Thin layer identification method to distinguish the origin and authenticity of Herba Euphorbiae

Abstract

The present invention provides a thin layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae chinensis, characterized in that the method includes the preparation of a test solution, the preparation of a control medicinal material solution and thin layer identification. The present invention uses a simple and fast preparation method to obtain a test solution and a control medicinal material solution, which are respectively spotted on the same thin layer plate, and after development, the thin layer chromatograms of Herba Euphorbiae chinensis with different origins and related products thereof can be obtained under ultraviolet light. The method is simple, fast, accurate, highly efficient, and low in cost, and can be well used for the quality control of Herba Euphorbiae chinensis and related products thereof, and can also effectively distinguish common counterfeit products in the market, such as Euphorbia microphylla.

Description

Technical Field

[0001] The invention relates to the field of quality control of traditional Chinese medicines, and in particular to a thin layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae Herba. Background Art

[0002] Euphorbia humifusa Willd., also known as grass blood-dried grass, blood-seeing grass, etc., is a whole herb from the Euphorbiaceae family. It has the effects of clearing heat, promoting dampness, jaundice, and stopping bleeding. It also has significant antibacterial, antiparasitic, antioxidant, liver-protecting, and detoxifying effects. The Chinese Pharmacopoeia 2020 Edition Volume 1 records that Euphorbia humifusa Willd. is the dried whole herb of Euphorbia humifusa Willd. or Euphorbia maculate L., a plant of the Euphorbiaceae family. In addition, Euphorbia humifusa Willd. is also a Mongolian medicine, called "Malagan-Zara-Ubus", and the fixed base is Euphorbia humifusa Willd. According to the origin survey, in actual application, in addition to the two bases of Euphorbia humifusa Willd. and Euphorbia maculate, there are also Euphorbia makinoi Hayata. Because of their extremely similar appearance to Euphorbia humifusa Willd., they are often mixed and collected together, and are also used as medicine for Euphorbia humifusa Willd. in some areas. However, in the standards of preparations (such as formulated granules), the base needs to be fixed to one type and cannot be mixed. While ensuring the use of the medicine, it is more conducive to the supervision of the medicine. Because there is a need to fix the base of Herba Epimedii in the actual production process, the prior art has also carried out a series of development and research on the identification method of Herba Epimedii for this purpose.

[0003] In the research on identification methods of different origins of Parthenocissus, reference 1 disclosed that observation of leaves under a microscope could distinguish Parthenocissus and Parthenocissus variegatus, but this method is not suitable for identification of related products of Parthenocissus that have lost their leaf morphology.

[0004] In the study of thin-layer identification methods for Herba Euphorbiae and its preparations, in addition to the provisions in the first volume of the Chinese Pharmacopoeia (2020 edition), Reference 2 also discloses a detection method for Herba Euphorbiae Chinese medicine preparations, using quercetin as a control to identify the medicinal ingredients in Herba Euphorbiae tablets; Reference 3 also discussed quercetin, an active ingredient in Herba Euphorbiae, selecting ethanol as the solvent for Soxhlet extraction, followed by water boiling, alcohol precipitation, and acid hydrolysis extraction, and then thin-layer identification. However, the pharmacopoeia method and references 2 and 3 all adopted acid hydrolysis in the preparation of the test solution, in which the flavonoid glycosides in Herba Euphorbiae were hydrolyzed into flavonoid aglycones (quercetin), and then the thin layer spots of a single quercetin reference substance were used for identification, evaluation and quality control. This method cannot fully reflect the original chemical composition information in Herba Euphorbiaee, and the amount of information on thin layer chromatography spots is limited, which is not conducive to comprehensive quality evaluation and control of Herba Euphorbiaee and its related products. In addition, quercetin is contained in many traditional Chinese medicines. Therefore, this method cannot distinguish between confusing or counterfeit products containing quercetin.

[0005] Reference 4 discloses a thin layer identification method for Herba Dichrysanthemi and its preparation. Although the method does not use the step of acid hydrolysis in the preparation process of the test solution, the amount of information of thin layer chromatography is increased, but a polyamide thin layer plate is used to analyze non-hydrolyzed components, not for base and authenticity identification. Although from the results, there are differences in the chromatographic spots of different bases and authenticity, the spots on the chromatogram are not completely separated, and the single spots are not round and tailing is serious, indicating that the method is not suitable for Herba Dichrysanthemi of different bases and authenticity identification of Herba Dichrysanthemi. Reference 5 discloses a thin layer chromatography method for Herba Dichrysanthemi, and its chromatogram clarity is poor, and it also does not pay attention to the base and authenticity identification of Herba Dichrysanthemi. In addition, the developing agent used in the above-mentioned prior art mostly contains toluene, which is not conducive to environmental safety and human health in production practice, and needs to be further improved.

[0006] Although the above-mentioned prior art provides some quality control methods for Herba Euphorbiae and its preparations, and the thin layer identification method has been continuously explored and improved, there is still a need for further improvement and development to provide a simple, safe and low-cost thin layer identification method for the origin and authenticity of Herba Euphorbiae.

[0007] References:

[0008] Reference 1: Wei Shenghua, Meng Na, Chai Ruijuan. Study on microscopic identification of brocade and brocade[J]. Hunan Agricultural Science, 2012, (1): 42-43, 49.

[0009] Reference 2: CN102078363A

[0010] Reference 3: Yu Lin. Thin-layer analysis of quercetin in Herba Euphorbiae [J]. Journal of Nanchang University (Engineering Science Edition), 1996, 18(3): 85-87.

[0011] Reference 4: CN115436557A

[0012] Reference 5: CN109085285B Summary of the invention

[0013] Problem that the invention aims to solve

[0014] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to establish a thin layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae ivy. The method is efficient, safe, accurate, simple and easy to operate, has obvious identification characteristics, is easy to judge the results, has good specificity, can better reflect the overall quality of Herba Euphorbiae ivy and its preparations, and can also distinguish the origin and authenticity of Herba Euphorbiae ivy at the same time.

[0015] Solutions for solving problems

[0016] [1] The present invention primarily provides a thin layer chromatography identification method for distinguishing the origin and authenticity of Euphorbia pulegiae, the method comprising the preparation of a test solution, the preparation of a control medicinal material solution and thin layer chromatography identification,

[0017] The preparation of the test solution comprises taking the test sample, adding water for treatment, filtering, extracting the filtrate by shaking with an organic solvent, standing to separate the layers, taking the supernatant and concentrating it to dryness, and dissolving the residue by adding an alcohol solvent;

[0018] The preparation of the control medicinal material solution includes taking a control medicinal material of Herba Dijin, boiling it with water or heating it under reflux, filtering it, extracting the filtrate by shaking it with an organic solvent, standing it to separate layers, taking the supernatant and concentrating it to dryness, and dissolving the residue by adding an alcohol solvent; the Herba Dijin control medicinal materials include Herba Dijin (Ban Dijin) medicinal materials and Herba Dijin (Dijin) medicinal materials;

[0019] The thin layer identification includes taking a test sample solution and a control medicinal material solution, spotting them on a thin layer plate respectively, developing them with chloroform-ethyl acetate-methanol-formic acid as a developing agent, and inspecting them.

[0020] [2]. The thin layer identification method according to [1], wherein the test samples include Herba Dichrysanthemi of different origins and related products, Herba Dichrysanthemi counterfeit herbs and related products; the Herba Dichrysanthemi of different origins include Herba Dichrysanthemi (Dichrysanthemi) and Herba Dichrysanthemi (Dichrysanthemi scaly); the Herba Dichrysanthemi counterfeit herbs are Euphorbia microphylla herbs; the related products are selected from one or more of the group consisting of decoction pieces, standard decoctions and formula granules.

[0021] [3]. The thin layer identification method according to [2], wherein, in the preparation of the test sample solution, the water treatment includes any one of water dissolution, water decoction and heating reflux treatment; when the related product is selected from medicinal slices, the mass volume ratio of the medicinal slices to the water used for the water treatment is 1g: (10-25) mL; when the related product is selected from standard decoctions, the mass volume ratio of the standard decoction to the water used for the water treatment is 1g: (20-60) / mL; when the related product is selected from formula granules, the mass volume ratio of the formula granules to the water used for the water treatment is 1g: (20-60) / mL; the time for the water treatment is 10-20 min.

[0022] [4]. The thin layer identification method according to any one of [1] to [3], wherein, in the preparation of the sample solution, the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; the shaking extraction is performed at least once, and the volume ratio of the filtrate to the organic solvent is (1-2): (1-1.5); and the alcohol solvent is methanol or an aqueous solution of methanol.

[0023] [5] The thin layer identification method according to any one of [1] to [4], wherein, in the preparation of the reference medicinal material solution, the mass volume ratio of the Herba Euphorbiae Ichthyophthirius reference medicinal material to the water used for the decoction or heating reflux treatment is 1 g: (100-250) mL, and the decoction or heating reflux treatment time is 10-20 min.

[0024] [6]. A thin layer identification method according to any one of [1] to [5], wherein, in the preparation of the reference medicinal material solution, the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; the shaking extraction is performed at least once, and the volume ratio of the filtrate to the organic solvent is (1 to 2): (1 to 1.5).

[0025] [7]. The thin layer identification method according to any one of [1] to [6], wherein, in the spotting operation of the thin layer identification, the samples are spotted on the same high-efficiency silica gel thin layer plate, the spotting amount of the test solution is 1 to 5 μL, and the spotting amount of the control medicinal material solution is 1 to 5 μL; in the thin layer identification, chloroform-ethyl acetate-methanol-formic acid with a volume ratio of (4 to 7): (2 to 3.5): (1 to 3): (0.2 to 0.9) is used as the developing agent; preferably, chloroform-ethyl acetate-methanol-formic acid with a volume ratio of 6:2.5:2:0.5 is used as the developing agent.

[0026] [8]. The thin layer identification method according to any one of [1] to [7], wherein the thin layer identification further comprises unfolding and drying, spraying with a color developer, heating until the spots are clearly colored, and then inspecting under ultraviolet light; preferably, the color developer is an ethanol solution of aluminum chloride with a concentration of 1% to 5%; preferably, the inspection is inspection under ultraviolet light with a wavelength of 365nm.

[0027] [9] The thin-layer identification method according to any one of [1] to [8], wherein in the thin-layer identification, the inspection includes comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value;

[0028] Preferably,

[0029] If the fluorescent spots are shown in the Rf value range of 0.19 to 0.24, and no fluorescent spots are shown in the Rf value range of 0.59 to 0.65, then the sample is Herba Dijin (Dijin),

[0030] If no fluorescent spots are shown in the Rf value range of 0.19 to 0.24, but fluorescent spots are shown in the Rf value range of 0.59 to 0.65, then the sample is Herba Dichondrium (Dichondrium scaly).

[0031] If no fluorescent spots are shown in the Rf value range of 0.19-0.24 and 0.59-0.65, the test sample is Euphorbia microphylla;

[0032] More preferably,

[0033] If the fluorescent spots are displayed in the Rf value range of 0.30-0.35, and no fluorescent spots are displayed in the Rf value range of 0.65-0.70, then the test sample is the herbal slices of Dijincao (Dijin),

[0034] If no fluorescent spots are shown in the Rf value range of 0.30-0.35 and 0.65-0.70, the sample is the herbal slice of Dijincao (Dijincao scabra).

[0035] If no fluorescent spots are shown in the Rf value range of 0.30 to 0.35, but fluorescent spots are shown in the Rf value range of 0.65 to 0.70, the test sample is a Euphorbia microphylla decoction piece.

[0036]

[10] . Application of the thin-layer identification method described in any one of [1] to [9] in distinguishing the origin and / or counterfeit of Herba Dichondrium and its related products, wherein the related products are selected from one or more of medicinal slices, standard decoctions and formula granules; the origin includes Herba Dichondrium (Dichondrium chinense) and / or Herba Dichondrium (Dichondrium maculatum); the counterfeit is Euphorbia microphylla; preferably, the method is used in simultaneously distinguishing the origin and / or counterfeit of Herba Dichondrium and its related products.

[0037] Effects of the Invention

[0038] The thin-layer identification method for distinguishing the origin and authenticity of Euphorbia pulex provided by the present invention can well develop and separate compounds with greater polarity in Euphorbia pulex and its related products. After color development, the spots are rich, clear, and have high brightness, with good separation, and the spots are within the Rf value range specified in the Chinese Pharmacopoeia. The identification characteristics are obvious, the results are easy to determine, and the specificity is good.

[0039] In addition, compared with the pharmacopoeia method and the method used in the above-mentioned prior art, the thin layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae provided by the present invention is simple and rapid to operate, does not require an acid hydrolysis step, does not destroy the chemical components of Herba Euphorbiae, presents the prototype components of Herba Euphorbiae to the greatest extent, and evaluates the consistency with the control medicinal material, which is more accurate and scientific.

[0040] Furthermore, the thin-layer identification method for distinguishing the origin and authenticity of Euphorbia pulex provided by the present invention can distinguish the origin and / or counterfeits of Euphorbia pulex and its related products, wherein the related products include medicinal materials, decoction pieces, standard decoctions and formula granules, and the origin includes Euphorbia pulex (Euphorbia pulex) and / or Euphorbia pulex (Euphorbia pulex scaly); the counterfeit is Euphorbia microphylla. Furthermore, the method can simultaneously distinguish the origin and / or counterfeits of Euphorbia pulex and its related products, providing a basis for establishing quality control standards for Euphorbia pulex and its related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The figure shows the TLC spectrum obtained by using silica gel GF254 thin layer plate and cyclohexane:ethyl acetate:glacial acetic acid (8:8:0.2) as the developing solvent (1-3: Radix Dijincao (Dijinban) formula granules (19120199, 19120209, 19120219); S: Radix Dijincao (Dijinban) control medicinal material.

[0042] Figure 2 The thin layer chromatography (TLC) spectrum obtained by the method disclosed in Reference 4 is shown (1-3: Radix Dijincao (Dijinban) formula granules (19120199, 19120209, 19120219); S: Radix Dijincao (Dijinban) control medicinal material).

[0043] Figure 3 The figure shows the thin layer analysis (TLC) spectrum obtained by using silica gel G thin layer plate with acetone:toluene:chloroform (5:2.5:2.5) as the developing solvent (Dijincao (Ban Dijin) formula granules (19120199, 19120209, 19120219); S: Dijincao (Ban Dijin) control medicinal material.

[0044] Figure 4 The thin layer chromatography (TLC) spectrum of the study of the developing agent ratio is shown (1: Radix Dijincao (Ban Dijin) formula granules (19120199); S: Radix Dijincao (Ban Dijin) control medicinal material).

[0045] Figure 5 The thin layer chromatography (TLC) spectrum of the developer ratio determined after investigation is shown (1: Radix Dijincao (Ban Dijin) formula granules (19120199); S: Radix Dijincao (Ban Dijin) control medicinal material).

[0046] Figure 6 The thin layer chromatography (TLC) spectra using different spotting amounts are shown (1-4: the spotting amounts of the test solution of Herba Euphorbiae Ichthyophthirius are 1 μL, 2 μL, 3 μL, and 4 μL, respectively; 5-8: the spotting amounts of the Herba Euphorbiae Ichthyophthirius control solution are 1 μL, 2 μL, 3 μL, and 4 μL, respectively).

[0047] Figure 7 The thin layer chromatography (TLC) spectra using different development temperatures are shown (1-3: Herba Euphorbiae chinensis test solution; S: Herba Euphorbiae chinensis control drug solution).

[0048] Figure 8 The TLC spectra using different development relative humidity are shown (1-3: Herba Euphorbiae Herba test solutions; S: Herba Euphorbiae Herba control medicinal material solution).

[0049] Fig. 9 The thin layer chromatography (TLC) spectrum of the Herba Dijincao (Dijinban) formula granules in Example 2 of the present invention is shown (1-3: Herba Dijincao (Dijinban) formula granules test solution; S: Herba Dijincao (Dijinban) control medicinal material solution).

[0050] Fig.10 The thin layer analysis (TLC) spectrum of the standard decoction of Herba Euphorbiae (Dijin) and the standard decoction of Herba Euphorbiae (Dijin) in Example 2 of the present invention is shown (1-3: test solution of standard decoction of Herba Euphorbiae (Dijin); 4-6: test solution of standard decoction of Herba Euphorbiae (Dijin); S: control medicinal material solution of Herba Euphorbiae (Dijin)).

[0051] Fig.11The thin layer analysis (TLC) spectra of the standard decoction of Euphorbia microphylla and the standard decoction of Herba Euphorbiae (Euphrasia officinalis) in Example 2 of the present invention are shown (1-3: test solution of the standard decoction of Euphorbia microphylla; 4-6: test solution of the standard decoction of Herba Euphorbiae (Euphrasia officinalis); S is the control medicinal material solution of Herba Euphorbiae (Euphrasia officinalis)).

[0052] Fig.12 The thin layer analysis (TLC) spectra of the decoction pieces of Herba Euphorbiae Dijin (Dijin), Herba Euphorbiae Dijin (Ban Dijin) and Herba Euphorbiae Microphyllae in Example 2 of the present invention are shown (1-3: test solution of Herba Euphorbiae Dijin (Dijin) decoction pieces; 4-6: test solution of Herba Euphorbiae Dijin (Ban Dijin) decoction pieces; 7-9: test solution of Herba Euphorbiae Microphyllae decoction pieces; S1: Herba Euphorbiae Dijin (Dijin) control medicinal material solution; S2: Herba Euphorbiae Dijin (Ban Dijin) control medicinal material solution).

[0053] Fig.13 It is a thin layer chromatogram of the Herba Euphorbiae (Dijinbanjin) decoction pieces in Comparative Example 1 of the present invention (1-12: multiple batches of Herba Euphorbiae (Dijinbanjin) decoction pieces test solution; T: quercetin reference solution).

[0054] Fig.14 It is a thin layer chromatogram of the standard decoction of Dijincao (Dijincao scabra), the fake microphylla Euphorbia and the standard decoction of Dijincao (Dijincao) in Comparative Example 2 of the present invention (1-3: test solution of standard decoction of Dijincao (Dijincao scabra); 4-6: test solution of standard decoction of Euphorbia microphylla; 7-9: test solution of standard decoction of Dijincao (Dijincao); S: reference medicinal material chromatographic solution of Dijincao (Dijincao scabra); T: quercetin reference solution).

[0055] Fig.15 It is a thin layer chromatogram of the Herba Euphorbiae (Dijinban) formula granules in Comparative Example 3 of the present invention (1-3: Herba Euphorbiae (Dijinban) formula granules test solution; T: quercetin reference solution).

[0056] Fig.16 It is a thin layer chromatogram of the standard decoctions of Euphorbia microphylla, Herba Euphorbiae (Dijin) and Herba Euphorbiae (Dijin scaly) in Comparative Example 4 of the present invention (1-3: test solution of standard decoction of Euphorbia microphylla; 4-6: test solution of standard decoction of Herba Euphorbiae (Dijin); 7-9: test solution of standard decoction of Herba Euphorbiae (Dijin scaly)).

[0057] Fig.17 It is a thin layer chromatogram of the standard decoctions of Euphorbia microphylla, Herba Euphorbiae Radix (Dijin) and Herba Euphorbiae Radix (Dijin scaly) in Comparative Example 5 of the present invention (1-3: test solution of standard decoction of Euphorbia microphylla; 4-6: test solution of standard decoction of Herba Euphorbiae Radix (Dijin); 7-9: test solution of standard decoction of Herba Euphorbiae Radix (Dijin scaly)).

[0058] Fig.18It is a thin layer chromatogram of the standard decoction of the counterfeit Euphorbia microphylla, Herba Euphorbiae Rugosae (Dijin) and Herba Euphorbiae Rugosae (Dijin scaly) in Comparative Example 6 of the present invention (1-3: test solution of standard decoction of Euphorbia microphylla; 4-6: test solution of standard decoction of Herba Euphorbiae Rugosae (Dijin); 7-9: test solution of standard decoction of Herba Euphorbiae Rugosae (Dijin scaly)).

[0059] Description of Reference Numerals

[0060] Area A: Rf value is within the range of 0.59 to 0.65

[0061] Area B: Rf value is within the range of 0.19 to 0.24

[0062] A' zone: Rf value is within the range of 0.65 to 0.70

[0063] B' area: Rf value is within the range of 0.30 to 0.35 DETAILED DESCRIPTION

[0064] In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0065] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0066] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0067] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0068] It should be understood that the singular article "a," "an," and "the" as used in the specification and claims of this application include plural referents unless the context clearly dictates otherwise.

[0069] In this specification, the references to "one or some specific / preferred embodiments / schemes", "another or some other specific / preferred embodiments / schemes", "one or another embodiment / scheme", "one or another technical solution", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) related to the embodiment described are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0070] The term "comprising" and any variations thereof in the specification and claims of the present invention and the above drawings are intended to cover non-exclusive inclusions. For example, a process, method or system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0071] Unless otherwise specified, the term "medicinal materials" used in this article refers to raw materials of Chinese medicinal materials that have not been processed or made into finished products; "decoction pieces" refer to Chinese medicinal materials that are processed according to needs and are used for prescriptions, or Chinese medicinal materials that can be directly used in Chinese medicine clinical practice; "standard decoctions" refer to water decoctions of single-flavor Chinese medicinal pieces prepared through standardized processes guided by Chinese medicine theory, based on clinical application, and with reference to modern extraction methods; "formulated granules" are granules made from single-flavor Chinese medicinal pieces processed according to traditional standards and extracted and concentrated for use in Chinese medicine clinical prescriptions; "control medicinal materials" (or "reference medicinal materials") refer to standard medicinal materials that have completed species identification and are used to identify the samples to be tested.

[0072] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0073] Unless otherwise specified, the term "thin layer chromatography" used in this article is a trace, rapid and simple separation and analysis method. A suitable stationary phase is coated on a glass plate, plastic or aluminum substrate to form a uniform thin layer. After spotting and development, the relative shift value (Rf) is compared with the relative shift value of the chromatogram obtained by the same method with a suitable reference substance, and is used for drug identification, impurity inspection or content determination. This method is a very important experimental technique for rapid separation and qualitative analysis of small amounts of substances, and is also used to track the progress of reactions. It has the characteristics of short development time, strong separation ability and high sensitivity. Thin layer chromatography is one of the important inspection items for traditional Chinese medicines. By comparing the chromatographic behavior of the test sample and the reference medicinal materials and reference substances, the authenticity and quality of the medicinal materials can be judged.

[0074] Unless otherwise specified, the term "spotting" used herein refers to the process of adding droplets of sample solution to be separated and identified onto a thin layer plate.

[0075] Unless otherwise specified, the term "development" used herein refers to the process in which the developing agent carries the sample components to migrate a certain distance on the thin layer plate by the capillary action of the stationary phase on the thin layer plate.

[0076] The invention provides a thin layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae, the method comprising the steps of preparing a test solution, preparing a reference medicinal material solution and thin layer identification; a test solution and a reference medicinal material solution are obtained by a simple and quick preparation method, and are spotted on the same thin layer chromatography plate respectively, and after development, a multi-information Herba Euphorbiae thin layer chromatogram is obtained by inspection under ultraviolet light. The method uses Herba Euphorbiae reference medicinal materials as a reference, has strong specificity, is suitable for production practice, and provides a basis for establishing quality control standards for Herba Euphorbiae and related products.

[0077] <Preparation of test solution>

[0078] The inventors investigated the preparation method of the test solution and compared the final extraction results, and finally determined the preparation method of the test solution, which specifically includes taking the test sample, adding water to treat, filtering, shaking the filtrate with an organic solvent to extract, standing to allow stratification, taking the supernatant and evaporating it, and adding an alcohol solvent to dissolve the residue.

[0079] In some specific embodiments of the present invention, the test samples include Radix Dijincao medicinal materials of different origins and related products thereof, Radix Dijincao counterfeit medicinal materials and related products thereof; the Radix Dijincao medicinal materials of different origins include Radix Dijincao (Dijin) medicinal materials and Radix Dijincao (Dijinban) medicinal materials; the Radix Dijincao counterfeit medicinal materials are Euphorbia microphylla medicinal materials; the related products are selected from one or more of decoction pieces, standard decoctions and formula granules. Specifically, the test samples of the present application include one or more selected from Radix Dijincao (Dijin) medicinal materials, Radix Dijincao (Dijin) decoction pieces, Radix Dijincao (Dijin) standard decoctions, Radix Dijincao (Dijin) formula granules, and one or more selected from Radix Dijincao (Dijinban) medicinal materials, Radix Dijincao (Dijinban) decoction pieces, Radix Dijincao (Dijin) standard decoctions, and Radix Dijincao (Dijinban) formula granules.

[0080] Regarding the preparation of the test solution, in some specific embodiments of the present invention, the water treatment includes any one of water dissolution, water decoction and heating reflux treatment.

[0081] The present invention has no particular restrictions on the amount of water used for water treatment. In order to ensure the efficiency of extraction and better extract the components to be identified, when the related products are selected from the decoction pieces, the mass volume ratio of the decoction pieces to the water used for the water treatment is 1g: (10-25) mL, preferably 1g: (10-20) mL, and more preferably 1g: 20 mL; when the related products are selected from the standard decoction, the mass volume ratio of the standard decoction to the water used for the water treatment is 1g: (20-60) / mL; preferably 1g: (20-55) / mL; more preferably 1g: 50mL; when the related products are selected from the formula granules, the mass volume ratio of the formula granules to the water used for the water treatment is 1g: (20-60) / mL; preferably 1g: (20-55) / mL; more preferably 1g: 50mL. The present invention has no particular restrictions on the time of water treatment. In order to ensure the efficiency of extraction and better extract the components to be identified, the time of water treatment is 10-20min, preferably 5-15min, and most preferably 15min.

[0082] In order to facilitate the subsequent extraction process, it is preferred to cool after adding water and then perform a filtering step. The present invention does not specifically limit the filtering method, and it can be any conventional filtering means in the art, such as filter paper, cotton filtering, etc.

[0083] After filtration, the filtrate is shaken and extracted with an organic solvent. Compared with extraction methods such as ultrasonic treatment, shaking extraction is simpler and easier. The organic solvent used can be one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate. In some specific embodiments of the present invention, ethyl acetate is preferred. In order to make the extraction more complete and the extraction effect better, the number of extractions is at least 1, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); preferably 1:1.

[0084] After that, the mixture is allowed to stand for separation, and the supernatant is concentrated to dryness. The present invention has no particular restrictions on the method for concentration to dryness. In some specific embodiments of the present invention, the method can be carried out by vacuum distillation and drying. After evaporation, an alcohol solvent is added to the residue. In some specific embodiments of the present invention, the alcohol solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. In terms of solubility, methanol is more preferred. In terms of dissolution efficiency, in some specific embodiments of the present invention, the ratio of the sample to the alcohol solvent is (0.3-1.3) g: (0.5-2) mL, preferably (0.4-1.1) g: (0.7-2) mL, and more preferably (0.5-1) g: 1 mL.

[0085] <Preparation of control medicinal material solution>

[0086] The preparation of the control medicinal material solution of the present invention comprises taking a control medicinal material of Herba Dijin, decocting it with water or heating it under reflux, filtering it, extracting the filtrate by shaking it with an organic solvent, standing it to separate layers, taking the supernatant and concentrating it to dryness, and dissolving the residue by adding an alcohol solvent; wherein the Herba Dijin control medicinal materials include Herba Dijin (Ban Dijin) medicinal materials and Herba Dijin (Dijin) medicinal materials.

[0087] In some specific embodiments of the present invention, the method of decocting with water or heating and refluxing is selected to treat the Herba Euphorbiae Herba control medicinal materials because water extraction is closer to the decoction method of traditional Chinese medicine decoctions than alcohol extraction (methanol, ethanol, etc.), and can fully extract the water-soluble components in the medicinal materials, and can better characterize the clinical quality of the medicinal materials; the present invention has no special restrictions on the amount of water used for decocting with water or heating and refluxing. In order to ensure the efficiency of extraction and better extract the components to be identified, the mass volume ratio of the Herba Euphorbiae Herba control medicinal materials to the water used for decocting with water or heating and refluxing is 1g: (100-250) mL, preferably 1g: (100-200) mL, and more preferably 1g: 200 mL. The present invention has no special restrictions on the time of water treatment. In order to ensure the efficiency of extraction and better extract the components to be identified, the time of decocting with water or heating and refluxing is 10-20 minutes, preferably 5-15 minutes, and most preferably 15 minutes.

[0088] In order to facilitate the subsequent extraction process, it is preferred to boil with water or heat under reflux and then cool, and then perform a filtering step. The present invention does not particularly limit the filtering method, and it can be any conventional filtering means in the art, such as filter paper, cotton filtering, etc.

[0089] After filtration, the filtrate is extracted with an organic solvent. The organic solvent used may be one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate. In some specific embodiments of the present invention, ethyl acetate is preferred. In order to make the extraction more complete and the extraction effect better, the extraction is performed at least once, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); preferably 1:1.

[0090] After that, the mixture is allowed to stand for separation, and the supernatant is concentrated to dryness. The present invention has no particular restrictions on the method for concentration to dryness. In some specific embodiments of the present invention, the mixture can be dried by distillation under reduced pressure. After evaporation, an alcohol solvent is added to the residue. In some specific embodiments of the present invention, the alcohol solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. In terms of solubility, methanol is more preferred. In terms of dissolution efficiency, in some specific embodiments of the present invention, the ratio of the amount of the herb Dijin herb to the alcohol solvent is (0.05-0.2) g: (0.8-2) mL, preferably (0.07-0.18) g: (0.9-1.5) mL, and more preferably 0.1 g: 1 mL.

[0091] <Thin layer identification step>

[0092] The thin layer identification step of the present invention comprises taking a test sample solution and a control medicinal material solution, spotting them on a thin layer plate respectively, developing them with chloroform-ethyl acetate-methanol-formic acid as a developing agent, drying them after development, spraying them with a color developer, heating them until the spots are clearly colored, and inspecting them under ultraviolet light.

[0093] In some specific embodiments of the present invention, the present invention has no special restrictions on the material of the thin layer plate, and can select silica gel thin layer plate, polyamide thin layer plate, ordinary prefabricated plate, high-efficiency prefabricated plate, etc. In order to fully separate the components in the herb, the present invention preferably uses a silica gel thin layer plate, and more preferably a silica gel high-efficiency G thin layer plate. Further, in order to facilitate inspection and reduce the differences caused by the stationary phase, it is preferred to take the test solution and the control medicinal material solution and spot them on the same silica gel thin layer plate respectively; in some specific embodiments of the present invention, the spotting amount of the test solution is 1 to 5 μL, preferably 1 to 4 μL, more preferably 2 to 4 μL; the spotting amount of the control medicinal material solution is 1 to 5 μL, preferably 1 to 4 μL, more preferably 2 to 4 μL; if the spotting amount is too low, the spot separation is relatively poor, and the spots cannot be clearly displayed; the present invention uses chloroform-ethyl acetate-methanol-formic acid as a developing agent for development, which is conducive to the good separation of various chemical components of herb and its related products of different origins and herb control medicinal materials on the thin layer plate. In some preferred embodiments of the present invention, in order to obtain better separation, the developing agent of the present invention is preferably calculated by volume ratio of chloroform: ethyl acetate: methanol: formic acid = (4-7): (2-3.5): (1-3): (0.2-0.9), further 6.5:2.5:2:0.5.

[0094] The present invention has no particular limitation on the temperature during development, and it can be carried out at 20-40°C or 3-8°C, and different temperatures have little effect on the separation effect; the present invention has no particular limitation on the relative humidity during development, and it can be carried out at a relative humidity of 18%-88%, and different relative humidity has little effect on the separation effect. In some specific embodiments of the present invention, the thin layer developer is selected from an ethanol solution of aluminum chloride, and the concentration of the ethanol solution of aluminum chloride is preferably 1%-5%, more preferably 2%-4%, and most preferably 3%, so as to obtain a better color development effect.

[0095] After that, heating is performed. The present invention does not particularly limit the heating method. The heating method can be performed by conventional methods in the art, preferably by hot air drying. The heating temperature is 100-110°C, preferably 105°C. The present invention is inspected under ultraviolet light (365nm). Compared with inspection under sunlight, the spots under ultraviolet light are clear and colorful. In some specific embodiments of the present invention, the chromatogram of the test sample is compared with the chromatogram of the control medicinal material to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

[0096] In some specific embodiments of the invention,

[0097] If the fluorescent spots are shown in the Rf value range of 0.19 to 0.24, and no fluorescent spots are shown in the Rf value range of 0.59 to 0.65, then the sample is Herba Dijin (Dijin),

[0098] If no fluorescent spots are shown in the Rf value range of 0.19 to 0.24, but fluorescent spots are shown in the Rf value range of 0.59 to 0.65, then the sample is Herba Dichondrium (Dichondrium scaly).

[0099] If no fluorescent spots are shown in the Rf value ranges of 0.19-0.24 and 0.59-0.65, the test sample is Euphorbia microphylla.

[0100] By adopting the thin-layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae Herba-Lingues provided by the present invention, a chromatogram of the test sample of Herba Euphorbiae Herba-Lingues and its related products which is consistent with the spots in the chromatogram of the Herba Euphorbiae Herba-Lingues control medicinal material can be obtained, the separation degree and the point nature in the chromatogram are good, the development time is short, and the inspection is clear; in addition, different origins of Herba Euphorbiae Herba-Lingues and the counterfeit Euphorbia microphylla can also be distinguished. By using the method provided by the present application, the quality of Herba Euphorbiae Herba-Lingues and its related products can be better controlled and their authenticity can be distinguished.

[0101] Example

[0102] The technical solutions of the present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to explain and illustrate the present invention, and are not used to limit the scope of protection of the present invention.

[0103] <Instruments and Reagents>

[0104] Instruments: CAMAG TLC VISUALIZER, METTLERTOLEDO, KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), silica gel G thin layer plate (Qingdao Ocean Chemical Plant), polyamide film (Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province), GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.), AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.), high-efficiency silica gel G thin layer plate (Qingdao Ocean Chemical Plant).

[0105] Reagents: ether (Sinopharm Chemical Reagent Co., Ltd.), toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), formic acid (Sinopharm Chemical Reagent Co., Ltd.), methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), and hydrochloric acid (Sinopharm Chemical Reagent Co., Ltd.) were all of analytical grade; water was ultrapure water.

[0106] The reference medicinal materials of Herba Dijincao (Dijincao scabra) (batch number: 121552-200902), Herba Dijincao (Dijincao scabra) (batch number: 121167-201805), and quercetin reference substance (batch number: 100081-201610) were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products.

[0107] The formula granules of Radix Euphorbiae (Dijincao Ban) (batch numbers: 19120199, 19120209, 19120219), standard decoction of Radix Euphorbiae (Dijincao Ban) (batch numbers: DG1905128, DG1911039, DG121909136), decoction pieces of Radix Euphorbiae (Dijincao Ban) (batch numbers: YP1905128, YP1911039, YP121909136), standard decoction of Radix Euphorbiae (Dijincao Ban) (batch numbers: DG1811006, DG2111047, DG2111048), and decoction pieces of Radix Euphorbiae (Dijincao Ban) (batch numbers: YP1811006, YP2111047, YP2111048) were prepared and provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0108] Example 1: Construction of a thin layer identification method for distinguishing the origin and authenticity of Herba Euphorbiae

[0109] <Investigation of thin layer identification method>

[0110] Study on the developing agent

[0111] In the process of investigating the developing agent, the effects of different stationary phases, the combination of multiple developing agents and different ratios of developing agents on the developing effect were investigated. The specific contents are as follows:

[0112] (1) Using silicone GF 254 Thin layer plate, using cyclohexane: ethyl acetate: glacial acetic acid (8:8:0.2) as the developing solvent, the chromatogram obtained has a single spot and less information. The chromatogram results are shown in Figure 1 .

[0113] (2) The method disclosed in Reference 4 was used, i.e., a polyamide plate was used and toluene:ethyl acetate:formic acid:water (1:8:2:1) was used as the developing solvent. The chromatogram obtained had less rounded spot information and a single spot had tailing. The results are shown in Figure 2 .

[0114] (3) Silica gel G thin layer plate was used, and acetone: toluene: chloroform (5:2.5:2.5) was used as the developing solvent. The chromatogram obtained had less rounded spot information, and a single spot had tailing. The results are shown in Figure 3 .

[0115] After the above investigation, the chloroform-ethyl acetate-methanol-formic acid system was finally selected. However, when the composition ratio was (6:2.5:0.5:0.2), the developing solvent had a smaller polarity and the spot Rf value was lower. The chromatogram results are shown in Figure 4 On this basis, the proportion of methanol was increased to improve the polarity of the developing agent. When the composition ratio was (6.5:2.5:1.5:0.5), the spot Rf value was more moderate and there were more round spots. The results are shown in Figure 5 .

[0116] Investigation of spotting quantity

[0117] Take the formula granules of Dijincao (Ban Dijincao) and the control medicinal material of Dijincao (Ban Dijincao), prepare the test solution and the control medicinal material solution respectively according to the above method, and take 1μL, 2μL, 3μL, 4μL of the test solution of Dijincao formula granules and 1μL, 2μL, 3μL, 4μL of the control medicinal material solution of Dijincao respectively, spot them on the same silica gel high-efficiency G thin layer plate, use chloroform-ethyl acetate-methanol-formic acid (6.5:2.5:2:0.5) as the developing agent, develop, then take out, dry, spray with 3% aluminum chloride ethanol test solution, blow dry with hot air, and inspect under ultraviolet light (365nm). The results are shown in Figure 6 , Figure 6 1 to 4 correspond to the chromatograms of 1 μL, 2 μL, 3 μL, and 4 μL of the test sample of the formula granules of Dijincao (Ban Dijin), and 5 to 8 correspond to the chromatograms of 1 μL, 2 μL, 3 μL, and 4 μL of the reference medicinal material of Dijincao (Ban Dijin). Figure 6It can be seen that when the sample volume is 2μL~4μL, the chromatogram of the formula particles of Dijincao (Dijincao scabiosaefolia) and the chromatogram of the reference medicinal material show clear spots at corresponding positions without other interference.

[0118] Temperature Investigation

[0119] Take the formula granules of Dijincao (Banded Dijincao) and the control medicinal material of Dijincao (Banded Dijincao), prepare the test solution and the control medicinal material solution respectively according to the above method, and take 2μL of the test solution of the formula granules of Dijincao (Banded Dijincao) and 2μL of the control medicinal material solution of Dijincao (Banded Dijincao) respectively, and spot them on the same silica gel high-efficiency G thin layer plate, use chloroform-ethyl acetate-methanol-formic acid (6.5:2.5:2:0.5) as the developing agent, and develop them under low temperature (T: 4℃, RH: 75%), room temperature (T: 22.1℃, RH: 75%) and high temperature (T: 35℃, RH: 75%) conditions, then take them out, dry them, spray them with 3% aluminum chloride ethanol test solution, blow dry with hot air, and inspect them under ultraviolet light (365nm). The results are shown in Figure 7 , Figure 7 1 to 3 correspond to the chromatograms of the test sample of the formula granules of Dijincao (Ban Dijin), and S corresponds to the chromatogram of the reference medicinal material of Dijincao (Ban Dijin). Figure 7 It can be seen that under different temperature conditions, the chromatograms of the test and control medicinal materials of the formula granules of Dijincao (Ban Dijin) show fluorescent spots of the same color at the corresponding positions, and the separation effect is good. The experimental results show that temperature has little effect on the thin layer identification of the formula granules of Dijincao (Ban Dijin), indicating that the thin layer identification method has good durability at different temperatures.

[0120] Humidity survey

[0121] Take the formula granules of Herba Dijincao (Banded Dijincao) and the control medicinal material of Herba Dijincao (Banded Dijincao), prepare the test solution and the control medicinal material solution respectively according to the above method, and take 2μL of the test solution of Herba Dijincao (Banded Dijincao) and 2μL of the control medicinal material solution of Herba Dijincao (Banded Dijincao) respectively, spot them on the same silica gel high-efficiency G thin layer plate, use chloroform-ethyl acetate-methanol-formic acid (6.5:2.5:2:0.5) as the developing agent, develop under different humidity conditions (RH: 18%, 75%, 88%), then take out, dry, spray with 3% aluminum chloride ethanol test solution, blow dry with hot air, and inspect under ultraviolet light (365nm). The results are shown in Figure 8 , Figure 8 1 to 3 correspond to the chromatograms of the test sample of the formula granules of Dijincao (Ban Dijin), and S corresponds to the chromatogram of the reference medicinal material of Dijincao (Ban Dijin). Figure 8It can be seen that under different humidity conditions, the chromatograms of the test and control medicinal materials of the formula granules of Dijincao (Ban Dijin) show fluorescent spots of the same color at the corresponding positions, and the separation effect is good. The experimental results show that humidity has little effect on the thin layer identification of the formula granules of Dijincao (Ban Dijin), indicating that the thin layer identification method has good durability under different humidity.

[0122] Example 2: Application of thin layer identification method in distinguishing the origin and authenticity of Herba Euphorbiae

[0123] <Preparation of test solution>

[0124] Preparation of test solution of Herba Euphorbiae Herba Decoction Pieces:

[0125] Take 1 g of Herba Euphorbiae Radix Pseudostellariae powder, add 20 mL of water, heat and reflux for 15 minutes, extract once with 20 mL of ethyl acetate by shaking, collect the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the Herba Euphorbiae Radix Pseudostellariae test solution.

[0126] Preparation of test solution of Herba Euphorbiae Herba Granules / Standard Decoction:

[0127] Take 0.5 g of Herba Dijincao granules or Herba Dijincao standard decoction, add 25 mL of water, slightly heat to dissolve, cool, shake and extract once with 25 mL of ethyl acetate, collect the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, and use this as the test solution of Herba Dijincao granules / standard decoction.

[0128] Preparation of control drug solution:

[0129] Take 0.1 g of Herba Dijincao reference medicinal materials (Herba Dijincao (Dijincao scabra) reference medicinal materials and / or Herba Dijincao (Dijincao) reference medicinal materials), add 20 mL of water, boil for 15 min, and boil slightly. Cool, and extract with 20 mL of ethyl acetate by shaking once, 20 mL each time. Collect the ethyl acetate solution, evaporate to dryness, and add 1 mL of methanol to the residue to dissolve it, as the Herba Dijincao reference medicinal material solution.

[0130] TLC identification steps:

[0131] Pipette 2 μL of the test solution and the control drug solution, respectively, and spot them on the same silica gel high-efficiency G thin layer plate, use chloroform-ethyl acetate-methanol-formic acid (6.5:2.5:2:0.5) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, blow dry with hot air, and examine under ultraviolet light (365nm).

[0132] Evaluation requirements: In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0133] like Fig. 9As shown, 1 to 3 correspond to the test sample chromatograms of different batches of Herba Dijincao (Ban Dijin) formula granules, and S is the Herba Dijincao (Ban Dijin) reference medicinal material chromatogram. It can be seen that the main fluorescent spots of the same color in the Herba Dijincao (Ban Dijin) formula granules and the Herba Dijincao (Ban Dijin) reference medicinal material chromatograms can correspond one to one.

[0134] like Fig.10 As shown, 1 to 3 correspond to the test sample chromatograms of different batches of standard decoction of Dijincao (Dijin) (batch numbers: DG1811006, DG2111047, DG2111048), 4 to 6 correspond to the test sample chromatograms of different batches of standard decoction of Dijincao (Ban Dijin) (batch numbers: DG1905128, DG1911039, DG121909136), and S is the reference medicinal material chromatogram of Dijincao (Ban Dijin). It can be seen that there are differences between Herba Dijin (Dijin) and Herba Dijin (Dijin pied) in area A (Rf value 0.59-0.65) and area B (Rf value 0.19-0.24). Herba Dijin (Dijin) does not show fluorescent spots in area A, but shows fluorescent spots in area B; Herba Dijin (Dijin pied) shows fluorescent spots in area A, but does not show fluorescent spots in area B. This thin layer chromatography method can distinguish standard decoctions of Herba Dijin of different origins.

[0135] like Fig.11 As shown, 1 to 3 correspond to the chromatograms of the test samples of different batches of standard decoction of Euphorbia microphylla (Batch No.: DG1905076, DG1905079, DG1905080), 4 to 6 correspond to the chromatograms of the test samples of different batches of standard decoction of Herba Dijincao (Batch No.: DG1905128, DG1911039, DG121909136), and S is the chromatogram of the reference medicinal material of Herba Dijincao (Batch No.: DG1905128, DG1911039, DG121909136). It can be seen that there are differences between Euphorbia microphylla and Herba Ornithophila (Echinochloa spp.) in area A (Rf value 0.59-0.65) and area B (Rf value 0.19-0.24). Euphorbia microphylla does not show fluorescent spots in areas A and B; Herba Ornithophila (Echinochloa spp.) shows fluorescent spots in area A but not in area B. This thin layer chromatography method can distinguish the fake (Euphorbia microphylla) from the authentic Herba Ornithophila (Echinochloa spp.).

[0136] like Fig.12As shown, 1 to 3 correspond to the test sample chromatograms of different batches of Herba Dijincao (Dijin) slices (batch numbers: YP1811006, YP2111047, YP2111048), 4 to 6 correspond to the test sample chromatograms of different batches of Herba Dijincao (Dijinban) slices (batch numbers: YP1905128, YP1911039, YP121909136), 7 to 9 correspond to different batches of Herba Euphorbiae Microphyllae slices (batch numbers: YP1905076, YP1905079, YP1905080); S1 is the reference medicinal material chromatogram of Herba Dijincao (Dijin), and S2 is the reference medicinal material chromatogram of Herba Dijincao (Dijinban). It can be seen that there are significant differences between Herba Dijinzi (Dijin), Herba Dijinzi (Bian Dijin) and Euphorbia microphylla. Specifically, there are differences between Herba Dijinzi (Dijin), Herba Dijinzi (Bian Dijin) and Euphorbia microphylla slices in A' area (Rf value 0.65-0.70) and B' area (Rf value 0.30-0.35). Among them, Herba Dijinzi (Dijin) slices show clear fluorescent spots in B' area, while the other two almost do not show fluorescent spots; while Euphorbia microphylla slices show clear fluorescent spots in A' area, while the other two almost do not show fluorescent spots. It can be seen that the thin layer chromatography method can distinguish between counterfeit products (Euphorbia microphylla) and Herba Dijinzi slices of different origins. Comparative Example 1: Identification method specified in Part 1 of the 2020 edition of the Chinese Pharmacopoeia

[0137] This comparative example adopts the method of the 2020 edition of the Chinese Pharmacopoeia to perform thin layer chromatography on the decoction pieces of Dijincao (Dijinban), and the specific process is as follows:

[0138] Preparation of test solution: Take 1g of Herba Dijincao (Dijincao) decoction pieces powder, add 50ml of 80% methanol, heat and reflux for 1 hour, cool, filter, evaporate the filtrate, add 60ml of water-ether (1:1) mixed solution to dissolve the residue, let stand and stratify, discard the ether solution, extract the aqueous solution with ether twice, 20ml each time, discard the ether solution, add 5ml of hydrochloric acid to the aqueous solution, hydrolyze in a water bath for 1 hour, take out, cool rapidly, extract with ether twice, 20ml each time, combine the ether solutions, wash with 30ml of water, discard the aqueous solution, evaporate the ether solution, dissolve the residue in 1ml of ethanol, and use it as the test solution.

[0139] Preparation of reference solution: Take quercetin reference substance and add ethanol to make a solution containing 1 mg per 1 ml as the reference solution.

[0140] Thin layer identification steps: Pipette 10μl of the test solution and 2μl of the reference solution, respectively spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (5:4.5:0.5) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105℃ for several minutes, and examine under ultraviolet light (365nm).

[0141] Evaluation requirements: In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the reference sample chromatogram.

[0142] Results Fig.13 , Fig.13 T in the middle corresponds to the quercetin reference substance, and 1 to 12 correspond to the chromatograms of multiple batches of Herba Dijincao (Dijin) decoction pieces. This method has only two chromatographic spots in the sample, and can provide less chromatographic information. Comparative Example 2: Identification method specified in the 2020 edition of the Chinese Pharmacopoeia

[0143] This comparative example refers to the thin layer chromatography development conditions of the 2020 edition of the Chinese Pharmacopoeia to perform thin layer chromatography on the standard decoction of Herba Dijincao (Dijin), the standard decoction of the fake Euphorbia microphylla, and the standard decoction of Herba Dijincao (Dijin), and the specific process is as follows:

[0144] Preparation of test solution: Take 0.5 g each of the standard decoction of Herba Euphorbiae Dijin (Dijin), the standard decoction of the fake Euphorbia microphylla and the standard decoction of Herba Euphorbiae Dijin (Dijinban), grind them into powder, add 30 ml of 80% methanol, treat with ultrasound for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, extract twice with ether by shaking, 10 ml each time, discard the ether solution, add 10 ml of dilute hydrochloric acid to the aqueous solution, hydrolyze in a water bath for 1 hour, take out, cool rapidly, extract twice with ether, 20 ml each time, combine the ether solutions, wash with 30 ml of water, discard the water solution, evaporate the ether solution to dryness, dissolve the residue in 1 ml of ethanol, and use as the test solution.

[0145] Preparation of control medicinal material solution: Take 1 g of Herba Dijincao (Dijincao scabiosaefolia) control medicinal material, add 50 ml of 80% methanol, heat and reflux for 1 hour, cool, filter, evaporate the filtrate, add 60 ml of water-ether (1:1) mixed solution to the residue to dissolve, let stand and stratify, discard the ether solution, extract the aqueous solution with ether twice, 20 ml each time, discard the ether solution, add 5 ml of hydrochloric acid to the aqueous solution, hydrolyze in a water bath for 1 hour, take out, cool rapidly, extract with ether twice, 20 ml each time, combine the ether solutions, wash with 30 ml of water, discard the aqueous solution, evaporate the ether solution, add 1 ml of ethanol to the residue to dissolve, and use it as the control medicinal material solution.

[0146] Preparation of reference solution: Take quercetin reference substance and add ethanol to make a solution containing 1 mg per 1 ml as the reference solution.

[0147] Thin layer identification steps: 5 μl of the test solution and the reference medicinal material solution and 2 μl of the reference solution are respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (5:4.5:0.5) is used as the developing agent. After development, the plate is taken out, dried, sprayed with 3% aluminum chloride ethanol solution, heated at 105°C for several minutes, and examined under ultraviolet light (365nm).

[0148] Results Fig.14 , Fig.14 1 to 3 correspond to the chromatogram of the standard decoction of Dijin (Dijin scabra), 4 to 6 correspond to the chromatogram of the standard decoction of Euphorbia microphylla, 7 to 9 correspond to the chromatogram of the standard decoction of Dijin (Dijin), S corresponds to the chromatogram of the reference medicinal material of Dijin (Dijin scabra), and T corresponds to the quercetin reference substance. This thin-layer identification condition cannot effectively distinguish the standard decoction of Dijin (Dijin), the fake (Euphorbia microphylla) and Dijin (Dijin scabra).

[0149] Comparative Example 3: Identification Method Used in Reference 2

[0150] This comparative example adopts the method used in reference 2 (CN102078363A) to perform thin layer chromatography on the formula granules of Herba Dijincao (Dijincao scabra). The specific process is as follows:

[0151] Take about 1.0g of this product, grind it into powder, add 50-90ml of 80% methanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate, add 90ml of water-ether (1:1) to dissolve the residue, let it stand for stratification, discard the ether solution, extract the aqueous solution with ether twice, 30ml each time, discard the ether solution, add 10ml of hydrochloric acid to the aqueous solution, place it in a water bath for hydrolysis for 1 hour, take it out, cool it quickly, extract it with ether twice, 30ml each time, combine the ether solution, wash with 50ml of water, discard the aqueous solution, evaporate the ether solution, add 1ml of ethanol to dissolve the residue, as the test solution, take another quercetin reference substance, add ethanol to make a solution containing 1mg per 1ml as the reference substance solution.

[0152] Thin layer chromatography: Test, take 5ul of the test solution and 2ul of the reference solution, spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (5:4:0.5) as the developing solvent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, heat at 105℃ for several minutes, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference.

[0153] Results Fig.15 , Fig.15 1 to 3 correspond to the chromatogram of the test sample of Dijincao (Dijincao) formula granules, and T corresponds to the quercetin reference substance. The patented developing agent is consistent with the pharmacopoeia method, and there are only 2 spots in the chromatogram of the test sample, which can provide less chromatographic information.

[0154] Comparative Example 4: Identification method used in reference 3

[0155] This comparative example adopts the thin layer identification method used in reference 3 (thin layer analysis of quercetin in Herba Euphorbiae) to perform thin layer chromatography on the standard decoction of Euphorbia microphyllae, the standard decoction of Herba Euphorbiae (Dijin) and the standard decoction of Herba Euphorbiae (Dijin), and the specific process is as follows:

[0156] Preparation of standard decoction test solution: Take 0.5 g each of the standard decoctions of Euphorbia microphylla, Herba Euphorbiae rutaecarpa (Dijin) and Herba Euphorbiae rutaecarpa (Dijin maculatum), add 25 mL of water, heat slightly to dissolve, cool, extract once with 25 mL of ethyl acetate by shaking, collect the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0157] Thin layer identification steps: aspirate 2μl of the test solution and spot them on the same thin layer plate respectively, use acetone-toluene-chloroform (5:2.5:2.5) as the developing agent, develop, take out, dry, and observe the fluorescent spots under ultraviolet light.

[0158] Results Fig.16 , Fig.16 1 to 3 correspond to the chromatograms of the standard decoction of the fake Euphorbia microphylla, 4 to 6 correspond to the chromatograms of the standard decoction of Herba Dijin (Dijin), and 7 to 9 correspond to the chromatograms of the standard decoction of Herba Dijin (Dijin). The results show that the chromatograms obtained by the thin-layer identification method can provide less chromatographic information and cannot effectively distinguish the fake Euphorbia microphylla, Herba Dijin (Dijin) and Herba Dijin (Dijin).

[0159] Comparative Example 5: Identification Method Used in Reference 4

[0160] This comparative example adopts the thin layer development system in reference 4 (CN115436557A) to perform thin layer chromatography on the standard decoction of Euphorbia microphylla, the standard decoction of Herba Dijin (Dijin) and the standard decoction of Herba Dijin (Dijin scaly). The specific process is as follows:

[0161] Preparation of standard decoction test solution: Take 0.5 g each of the standard decoctions of Euphorbia microphylla, Dijin (Dijin) and Dijin (Dijin maculate), add 25 mL of water, heat slightly to dissolve, cool, extract once with 25 mL of ethyl acetate by shaking, collect the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0162] Thin layer identification steps: aspirate 2μl of the test solution and spot them on the same polyamide film respectively, use toluene-ethyl acetate-formic acid-water (1:8:2:1) as the developing agent, develop, take out, dry, spray with aluminum chloride ethanol solution, blow dry with hot air, and examine under ultraviolet light (365nm).

[0163] Results Fig.17 , Fig.171 to 3 correspond to the standard decoction chromatogram of Euphorbia microphylla, 4 to 6 correspond to the standard decoction chromatogram of Dijin (Dijin), and 7 to 9 correspond to the standard decoction chromatogram of Dijin (Dijin scaly). In this thin layer chromatogram, the fake Euphorbia microphylla, Dijin (Dijin) and Dijin (Dijin scaly) show different chromatographic spots, but the chromatographic spots are not round enough and show tailing. In addition, the developing agent contains toluene, which is not healthy and environmentally friendly. In addition, according to the content under the above "Investigation of the developing agent", the method disclosed in the reference 4 was used to obtain Figure 2 Compared with the comparative example 5 Fig.17 Compared with the above, the chromatographic spots are not the same, indicating that the method has high requirements for operation and poor reproducibility. In addition, in actual operation, the polyamide film is very thin and easy to curl, and the thin layer chromatography is difficult and requires high requirements for operators, which is not easy to implement.

[0164] Comparative Example 6: Identification method used in reference 5

[0165] This comparative example adopts the thin layer development system of reference 5 (CN109085285B) to perform thin layer chromatography on the standard decoction of Euphorbia microphylla, the standard decoction of Dijin (Dijin) and the standard decoction of Dijin (Dijin scaly), and the specific process is as follows:

[0166] Preparation of standard decoction test solution: Take 0.5 g each of the standard decoctions of Euphorbia microphylla, Dijin (Dijin) and Dijin (Dijin maculate), add 25 mL of water, heat slightly to dissolve, cool, extract once with 25 mL of ethyl acetate by shaking, collect the ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0167] Thin layer identification steps: Pipette 2 μl of the test sample solution and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-glacial acetic acid (8:8:0.2) as the developing agent, develop, take out, dry, and spray with 10% sulfuric acid ethanol solution.

[0168] Results Fig.18 , Fig.18 In the middle, 1 to 3 correspond to the standard decoction chromatograms of Euphorbia microphylla, 4 to 6 correspond to the standard decoction chromatograms of Herba Dichrysanthemi (Dijin), and 7 to 9 correspond to the standard decoction chromatograms of Herba Dichrysanthemi (Dijin). In this thin layer chromatogram, no chromatographic spots were observed for the fake Euphorbia microphylla, Herba Dichrysanthemi (Dijin) and Herba Dichrysanthemi (Dijin), indicating that this method cannot distinguish fakes and Herba Dichrysanthemi of different origins.

[0169] The above embodiments are only used to illustrate several embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood that any limitation is imposed on the scope of the present invention. It should be clear that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A thin layer identification method for distinguishing the origin and authenticity of Euphorbia obesa, characterized in that: The method comprises the preparation of a test solution, the preparation of a reference medicinal material solution and thin layer identification. The preparation of the test solution includes taking the test sample, adding water for treatment, filtering, extracting the filtrate by shaking with an organic solvent, standing to separate the layers, taking the supernatant and concentrating it to dryness, and dissolving the residue by adding an alcohol solvent; the test sample includes different origins of Herba Dichondrium and related products, Herba Dichondrium counterfeit medicinal materials and related products; the Herba Dichondrium of different origins includes Herba Dichondrium Euphorbia humifusa Parthenocissus maculatus Euphorbia maculate L.; the fake Herba Euphorbiae Herba is Euphorbia microphylla; the organic solvent is selected from ethyl acetate; The preparation of the control medicinal material solution comprises taking a control medicinal material of Herba Dichrysanthemi, boiling it with water or heating it under reflux, filtering it, extracting the filtrate by shaking with an organic solvent, standing it to separate into layers, taking the supernatant and concentrating it to dryness, and dissolving the residue by adding an alcohol solvent; the Herba Dichrysanthemi control medicinal material comprises a base of Herba Dichrysanthemi Euphorbia maculate Parthenocissus chinensis L. Euphorbia humifusa Willd.'s Herba Dijin Herba; the organic solvent is selected from ethyl acetate; The thin layer identification comprises taking a test sample solution and a control medicinal material solution, spotting them on the same high-efficiency silica gel G thin layer plate, developing them with chloroform-ethyl acetate-methanol-formic acid in a volume ratio of 6:2.5:2:0.5 as a developing agent, and inspecting them; The related products are selected from one or more of medicinal pieces, standard decoctions and formula granules.

2. The thin layer identification method according to claim 1, characterized in that: In the preparation of the test solution, the water treatment includes any one of water dissolution, water decoction and heating reflux treatment; when the related product is selected from decoction pieces, the mass volume ratio of the decoction pieces to the water used for the water treatment is 1g: (10~25)mL; when the related product is selected from standard decoctions, the mass volume ratio of the standard decoction to the water used for the water treatment is 1g: (20~60)mL; when the related product is selected from formula granules, the mass volume ratio of the formula granules to the water used for the water treatment is 1g: (20~60)mL; the time for the water treatment is 10~20min.

3. The thin layer identification method according to claim 1 or 2, characterized in that: In the preparation of the test solution, the shaking extraction is performed at least once, and the volume ratio of the filtrate to the organic solvent is (1-2): (1-1.5); the alcohol solvent is methanol or an aqueous solution of methanol.

4. The thin layer identification method according to claim 1 or 2, characterized in that: In the preparation of the control medicinal material solution, the mass volume ratio of the Herba Euphorbiae Ichthyophthirius control medicinal material to the water used for the decoction or heating reflux treatment is 1 g: (100-250) mL, and the decoction or heating reflux treatment time is 10-20 min.

5. The thin layer identification method according to claim 1 or 2, characterized in that: In the preparation of the control medicinal material solution, the shaking extraction is performed at least once, and the volume ratio of the filtrate to the organic solvent is (1-2): (1-1.5).

6. The thin layer identification method according to claim 1 or 2, characterized in that: In the spotting operation of the thin layer identification, the spotting volume of the test solution is 1~5µL, and the spotting volume of the control medicinal material solution is 1~5µL.

7. The thin layer identification method according to claim 1 or 2, characterized in that: The thin layer identification also includes drying after unfolding, spraying with a color developer, heating until the spots are clearly colored, and then placing under ultraviolet light for inspection.

8. The thin layer identification method according to claim 7, characterized in that: The color developer is an ethanol solution of aluminum chloride with a concentration of 1% to 5%.

9. The thin layer identification method according to claim 7, characterized in that: The inspection is inspection under ultraviolet light with a wavelength of 365 nm.

10. The thin layer identification method according to claim 1 or 2, characterized in that: In the thin layer identification, the inspection includes comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

11. The thin layer identification method according to claim 10, characterized in that: If the fluorescent spots are shown in the Rf value range of 0.19-0.24, and no fluorescent spots are shown in the Rf value range of 0.59-0.65, then the sample is originally from the ground ivy. Euphorbia humifusa Willd.'s Standardized Decoction of Herba Euphorbiae, If no fluorescent spots are shown in the Rf value range of 0.19-0.24, but fluorescent spots are shown in the Rf value range of 0.59-0.65, then the sample is derived from the schizophyllum serrata. Euphorbia maculate Standardized decoction of Herba Euphorbiae L., If no fluorescent spots are shown in the Rf value ranges of 0.19-0.24 and 0.59-0.65, the test sample is the standard decoction of Euphorbia microphylla.

12. The thin layer identification method according to claim 10, characterized in that: If the fluorescent spots are shown in the Rf value range of 0.30-0.35, and no fluorescent spots are shown in the Rf value range of 0.65-0.70, then the sample is originally from ground ivy. Euphorbia humifusa Willd.'s Herba Euphorbiae rutaecarpae, If no fluorescent spots are shown in the Rf value range of 0.30-0.35 and 0.65-0.70, the sample is derived from the genus Psoralea corylifolia. Euphorbia maculate L.'s Herba Dijincao decoction pieces, If no fluorescent spots are shown in the Rf value range of 0.30-0.35, but fluorescent spots are shown in the Rf value range of 0.65-0.70, the test sample is Euphorbia microphylla decoction pieces.

13. Use of the thin layer identification method according to any one of claims 1 to 12 in distinguishing the original and / or counterfeit products of Herba Dioscoreae and its related products, wherein: The related products are selected from one or more of decoction pieces, standard decoctions and formula granules; the base includes Dijin Euphorbia humifusa Willd. and Parthenocissus Euphorbia maculate L.; The counterfeit products are Euphorbia microphylla medicinal materials, Euphorbia microphylla decoction pieces or Euphorbia microphylla standard decoction.

14. The use according to claim 13, characterized in that The application is an application in simultaneously distinguishing the original and counterfeit products of Herba Dichondrium and its related products.

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