Leech medicinal material, thin-layer chromatography method for primordial identification of leech medicinal material and application of leech medicinal material

Abstract

The invention discloses a leech medicinal material and a thin-layer chromatography method for prime identification thereof, and the thin-layer chromatography method comprises the following steps: (1) pre-treating a leech medicinal material to be detected to obtain a leech medicinal material test solution, and pre-treating the leech medicinal material to obtain a leech reference medicinal material solution; wherein the leeches are leeches or leeches in the leech family; and (2) carrying out thin-layer chromatography identification analysis on the to-be-detected leech medicinal material test solution and the leech control medicinal material solution so as to determine whether the to-be-detected leech is leech or leech, wherein in the step of thin-layer chromatography identification analysis, trichloromethane-methanol is used as a developing solvent, and the volume ratio of trichloromethane to methanol is (6-10): about 1; wherein the to-be-detected leeches are selected from one or more of leeches, leeches, leeches, poecilobdella manillensis, poecilobdella lubriosa, hirudinaria obscura, poecilobdella japonica and poecilobdella brueckii. The method is simple to operate, required instruments and equipment are low in cost, and detection results are visual, accurate and easy to identify.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a thin layer chromatography method for identifying leech medicinal materials and their origin and application thereof. Background Art

[0002] Leeches come from the dried body of Whitmaniapigra Whitman, Hirudo nipponicaWhitman or Whitmania acranulata Whitman, which are animals of the Hirudinidae family. They have the effects of breaking blood, promoting menstruation, removing blood stasis and eliminating symptoms. Modern pharmacological studies have shown that leeches have multiple effects such as anticoagulation, antithrombosis, anti-atherosclerosis, anti-platelet aggregation, anti-tumor, anti-inflammatory, improving blood rheology, and protecting against cerebral ischemia-reperfusion injury. However, there are more than 680 species of leeches distributed around the world, and more than 100 species in China. However, there are only three legal origins of leeches, namely leeches, leeches, and willow leeches. There is a great demand for leeches in the medicinal materials market. Their prices have been rising year by year and are in short supply. In addition, adulterated products are common. The main types of adulterated leeches include the Philippine cattle leech, the smooth golden thread leech, the eight-eye stone leech and the Japanese leech, which leads to uneven quality of leeches and seriously affects the clinical efficacy.

[0003] Leech identification methods include property identification, thin layer identification, molecular identification, protein electrophoresis identification and other methods. The authenticity identification methods (property identification, thin layer identification methods) of the legal standard for leeches (Pharmacopoeia of the People's Republic of China (hereinafter referred to as "TCM Pharmacopoeia") (2020 edition) are difficult to distinguish between leech species. Among the molecular identification methods, PCR has extremely high sensitivity and can amplify trace amounts of DNA templates. Cross-contamination of trace amounts of reagents, instruments, consumables, aerosols or samples may cause false positive results, while incorrect operation by the tester or failure of reagents and consumables can easily cause false negative results. Protein electrophoresis has not yet been studied to focus on the similarities and differences between small molecule proteins and polypeptides of different leech species. In addition, the use of molecular identification and protein electrophoresis identification requires the purchase of relevant instruments, and the skills of the experimental personnel are also required to be high.

[0004] The prior art does not involve the thin layer chromatography method and application of the leech medicinal material and its origin identification of the present invention. Summary of the invention

[0005] Based on this, the present invention provides a thin layer chromatography method for identifying leech medicinal materials and their origin, and the thin layer chromatography method comprises the following steps:

[0006] (1) pre-treating the leech medicinal material to be tested to obtain a leech medicinal material test solution, and pre-treating the leech medicinal material to obtain a leech control medicinal material solution; wherein the leech is a leech (Hirudo nipponica Whitman) or a leech (Whitmaniapigra Whitman) of the Hirudinidae family; and

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

[0008] Wherein, in the step of 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;

[0009] The leech to be tested is selected from one or more of the following: leeches, leeches, Whitmaniaacranulata Whitman, Poecilobdella manillensis Lesson, Whitmania laevis Baird, Erpobdella octoculata, Mimobdella japonica Blanchard and Poecilobdella javanica Wahlberg.

[0010] Furthermore, the method for preparing the leech medicinal material test solution includes: weighing an appropriate amount of leech medicinal material powder to be tested, adding a solvent, ultrasonically extracting, centrifuging, and taking a supernatant to obtain the leech medicinal material test solution to be tested.

[0011] Furthermore, the preparation method of the leech control medicinal material solution comprises: weighing an appropriate amount of leech medicinal material powder, adding a solvent, ultrasonically extracting, centrifuging, and taking a supernatant to obtain the leech control medicinal material solution.

[0012] Further, the solvent is alcohol, such as ethanol.

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

[0014] Furthermore, the mass / volume (g / ml) ratio of the leech powder to be tested to the solvent is 0.1-0.5, such as about 0.2.

[0015] Furthermore, the mass / volume (g / ml) ratio of the leech medicinal material powder to the solvent is 0.1-0.5, such as about 0.2.

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

[0017] Furthermore, the weight of the leech medicinal material powder is 0.5-2 g, for example, about 1 g.

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

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

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

[0021] Furthermore, the ultrasonic extraction time is 10 to 20 minutes, for example, about 15 minutes.

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

[0023] Furthermore, the preparation method of the leech medicinal material powder comprises: weighing an appropriate amount of leech medicinal material, crushing, and sieving to obtain the leech medicinal material powder.

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

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

[0026] Furthermore, 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] Furthermore, the thin layer chromatography identification analysis method comprises: spotting the leech medicinal material test solution and the leech control medicinal material solution on the same thin layer plate, developing, taking out the developed thin layer plate, drying it, and inspecting it under ultraviolet light.

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

[0030] Furthermore, the wavelength of the ultraviolet lamp is 10-400 nm, for example, about 365 nm.

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

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

[0033] Furthermore, the thin layer identification standard includes: checking whether the thin layer plate shows fluorescent spots of the same color and position at the corresponding positions of the leech medicinal material to be tested and the leech medicinal material; if the leech medicinal material to be tested has green fluorescent spots and the positions are the same as those of the fluorescent spots of the leech (Hirudo nipponica Whitman), the leech to be tested is the leech (Hirudonipponica Whitman); if the leech medicinal material to be tested has red fluorescent spots and the positions are the same as those of the fluorescent spots of the leech (WhitmaniapigraWhitman), the leech to be tested is the leech (Whitmaniapigra Whitman); if the fluorescent spots of the leech medicinal material to be tested are different from those of the leech medicinal material, the leech to be tested is the willow leaf leech (Whitmaniaacranulata Whitman) or other closely related counterfeit products.

[0034] Furthermore, the other closely related counterfeits include the Philippine cow leech (Poecilobdella manillensis Lesson), the smooth golden line leech (Whitmania laevis Baird), the eight-eye stone leech (Erpobdella octoculata), the Japanese leech (Mimobdella japonica Blanchard) and the rod-shaped cow leech (Poecilobdella javanica Wahlberg).

[0035] According to another aspect of the present invention, a thin layer chromatography method for identifying leech medicinal materials and their origin is provided, characterized in that it comprises:

[0036] Step 1, taking leech (leech or leech) control medicinal materials and samples to be tested, crushing and sieving to obtain leech sample powder, leech sample powder and sample powder to be tested, and weighing equal amounts of leech sample powder, leech sample powder and sample powder to be tested respectively;

[0037] Step 2, respectively extracting the above powders with ethanol solution by ultrasonic method, centrifuging, taking the supernatant, and obtaining a leech sample test solution, a leech sample test solution and a test solution of the sample to be detected;

[0038] Step 3: Spot the above test solutions on the same silica gel thin layer plate, develop it, and examine the thin layer chromatogram under ultraviolet light. Identify the sample to be tested based on the color and position of the fluorescent spots on the thin layer chromatogram.

[0039] According to another aspect of the present invention, there is provided a use of the above-mentioned thin layer chromatography method in distinguishing the original animals of leech medicinal materials.

[0040] According to another aspect of the present invention, a kit for distinguishing the original animal of leech medicinal materials is provided, which comprises 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] Furthermore, the volume ratio of the chloroform to the methanol is (6-10):about 1.

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

[0043] Furthermore, 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] Beneficial effects of the present invention:

[0046] The method establishes for the first time a thin layer identification method for distinguishing leeches and leech-based species. Specifically, the present invention subjects the leech medicinal material sample to ethanol ultrasonic extraction, centrifugation, plate spotting, and development, thereby obtaining the number and position of fluorescent spots of the sample on a silica gel thin layer plate under ultraviolet light. The sample to be tested can be identified according to the number and position of the fluorescent spots. The operation is simple, the cost of required instruments and equipment is low, and the test results are intuitive, accurate, and easy to identify. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0048] Figure 1 It is the thin layer chromatogram of Example 1 of the present invention.

[0049] Figure 2 It is the thin layer chromatogram of comparative example 1 of the present invention.

[0050] Figure 3 It is the thin layer chromatogram of comparative example 2 of the present invention.

[0051] Figure 4 It is the thin layer chromatogram of comparative example 3 of the present invention.

[0052] Figure 5 It is the thin layer chromatogram of comparative example 4 of the present invention. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

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

[0055] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0056] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0057] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0058] As described in the background technology section, the prior art does not involve a thin layer chromatography method for identifying leech medicinal materials and their origins and their applications. In order to solve the above problems, the present invention provides a thin layer chromatography method for identifying leech medicinal materials and their origins, and the thin layer chromatography method comprises the following steps:

[0059] (1) pre-treating the leech medicinal material to be tested to obtain a leech medicinal material test solution, and pre-treating the leech medicinal material to obtain a leech control medicinal material solution; wherein the leech is a leech (Hirudo nipponica Whitman) or a leech (Whitmaniapigra Whitman) of the Hirudinidae family; and

[0060] (2) performing thin layer chromatography identification analysis on the leech medicinal material test solution and the leech control medicinal material solution to determine whether the leech to be tested is a leech (Hirudo nipponica Whitman) or a leech (Whitmania pigra Whitman);

[0061] Wherein, in the step of 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] The leech to be tested is selected from one or more of the following: leeches, leeches, Whitmaniaacranulata Whitman, Poecilobdella manillensis Lesson, Whitmania laevis Baird, Erpobdella octoculata, Mimobdella japonica Blanchard and Poecilobdella javanica Wahlberg.

[0063] In the present invention, when volume, volume percentage concentration, ratio, mass, power, frequency, time, wavelength, concentration, or other value or parameter is expressed as a range, a preferred range, or a range limited 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 to 10" is disclosed, the described range should be interpreted as including the range "6 to 10", "6 to 9", "6 to 8", "6 to 7", "7 to 10", "7 to 9", "7 to 8", "8 to 10", "8 to 9", "9 to 10", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

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

[0065] In a preferred embodiment, the method for preparing the leech medicinal material test solution comprises: weighing an appropriate amount of leech medicinal material powder to be tested, adding a solvent, ultrasonically extracting, centrifuging, and taking a supernatant to obtain the leech medicinal material test solution to be tested.

[0066] In a preferred embodiment, the preparation method of the leech control medicinal material solution comprises: weighing an appropriate amount of leech medicinal material powder, adding a solvent, ultrasonically extracting, centrifuging, and taking a supernatant to obtain the leech control medicinal material solution.

[0067] In a preferred embodiment, the solvent is an 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 invention, "about" refers to a value within a range of ±5% of a particular 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 leech powder to be tested to the solvent is 0.1-0.5, such as about 0.2.

[0071] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 medicinal material powder to the solvent is 0.1-0.5, such as about 0.2.

[0073] In the present invention, "about" refers to a value within a range of ±5% of a specific 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 leech powder to be tested is 0.5-2 g, for example, about 1 g.

[0075] In the present invention, "about" refers to a value within the range of ±5% of a specific 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 medicinal material powder is 0.5-2 g, for example, about 1 g.

[0077] In the present invention, "about" refers to a value within the range of ±5% of a specific 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 1-10 ml, such as about 5 ml.

[0079] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 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 the present 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 30-50 kHz, for example, about 40 kHz.

[0083] In the present 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, such as about 15 minutes.

[0085] In the present 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 medicinal material powder to be tested comprises: weighing an appropriate amount of the leech medicinal material to be tested, crushing, and sieving to obtain the leech medicinal material powder to be tested.

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

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

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

[0090] In the present invention, "about" refers to a value within the 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 the chloroform to the methanol is (7.5-8.5):about 1.

[0092] In the present invention, "about" refers to a value within the 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 volume ratio of the chloroform to the methanol is about 8: about 1.

[0094] In the present invention, "about" refers to a value within the range of ±5% of a specific 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 thin layer chromatography identification analysis method comprises: spotting the leech medicinal material test solution and the leech medicinal material control solution on the same thin layer plate, developing, taking out the developed thin layer plate, drying, and inspecting under ultraviolet 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 ultraviolet lamp is 10-400 nm, for example, about 365 nm.

[0098] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 365" includes ±5% of 365, or from 346.75 to 383.25.

[0099] In a preferred embodiment, the thin layer chromatography identification analysis method does not include the step of using sulfuric acid ethanol solution for color development.

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

[0101] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10%" includes ±5% of 10%, or from 9.5% to 10.5%.

[0102] In a preferred embodiment, the thin layer identification standard includes: checking whether the thin layer plate shows fluorescent spots of the same color and position at the corresponding positions of the leech medicinal material to be tested and the leech medicinal material; if the leech medicinal material to be tested has green fluorescent spots and the positions are the same as those of the fluorescent spots of the leech (Hirudo nipponica Whitman), the leech to be tested is the leech (Hirudo nipponica Whitman); if the leech medicinal material to be tested has red fluorescent spots and the positions are the same as those of the fluorescent spots of the leech (Whitmania pigra Whitman), the leech to be tested is the leech (WhitmaniapigraWhitman); if the fluorescent spots of the leech medicinal material to be tested are different from those of the leech medicinal material, the leech to be tested is the willow leaf leech (Whitmania acranulata Whitman) or other closely related counterfeit products.

[0103] In a preferred embodiment, the other closely related counterfeits include Philippine cow leech (Poecilobdellamanillensis Lesson), smooth golden line leech (Whitmania laevis Baird), eight-eye stone leech (Erpobdellaoctoculata), Japanese leech (Mimobdella japonica Blanchard) and rod-patterned cow leech (Poecilobdellajavanica Wahlberg).

[0104] According to another aspect of the present invention, a thin layer chromatography method for identifying leech medicinal materials and their origin is provided, characterized in that it comprises:

[0105] Step 1, taking leech (leech or leech) control medicinal materials and samples to be tested, crushing and sieving to obtain leech sample powder, leech sample powder and sample powder to be tested, and weighing equal amounts of leech sample powder, leech sample powder and sample powder to be tested respectively;

[0106] Step 2, respectively extracting the above powders with ethanol solution by ultrasonic method, centrifuging, taking the supernatant, and obtaining a leech sample test solution, a leech sample test solution and a test solution of the sample to be detected;

[0107] Step 3: Spot the above test solutions on the same silica gel thin layer plate, develop it, and examine the thin layer chromatogram under ultraviolet light. Identify the sample to be tested based on the color and position of the fluorescent spots on the thin layer chromatogram.

[0108] According to another aspect of the present invention, there is provided a use of the above-mentioned thin layer chromatography method in distinguishing the original animals of leech medicinal materials.

[0109] According to another aspect of the present invention, a kit for distinguishing the original animal of leech medicinal materials is provided, which comprises 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 invention, "about" refers to a value within the range of ±5% of a specific 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 invention, "about" refers to a value within the range of ±5% of a specific 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 invention, "about" refers to a value within the range of ±5% of a specific 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 invention, "about" refers to a value within the range of ±5% of a specific 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 invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0119] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0120] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0121] Experimental instruments and reagents

[0122] KQ-250DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), CAMAG ATS 4 fully automatic spotter (Kama, Switzerland), 2020D UV / white light transilluminator (Cold Spring), TGL20MW desktop high-speed refrigerated centrifuge (Hunan Hercy Instrument Equipment Co., Ltd.), BSA-124S-CW one-tenth electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0123] Leech control medicinal materials (China Food and Drug Inspection Institute, batch number: 121061-202107), Philippine cattle leech control medicinal materials (China Food and Drug Inspection Institute, batch number: 121754-201901), silica gel G plate (Qingdao Ocean Chemical Plant, 10×20 cm), cyclohexane (Tianjin Zhiyuan Chemical Reagent Co., Ltd.), ethyl acetate (Tianjin Damao Chemical Reagent Factory), sulfuric acid (Beijing Tongguang Fine Chemical Company), ethanol, methanol (Sinopharm Chemical Reagent Co., Ltd.), chloroform (Beijing Tongguang Fine Chemical Company).

[0124] Experimental Materials

[0125] 28 batches of samples were collected from different regions of China, including 12 batches of leeches, 11 batches of leeches, 1 batch of bar-patterned leeches, 1 batch of Philippine leeches, 2 batches of Japanese leeches and 1 batch of bright golden line leeches. The specific information is as follows:

[0126] Sample 1: leech (Hirudo nipponica Whitman), origin: Jining County, Jining City, Shandong Province;

[0127] Sample 2: leech (Hirudo nipponica Whitman), origin: Sucheng District, Suqian City, Jiangsu Province;

[0128] Sample 3: leech (Hirudo nipponica Whitman), produced in Bamencheng Town, Baodi District, Tianjin;

[0129] Sample 4: leech (Hirudo nipponica Whitman), origin: Xihua County, Zhoukou City, Henan Province;

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

[0131] Sample 6: leech (Hirudo nipponica Whitman), origin: Sucheng District, Suqian City, Jiangsu Province;

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

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

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

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

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

[0137] Sample 12: leech (Hirudo nipponica Whitman), produced in Bamencheng Town, Baodi District, Tianjin;

[0138] Sample 13: leech (Whitmaniapigra Whitman), origin: Shandong (purchased from Anguo medicinal material market);

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

[0140] Sample 15: leech (Whitmaniapigra Whitman), origin: Sucheng District, Suqian City, Jiangsu Province;

[0141] Sample 16 leech (Whitmaniapigra Whitman), produced in Bamencheng Town, Baodi District, Tianjin;

[0142] Sample 17 leech (Whitmaniapigra Whitman), originating from Xihua County, Zhoukou City, Henan Province;

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

[0144] Sample 19 leech (Whitmaniapigra Whitman), origin: Sucheng District, Suqian City, Jiangsu Province;

[0145] Sample 20 leech (Whitmaniapigra Whitman), originating from De'an County, Jiujiang City, Jiangxi Province;

[0146] Sample 21 leech (Whitmaniapigra Whitman), origin: Yuanjiang City, Yiyang City, Hunan Province;

[0147] Sample 22: leech (Whitmania pigra Whitman), originating from Gong'an County, Jingzhou City, Hubei Province;

[0148] Sample 23: leech (Whitmania pigra Whitman), originating from Gong'an County, Jingzhou City, Hubei Province;

[0149] Sample 24: Poecilobdellajavanica Wahlberg, originating from Qinnan District, Qinzhou City, Guangxi;

[0150] Sample 25: Poecilobdella manillensis Lesson, originating from Zhongshan City, Guangdong Province;

[0151] Sample 26: Japanese leech (Mimobdella japonica Blanchard), originating from Xihua County, Zhoukou City, Henan Province;

[0152] Sample 27: Japanese leech (Mimobdella japonica Blanchard), produced in Xinhui District, Jiangmen City, Guangdong Province;

[0153] Sample 28: Whitmania laevis Baird, originating from De'an County, Jiujiang City, Jiangxi Province.

[0154] Example 1

[0155] Thin layer chromatography methods for leeches and leeches, including:

[0156] Step 1: Take leeches, leeches and samples to be tested, crush them, and pass them through a No. 3 sieve to obtain powders of leeches, leeches and samples to be tested, and then weigh 1g of the powders of leeches, leeches and samples to be tested respectively;

[0157] Step 2: The above powders were respectively subjected to ultrasonic extraction with 5 mL of 95% (V / V) ethanol solution at a power of 250 W and a frequency of 40 KHz for 15 min, centrifuged, and the supernatant was taken to obtain a leech sample test solution and a leech sample test solution;

[0158] Step 3: Spot the above test solutions on the same silica gel G thin layer plate, use chloroform-methanol (8:1, V / V) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). The results are as follows: Figure 1 shown. Figure 1 In the table, leeches and Philippine cattle leeches are control medicinal materials, 1 to 12 are leech samples, 13 to 23 are leech samples, 24 is a bar-patterned cattle leech sample, 25 is a Philippine cattle leech sample, 26 and 27 are Japanese leech samples, and 28 is a smooth golden line leech sample.

[0159] Depend on Figure 1 It can be seen that under ultraviolet light (365nm), leeches show green fluorescent spots (A) and leeches show red fluorescent spots (B).

[0160] Comparative Example 1

[0161] Thin layer chromatography methods for leeches and leeches, including:

[0162] Step 1: Take leeches, leeches and samples to be tested, crush them, and pass them through a No. 3 sieve to obtain powders of leeches, leeches and samples to be tested, and then weigh 1g of the powders of leeches, leeches and samples to be tested respectively;

[0163] Step 2: The above powders were respectively subjected to ultrasonic extraction with 5 mL of 95% (V / V) ethanol solution at a power of 250 W and a frequency of 40 KHz for 15 min, centrifuged, and the supernatant was taken to obtain a leech sample test solution and a leech sample test solution;

[0164] Step 3: Spot the above test solutions on the same silica gel G thin layer plate, use chloroform-methanol (8:1, V / V) as the developing agent, develop, take out, dry, develop with 10% (V / V) sulfuric acid ethanol solution, and examine under ultraviolet light (365nm). The results are as follows: Figure 2 shown. Figure 2 In the table, leeches and Philippine cattle leeches are control medicinal materials, 1 to 12 are leech samples, 13 to 23 are leech samples, 24 is a bar-patterned cattle leech sample, 25 is a Philippine cattle leech sample, 26 and 27 are Japanese leech samples, and 28 is a smooth golden line leech sample.

[0165] Depend on Figure 2 It can be seen that the characteristic spots that can distinguish leeches from leeches are: under ultraviolet light (365nm), leeches show green fluorescent spots (A), and leeches show red fluorescent spots (B). Comparing the results of the thin layer with and without the color developer, the fluorescent spots will weaken after the color developer is sprayed, so the detection condition is not to spray the color developer.

[0166] Comparative Example 2

[0167] Thin layer chromatography methods for leeches and leeches, including:

[0168] Step 1: Take leeches, leeches and samples to be tested, crush them, and pass them through a No. 3 sieve to obtain powders of leeches, leeches and samples to be tested, and then weigh 1g of the powders of leeches, leeches and samples to be tested respectively;

[0169] Step 2: The above powders were respectively subjected to ultrasonic extraction with 5 mL of 95% (V / V) ethanol solution at a power of 250 W and a frequency of 40 KHz for 15 min, centrifuged, and the supernatant was taken to obtain a leech sample test solution and a leech sample test solution;

[0170] Step 3: Spot the above test solutions on the same silica gel G thin layer plate, use chloroform-methanol (5:1, V / V) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). The results are as follows: Figure 3 shown. Figure 3 In the table, leeches and Philippine cattle leeches are control medicinal materials, 1 to 12 are leech samples, 13 to 23 are leech samples, 24 is a bar-patterned cattle leech sample, 25 is a Philippine cattle leech sample, 26 and 27 are Japanese leech samples, and 28 is a smooth golden line leech sample.

[0171] Depend on Figure 3 It can be seen that, using the thin layer development conditions 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 overlap with another spot and are located near the front. Therefore, using the thin layer development conditions of Comparative Example 2, it is impossible to simultaneously display the characteristic spots in leeches and leeches, indicating that the thin layer development effect is not good under the above-mentioned developing agent conditions.

[0172] Comparative Example 3

[0173] Thin layer chromatography methods for leeches and leeches, including:

[0174] Step 1: Take leeches, leeches and samples to be tested, crush them, and pass them through a No. 3 sieve to obtain powders of leeches, leeches and samples to be tested, and then weigh 1g of the powders of leeches, leeches and samples to be tested respectively;

[0175] Step 2: The above powders were respectively subjected to ultrasonic extraction with 5 mL of 95% (V / V) ethanol solution at a power of 250 W and a frequency of 40 KHz for 15 min, centrifuged, and the supernatant was taken to obtain a leech sample test solution and a leech sample test solution;

[0176] Step 3: Spot the above test solutions on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate (1:1, V / V) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). The results are as follows: Figure 4 shown. Figure 4In the table, leeches and Philippine cattle leeches are control medicinal materials, 1 to 12 are leech samples, 13 to 23 are leech samples, 24 is a bar-patterned cattle leech sample, 25 is a Philippine cattle leech sample, 26 and 27 are Japanese leech samples, and 28 is a smooth golden line leech sample.

[0177] Depend on Figure 4 It can be seen that under ultraviolet light (365nm), the leech shows a red fluorescent spot (B), but its position is at the origin, and there is no characteristic spot of the leech. Therefore, using the thin layer development conditions of Comparative Example 3, it is impossible to simultaneously display the characteristic spots in the leech and the leech, indicating that the thin layer development effect is not good under the above-mentioned developing agent conditions.

[0178] Comparative Example 4

[0179] Thin layer chromatography methods for leeches and leeches, including:

[0180] Step 1: Take leeches, leeches and samples to be tested, crush them, and pass them through a No. 3 sieve to obtain powders of leeches, leeches and samples to be tested, and then weigh 1g of the powders of leeches, leeches and samples to be tested respectively;

[0181] Step 2: The above powders were respectively subjected to ultrasonic extraction with 5 mL of 95% (V / V) ethanol solution at a power of 250 W and a frequency of 40 KHz for 15 min, centrifuged, and the supernatant was taken to obtain a leech sample test solution and a leech sample test solution;

[0182] Step 3: Spot the above test solutions on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate (4:1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and the results are as follows: Figure 5 shown. Figure 5 In the table, leeches and Philippine cattle leeches are control medicinal materials, 1 to 12 are leech samples, 13 to 23 are leech samples, 24 is a bar-patterned cattle leech sample, 25 is a Philippine cattle leech sample, 26 and 27 are Japanese leech samples, and 28 is a smooth golden line leech sample.

[0183] This method is the thin layer chromatography method of leech medicinal materials in the Chinese Pharmacopoeia (2020 edition). Figure 5 It can be seen that in all the chromatograms of the test products derived from leeches or leeches, the same purple-red spots ( Figure 5 a); under ultraviolet light (365nm), the same orange-red fluorescent spots appear ( Figure 5 b) This indicates that the thin layer identification method included in the pharmacopoeia cannot effectively distinguish between leeches and leeches.

[0184] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A thin layer chromatography method for identifying leech medicinal materials and their origin, characterized in that: The thin layer chromatography method comprises the following steps: (1) pre-treating the leech medicinal material to be tested to obtain a leech medicinal material test solution, and pre-treating the leech medicinal material to obtain a leech control medicinal material solution; wherein the leech is a leech (Hirudo nipponica Whitman) or a leech (Whitmaniapigra Whitman) of the Hirudinidae family; and (2) performing thin layer chromatography identification analysis on the leech medicinal material test solution and the leech control medicinal material solution to determine whether the leech to be tested is a leech (Hirudo nipponica Whitman) or a leech (Whitmania pigra Whitman); Wherein, in the step of 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; Wherein, the leech to be tested is selected from one or more of the following: leech, leech, willow leech (Whitmaniaacranulata Whitman), Philippine cattle leech (Poecilobdella manillensis Lesson), smooth golden line leech (Whitmania laevis Baird), eight-mesh stone leech (Erpobdella octoculata), Japanese leech (Mimobdellajaponica Blanchard) and rod-shaped cattle leech (Poecilobdellajavanica Wahlberg).

2. The thin layer chromatography method according to claim 1, characterized in that The method for preparing the leech medicinal material test solution to be tested comprises: weighing an appropriate amount of leech medicinal material powder to be tested, adding a solvent, ultrasonically extracting, centrifuging, and taking a supernatant to obtain the leech medicinal material test solution to be tested; Preferably, the preparation method of the leech control medicinal material solution comprises: weighing an appropriate amount of leech medicinal material powder, adding a solvent, ultrasonically extracting, centrifuging, and taking a supernatant to obtain the leech control medicinal material solution.

3. The thin layer chromatography method according to claim 2, characterized in that: The solvent is an alcohol, such as ethanol; Preferably, the volume percentage concentration of the ethanol is 90% to 100%, for example, about 95%; More preferably, the mass / volume (g / ml) ratio of the leech powder to be tested to the solvent is 0.1 to 0.5, for example, about 0.2; More preferably, the mass / volume (g / ml) ratio of the leech medicinal material powder to the solvent is 0.1 to 0.5, such as about 0.2; More preferably, the mass of the leech powder to be tested is 0.5 to 2 g, for example, about 1 g; More preferably, the mass of the leech medicinal material powder is 0.5 to 2 g, for example, about 1 g; Also preferably, the volume of the solvent is 1 to 10 mL, for example, about 5 mL; Still more preferably, the power of the ultrasonic extraction is 200-300W, for example, about 250W; Still more preferably, the frequency of the ultrasonic extraction is 30 to 50 kHz, for example, about 40 kHz; Even more preferably, the ultrasonic extraction time is 10 to 20 minutes, such as about 15 minutes.

4. The thin layer chromatography method according to claim 2, characterized in that: The preparation method of the leech medicinal material powder to be tested comprises: weighing an appropriate amount of the leech medicinal material to be tested, crushing, and sieving to obtain the leech medicinal material powder to be tested; Preferably, the method for preparing the leech medicinal material powder comprises: weighing an appropriate amount of leech medicinal material, crushing, and sieving to obtain the leech medicinal material powder; More preferably, the sieve is a No. 3 sieve.

5. The thin layer chromatography method according to claim 1, characterized in that: The volume ratio of the chloroform to the methanol is (7-9): about 1; Preferably, the volume ratio of the chloroform to the methanol is (7.5-8.5): about 1; More preferably, the volume ratio of the chloroform to the methanol is about 8:about 1.

6. The thin layer chromatography method according to claim 1, characterized in that: The thin layer chromatography identification analysis method comprises: applying the leech medicinal material test solution and the leech medicinal material control solution to the same thin layer plate, developing the plate, taking out the developed thin layer plate, drying it, and inspecting it under an ultraviolet lamp; Preferably, the thin layer plate is a silica gel G thin layer plate; More preferably, the wavelength of the ultraviolet lamp is 10-400 nm, for example, about 365 nm.

7. The thin layer chromatography method according to claim 6, characterized in that: The method of thin layer chromatography identification analysis does not include the step of using sulfuric acid ethanol solution for color development; Preferably, the concentration of the sulfuric acid ethanol solution is about 10% (V / V).

8. The thin layer chromatography method according to claim 1, characterized in that: The thin layer identification standard includes: checking whether the thin layer plate shows fluorescent spots of the same color and position at the corresponding positions of the leech medicinal material to be tested and the leech medicinal material; if the leech medicinal material to be tested has green fluorescent spots and the positions are the same as those of the fluorescent spots of the leech (Hirudo nipponica Whitman), the leech to be tested is the leech (Hirudo nipponica Whitman); if the leech medicinal material to be tested has red fluorescent spots and the positions are the same as those of the fluorescent spots of the leech (Whitmaniapigra Whitman), the leech to be tested is the leech (Whitmaniapigra Whitman); if the fluorescent spots of the leech medicinal material to be tested are different from those of the leech medicinal material, the leech to be tested is the willow leaf leech (Whitmania acranulata Whitman) or other closely related counterfeit products; Preferably, the other closely related counterfeits include Philippine cow leech (Poecilobdella manillensis Lesson), smooth golden line leech (Whitmania laevis Baird), eight-mesh stone leech (Erpobdella octoculata), Japanese leech (Mimobdellajaponica Blanchard) and rod-patterned cow leech (Poecilobdellajavanica Wahlberg).

9. Use of the thin layer chromatography method according to any one of claims 1 to 8 in distinguishing the original animals of leech medicinal materials.

10. A kit for distinguishing the original animal of leech medicinal materials, 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; Preferably, the volume ratio of the chloroform to the methanol is (6-10): about 1; More preferably, the volume ratio of the chloroform to the methanol is (7-9): about 1; Preferably, the volume ratio of the chloroform to the methanol is (7.5-8.5): about 1; Even more preferably, the volume ratio of the chloroform to the methanol is about 8:about 1.

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