellets slowly sink to the bottom or float to the surface of the coolants,collect the pellets and then wash and dry them.The manufacture of dripping pills is then accomplished.(Figure.3—11)
Figure.3—11 Basic process
5.2.1 To master the basic preparation procedures of dripping pills.
5.2.2 To be acquainted with the inf luential factors in the preparation of dripping pills.
5.2.3 To understand the basic components and corresponding functions of dripping pill machines.
Dripping pill machine; UV spectrophotometer; electronic balance; disintegration tester;constant temperature water bath.
Total flavonoids extract of Puerariae Lobatae Radix; puerarin (purity≥98%); PEG-4000;dimethyl silicone oil; 30 % ethanol.
5.5.1.1 Preparation of melting liquid of extracts and PEG-4000 Add total flavonoids extracts of Puerariae Lobatae Radix into little absolute ethyl alcohol.After the extracts are fully dissolved,add the solution into melted PEG4000 (drug:base =1:5 v/v),and mix evenly,until the ethanol is volatilized.Preserve it in a 80℃ water bath to remove bubbles.
5.5.1.2 Parameters set for the dripping pill machine Open the tank and add dimethyl silicone oil into it.Open the compressed air switch and general power switch.Set the relevant parameters as oil bath temperature of 80℃,liquid temperature of 80℃,tube temperature of 50℃and cooling temperature of 10℃.
5.5.1.3 Preparation of dripping pills Pour the melting liquid into the receiver of the dripping pill machine.Drop the pellets with the dripping speed of 20 drops /min and the dripping distance of 6cm in the temperature of 70—80℃.Collect the dripping pills at the outlet port,wash it to remove the coolant and then dry.
5.5.2.1 Appearance description The dripping pills should be spherical or spherical-like,with uniform size,and color and no adherence.
5.5.2.2 Weight variation Weigh accurately the total weight of 20 dripping pills,and calculate their average weight.Next weigh accurately each of the 20 dripping pills.Compare the weight of each pill with the average weight.According to the requirements stated in Tab.3—11,not more than 2 pills deviate outside the limit of weight variation,none should deviate outside 1 fold of the limit.
Tab.3—11 Requirments of limit of weight variation
5.5.2.3 Disintegration test Carry out the test as described under Disintegration test(General Requirements 0921,Volume IV, Chinese Pharmacopeia 2015 version.).Use a stainlesssteel wire gauze with apertures of 0.425mm in internal diameter.Carry out the test as described above using 6 dripping pills.All the dripping pills should disperse completely within 30 minutes.If 1 of the dripping pills fails to disperse completely,repeat the operation with another 6 dripping pills.
5.5.2.4 Determination of the content of total flavonoids in dripping pills Preparation of sample solution:Take 20 pills and ground them down.Take the powders of proper amount,and accurately weigh them.Place them into a 100mL triangular flask with a plug and add 50mL of 30% ethanol precisely,seal and weigh it.Ultrasonicate for 20min,cool and weigh it again.Complement the loss in weight with 30% ethanol,shake well and then filter off.
Preparation of reference solution:Weigh 10mg of control sample of puerarin into a volumetric flask of 10mL,and add some 30% ethanol to prepare the stock solution.Dilute it with proper multiples.
Method:With UV-spectrophotometry,determinate the absorbance (A) of the reference solution and the sample solution.Calculate the content (%) of the total flavonoids in the samples with the external standard method.
5.6.1 Describe the appearance of the made dripping pills constructed.
5.6.2 Record the weight of every dripping pill,and calculate the average weight and weight variation (Tab.3—12).
Tab.3—12 Results of weight variation
Total weight（g）
5.6.3 Record the disintegration time of the dripping pills (Tab.3—13).
Tab.3—13 Results of disintegration time
5.6.4 Determine the content of total flavonoids in the dripping pills with UV-spectrophotometry (Tab.3—14).
Tab.3—14 Results of content determination
Teachers:Lectures and demonstration.
Students:Hands-on practice and operation.
5.8.1 When the molten drug liquid and base are mixed evenly with no bubble inside,the dripping pills are highly dispersed with a smooth surface.
5.8.2 Heated oil bath is used to control the viscosity of the molten liquid,which should be dripped successfully.The dripping speed can be controlled by a valve.
5.8.3 The dripping distance,the height and temperature of the coolant are the main inf luential factors that can affect the appearance,adhesion degrees and tailing of pills,which should be set according to the degree of roundness.
5.8.4 In the preparation of dripping pills,the liquid temperature should be no less than 80℃,otherwise the drop will solidify too early to drip in the outlet port.
5.9.1 In the preparation of dripping pills,what are the key process parameters to be considered?
5.9.2 What are the inf luential factors for the formation,shape and weight of dripping pills?How would you control them in an experimental operation?
5.9.3 What are the main requirements in the quality evaluation of dripping pills?
For the general requirements for quality evaluation of dripping pills,please refer to General Requirements 0108,Volume IV,2015 edition of the Chinese Pharmacopoeia .
Glossary
滴丸 dripping pill
重量差异 weight variation
溶散时限 disintegration time
总黄酮 total flavonoids
质量评价 quality evaluation
【基本知识】
1.成型技术 任何药物在供临床使用之前，都必须根据药物的性质、用药目的和给药途径，加工制成适宜的应用形式。剂型作为药物应用的形式，可以保证药物的有效性，提高药物稳定性，降低药物毒、副作用，掩盖、改善药物不良嗅味，方便药物应用，发挥药物最大作用。药物剂型选择原则为：剂型应适应药物的性质，满足临床用药要求，结合生产等因素进行全面考虑。
2.溶出度测定方法 溶出度是指药物从片剂、胶囊剂或颗粒剂等制剂在规定条件下溶出的速率和程度。对于难溶性药物，其吸收是溶出速度限制过程，溶解速度快慢将直接影响到药物的生物利用度。
【目的要求】
1.掌握溶出度的测定方法。
2.通过愈风宁心片、滴丸的溶出度比较，充分理解剂型是影响疗效发挥的一个重要因素。
【仪器试剂】 药物溶出度仪、紫外可见分光光度计、蒸馏水、30%乙醇。
【实验材料】 愈风宁心片，愈风宁心滴丸，葛根素对照品（纯度≥98%）。
【实验内容】
1.用1000mL量筒量取900mL溶出介质，倒入溶出杯中，调节温度（37±0.5）℃后加热。
2.每组分别取3片愈风宁心片或3粒愈风宁心滴丸，分别投入3个干燥的转篮内，调节转速（100±1）r/min。
3.待温度达到设定温度后，按下转速开关和升降按钮，将转篮降入溶出杯中，自药品接触溶出介质起，开始计时。
4.至规定取样时间，取样（5mL/次），滤过（微孔滤膜），测定制剂中葛根总黄酮的含量（外标两点法）。
【实验结果及分析】 记录实验数据，绘制溶出曲线，标出T50、Td（表3—15、表3—16）。
表3—15 愈风宁心片的溶出变化
表3—16 愈风宁心滴丸的溶出变化
累积释放率（%）=某时间点释放量（mg）/药物总量
【教学方式】 教师讲授和演示及学生操作。
【注意事项】
1.溶出介质倒入溶出杯后不能直接将转篮置于其中，应先预热至设定温度。
2.取样位置应在转篮顶端至液面的中点，距离溶出杯壁10mm处。
3.取样误差应控制在±1%之内。
4.多次取样时，若取液总体积超过溶出介质的1%时，应及时补充溶出介质。
5.样品取样滤过的操作过程应在30秒内完成。
6.过滤时，应弃去初滤液，取续滤液测定含量。
【思考题】
1.欲将一个有效方剂制成成方制剂，应如何选择适宜的剂型？
2.在溶出度实验中，需要考虑哪些影响因素？
【知识链接】 2015年版《中华人民共和国药典》四部通则0931“溶出度与释放度检查法”项下相关内容。
Before the clinical use,any medicine should be made in a proper application form on the basis of its properties,medication purpose and administration route,in order to ensure the effectiveness of drugs,to enhance the stability of drugs,to reduce the toxicity or side-effects of drugs,to improve the smell or flavor of drugs and to facilitate the application of drugs.The proper dosage form should adapt to the properties of drugs,meet the clinical needs and prepared with comprehensive considerations on manufacturing and other factors.
Dissolution refers to the dissolution rate and extent of the drugs under specif ied conditions from solid preparations,such as tablets,capsule or particles.For the poorly soluble drug,the dissolution rate is the limiting process before its absorption.Therefore,the speed of dissolution directly affect the bioavailability of the drug.
6.2.1 To master the determination method in dissolution test.
6.2.2 To understand the importance of formulation on eff icacy through the dissolution comparison of tablets and dripping pills.
Instruments:UV-7504 UV/VIS spectrophotometer,drug dissolution tester.
Reagents:Redistilled water,30% ethanol.
Yufeng Ningxin Tablets; Yufeng Ningxin Dripping pills; Puerarin reference substance（purity≥98%）.
6.5.1 Take 900mL dissolution media by 1000mL cylinder,pour it into the dissolution cup,and adjust the temperature to 37±0.5℃.
6.5.2 Put the samples,respectively,into three dry rotating baskets.Adjust the rotating speed 100±1 r/min.
6.5.3 After reaching the set temperature,press the speed switch and elevator buttons.When the baskets are sunk into the dissolution cups,and the samples touch the dissolution media,start timing.
6.5.4 At the required sampling time point,sample (5mL/time),filter off (millipore) and determine the content of the total flavonoids in the samples (external reference method).
Draw the dissolution curves and estimate T50 and Td.(Tab.3—15 and Tab.3—16)
Tab.3—15 Dissolution Data of Yufeng Ningxin Tablets
Tab.3—16 Dissolution Data of Yufeng Ningxin Dripping Pills
Cumulative Release
Teachers:Lectures and demonstration.
Students:Hands-on practice and operation.
6.8.1 Do not place the baskets directly into the dissolution media.Please wait until the system reaches the set temperature.
6.8.2 The sampling location is in the mid-point between the top of the basket and the solution surface,where there is about 10mm away from the cup wall.
6.8.3 The sampling volume error should be controlled within ±1%.
6.8.4 In multiple sampling,if the total sampling volume is more than 1%,the dissolution media should be renewed in time.
6.8.5 The process of sampling and filtering should be finished within 30 seconds.
6.8.6 In filtering,the initial filtrate should be discarded.Take the continued filtrate as the sample to determine the concentration.
6.9.1 How would you choose the form of a drug in the preparation design of an effective prescription?
6.9.2 In the dissolution test,which operating factors should be considered？
For the dissolution evaluation,please refer to General Requirements 0931,Volume IV,2015 edition of the Chinese Pharmacopoeia .
Glossary
1.tablet 片剂
2.dripping pill 滴丸
3.dissolution 溶出度
4.dissolution medium 溶出介质
5.cumulative release rate 累积释放率
6.bioavailability 生物利用度
拓展试验是在完成了综合实验实训的前提下，并查阅了大量的与之相关的资料后，为开发学生的创新能力和各学科知识的综合运用能力而设计的后续实验，拓展实验应该是一个动态的自主设计实验。各学校可以根据不同的实际情况自行设计。本教材提供两方面的实验拓展思路。拓展试验作为本课程的一部分计入考核成绩。
拓展试验可以以葛根或粉葛为研究对象，假设有充足的科研经费和优良的实验条件，自主命题并设计出你感兴趣的药物研发或食品保健产品的研发路线和实验方案；也可以根据市场热点问题选择与本实验不相关的某一天然药物为研究对象，要求学生自主设计一可行性的研发路线和实验方案。
【目的要求】 拓展学生的科研思维。
【教学内容】 启发学生的想象力，鼓励学生在教学过程中自主设计出感兴趣的与该实验相关的新产品开发。
【教学方式】 学生自主设计，教师辅导完成拓展论文的写作。
For expanding an experiment,a follow-up and important training for medical students should be dynamic and self-designed.It is aimed at improving the innovation ability and integrated applied ability of multiple discipline knowledge on the premise of finishing the integrated experiment and retrieving related literature.Different universities could have their own experimental design depending on their situation.The present textbook provides two thoughts of expanding an experiment.As a part of this course,expanding an experiment will be assessed and included in the final exam result.
1.Students can take “Ge gen” and “Fen ge” as a subject to design their interested research plan and technical scheme of corresponding drug candidates or dietary supplements on the premise of suff icient research funding and excellent experimental conditions.
2.Students can also select other natural medicines,according to the current hot topic in the pharmaceutical market,as a research subject to propose the theoretically feasible research plan and technical scheme.
Expanding scientif ic thinking of students.
Inspiring the imagination of students,encouraging the students to design interested and experiment-related product by themselves.
Program design by students themselves,writing the article assisted with teachers.