A method for identifying Dytiscidae

Abstract

The invention provides a method for identifying water beetles, which includes property identification, microscopic identification and thin layer chromatography identification; the thin layer chromatography identification includes the following steps: (1) taking the powder of the product, adding ethanol for ultrasonic treatment, centrifuging, and taking the supernatant as a test solution; (2) taking leucine, lysine and valine reference substances, and adding ethanol to prepare leucine, lysine and valine reference substance solutions; (3) absorbing the test solution and the reference substance solution, respectively spotting them on the same silica gel G thin layer plate, using a mixed solution of n-butanol-glacial acetic acid-water with a volume ratio of 3:1:1 as a developing agent, developing, taking out, drying, spraying with ninhydrin test solution, and heating at 105°C until the spots are clearly colored. The identification method includes identifying water beetles from multiple aspects in terms of appearance, properties and components, has high accuracy and good durability, and the result is not affected by the detection temperature or humidity, and can be used as a medicinal identification standard for water beetles.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material identification, and particularly relates to a method for identifying water slugs. Background Art

[0002] Dytiscidae, also known as Zelao, black shell worm, water turtle, water turtle, and urine-shooting turtle. The adult body is 13 to 45 mm long, oval or long oval, slightly narrow at the front, black or dark brown; the head is slightly flat, and the head is retracted into the prothorax; the dorsal and ventral sides of the body are arched, the compound eyes are prominent, the antennae are filamentous, 11 segments, and the mandibles are short; there are 3 pairs of legs, the front legs are small, the hind legs are flattened laterally, and there are long hairs. The hind legs are swimming legs, the posterior coxa and the metathoracic ventral plate occupy more than half of the ventral surface, and there are no thorns on the ventral surface of the chest. The body of Dytiscidae mainly contains protein, crude fat, total sugar, etc. There are many fatty acids in the body of Dytiscidae, mainly unsaturated fatty acids. The body of Dytiscidae contains many trace elements necessary for the human body, such as Cu, Co, Mn, Se, S, Ti, K, Na, Mg, etc.

[0003] Nature and flavor of Dytiscus and its meridians: sweet, slightly salty, neutral in nature, and enters the kidney meridian and bladder meridian; Functions and indications: tonifying the kidney, reducing urine, and activating blood circulation. It is mainly used to treat enuresis in children, frequent enuresis in the elderly, and brown spots on the face.

[0004] At present, water beetles are mainly used as food and have not yet been included in the legal standards for traditional Chinese medicine. No legal identification method for water beetles has been established. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a method for identifying water slugs, which includes property identification, microscopic identification and thin layer chromatography identification, has the advantages of high accuracy and good durability, and can be used as a medicinal identification standard for water slugs.

[0006] To achieve the above object, the present invention adopts the following technical solution.

[0007] A method for identifying a water beetle, the method comprising the following steps:

[0008] S1. Identification of properties: The dried insect body is long and oval, shiny all over; the back is black; the edge of the elytra has a narrow brown-yellow edge; after removing the elytra, a pair of light-colored membranous wings can be seen; the ventral surface is reddish brown to dark brown; there are horizontal stripes on the abdomen; the texture is crisp, smelly, and slightly salty;

[0009] S2. Microscopic identification: The powder of this product is gray-brown or brown; the body wall fragments are dark brown or yellow, with irregular textures on the surface, occasionally densely covered with fine particles, round hair pits and tumor-like protrusions, on which short, thick or slender bristles grow; the bristles are brown-yellow or yellow, mostly broken, with sharp or blunt tips, some with vertical straight textures, and the medullary cavity is narrow; the striated muscle fibers are colorless or light yellow, often broken, with fine and dense vertical stripes, straight or wavy, with bright bands and dark bands wider;

[0010] S3. Thin layer chromatography identification: (1) Take the powder of the product, add ethanol to ultrasonic treatment, centrifuge, and take the supernatant as the test solution; (2) Take leucine, lysine and valine reference substances, add ethanol to prepare leucine, lysine and valine reference substance solutions respectively; (3) Pipette the test solution and reference substance solution, respectively spot them on the same silica gel G thin layer plate, use a mixture of n-butanol-glacial acetic acid-water in a volume ratio of 3:1:1 as the developing agent, develop, take out, dry, spray with ninhydrin test solution, and heat at 105°C until the spots are clearly colored.

[0011] In some embodiments, the liquid ratio of the powder of the product to ethanol in step (1) is 1:10 g / mL.

[0012] In some embodiments, the ultrasonic treatment time in step (1) is 15 minutes.

[0013] In some of the embodiments, the concentrations of the leucine, lysine and valine reference solutions in step (2) are 1 mg / mL, 1 mg / mL and 0.5 mg / mL, respectively.

[0014] In some embodiments, the volume fraction of ethanol in step (1) is 75%.

[0015] In some embodiments, the volume fraction of ethanol in step (2) is 75%.

[0016] In some embodiments, the spotting volumes of the test solution and the reference solution in step (3) are 5-8 μl and 1 μl, respectively.

[0017] The present invention provides a method for identifying water beetles, which identifies water beetles in all aspects, including shape, specification, size, surface or cross-section color, characteristics, texture, fracture phenomenon, smell and composition, thereby improving the specificity and accuracy of the identification method. In the method, thin layer chromatography identification uses leucine, lysine and valine as reference substances, has good specificity, and further optimizes the corresponding thin layer chromatography identification conditions: a mixed solution of n-butanol-glacial acetic acid-water with a volume ratio of 3:1:1 is used as a developing agent, so that the three amino acid standards can all obtain suitable Rf values, and the spots on the thin layer chromatogram are clearly distinguishable. The identification method of the present invention has high accuracy and good durability, and the result is not affected by the detection temperature or humidity, and can be used as a medicinal identification standard for water beetles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the characteristics diagram of the 19009 batch of water slugs.

[0019] Figure 2 This is the characteristics diagram of the 19010 batch of water slugs.

[0020] Figure 3 This is the characteristics diagram of the 19013 batch of water slugs.

[0021] Figure 4 This is the characteristics diagram of the 19014 batch of water slugs.

[0022] Figure 5 This is the characteristics diagram of the 19015 batch of water slugs.

[0023] Figure 6 This is the microscopic characteristic diagram of water beetle powder; among them, 1 is the body wall fragment, 2 is the bristle, and 3 is the striated muscle fiber.

[0024] Figure 7 This is a picture of thin layer chromatography identification results.

[0025] Figure 8 This is a graph showing the heavy metal content test results. DETAILED DESCRIPTION

[0026] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0027] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0028] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0029] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0030] The following describes the invention in conjunction with specific embodiments.

[0031] The product described in the present invention is the whole insect of the Dytiscidae insect, which can be caught all year round. After being caught, it is scalded to death with boiling water and dried in the sun.

[0032] The information of the water beetle samples involved in the present invention is shown in Table 1:

[0033] Table 1 Sample information table

[0034]

[0035] The properties are described according to the actual situation of the samples. The properties of the above 5 batches of samples are shown in Figures 1 to 5 .

[0036] Example 1

[0037] This example identifies the samples in Table 1.

[0038] 1. Characteristic identification

[0039] The samples in Table 1 are all dried insect bodies, long and oval, shiny all over; black on the back; narrow brown-yellow edges on the elytra; a pair of light-colored membranous wings can be seen after removing the elytra; reddish brown to dark brown on the ventral surface; horizontal stripes on the abdomen; crispy, fishy, ​​and slightly salty. They meet the identification standards for the characteristics of Dytiscidae.

[0040] 2. Microscopic identification

[0041] The powder of this product is gray-brown or brown. The body wall fragments are dark brown or yellow, with irregular textures on the surface, occasionally densely covered with fine particles, round hair pits and tumor-like protrusions, on which short thick or slender bristles grow; the bristles are brown-yellow or yellow, mostly broken, with sharp or blunt tips, some with vertical straight textures, and narrow medullary cavity. Striated muscle fibers are colorless or light yellow, often broken, with fine vertical stripes, straight or micro-wave-shaped, and the light band is wider than the dark band. The microscopic characteristics of the powder are shown in the figure below. Figure 6 shown.

[0042] 3. Thin layer chromatography identification

[0043] (1) Take 1g of the powder, add 10ml of 75% ethanol, ultrasonically treat for 15 minutes, centrifuge, and take the supernatant as the test solution;

[0044] (2) Separately, take a leucine reference substance, a lysine reference substance, and a valine reference substance, and add 75% ethanol to prepare solutions containing 1 mg, 1 mg, and 0.5 mg per ml, respectively, as reference substance solutions;

[0045] (3) According to the thin layer chromatography method (General Method 0502), 5-8 μl of the test solution and 1 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, and n-butanol-glacial acetic acid-water (volume ratio of 3:1:1) is used as the developing agent. The plate is developed, removed, dried, sprayed with ninhydrin test solution, and heated at 105°C until the spots are clearly colored.

[0046] The results are as follows Figure 7As shown, in the chromatogram of the test sample, spots of the same color are shown at the corresponding positions of the chromatogram of the reference sample, and the spots of each sample are clearly distinguishable. In this embodiment, leucine, lysine and valine are used as reference samples, and the specificity is good.

[0047] Example 2

[0048] In this example, the moisture content, total ash content, heavy metal content and alcohol-soluble extracts of each batch of water lice samples in Table 1 were tested.

[0049] 1. Moisture

[0050] The moisture content was determined according to the water content determination method (Method 2, General Rules 0832 of the 2020 edition of the Chinese Pharmacopoeia). Five batches of samples were measured, and the results were 4.9% to 12.6%, with an average value of 9.4% (see Table 2).

[0051] Table 2 Water content determination results of Dytiscidae

[0052]

[0053] 2. Total ash

[0054] Ash content was determined according to the ash content determination method (General Rules 2302 of the 2015 edition of the Chinese Pharmacopoeia). Five batches of samples were measured, and the results were 2.4% to 3.9%, with an average value of 3.1% (see Table 3).

[0055] Table 3 Determination results of total ash content of Dytiscidae

[0056]

[0057]

[0058] 3. Heavy metals

[0059] Determined according to the heavy metal inspection method (General Rule 0821 Method 2). Five batches of samples were tested, and the results are shown in Figure 8 The color of the sample colorimetric tubes is lighter than that of the standard colorimetric tubes with a heavy metal content of 30 mg / kg, and the color of the colorimetric tubes of batch 1 (batch number 19010) is darker than that of the standard colorimetric tubes with a heavy metal content of 20 mg / kg.

[0060] 4. Alcohol-soluble extract

[0061] Using ethanol as solvent, the hot soaking method under the alcohol-soluble extract (General Rules 2201 of the 2015 edition of the Chinese Pharmacopoeia) was used for determination. Five batches of samples were measured, and the results were 21.1-32.7%, with an average value of 24.8% (see Table 4).

[0062] Table 4 Determination results of Dytiscidae extract

[0063]

[0064] The above results show that the above 5 batches of samples are of high quality, indicating that the method of the present invention can be used to identify and screen out water lice of good quality.

[0065] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for identifying water beetles, characterized in that: The identification method comprises the following steps: S1. Identification of properties: The dried insect body is long and oval, shiny all over; the back is black; the edge of the elytra has a narrow brown-yellow edge; after removing the elytra, a pair of light-colored membranous wings can be seen; the ventral surface is reddish brown to dark brown; there are horizontal stripes on the abdomen; the texture is crisp, smelly, and slightly salty; S2. Microscopic identification: The powder of this product is gray-brown or brown; the body wall fragments are dark brown or yellow, with irregular textures on the surface, occasionally densely covered with fine particles, round hair pits and tumor-like protrusions, on which short, thick or slender bristles grow; the bristles are brown-yellow or yellow, mostly broken, with sharp or blunt tips, some with vertical straight textures, and the medullary cavity is narrow; the striated muscle fibers are colorless or light yellow, often broken, with fine and dense vertical stripes, straight or wavy, with bright bands and dark bands wider; S3. Thin layer chromatography identification: (1) Take the powder of the product, add ethanol to ultrasonic treatment, centrifuge, and take the supernatant as the test solution; (2) Take leucine, lysine and valine reference substances, add ethanol to prepare leucine, lysine and valine reference substance solutions respectively; (3) Pipette the test solution and reference substance solution, respectively spot them on the same silica gel G thin layer plate, use a mixture of n-butanol-glacial acetic acid-water in a volume ratio of 3:1:1 as the developing agent, develop, take out, dry, spray with ninhydrin test solution, and heat at 105°C until the spots are clearly colored.

2. The method for identifying a water beetle according to claim 1, wherein: The solid-liquid ratio of the product powder to ethanol in step (1) is 1:10 g / mL.

3. The method for identifying water beetles according to claim 1, characterized in that: The ultrasonic treatment time in step (1) is 15 minutes.

4. The method for identifying water beetles according to claim 1, characterized in that: The concentrations of the leucine, lysine and valine reference substance solutions in step (2) are 1 mg / mL, 1 mg / mL and 0.5 mg / mL, respectively.

5. The method for identifying water beetles according to claim 1, characterized in that: The volume fraction of ethanol in step (1) is 75%.

6. The method for identifying water beetles according to claim 1, characterized in that: The volume fraction of ethanol in step (2) is 75%.

7. The method for identifying a water beetle according to claim 1, wherein: In step (3), the spotting volumes of the test solution and the reference solution are 5-8 μl and 1 μl, respectively.

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