A method for identifying medicinal material of hirudo and its original source by thin layer chromatography and application thereof

Abstract

The application discloses a kind of leech medicinal materials and its original identification thin layer chromatography method, the thin layer chromatography method includes the following steps: (1) the leech medicinal materials to be measured is pretreated to obtain leech medicinal materials test solution, leech medicinal materials are pretreated to obtain leech control medicinal solution;Wherein, the leech is Hirudinidae leech or leech worm;And (2) the leech medicinal materials test solution and the leech control medicinal solution are identified and analyzed by thin layer chromatography, to determine whether the leech to be measured is leech or leech worm;Wherein, the step of thin layer chromatography identification analysis adopts chloroform-methanol as developing agent, the volume ratio of chloroform and methanol is (6-10): about 1;Wherein, the leech to be measured is selected from one or more of the following: leech, leech worm, willow leaf leech, flatworm, light-rubbing golden line leech, eight-eye stone leech, Japanese leech and stick leech.The method is simple in operation, and the required instrument equipment cost is low, and the detection result is intuitive, accurate and easy to identify.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a medicinal material of Whitmania and a thin layer chromatography method for identifying the base origin thereof and application thereof. BACKGROUND

[0002] Whitmania is derived from the dried body of Whitmaniapigra Whitman, Hirudo nipponica Whitman or Whitmania acranulata Whitman of the Hirudinidae family, and has the effects of breaking blood, unblocking channels, removing blood stasis and resolving masses. Modern pharmacological studies have shown that Whitmania has multiple effects such as anticoagulation, antithrombosis, anti-atherosclerosis, anti-platelet aggregation, anti-tumor, anti-inflammatory, improving hemorheology, and protecting against cerebral ischemia-reperfusion injury. However, there are more than 680 species of Whitmania distributed worldwide, and more than 100 species in China. However, the legal base origin of Whitmania is only three species, namely Whitmania, Whitmania pigra Whitman and Whitmania acranulata Whitman. The demand for Whitmania is large on the medicinal material market, and its price has been rising year by year and is in short supply. Moreover, counterfeit products are common, and the main adulterants are Philodina sp., Whitmania pigra Whitman, Hirudo nipponica Whitman, and Japanese species Whitmania, etc., which leads to uneven quality of Whitmania and seriously affects the clinical efficacy.

[0003] The identification methods of Whitmania include characteristics identification, thin layer identification, molecular identification, and protein electrophoresis identification. The true and false identification methods (characteristics identification and thin layer identification method) of the legal standard of Whitmania (Chinese Pharmacopoeia (2020 edition)) are difficult to distinguish the species of Whitmania. PCR in the method of molecular identification has extremely high sensitivity, can amplify trace DNA templates, and cross contamination of trace reagents, instruments, consumables, aerosols or samples may cause false positive results, while the wrong operation of the detection personnel or the failure of the reagents and consumables is easy to cause false negative results. Protein electrophoresis has not been concerned about the similarities and differences of small molecular proteins and polypeptides of different species of Whitmania. Moreover, molecular identification and protein electrophoresis identification require the purchase of related instruments, and the skill requirements for experiment personnel are also high.

[0004] The prior art does not involve the thin layer chromatography method for identifying a medicinal material of Whitmania and the base origin thereof and application thereof. SUMMARY

[0005] Therefore, the present application provides a thin layer chromatography method for identifying a medicinal material of Whitmania and the base origin thereof, which comprises the following steps:

[0006] (1) pre-treating the to-be-tested leech medicinal material to obtain a to-be-tested leech medicinal material sample solution, and pre-treating the water leech medicinal material to obtain a water leech control medicinal material solution; wherein the water leech is Hirudo nipponica Whitman or Whitmania pigra Whitman; and

[0007] (2) performing thin layer chromatography identification analysis on the to-be-tested leech medicinal material sample solution and the water leech control medicinal material solution, so as to determine whether the to-be-tested leech is Hirudo nipponica Whitman or Whitmania pigra Whitman;

[0008] In the step of the thin layer chromatography identification analysis, chloroform-methanol is used as the developing agent, and the volume ratio of the chloroform to the methanol is (6-10): about 1.

[0009] In the step of the thin layer chromatography identification analysis, chloroform-methanol is used as the developing agent, and the volume ratio of the chloroform to the methanol is (6-10): about 1.

[0010] Further, the preparation method of the to-be-tested leech medicinal material sample solution comprises: weighing an appropriate amount of to-be-tested leech medicinal material powder, adding a solvent, ultrasonic extraction, centrifugation, taking the supernatant, and obtaining the to-be-tested leech medicinal material sample solution.

[0011] Further, the preparation method of the to-be-tested leech medicinal material sample solution comprises: weighing an appropriate amount of to-be-tested leech medicinal material powder, adding a solvent, ultrasonic extraction, centrifugation, taking the supernatant, and obtaining the to-be-tested leech medicinal material sample solution.

[0012] Further, the solvent is alcohol, for example, ethanol.

[0013] Further, the volume percentage concentration of the ethanol is 90%-100%, for example, about 95%.

[0014] Further, the mass / volume (g / ml) ratio of the to-be-tested leech medicinal material powder to the solvent is 0.1-0.5, for example, about 0.2.

[0015] Further, the ratio of the mass / volume (g / ml) of the medicinal material powder of Leech to the solvent is 0.1-0.5, for example, about 0.2.

[0016] Further, the mass of the medicinal material powder of Leech to be tested is 0.5-2 g, for example, about 1 g.

[0017] Further, the mass of the medicinal material powder of Leech to be tested is 0.5-2 g, for example, about 1 g.

[0018] Further, the volume of the solvent is 1-10 mL, for example, about 5 mL.

[0019] Further, the power of the ultrasonic extraction is 200-300 W, for example, about 250 W.

[0020] Further, the frequency of the ultrasonic extraction is 30-50 kHz, for example, about 40 kHz.

[0021] Further, the time of the ultrasonic extraction is 10-20 min, for example, about 15 min.

[0022] Further, the preparation method of the medicinal material powder of Leech to be tested comprises: weighing an appropriate amount of medicinal material of Leech to be tested, crushing, sieving, and obtaining the medicinal material powder of Leech to be tested.

[0023] Further, the preparation method of the medicinal material powder of Leech to be tested comprises: weighing an appropriate amount of medicinal material of Leech to be tested, crushing, sieving, and obtaining the medicinal material powder of Leech to be tested.

[0024] Further, the sieve is a No. 3 sieve.

[0025] Further, the volume ratio of the chloroform to the methanol is (7-9): about 1.

[0026] Further, the volume ratio of the chloroform to the methanol is (7.5-8.5): about 1.

[0027] Further, the volume ratio of the chloroform to the methanol is about 8: about 1.

[0028] Further, the method of the thin layer chromatography identification analysis comprises: spotting the test sample solution of the medicinal material of Leech to be tested and the Leech medicinal material solution on the same thin layer plate, developing, taking out the developed thin layer plate, drying, and observing under a UV light.

[0029] Further, the thin layer plate is a silica gel G thin layer plate.

[0030] Further, the wavelength of the UV light is 10-400 nm, for example, about 365 nm.

[0031] Further, the method of the thin layer chromatography identification analysis does not comprise the step of using sulfuric acid ethanol solution for color development.

[0032] Further, the concentration of the ethanol solution of sulfuric acid is about 10% (V / V).

[0033] Further, the standard of the thin layer identification includes: checking whether the thin layer plate shows the same color and position of fluorescent spots on the corresponding positions of the to-be-tested leech medicinal material and the water leech medicinal material; if the to-be-tested leech medicinal material has green fluorescent spots and the position of the fluorescent spots is the same as that of the water leech (Hirudo nipponica Whitman), the to-be-tested leech is the water leech (Hirudo nipponica Whitman); if the to-be-tested leech medicinal material has red fluorescent spots and the position of the fluorescent spots is the same as that of the Whitmania pigra Whitman, the to-be-tested leech is the Whitmania pigra Whitman; if the fluorescent spots of the to-be-tested leech medicinal material are not the same as those of the water leech medicinal material, the to-be-tested leech is the Whitmania acranulata Whitman or other related adulterants.

[0034] Further, the other related adulterants include Poecilobdella manillensis Lesson, Whitmania laevis Baird, Erpobdella octoculata, Mimobdella japonica Blanchard and Poecilobdella javanica Wahlberg.

[0035] According to another aspect of the present application, a thin layer chromatography method for identifying the medicinal material of water leech and its original source is provided, which is characterized by comprising:

[0036] Step one, taking the control medicinal material of water leech (water leech or Whitmania pigra) and the sample to be detected, crushing, sieving, obtaining the sample powder of water leech, the sample powder of Whitmania pigra and the sample powder to be detected, and taking equal amounts of the sample powder of water leech, the sample powder of Whitmania pigra and the sample powder to be detected respectively;

[0037] Step two, ultrasonically extracting and centrifuging the above powders with ethanol solution respectively, taking the supernatant, obtaining the test solution of water leech sample, the test solution of Whitmania pigra sample and the test solution of the sample to be detected;

[0038] Step three, spotting the above test solutions on the same silica gel thin layer plate respectively, developing, and observing the thin layer chromatography under the ultraviolet lamp, and identifying the sample to be detected according to the color and position of the fluorescent spots on the thin layer chromatography.

[0039] According to another aspect of the present application, there is provided a use of the above thin layer chromatography method for distinguishing the animal species of medicinal leech.

[0040] According to another aspect of the present application, there is provided a kit for distinguishing the animal species of medicinal leech, comprising a thin layer plate and a developing agent, wherein the developing agent is chloroform-methanol, and the thin layer plate is a silica gel G thin layer plate.

[0041] Further, the volume ratio of the chloroform to the methanol is (6-10): about 1.

[0042] Further, the volume ratio of the chloroform to the methanol is (7-9): about 1.

[0043] Further, the volume ratio of the chloroform to the methanol is (7.5-8.5): about 1.

[0044] Further, the volume ratio of the chloroform to the methanol is about 8: about 1.

[0045] Advantages of the present application:

[0046] The method first establishes a thin layer identification method for distinguishing the animal species of medicinal leech and leech worm, and specifically, the present application can obtain the number and position of fluorescent spots of the sample on the silica gel thin layer plate under the ultraviolet lamp by ultrasonic extraction, centrifugation, spotting, and development of the leech medicinal material sample to be tested, and the sample to be tested can be identified according to the number and position of the fluorescent spots, which is simple to operate, low in cost of required instrument and equipment, and intuitive, accurate, and easy to identify in detection results. BRIEF DESCRIPTION OF DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to these drawings without exceeding the scope of the present application.

[0048] Figure 1 The thin layer chromatogram of Example 1 of the present application.

[0049] Figure 2 The thin layer chromatogram of Comparative Example 1 of the present application.

[0050] Figure 3 The thin layer chromatogram of Comparative Example 2 of the present application.

[0051] Figure 4 The thin layer chromatogram of Comparative Example 3 of the present application.

[0052] Figure 5 The thin layer chromatogram of Comparative Example 4 of the present application. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0055] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0056] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0057] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0058] As described in the background section, the prior art does not involve a thin-layer chromatography method for identifying leeches and their origins, nor its application. To address the above problems, the present invention provides a thin-layer chromatography method for identifying leeches and their origins, which includes the following steps:

[0059] (1) The leech material to be tested is pretreated to obtain a test solution of the leech material, and the leech material is pretreated to obtain a reference solution of the leech material; wherein the leech is Hirudo nipponica Whitman or Whitmania pigra Whitman; and

[0060] (2) the test leech medicinal material sample solution and the Hirudo nipponica Whitman control medicinal material solution are subjected to thin layer chromatography identification analysis, so as to determine whether the test leech is Hirudo nipponica Whitman or Whitmania pigra Whitman;

[0061] In the step of the thin layer chromatography identification analysis, chloroform-methanol is used as a developing agent, and the volume ratio of the chloroform to the methanol is (6-10): about 1.

[0062] In the step of the thin layer chromatography identification analysis, chloroform-methanol is used as a developing agent, and the volume ratio of the chloroform to the methanol is (6-10): about 1.

[0063] In the present application, when a volume, a volume percentage concentration, a ratio, a mass, a power, a frequency, a time, a wavelength, a concentration, or other value or parameter is expressed in a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "6-10" is disclosed, the described range should be interpreted as including the ranges "6-10", "6-9", "6-8", "6-7", "7-10", "7-9", "7-8", "8-10", "8-9", "9-10", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include the end values and all integers and fractions within the range.

[0064] In the present application, "about" refers to a value within ±5% of a particular value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0065] In a preferred embodiment, the preparation method of the test leech medicinal material sample solution comprises: weighing an appropriate amount of test leech medicinal material powder, adding a solvent, ultrasonic extraction, centrifugation, taking the supernatant, and obtaining the test leech medicinal material sample solution.

[0066] In a preferred embodiment, the preparation method of the Leech Reference Drug Material Solution comprises: weighing a proper amount of Leech Drug Material Powder, adding a solvent, ultrasonic extraction, centrifugation, taking supernatant, and obtaining the Leech Reference Drug Material Solution.

[0067] In a preferred embodiment, the solvent is alcohol, such as ethanol.

[0068] In a preferred embodiment, the volume percentage concentration of the ethanol is 90% to 100%, such as about 95%.

[0069] In the present application, "about" refers to a value within ±5% of a specified value. For example, "about 95%" includes ±5% of 95%, or from 90.25% to 99.75%.

[0070] In a preferred embodiment, the mass / volume (g / ml) ratio of the to-be-tested Leech Drug Material Powder to the solvent is 0.1 to 0.5, such as about 0.2.

[0071] In the present application, "about" refers to a value within ±5% of a specified value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0072] In a preferred embodiment, the mass / volume (g / ml) ratio of the Leech Drug Material Powder to the solvent is 0.1 to 0.5, such as about 0.2.

[0073] In the present application, "about" refers to a value within ±5% of a specified value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0074] In a preferred embodiment, the mass of the to-be-tested Leech Drug Material Powder is 0.5 to 2g, such as about 1g.

[0075] In the present application, "about" refers to a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0076] In a preferred embodiment, the mass of the Leech Drug Material Powder is 0.5 to 2g, such as about 1g.

[0077] In the present application, "about" refers to a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0078] In a preferred embodiment, the volume of the solvent is 1 to 10ml, such as about 5ml.

[0079] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0080] In a preferred embodiment, the power of the ultrasonic extraction is 200-300W, for example, about 250W.

[0081] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0082] In a preferred embodiment, the frequency of the ultrasonic extraction is 30 to 50 kHz, for example, about 40 kHz.

[0083] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0084] In a preferred embodiment, the ultrasonic extraction time is 10 to 20 minutes, for example, about 15 minutes.

[0085] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0086] In a preferred embodiment, the method for preparing the leech powder to be tested includes: weighing an appropriate amount of leech, crushing it, and sieving it to obtain the leech powder to be tested.

[0087] In a preferred embodiment, the method for preparing the leech medicinal powder includes: weighing an appropriate amount of leech medicinal material, crushing it, and sieving it to obtain the leech medicinal powder.

[0088] In a preferred embodiment, the sieve is a No. 3 sieve.

[0089] In a preferred embodiment, the volume ratio of the trichloromethane to the methanol is (7-9): about 1.

[0090] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0091] In a preferred embodiment, the volume ratio of chloroform to methanol is (7.5 to 8.5): about 1.

[0092] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0093] In a preferred embodiment, the ratio of the volume of chloroform to the volume of methanol is about 8: about 1.

[0094] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0095] In a preferred embodiment, the method of thin layer chromatographic identification analysis comprises: spotting the test solution of the leech medicinal material and the control solution of the water leech medicinal material on the same thin layer plate, developing, taking out the developed thin layer plate, drying, and observing under a UV light.

[0096] In a preferred embodiment, the thin layer plate is a silica gel G thin layer plate.

[0097] In a preferred embodiment, the wavelength of the UV light is 10-400 nm, such as about 365 nm.

[0098] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 365" includes ±5% of 365, or from 346.75 to 383.25.

[0099] In a preferred embodiment, the method of thin layer chromatographic identification analysis does not comprise the step of developing with sulfuric acid ethanol solution.

[0100] In a preferred embodiment, the concentration of the sulfuric acid ethanol solution is about 10% (V / V).

[0101] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 10%" includes ±5% of 10%, or from 9.5% to 10.5%.

[0102] In a preferred embodiment, the standard for the thin layer identification includes: checking whether the thin layer plate shows the same color and position fluorescent spots on the corresponding positions of the to-be-tested leech medicinal material and the water leech medicinal material; if the to-be-tested leech medicinal material has green fluorescent spots and the position of the fluorescent spots is the same as that of the water leech (Hirudo nipponica Whitman), the to-be-tested leech is the water leech (Hirudo nipponica Whitman); if the to-be-tested leech medicinal material has red fluorescent spots and the position of the fluorescent spots is the same as that of the Whitmania pigra Whitman, the to-be-tested leech is the Whitmania pigra Whitman; if the fluorescent spots of the to-be-tested leech medicinal material are not the same as those of the water leech medicinal material, the to-be-tested leech is the Whitmania acranulata Whitman or other related and fake products.

[0103] In a preferred embodiment, the other related and fake products include the Poecilobdella manillensis Lesson, the Whitmania laevis Baird, the Erpobdella octoculata, the Mimobdella japonica Blanchard and the Poecilobdella javanica Wahlberg.

[0104] According to another aspect of the present application, a thin layer chromatography method for identifying the medicinal material of water leech and its original animal is provided, which is characterized by comprising:

[0105] Step one, taking the control medicinal material of water leech (water leech or Whitmania pigra Whitman) and the to-be-tested sample, crushing, sieving, obtaining the water leech sample powder, the Whitmania pigra Whitman sample powder and the to-be-tested sample powder, and taking equal amounts of the water leech sample powder, the Whitmania pigra Whitman sample powder and the to-be-tested sample powder respectively;

[0106] Step two, ultrasonically extracting and centrifuging the powders respectively with the ethanol solution, taking the supernatant, obtaining the water leech sample test solution, the Whitmania pigra Whitman sample test solution and the to-be-tested sample test solution;

[0107] Step three, spotting the test solutions on the same silica gel thin layer plate respectively, developing, and observing the thin layer chromatography under the ultraviolet lamp, and identifying the to-be-tested sample according to the color and position of the fluorescent spots on the thin layer chromatography.

[0108] According to another aspect of the present application, the use of the above thin layer chromatography method in distinguishing the original animal of the medicinal material of water leech is provided.

[0109] According to another aspect of the present application, there is provided a kit for differentiating the animal origin of Leech medicinal material, comprising a thin layer plate and a developing agent, wherein the developing agent is chloroform-methanol and the thin layer plate is a silica gel G thin layer plate.

[0110] In a preferred embodiment, the volume ratio of the chloroform to the methanol is (6-10): about 1.

[0111] In the present application, "about" means a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0112] In a preferred embodiment, the volume ratio of the chloroform to the methanol is (7-9): about 1.

[0113] In the present application, "about" means a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0114] In a preferred embodiment, the volume ratio of the chloroform to the methanol is (7.5-8.5): about 1.

[0115] In the present application, "about" means a value within ±5% of a specified value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0116] In a preferred embodiment, the volume ratio of the chloroform to the methanol is about 8: about 1.

[0117] In the present application, "about" means a value within ±5% of a specified value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0118] The present application is further described in conjunction with the following examples. It should be understood that these examples are merely for the purpose of illustration and are not intended to limit the scope of the present application. The experimental methods in the following examples, unless otherwise stated, were carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials are described herein.

[0120] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, can be combined in any combination. All features disclosed in the present patent specification can be used in any combination, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless specifically stated, the disclosed features are only general examples of equivalent or similar features.

[0121] Experimental instruments and reagents

[0122] KQ-250DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), CAMAG ATS 4 full-automatic sample applicator (Switzerland), 2020D ultraviolet / white light transmission instrument (Cold Spring), TGL20MW table type high-speed refrigerated centrifuge (Huxi Instrument and Equipment Co., Ltd. in Hunan), BSA-124S-CW type one-hundred-thousandth electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0123] Leech (Limnatis humilis) control medicinal material (China Institute for Food and Drug Control, batch number: 121061-202107), Lampreys control medicinal material (China Institute for Food and Drug Control, batch number: 121754-201901), Silica gel G plate (Qingdao Marine Chemical Plant, 10x20cm), Cyclohexane (Tianjin Cheryuan Chemical Reagent Co., Ltd.), Ethyl acetate (Tianjin Damao Chemical Reagent Factory), Sulfuric acid (Beijing Tongguang Fine Chemicals Co., Ltd.), Ethanol, methanol (National Pharmaceutical Group Chemical Reagent Co., Ltd.), Trichloromethane (Beijing Tongguang Fine Chemicals Co., Ltd.).

[0124] Experimental materials

[0125] 28 batches of samples were collected from different regions in China, including 12 batches of Limnatis humilis, 11 batches of Limnatis humilis, 1 batch of Limnatis humilis, 1 batch of Lampreys, 2 batches of Japanese leeches and 1 batch of light-rubbing golden leeches. The specific information is as follows:

[0126] Sample 1 Limnatis humilis (Hirudo nipponica Whitman), produced in Jining County, Jining City, Shandong Province;

[0127] Sample 2 Limnatis humilis (Hirudo nipponica Whitman), produced in Suzhou City, Suzhou City, Jiangsu Province;

[0128] Sample 3 Limnatis humilis (Hirudo nipponica Whitman), produced in Bamen Town, Baodi District, Tianjin City;

[0129] Sample 4 Limnatis humilis (Hirudo nipponica Whitman), produced in Xihua County, Zhoukou City, Henan Province;

[0130] Sample 5 Hirudo nipponica Whitman, origin: Jining County, Jining City, Shandong Province;

[0131] Sample 6 Hirudo nipponica Whitman, origin: Suzhou District, Suqian City, Jiangsu Province;

[0132] Sample 7 Hirudo nipponica Whitman, origin: De'an County, Jiujiang City, Jiangxi Province;

[0133] Sample 8 Hirudo nipponica Whitman, origin: Yuanjiang City, Yiyang City, Hunan Province;

[0134] Sample 9 Hirudo nipponica Whitman, origin: Jining County, Jining City, Shandong Province;

[0135] Sample 10 Hirudo nipponica Whitman, origin: Gong'an County, Jingzhou City, Hubei Province;

[0136] Sample 11 Hirudo nipponica Whitman, origin: Gong'an County, Jingzhou City, Hubei Province;

[0137] Sample 12 Hirudo nipponica Whitman, origin: Bamiancheng Town, Baodi District, Tianjin;

[0138] Sample 13 Whitmania pigra Whitman, origin: Shandong (purchased from Anguo Medicine Market);

[0139] Sample 14 Whitmania pigra Whitman, origin: Jining County, Jining City, Shandong Province;

[0140] Sample 15 Whitmania pigra Whitman, origin: Suzhou District, Suqian City, Jiangsu Province;

[0141] Sample 16 Whitmania pigra Whitman, origin: Bamiancheng Town, Baodi District, Tianjin;

[0142] Sample 17 Whitmania pigra Whitman, origin: Xihua County, Zhoukou City, Henan Province;

[0143] Sample 18 Whitmania pigra Whitman, origin: Jining County, Jining City, Shandong Province;

[0144] Sample 19 Whitmania pigra Whitman, produced in Suqian City, Jiangsu Province;

[0145] Sample 20 Whitmania pigra Whitman, produced in De'an County, Jiujiang City, Jiangxi Province;

[0146] Sample 21 Whitmania pigra Whitman, produced in Yuanjiang City, Yiyang City, Hunan Province;

[0147] Sample 22 Whitmania pigra Whitman, produced in Gong'an County, Jingzhou City, Hubei Province;

[0148] Sample 23 Whitmania pigra Whitman, produced in Gong'an County, Jingzhou City, Hubei Province;

[0149] Sample 24 Poecilobdella javanica Wahlberg, produced in Qinnan District, Qinzhou City, Guangxi;

[0150] Sample 25 Poecilobdella manillensis Lesson, produced in Zhongshan City, Guangdong Province;

[0151] Sample 26 Mimobdella japonica Blanchard, produced in Xihua County, Zhoukou City, Henan Province;

[0152] Sample 27 Mimobdella japonica Blanchard, produced in Xinhui District, Jiangmen City, Guangdong Province;

[0153] Sample 28 Whitmania laevis Baird, produced in De'an County, Jiujiang City, Jiangxi Province.

[0154] Example 1

[0155] The thin layer chromatography method of leeches and leeches includes:

[0156] Step one, take leeches, leeches and samples to be tested, crush, pass through a 3# pharmaceutical sieve, obtain leech, leech and sample powder to be tested, then respectively take 1g of leech, leech and sample powder to be tested;

[0157] Step two, the above powders are respectively extracted with 5mL 95%(V / V) ethanol solution under the power of 250w and the frequency of 40KHz for 15min, centrifuged, take the supernatant, obtain leech sample test solution and leech sample test solution;

[0158] Step three, the above test solution was respectively spotted on the same silica gel G thin layer plate, with chloroform-methanol (8:1, V / V) as developing agent, developed, taken out, air dried, and observed under ultraviolet light (365nm), the results were shown in Table 1. Figure 1 Figure 1 Among them, leech, cattle leech as control medicinal materials, 1-12 for leech samples, 13-23 for cattle leech samples, 24 for Haemadipsa ruffescens sample, 25 for cattle leech sample, 26, 27 for Japanese leech sample, 28 for golden line leech sample.

[0159] It can be seen that, under ultraviolet light (365nm), leech showed green fluorescent spots (A), and cattle leech showed red fluorescent spots (B). Figure 1

[0160] Comparative example 1

[0161] The thin layer chromatography method of leech and cattle leech includes:

[0162] Step one, taking leech, cattle leech and samples to be detected, crushing, passing through No. 3 drug sieve, obtaining leech, cattle leech and sample powder, then respectively taking 1g of leech, cattle leech and sample powder;

[0163] Step two, the above powder was respectively ultrasonically extracted with 5mL 95%(V / V) ethanol solution under the power of 250w and the frequency of 40KHz for 15min, centrifuged, taking supernatant, obtaining leech sample test solution and cattle leech sample test solution;

[0164] Step three, the above test solution was respectively spotted on the same silica gel G thin layer plate, with chloroform-methanol (8:1, V / V) as developing agent, developed, taken out, air dried, and observed under ultraviolet light (365nm), the results were shown in Table 1. Figure 2 Figure 2 Among them, leech, cattle leech as control medicinal materials, 1-12 for leech samples, 13-23 for cattle leech samples, 24 for Haemadipsa ruffescens sample, 25 for cattle leech sample, 26, 27 for Japanese leech sample, 28 for golden line leech sample.

[0165] It can be seen that, under ultraviolet light (365nm), leech showed green fluorescent spots (A), and cattle leech showed red fluorescent spots (B). Figure 2

[0166] Comparative example 2

[0167] The thin layer chromatography method of leech and cattle leech includes:

[0168] ​​​​Step one, take leeches, leeches and samples to be tested, crush, pass through No. 3 drug sieve, get leeches, leeches and sample powder, then take 1g of leeches, leeches and sample powder respectively;

[0169] Step two, the above powder is respectively extracted with 5mL 95%(V / V) ethanol solution under the power of 250w and the frequency of 40KHz for 15min, centrifuged, and the supernatant is taken to get leech sample test solution and leech sample test solution;

[0170] Step three, the above test solution is respectively spotted on the same silica gel G thin layer plate, with chloroform-methanol(5:1, V / V) as developing agent, developed, taken out, dried, and observed under ultraviolet light(365nm), the results are shown in Figure 3 . Figure 3 Among them, leeches and leeches are control medicinal materials, 1-12 are leech samples, 13-23 are leech samples, 24 is a stick pattern leech sample, 25 is a leech sample, 26 and 27 are Japanese leech samples, and 28 is a light smooth golden leech sample.

[0171] From Figure 3 It can be seen that under the thin layer developing condition of comparative example 2, under ultraviolet light(365nm), leeches show green fluorescent spots(A), and leeches show red fluorescent spots(B), but the red fluorescent spots of leeches are overlapped with another spot, and the position is close to the front. Therefore, the thin layer developing condition of comparative example 2 cannot develop the characteristic spots in leeches and leeches at the same time, which shows that the thin layer developing effect is not good under the above developing agent condition.

[0172] Comparative example 3

[0173] The thin layer chromatography method of leeches and leeches comprises:

[0174] Step one, take leeches, leeches and samples to be tested, crush, pass through No. 3 drug sieve, get leeches, leeches and sample powder, then take 1g of leeches, leeches and sample powder respectively;

[0175] Step two, the above powder is respectively extracted with 5mL 95%(V / V) ethanol solution under the power of 250w and the frequency of 40KHz for 15min, centrifuged, and the supernatant is taken to get leech sample test solution and leech sample test solution;

[0176] Step three, the above test solution is respectively spotted on the same silica gel G thin layer plate, with chloroform-methanol(5:1, V / V) as developing agent, developed, taken out, dried, and observed under ultraviolet light(365nm), the results are shown in Figure 4 . Figure 4In the experiment, leech, limnetes philippinensis was used as control medicine, 1-12 was leech sample, 13-23 was limnetes philippinensis sample, 24 was limnetes marginatus sample, 25 was limnetes philippinensis sample, 26, 27 was limnetes japonicus sample, and 28 was limnetes luminosus sample.

[0177] It can be seen that under the ultraviolet light (365 nm), limnetes philippinensis showed red fluorescent spots (B), but its position was at the origin, and there was no characteristic spot of leech. Therefore, under the thin layer development condition of comparative example 3, the characteristic spots in leech and limnetes philippinensis cannot be developed at the same time, indicating that the thin layer development effect is not good under the above development agent condition. Figure 4 Comparative example 4

[0178] The thin layer chromatography method of leech and limnetes philippinensis comprises the following steps:

[0179] Step one, taking leech, limnetes philippinensis and the sample to be detected, crushing, passing through No. 3 drug sieve to obtain leech, limnetes philippinensis and sample powder, and then taking 1 g of leech, limnetes philippinensis and sample powder respectively;

[0180] Step two, the above powders are respectively ultrasonically extracted with 5 mL of 95% (V / V) ethanol solution under the power of 250 w and the frequency of 40 KHz for 15 min, centrifuged, and the supernatant is taken to obtain leech sample test solution and limnetes philippinensis sample test solution;

[0181] Step three, the above test solutions are respectively spotted on the same silica gel G thin layer plate, and cyclohexane-ethyl acetate (4:1) is used as the developing agent, and then developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105 DEG C until the spots are colored clearly, and the results are shown in

[0182] Figure 5 In the experiment, leech, limnetes philippinensis was used as control medicine, 1-12 was leech sample, 13-23 was limnetes philippinensis sample, 24 was limnetes marginatus sample, 25 was limnetes philippinensis sample, 26, 27 was limnetes japonicus sample, and 28 was limnetes luminosus sample. Figure 5 This method is the thin layer chromatography method of leech medicine in Chinese Pharmacopoeia (2020 edition). It can be seen from

[0183] that all test samples derived from leech or limnetes philippinensis show the same purple red spots (a) at the positions corresponding to the chromatogram of the control medicine, and the same orange red fluorescent spots (b) under the ultraviolet light (365 nm), indicating that the thin layer identification method recorded in the pharmacopoeia cannot effectively distinguish leech and limnetes philippinensis. Figure 5 Figure 5 (a); orange red fluorescent spots (b) under the ultraviolet light (365 nm), indicating that the thin layer identification method recorded in the pharmacopoeia cannot effectively distinguish leech and limnetes philippinensis. Figure 5

[0184] ​​​The above has carried out the detailed introduction to the embodiment of the application, the principle and implementation mode of the application have been described in this paper by applying specific examples, the above embodiment description is only used for helping understanding the method of the application and its core idea. Meanwhile, the changes or deformations made by the person skilled in the art according to the idea of the application, based on the specific implementation mode and application range of the application, all belong to the protection range of the application. In summary, the content of the specification should not be understood as the limitation of the application.

Claims

1. A thin layer chromatography identification method of Hirudo nipponica Whitman and Whitmania pigra Whitman, characterized in that, The thin layer chromatography identification method comprises the following steps: (1) The test leech medicinal material is pretreated to obtain a test leech medicinal material sample solution, and the water leech medicinal material is pretreated to obtain a water leech control medicinal material solution; wherein the water leech is Hirudo nipponica Whitman of the water leech family; and (2) The test leech medicinal material sample solution and the water leech control medicinal material solution are subjected to thin layer chromatography identification analysis, so as to determine whether the test leech is Hirudo nipponica Whitman; In the step of the thin layer chromatography identification analysis, chloroform-methanol is used as a developing agent, and the volume ratio of the chloroform to the methanol is 8:

1. The test leech is Hirudo nipponica Whitman and Whitmania pigra Whitman. The standard of the thin layer identification is that whether the thin layer plate shows the same color and position fluorescent spots at the corresponding positions of the test leech medicinal material and the water leech medicinal material; if the test leech medicinal material has green fluorescent spots and the positions of the fluorescent spots are the same as those of the water leech Hirudo nipponica Whitman control medicinal material, the test leech is Hirudo nipponica Whitman. The preparation method of the test leech medicinal material sample solution comprises the following steps: a proper amount of test leech medicinal material powder is weighed, 95% volume percentage ethanol is added, ultrasonic extraction is performed, centrifugation is performed, and the supernatant is taken to obtain the test leech medicinal material sample solution. The method of the thin layer chromatography identification analysis comprises the following steps: the test leech medicinal material sample solution and the water leech control medicinal material solution are spotted on the same thin layer plate, development is performed, the developed silica gel G thin layer plate is taken out, air-dried, and then observed under a wavelength of 365 nm ultraviolet light.

2. The thin layer chromatography discrimination method according to claim 1, characterized in that, The preparation method of the water leech control medicinal material solution comprises the following steps: a proper amount of water leech medicinal material powder is weighed, 95% volume percentage ethanol is added, ultrasonic extraction is performed, centrifugation is performed, and the supernatant is taken to obtain the water leech control medicinal material solution.

3. The thin layer chromatography discrimination method according to claim 1, characterized in that, The mass / volume ratio of the test leech medicinal material powder to the 95% volume percentage ethanol is 0.1-0.5 g / ml.

4. The thin layer chromatography discrimination method according to claim 3, characterized in that, The mass / volume ratio of the test leech medicinal material powder to the 95% volume percentage ethanol is 0.19-0.21 g / ml.

5. The thin layer chromatography discrimination method according to claim 2, wherein The mass / volume ratio of the water leech medicinal material powder to the 95% volume percentage ethanol is 0.1-0.5 g / ml.

6. The thin layer chromatography discrimination method according to claim 5, wherein, The mass / volume ratio of the water leech medicinal material powder to the 95% volume percentage ethanol is 0.19-0.21 g / ml.

7. The thin layer chromatography discrimination method according to claim 1, wherein The mass of the test leech medicinal material powder is 0.5-2 g.

8. The thin layer chromatography discrimination method according to claim 7, characterized in that, The mass of the test leech medicinal material powder is 0.95-1.05 g.

9. The thin layer chromatography discrimination method according to claim 2, wherein, The mass of the water leech medicinal material powder is 0.5-2 g.

10. The thin layer chromatography discrimination method according to claim 9, characterized in that, The mass of the water leech medicinal material powder is 0.95-1.05 g.

11. The thin layer chromatography discrimination method according to claim 1 or 2, characterized by, The volume of the 95% volume percentage concentration of ethanol is 1-10 mL.

12. The thin layer chromatography discrimination method according to claim 11, wherein, The volume of the 95% volume percentage concentration of ethanol is 4.75-5.25 mL.

13. The thin layer chromatography discrimination method according to claim 1 or 2, characterized by, The power of the ultrasonic extraction is 200-300 W.

14. The thin layer chromatography discrimination method according to claim 13, wherein, The power of the ultrasonic extraction is 237.5-262.5 W.

15. The thin layer chromatography discrimination method according to claim 1 or 2, characterized by, The frequency of the ultrasonic extraction is 30-50 kHz.

16. The thin layer chromatography discrimination method according to claim 15, wherein, The frequency of the ultrasonic extraction is 38-42 kHz.

17. The thin layer chromatography discrimination method according to claim 1 or 2, characterized by, The time of the ultrasonic extraction is 10-20 min.

18. The thin layer chromatography discrimination method according to claim 17, wherein, The time of the ultrasonic extraction is 14.25-15.75 min.

19. The thin layer chromatography discrimination method according to claim 1, wherein, The preparation method of the leech medicinal material powder comprises the following steps: weighing a proper amount of leech medicinal material, crushing, sieving, and obtaining the leech medicinal material powder.

20. The thin layer chromatography discrimination method according to claim 2, wherein, The preparation method of the leech medicinal material powder comprises the following steps: weighing a proper amount of leech medicinal material, crushing, sieving, and obtaining the leech medicinal material powder.

21. The thin layer chromatography discrimination method according to claim 19 or 20, characterized in that, The sieve is a No. 3 sieve.

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