阅读说明：本技术 一种中药土鳖虫的鉴定试剂盒及其鉴定方法 (Identification kit and identification method for traditional Chinese medicine ground beetles ) 是由 鞠康 马灵珍 郭宣宣 郭伟娜 吴小菲 赵瑞瑞 李清文 于 2019-11-08 设计创作，主要内容包括：本发明基于薄层色谱法鉴定中药土鳖虫的原理,公开了一种中药土鳖虫的鉴定试剂盒及其鉴定方法,属于中药材鉴定技术领域,所述鉴定试剂盒包括样品溶剂、对照品溶液、展开剂、显色剂和检测试剂条,所述检测试纸条由上至下依次包括上载板、硅胶层、中载板、吸水层和下载板。与现有技术相比,该鉴定试剂盒及其方法无需额外干燥或加热等设备辅助,整个鉴定过程通过检测试剂条在室内常规条件下操作即可,此外,点样槽的设计,相比较传统薄层色谱过程中用毛细管人工点样,计量把握更精确,对操作员的经验要求较低,获得的薄层色谱斑点清晰规整。(The invention discloses an identification kit and an identification method for traditional Chinese medicine ground beetles based on the principle of identifying traditional Chinese medicine ground beetles by thin-layer chromatography, and belongs to the technical field of traditional Chinese medicine identification. Compared with the prior art, the identification kit and the method thereof do not need additional drying or heating and other equipment assistance, the whole identification process can be operated under indoor conventional conditions through the detection reagent strip, in addition, the design of the sample application groove is compared with the traditional thin-layer chromatography process for capillary artificial sample application, the measurement is more accurate, the experience requirement on operators is lower, and the obtained thin-layer chromatography spots are clear and regular.)

1. The identification kit for the traditional Chinese medicine ground beeltle is characterized by comprising a sample solvent, a reference solution, a developing agent, a color developing agent and a detection reagent strip, wherein the detection reagent strip sequentially comprises an upper support plate, a silica gel layer, a middle support plate, a water absorbing layer and a lower support plate from top to bottom, the upper support plate is sequentially provided with a developing agent groove, a sample application groove and an observation window along the long axis direction, the developing agent groove is communicated with one end of the silica gel layer, the sample application groove is 4-6mm away from the developing agent groove, the bottom of the sample application groove is provided with a sample application needle communicated with the silica gel layer, the observation window is used for observing the chromatographic condition of a sample, and the observation window is provided with; the middle carrier plate is provided with a plurality of water permeable holes communicated with the silica gel layer and the water absorbing layer, the middle carrier plate is provided with a drawing diaphragm, the drawing diaphragm covers the water permeable holes of the middle carrier plate, and the drawing diaphragm comprises a drawing section extending out of the middle carrier plate; and calcium oxide is filled in the water absorption layer.

2. The kit for identifying the Eupolyphaga Seu Steleophaga as claimed in claim 1, wherein the upper, middle and lower carrier plates are glass plates or PVC plates.

3. The kit for identifying the traditional Chinese medicine ground beetle according to claim 1, wherein the silica gel layer is made of silica gel G.

4. The kit for identifying Eupolyphaga Seu Steleophaga as claimed in claim 1, wherein the sample application grooves comprise at least 1 standard sample application groove and at least 1 sample application groove, and the sample application needles in the sample application grooves have equal pore volume.

5. The kit for identifying Eupolyphaga Seu Steleophaga as claimed in claim 1, wherein a stop line is disposed at an end of the silica gel layer in the observation window away from the developer tank, and anhydrous cupric sulfate is coated in the stop line.

6. The kit for identifying a Chinese herbal medicine ground beetle according to claim 1, wherein the sample solvent is a 50% methanol solution by volume.

7. The kit for identifying the traditional Chinese medicine ground beetle according to claim 1, wherein the preparation method of the reference solution comprises the following steps: taking a plurality of standard products, adding a sample solvent to prepare a mixed solution containing 0.5mg of the standard products per 1ml of the sample solvent, wherein the standard products comprise arginine, glycine, valine and phenylalanine standard products.

8. The traditional Chinese medicine ground beetle identification kit according to claim 1, wherein the developing solvent is a mixture of 5 volume percent: 5: 0.5 toluene-dichloromethane-acetone solution or 3 volume percent: 1: 1, and the color developing agent is vanillin sulfuric acid test solution or ninhydrin solution.

9. The identification method of the traditional Chinese medicine ground beetle is characterized by comprising the following steps:

step S1, preparation of a test solution: adding 25ml of sample solvent into 1g of Eupolyphaga Seu Steleophaga powder to be detected, performing ultrasonic treatment, filtering, evaporating filtrate, and dissolving residue with 5ml of sample solvent to obtain sample solution;

step S2, spotting: adding a sample solution into a sample application groove of the sample, adding a reference solution into a sample application groove of a standard sample, keeping each solution in the sample application groove for 3-6 seconds, and discarding the residual solution in the sample application groove;

step S3, thin layer expansion: adding the developing agent into a developing agent groove, standing and developing, and discarding the residual developing agent in the developing agent groove when the termination line turns blue;

step S4, dry color development: tearing a transparent diaphragm of the observation window, spraying a color developing agent on the surface of the silica gel layer, simultaneously drawing out the drawing diaphragm, and obtaining chromatograms of a test solution and a reference solution when the silica gel layer develops color;

step S5, result determination: if the test solution has spots with the same color at the position corresponding to the chromatogram of the reference solution, the test solution is qualified Chinese medicinal Eupolyphaga Seu Steleophaga, otherwise, the test solution is unqualified.

Technical Field

The invention relates to the technical field of traditional Chinese medicine material identification, in particular to an identification kit and an identification method for a traditional Chinese medicine ground beetle.

Background

The Chinese medicinal Eupolyphaga Seu Steleophaga is female dry body of Eupolyphaga Seu Steleophaga (Eupolyphaga sinensis Walker) or Eupolyphaga Seu Steleophaga (Polyphaga planchyi Bolivar) belonging to family Cyperaceae, and the insect lives in the ground, so it is named as Eupolyphaga Seu Steleophaga or Eupolyphaga Seu Steleophaga. The Chinese medicinal Eupolyphaga comprises Eupolyphaga Seu Steleophaga or winged Eupolyphaga Seu Steleophaga, which is flat, oval, narrow in front end, wide in back end, purple brown in back, glossy, without wings, developed in front and back plate, covering head, 9 joints of abdomen and back plate, arranged in covering tile shape, red brown on abdomen, small in head, 1 pair of filiform tentacles, and usually falling off, 3 pairs of feet on chest, fine hair and thorns, transverse link on abdomen, crisp, fragile, fishy smell, and slightly salty taste. The back of the winged euonymus is dark brown, and light yellow-brown plaques and black dots are usually formed on the edge of the winged euonymus. The powder of the Chinese medicinal ground beetle is grayish brown, fragments on the body wall are dark brown or yellow, irregular textures are arranged on the surface, short and thick or slender setae are grown on the surface, and a circular hair pit with the diameter of 5-32 mu m after the setae fall off is often seen; the setae are brown yellow or yellow, the tips of the setae are sharp or blunt, the setae are 12-270 mu m long, the diameters of the setae are 10-32 mu m, the setae have vertical and straight textures, the striated muscle fibers are colorless or light yellow, the setae are broken frequently, the setae have fine striations, the setae are straight or microwave-shaped, and the bright bands are wider than the dark bands.

The Chinese medicinal material Eupolyphaga Seu Steleophaga mainly comprises protein, volatile oil, fatty acid, amino acids, sterol and various microelements. In addition, the medicine contains various fatty aldehyde, aromatic aldehyde, dichlorobenzene, dimethyl disulfide and other components which are not common in traditional Chinese medicines. The caught ground beetle can be used as a medicine after being killed by boiling water, dried in the sun or dried, has higher medicinal value, can break blood stasis and reunite bones, and is a commonly used medicine for treating fracture of bones and muscles, amenorrhea due to blood stasis and abdominal mass.

At present, the identification method of the traditional Chinese medicine ground beetle mainly comprises morphological identification and physicochemical identification, the morphological identification is visual, but the error is large, and accurate judgment can be made only by depending on identification personnel with rich experience. The physicochemical identification method mainly comprises liquid chromatography or thin-layer chromatography, and achieves the identification purpose by identifying the characteristic components therein, for example, the method for identifying the Chinese medicinal material of the ground beetle by the thin-layer chromatography disclosed in the Chinese pharmacopoeia 2015 edition comprises the following steps: taking Eupolyphaga Seu Steleophaga powder, adding methanol, ultrasonic treating, evaporating filtrate to dryness, dissolving residue with methanol to obtain sample, taking Eupolyphaga Seu Steleophaga control medicinal material, preparing control medicinal material solution by the same method, performing thin layer chromatography test, dropping the two solutions on the same silica gel G thin layer plate, developing with toluene-dichloromethane-acetone (5: 5: 0.5) developer, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution; spraying vanillin-sulfuric acid solution, and heating at 105 deg.C until the spots are clear and have the same color. Although the physical and chemical identification method is more accurate than morphological identification, the operation is more complex, more instruments and equipment are required, and the identification time is long.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides an identification kit for Chinese medicinal ground beetles and an identification method thereof, and aims to solve the technical problems of low accuracy, complex operation, high instrument dependence and the like of the conventional identification method.

The invention is realized by the following technical scheme:

the invention provides an identification kit for Chinese medicinal ground beetles, which comprises a sample solvent, a reference solution, a developing agent, a color developing agent and a detection reagent strip, wherein the detection reagent strip sequentially comprises an upper support plate, a silica gel layer, a middle support plate, a water absorbing layer and a lower support plate from top to bottom, the upper support plate is sequentially provided with a developing agent groove, a sample application groove and an observation window along the long axis direction, the developing agent groove is communicated with one end of the silica gel layer, the sample application groove is 4-6mm away from the developing agent groove, the bottom of the sample application groove is provided with a sample application needle communicated with the silica gel layer, the observation window is used for observing the chromatographic condition of a sample, and the observation window is provided with a transparent diaphragm for isolating the silica gel layer; the middle carrier plate is provided with a plurality of water permeable holes communicated with the silica gel layer and the water absorbing layer, the middle carrier plate is provided with a drawing diaphragm, the drawing diaphragm covers the water permeable holes of the middle carrier plate and is used for preventing the water absorbing layer from absorbing water of the silica gel layer, and the drawing diaphragm comprises a drawing section extending out of the middle carrier plate so as to be convenient to draw out the drawing diaphragm and open a water absorbing channel of the water absorbing layer; the water absorption layer is filled with calcium oxide and used for absorbing water of the silica gel layer and releasing heat.

As a further optimized scheme of the invention, the upper, middle and lower carrier plates are glass plates or PVC plates.

As a further optimized scheme of the invention, the silica gel layer is made of silica gel G.

As a further optimization scheme of the invention, the sample application grooves are provided with a plurality of sample application grooves, wherein the sample application grooves comprise at least 1 standard sample application groove and at least 1 sample application groove, the pore volumes of the sample application needles in the sample application grooves are equal, and preferably, the pore volume of the sample application needle in each sample application groove is 0.5-5 mu l.

As a further optimization scheme of the invention, one end of the silica gel layer in the observation window, which is far away from the developing agent groove, is provided with a termination line, and anhydrous copper sulfate is coated in the termination line.

As a further optimization scheme of the invention, the sample solvent is a methanol solution with the volume ratio of 50%.

As a further optimized scheme of the present invention, the preparation method of the reference solution comprises: taking a plurality of standard products, adding a sample solvent to prepare a mixed solution containing 0.5mg of the standard products per 1ml of the sample solvent, wherein the standard products comprise arginine, glycine, valine and phenylalanine standard products.

As a further optimization scheme of the invention, the developing agent is a mixture of 5 volume percent: 5: 0.5 toluene-dichloromethane-acetone solution or 3 volume percent: 1: 1, and the color developing agent is vanillin sulfuric acid test solution or ninhydrin solution.

The invention also provides an identification method of the traditional Chinese medicine ground beetle, which comprises the following steps:

step S1, preparation of a test solution: adding 25ml of sample solvent into 1g of Eupolyphaga Seu Steleophaga powder to be detected, performing ultrasonic treatment, filtering, evaporating filtrate, and dissolving residue with 5ml of sample solvent to obtain sample solution;

step S2, spotting: adding a sample solution into a sample application groove of the sample, adding a reference solution into a sample application groove of a standard sample, keeping each solution in the sample application groove for 3-6 seconds, and discarding the residual solution in the sample application groove;

step S3, thin layer expansion: adding the developing agent into a developing agent groove, standing and developing, and discarding the residual developing agent in the developing agent groove when the termination line turns blue;

step S4, dry color development: tearing a transparent diaphragm of the observation window, spraying a color developing agent on the surface of the silica gel layer, and simultaneously drawing out the drawn diaphragm, wherein at the moment, calcium oxide in the water absorption layer can absorb water in the silica gel layer and release heat, and the silica gel layer can rapidly develop color while drying the silica gel layer, so that chromatograms of a test solution and a reference solution are obtained;

step S5, result determination: if the test solution has spots with the same color at the position corresponding to the chromatogram of the reference solution, the test solution is qualified Chinese medicinal Eupolyphaga Seu Steleophaga, otherwise, the test solution is unqualified.

The invention provides an identification kit of Chinese medicinal ground beetles and an identification method thereof based on the thin-layer chromatography identification principle disclosed by Chinese pharmacopoeia. In addition, the design of point sample groove compares traditional thin layer chromatography in-process with the artifical point sample of capillary, and the measurement is held more accurately, and is lower to operator's experience requirement, and the thin layer chromatography spot that obtains is clear regular.

Drawings

FIG. 1 is a schematic view of the overall structure of a test strip;

FIG. 2 is a schematic diagram of a cross-sectional structure of a test strip in a long axis direction;

in the figure: 1-upper carrier plate, 2-silica gel layer, 3-middle carrier plate, 4-water absorbing layer, 5-lower carrier plate, 11-developing agent groove, 12-sample application groove, 13-sample application needle, 14-observation window, 15, transparent diaphragm, 21-stop line, 31-water permeable hole and 32-drawing diaphragm.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.