一、简介1. Introduction

自然界中有丰富的动植物和微生物资源，从中筛选和提取生物活性物质一直是国内外研究的重点。生物学中的化学成分是多种多样的，结构也完全不同，但具有生物活性的成分比例较小。因此，生物活性成分的快速检测和筛选对于天然活性物质的研究至关重要。然而，传统的从天然产物中分离活性成分的方法也需要通过天然产物中的化合物的分离，活性化合物的筛选分两步进行。一般评价的活动应该在动物或器官的整个历史上进行，因为活性物质经常出现孤立的化合物，There are abundant resources of animals, plants and microorganisms in nature, from which the screening and extraction of bioactive substances has always been the focus of research at home and abroad. The chemical constituents in biology are manifold and structure utterly different, but which with biological activity are less proportion. Therefore, detecting and screening of biological active components rapidly is vital to the study of natural active substances. However, the traditional method of separation of active ingredients from natural products also need to pass to isolate compounds in natural products and screening of the compound active two step, the activity of general appraisal should be carried out in the entire history of the animals or organ, so often appear isolated compounds for the active substance or quantity is not enough to a large number of active screening[ 1 ]。因此，建立一种快速、准确的活性物质筛选方法，对于活性物质的分离、新化合物的发现、质量标准的控制和组分应用范围的扩大具有重要意义。.Consequently, to establish a rapid and accurate screening method for active substances plays an important role in the separation of active substances, the discovery of new compounds, the control of quality standards and the expansion of the application range of components.

薄层生物自显影是指参照方法学技术，在活动指导的基础上，对自然生物的活性成分进行分离、定位和评价，具有操作简单、成本低、灵敏度和特异性高的优点。该技术将薄层色谱分离与生物活性测定相结合；薄层色谱是一种现代色谱技术，通过涂覆在支撑板表面的吸附材料作为固定相，洗脱液作为流动相来分离混合物。通过利用固定相中各组分不同的吸附能力，在流动相洗脱过程中产生连续的吸附和解吸，实现不同组分的分离。薄层色谱作为一种经典的分析技术于1938年首次开发，具有成本低、开发时间快、重现性好、灵敏度高等优点。随着技术的发展，在此基础上衍生出二维薄层色谱（2D-TLC）和高效薄层色谱（HP bioautography is used to isolate, locate, and evaluate the active constituents of natural creatures based on the guidance of activity refers to a methodological technique, and has the advantages of operation simply, cost lowly and high levels of sensitivity and specificity. TLC），并且这类检测技术的应用范围不断扩大。天然磷脂已通过2D-his technique combines TLC定量，验证了该技术的适用性 separation with biological activity determination, 并且这种检测技术的应用范围正在不断扩大。天然磷脂已通过2D-TLC定量，验证了该技术的适用性 is a modern chromatography technology in which mixtures are separated by adsorption materials coated on the surface of the supporting plate as the stationary phase and eluent as the mobile phase. By means of utilizing the different adsorption capacities of each component on the stationary phase, continuous adsorption and desorption can be generated during the mobile phase elution process to achieve the separation of different 并且这种检测技术的应用范围正在不断扩大。天然磷脂已通过2D-TLC定量，验证了该技术的适用性. component. Bioautography is a method that uses chemical components to react with substrates and chromogenic agents to form disparate color contrasts, so as to observe contrast spots in a chromogenic background to track active components ^[2]. TLC combined with bioautography can reduce the blindness of compound separation and reduce the use of active animals compared with traditional methodology. On the one hand, it can reflect the difference of chemical composition between samples. On the other hand, this assay format allows preassigning of the biological activity, observed in a mixture, to one or a few of its components through the spots that they produce ^[3]. It determines the presence, absence or strength of the biological activity of the detected substance by showing spots with different colors from the inactive area under certain conditions, which is especially suitable for the screening and discovery of active ingredients in complex mixture systems.

2. Classification

Three basic types of TLC bioautography can be distinguished: agar diffusion, direct bioautography and agar overlay bioautography, and, in addition, HPTLC bioautography and 2D-TLC bioautography, which have emerged in recent years.

Agar Diffusion, also known as the agar contact method, agar diffusion is also the least-employed one of these techniques. In this method, medium inoculated with pathogenic microorganisms is used as the carrier, and a thin-layer plate adsorbed with the compounds is face down and in contact with the surface of the medium. The compound is placed in contact with the surface of the medium for a certain time to diffuse it, and, then, after removal of the thin-layer plate, the medium is cultured overnight at about 37 °C for bioautography. Agar is prone to adhere to silica gel due to the prominent adsorption between them; therefore, it is easy to damage the agar layer and affect the observation of the spots after removal of the thin plate; thus, adding a wet filter paper between the plate and the agar layer could reduce this error to a certain extent. In addition, another defect of the agar diffusion is that compounds will be lost during the transfer from the thin-layer plate to the culture medium, which is not conducive to the precise determination of the concentration and brings about a low level of sensitivity and specificity, cooperatively ensuring the utilization rate of this method is on the decline.

Direct bioautography (DB) is the most commonly used and easiest method among the three methods. This method, using a thin-layer plate as the carrier, sprays the pathogenic microorganism suspension with a certain concentration of specific nutrient solution directly onto the unfolded thin-layer plate, or directly places the plate into the suspension. It can observe the experimental result directly or under the appropriate color reagent after cultivation in a humid and dark environment for a period of time. In this method, separation, pretreatment, cultivation and development are performed directly on the plate. The action duration of this method is longer, which is conducive to the preservation of the experimental results. This is the simplest method for the detection of antifungal substances, such as 

Agar Overlay Bioautography, also known as immersion bioautography, agar overlay bioautography is a hybrid of both previous methods ^[4]. In order to allow a good diffusion of the tested compounds into the agar medium, the plates can be placed in a low temperature for a few hours before incubation. The melt agar medium inoculated with microorganisms is uniformly coated on the thin plate. After the agar solidifies, the thin plate is cultured overnight at about 30 °C and dyed with a color developing agent, and then the experimental results can be observed ^[5]. The advantage of this method is to reduce the influence of the experimental steps on the results. The agar overlay method is suitable for broad-spectrum microorganisms, especially for yeast and bacteria. This method has also been well developed over the decades. For instance, Rabia Tanvir et al. applied NP silica plates to non-polar to medium-polar compounds; then, the RP silica plate was polarized 

3. Detection Technique

TLC bioautography can only select materials that contain active ingredients, and it is therefore crucial that follow-up tests are performed for the determination of specific components. Commonly used techniques to determine the structure of the compound technology involves ex situ and in situ techniques; the former mainly includes nuclear magnetic resonance (NMR), electron ionization mass spectrometry (EI-MS) and electrospray ionization mass spectrometry (ESI-MS), which are common detection techniques and suitable for the detection of most compounds. Traditional MS mostly uses closed ionization technology, which requires complex pretreatment operation before sample analysis, which limits the wide application of this technology. In situ ionization MS is a kind of direct ionization and mass analysis technology for samples under normal temperature and pressure conditions without sample pretreatment ^[6]. The advent of this technology is a major change in the field of mass spectrometry analysis, which can realize in situ, rapid, nondestructive and direct mass spectrometry analysis of trace components on the surface of objects, greatly expanding the application range of mass spectrometry analysis. In situ ionization mass spectrometry can be used to identify the structure of chemical components on the thin-layer surface; solve the problem of compound analysis after the active components are screened out by TLC bioautography; and realize in situ, real-time and direct mass spectrometry analyses of target components in spots with inhibitory effects under the background of active color on the thin layer. In particular, the direct analysis in real-time mass spectrometry (DART-MS) and desorption electrospray ionization mass spectrometry (DESI-MS) developed in recent years have attracted extensive attention for their rapid in situ analysis of samples free of preparation.

4. Biological Applications

At present, the commonly used microbiological activity determination methods include the microbial inhibition method, enzymatic method, immunoassay method, radio immune assay (RIA) method, etc. These methods have their own advantages and disadvantages. For example, although the microbial inhibition method is simple in operation and low in cost, its specificity and sensitivity are not high, and it is easy to produce false positive results. However, highly sensitive, enzymatic and radio immune assays are expensive ^[7]. Broth 肉汤微量稀释法是筛选具有抗菌活性化合物的常用方法，但需要将化合物与培养基和微生物共培养，耗时长，应用范围缩小，常用检测细菌或真菌耐药性和药物敏感性microdilution method is a commonly used method for screening compounds with antimicrobial activity, it needs to co-culture the compounds with the culture medium and microorganism, which take a lot of time and narrowed the scope of application, which is used to detect bacterial or fungal resistance and drug sensitivity[ 8 ] [ 9 ]. 与上述方法相比，Compared with the above methods, TLC生物自显影是一种优良的生物活性测定方法。用最少的实验室设备和仪器就可以做到这一点，而且操作更简单，实验成本更低，灵敏度和特异性更高，同时可以提高化合物的代谢物或转化物的生物活性。由 bioautography is an excellent method for the determination of biological activity. It is possible with a minimum amount of laboratory equipment and apparatus, operation simpler, experimental cost lower, sensitivity and specificity higher, at the same time, the bioactivity of metabolites or converts of compounds can be determined by TLC 生物自显影测定。但是，bioautography. However, TLC生物自显影也有一定的局限性。一个这样的障碍是细胞毒性化合物的不敏感性，例如喜树碱、类黄酮和木脂素。大多数这些化合物很难在薄层板上显示抑制点。另外，由于 bioautography also has certain limitations. One such obstacle is the insensitivity of cytotoxic compounds, such as camptothecin, quassinoid and lignans, most of which are difficult to show inhibitory spots on the thin-layer plate. In addition, as the microbial culture medium used by TLC生物自显影所用的微生物培养基一般以水为溶剂，极性比较大；因此，当被测化合物极性过大时，容易扩散过快，导致抑制点形成困难。极性过小导致化合物有效性的问题是该技术应用的障碍，因为它们不易进入介质，也不能形成抑制点​​。这个缺陷可以通过后来建立的 bioautography is generally used as a solvent with water, the polarity is relatively large, so when the measured compound is too polar, it is easy to spread too fast, then lead to difficult to form inhibition spots, questions regarding the validity of too small polarity results in compounds is a barrier to the application in this technology, because of they are not easy to enter the medium and also cannot form inhibition spots. This defect can be improved by 2D-TLC 生物自显影来改善。bioautography established later.