Chromatographic method for identifying authenticity of sedum sarmentosum

Abstract

The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a chromatographic method for identifying authenticity of sedum sarmentosum. The method comprises the following steps: firstly, establishing a sedum sarmentosum reference substance characteristic chromatogram through a liquid chromatography-mass spectrometry technology, attributing characteristic peaks in the characteristic chromatogram according to chromatographic peaks of index component reference substances, and then detecting and identifying a test sample. According to the method, the characteristics of the sedum sarmentosum and the fake sedum sarmentosum are comprehensively reflected, a new analysis means is provided for internal quality control of the sedum sarmentosum medicinal material, and the method is not only suitable for the sedum sarmentosum and the fake sedum sarmentosum medicinal material, but also suitable for preparations such as decoction pieces, standard decoction and / or formula granules. The method is simple, accurate, reliable, convenient to operate, short in time, small in solvent consumption and small in environmental pollution. Meanwhile, the method has the advantages of strong specificity, high stability, good repeatability and the like, and is particularly suitable for detection and monitoring of large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a chromatographic method for identifying the authenticity of Herba Creepersii. Background Art

[0002] Sedum sarmentosum Bunge is the dried whole herb of Sedum sarmentosum Bunge, a plant of the Crassulaceae family. It is a common folk medicine. There is no clear record of it in ancient documents. The Chinese Pharmacopoeia was first recorded in 1977. Sedum sarmentosum Bunge is cool in nature, sweet and light in taste. It enters the liver, gallbladder and small intestine meridians. It has the effects of removing dampness and jaundice, clearing away heat and detoxifying. It is used for damp-heat jaundice, dysuria, carbuncle and ulcer (National Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China [M]. China Medical Science and Technology Press, 2015). Modern pharmacological studies have shown that Sedum sarmentosum Bunge has liver protection, anti-tumor, immunomodulatory, estrogen-like, angiotensin converting enzyme (ACE) inhibition, and muscle strength enhancement. In addition, its injection has antibacterial effects, and its extract has anti-inflammatory, antioxidant, and cell aging delaying effects. Clinically, Herba Crepisae is mostly used to treat various acute and chronic liver diseases. Granules are used for preventive treatment of intrahepatic cholestasis of pregnancy, injections are used to treat corneal ulcers, oral and topical applications can be used to treat patients with summer boils and carbuncles, fresh Herba Crepisae can be used to treat bedsores and herpes zoster in the elderly, and external applications can also be used to treat burns caused by water or fire, snake bites, etc. (Li Huijuan et al. Research progress on Herba Crepisae [J]. Pharmaceutical Research, 2015, 34(11): 661-663).

[0003] Due to the increase in market demand in recent years, some confusion has occurred in the medicinal material market, which has seriously affected the quality of the medicinal materials of Creeping Grass and caused a decline in clinical efficacy. Common counterfeits of Creeping Grass on the market include Eclipta prostrata, Portulaca oleracea, Sedum notoginseng, Sedum serrata, etc. At present, the identification of Creeping Grass and its counterfeits mainly adopts traditional trait identification, microscopic identification and new DNA barcode identification methods. Among them, Wu Fuxing used the traditional trait method to distinguish Creeping Grass from its common confused products and counterfeits, including Sedum serrata, Artemisia serrata, Commelina communis, Eclipta prostrata, and Portulaca oleracea (Wu Fuxing. Identification of Creeping Grass and Its Confusion Products and Counterfeits [J]. Yunnan Journal of Traditional Chinese Medicine, 2008 (29): 28). Lü Jiani et al. used the method of microscopic identification to distinguish between two representative plants of the Crassulaceae family, Crassulaceae and Sedum truncatum, and the representative plants of the Portulacaceae family, Portulaca oleracea and Portulaca grandiflora. Through comparative studies of two groups of medicinal plants, Crassulaceae and Portulaca grandiflora, and Sedum truncatum and Portulaca oleracea, they found that although they were similar in appearance (before flowering), they had obvious differences in microscopic structure (Lü Jiani et al. Identification of two groups of easily confused crude drugs of Crassulaceae and Portulaca oleracea [J]. Zhejiang Medical Education, 2008(2):4). Liu Meizi et al. obtained the ITS2 gene fragment by PCR amplification of the DNA of the creeping sedge, performed bidirectional sequencing on the purified PCR products, used CodonCode Aligner to splice the sequences, performed sequence analysis using MEGA5.0 software, and constructed an NJ tree of the K2P model. The results showed that there was obvious variation in the ITS2 sequences of the creeping sedge and its adulterants, and the intraspecific genetic distance was smaller than the interspecific genetic distance. It can be clearly seen from the NJ tree that the creeping sedges from different sources are clustered into one branch, which can be well distinguished from the adulterants (Liu Meizi et al. Application of ITS2 sequences to identify creeping sedge and its adulterants [J]. Modern Chinese Medicine, 2011).

[0004] However, there are few studies on the differences between creeping grass and its counterfeits, such as Eclipta prostrata, Portulaca oleracea, Sedum notoginseng, and Sedum truncatum. In addition, the above studies have some defects in distinguishing creeping grass and its counterfeits: First, with the development of society and changes in the environment, the corresponding trait identification indicators have not yet been established. In addition, trait identification requires the identifier to have rich practical experience and long-term knowledge accumulation, which makes it difficult to promote. Secondly, microscopic identification has the disadvantage of low resolution, which makes it difficult to identify medicinal materials with multiple origins and confusing varieties. In addition, DNA barcode identification has the disadvantages of insufficient sensitivity and poor operability.

[0005] Therefore, it is necessary to establish a method that can distinguish between creeping grass and its counterfeits such as Eclipta prostrata, Portulaca oleracea, Sedum notoginseng, and Sedum truncatum, and can specifically evaluate the quality of creeping grass. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] In order to solve the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a chromatographic method for identifying the authenticity of Creeping Jasmine. The method is applicable to medicinal materials, decoction pieces, standard decoctions and / or formula granules of Creeping Jasmine and its counterfeits. Creeping Jasmine can be identified from its counterfeits including Eclipta prostrata, Portulaca oleracea, Sedum notoginseng and Sedum truncatum, providing a new analytical means for the intrinsic quality control of Creeping Jasmine medicinal materials.

[0008] Solutions for solving problems

[0009] [1]. The present invention provides a chromatographic method for identifying the authenticity of Creeping Herb, wherein the method comprises the following steps:

[0010] (1) Preparation of a Herba Crepisae reference substance solution: extracting the Herba Crepisae reference substance with an extraction solvent, collecting the extract, and obtaining a Herba Crepisae reference substance solution;

[0011] The Herba Crepisae reference substance is selected from one or more of Herba Crepisae medicinal materials, Herba Crepisae decoction pieces, Herba Crepisae standard decoctions and Herba Crepisae formula granules;

[0012] (2) Preparation of reference solution: vanillic acid 4-β-D-glucoside and isorhamnetin-3,7-O-diglucoside, as reference substances, are respectively dissolved in a solvent to obtain a reference solution;

[0013] (3) Establishment of characteristic spectrum: using ultra-high performance liquid chromatography to detect the Herba Crepisae reference substance solution described in step (1) and the reference substance solution described in step (2), and after obtaining the Herba Crepisae reference substance detection spectrum and the reference substance detection spectrum respectively, establish the Herba Crepisae reference substance characteristic spectrum, and attribute the characteristic peaks in the characteristic spectrum according to the chromatographic peaks of the reference substance;

[0014] (4) Processing and identification of the test sample: taking the test sample, obtaining a test sample solution according to the method in step (1), and then establishing a test sample characteristic spectrum according to the method in step (3); comparing the test sample characteristic spectrum with the characteristic spectrum of the Herba Crepisae reference substance in step (3) to identify the test sample as Herba Crepisae or a counterfeit;

[0015] The test sample is selected from one or more of the tested medicinal materials, decoction pieces, standard decoctions and formula granules.

[0016] [2] The method according to [1], wherein in step (1), the extraction solvent is methanol, water or a mixture of the two; preferably, the extraction solvent is a 30% to 70% v / v methanol aqueous solution;

[0017] and / or,

[0018] The extraction method is ultrasonic treatment, shaking extraction, heating reflux extraction; preferably, the extraction method is ultrasonic extraction, the power of the ultrasonic extraction is 200-300W, the frequency of the ultrasonic extraction is 35-45kHz, and the time of the ultrasonic extraction is 30-120min;

[0019] and / or,

[0020] The mass volume ratio of the Creeping Herb reference substance to the extraction solvent is 1 g: (5-50 mL).

[0021] [3] The method according to [1] or [2], wherein in step (2), the solvent is methanol or an aqueous solution thereof; preferably, the solvent is methanol or a methanol aqueous solution of 50% v / v or more,

[0022] and / or,

[0023] The concentration of the reference solution is 5-40 μg / mL.

[0024] [4] The method according to any one of the technical solutions [1] to [3], wherein in step (3),

[0025] The chromatographic conditions of the ultra-high performance liquid chromatography include:

[0026] Stationary phase: chromatographic column with octadecylsilane bonded silica gel as filler;

[0027] Mobile phase: acetonitrile as mobile phase A, formic acid aqueous solution as mobile phase B;

[0028] Elution mode: gradient elution.

[0029] [5] The method according to [4], wherein in step (3), a 0.05% to 0.20% v / v formic acid aqueous solution is used as mobile phase B;

[0030] and / or,

[0031] The elution procedure of the gradient elution is as follows:

[0032]

[0033] [6] The method according to [4] or [5], wherein the chromatographic conditions of the ultra-high performance liquid chromatography method further include: a column temperature of 20 to 35°C, and / or a flow rate of 0.25 to 0.30 mL / min, and / or a detection wavelength of 220 to 300 nm.

[0034] [7] The method according to any one of the technical solutions [1] to [6], wherein in step (3),

[0035] The characteristic spectrum of the Herba Crepisae reference substance includes 9 characteristic peaks, wherein characteristic peak 3 corresponds to the chromatographic peak of the vanillic acid-4-β-D-glucoside reference substance, and characteristic peak 9 corresponds to the chromatographic peak of the isorhamnetin-3,7-O-diglucoside reference substance;

[0036] Preferably, the relative retention time of characteristic peak 1 and characteristic peak 2 is calculated with characteristic peak 3 as the reference peak, and the relative retention time should be within ±10% of the specified value, and the specified values ​​are 0.46 and 0.64 respectively;

[0037] Taking characteristic peak 9 as the reference peak, calculate the relative retention times of characteristic peak 4, characteristic peak 5, characteristic peak 6, characteristic peak 7 and characteristic peak 8. The relative retention times should be within ±10% of the specified values, and the specified values ​​are 0.56, 0.71, 0.77, 0.82 and 0.97 respectively.

[0038] [8] The method according to any one of the technical solutions [1] to [7], wherein in step (4),

[0039] The identification criteria include: if the characteristic spectrum of the sample contains characteristic peak 9, the sample is Sedum sibiricum; otherwise, it is a fake;

[0040] Preferably, the identification criteria also include:

[0041] If the characteristic spectrum of the sample does not contain characteristic peak 1, the sample is Portulaca oleracea or Eclipta prostrata;

[0042] If the characteristic spectrum of the test sample includes characteristic peak 1 and characteristic peak 3, the test sample is Sedum serrata;

[0043] If the characteristic spectrum of the test sample includes characteristic peak 1 and characteristic peak 5, the test sample is Sedum notoginseng.

[0044] [9]. The method according to any one of the technical solutions [1] to [8], wherein the counterfeit products include one or more of Portulaca oleracea, Eclipta prostrata, Sedum truncatum and Sedum notoginseng.

[0045]

[10] . The present invention also provides the application of the method described in any one of the technical solutions [1] to [9] in the quality detection of creeping hyacinth and its counterfeits.

[0046] Effects of the Invention

[0047] The present invention establishes a characteristic spectrum of a reference substance of the creeping vine by liquid chromatography-mass spectrometry technology, and provides a chromatographic method for distinguishing the authenticity of the creeping vine. The chromatographic conditions are reasonably controlled by ultra-high performance liquid chromatography, and 9 characteristic peaks of the creeping vine reference substance are determined, which constitutes the overall picture of the characteristic spectrum of the creeping vine medicinal materials and their preparations. At the same time, a comparative study is also conducted on the counterfeit products of the creeping vine, the preparations of Eclipta prostrata, Portulaca oleracea, Sedum sedum and Sedum serrata, etc., which can more comprehensively reflect the characteristics of the creeping vine medicinal materials and their counterfeit products, and achieve the purpose of distinguishing the authenticity.

[0048] The present invention controls the intrinsic overall quality of the Herbaceous Creeping Grass materials, reflects the characteristics of the Herbaceous Creeping Grass materials and their counterfeits in a relatively comprehensive manner, achieves the purpose of simultaneously identifying the Herbaceous Creeping Grass and its counterfeits, and provides a new analytical means for the intrinsic quality control of the Herbaceous Creeping Grass materials, herbal slices, standard decoctions and / or formula granules, etc.

[0049] The method of the present invention is simple, accurate, reliable, easy to operate, short in time, with low solvent consumption and little environmental pollution. It also has the advantages of strong specificity, high stability, good reproducibility, etc., and is particularly suitable for detection and monitoring of large-scale production.

[0050] It should be noted that the above description does not disclose all embodiments of the present invention and all advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The DAD-3D image of the herbaceous plant in Example 1

[0052] Figure 2 The chromatograms of the Herba Lysimachiae at different wavelengths in Example 1 are

[0053] Figure 3 This is the chromatogram of Herba Tetrapanacissima when eluted according to Gradient 1.

[0054] Figure 4 This is the chromatogram of Herba Tetrapanacissima when eluted according to gradient 2.

[0055] Figure 5 This is the chromatogram of Herba Tetrapanacissima when eluted according to Gradient 3.

[0056] Figure 6 This is the chromatogram of Herba Tetrapanacissima when eluted according to Gradient 4.

[0057] Figure 7 This is the chromatogram of Herba Tetrapanacis when eluted according to Gradient 5.

[0058] Figure 8 This is the chromatogram of Herba Tetrapanacissima when eluted according to Gradient 6.

[0059] Fig. 9This is the chromatogram of Herba Tetrapanacissima when eluted according to Gradient 7.

[0060] Fig.10 Chromatograms of Herba Lysimachiae on different chromatographic columns

[0061] Fig.11 Chromatograms of Herba Lysimachiae at different column temperatures

[0062] Fig.12 Chromatograms of Herba Lysimachiae at different flow rates

[0063] Fig.13 This is the characteristic spectrum of the reference substance of Example 1

[0064] Fig.14 This is a comparative characteristic spectrum of the reference substance of the creeping hyacinth and its counterfeit substance in Example 1

[0065] Fig.15 Characteristic spectrum for the reference substance of Example 1

[0066] Fig.16 The characteristic spectrum of Example 2:

[0067] Fig.17 This is the characteristic spectrum of the sample in Example 3 DETAILED DESCRIPTION

[0068] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and embodiments obtained by appropriately combining the technical means disclosed in different embodiments and embodiments are also included in the technical scope of the present invention. In addition, all the documents recorded in this specification are cited in this specification as references.

[0069] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0070] In the context of describing this specification (especially in the context of the following claims), the terms "a", "an", and "the" and similar language are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0071] In this specification, the numerical range expressed using "numerical value A to numerical value B" or "numerical value A-numerical value B" means a range including the endpoints numerical values ​​A and B.

[0072] In this specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes the situation where the event occurs and the situation where the event does not occur.

[0073] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "some specific / preferred technical solutions", "other specific / preferred technical solutions", etc. refer to the specific elements (e.g., features, structures, properties and / or characteristics) related to the embodiments described herein being 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.

[0074] The term "comprising" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, 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.

[0075] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0076] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0077] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0078] Unless otherwise specified, the terms "precision weighing or precision weighing" used in this article mean that the weight should be weighed accurately to one thousandth of the weight taken, the term "weighing" means that the weight should be weighed accurately to one hundredth of the weight taken, the term "precision addition" means that the volume should be measured accurately to one thousandth of the volume taken, and the term "precision aspiration" refers to the operation method of accurately measuring samples through a microinjector.

[0079] Unless otherwise specified, the term "subsequent filtrate" used herein refers to the filtrate collected after the primary filtrate is discarded during filtration. Compared with the primary filtrate, the subsequent filtrate is closer to the actual concentration of the sample, because the filter medium (such as filter membrane, filter paper, etc.) may adsorb solutes, resulting in a lower sample concentration in the primary filtrate; in addition, the subsequent filtrate is cleaner, because the solutes adsorbed by the filter medium can form a filter cake, which reduces the filter pore size and can thus intercept smaller particles.

[0080] Unless otherwise specified, the term "reference peak" (or "control peak") used herein refers to the chromatographic peak corresponding to the standard sample when calculating the relative retention time.

[0081] Unless otherwise specified, the percentage "%" in this document referring to solution concentration refers to volume percentage.

[0082] Unless otherwise specified, the term "reference substance" used in this article refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance, which is usually reviewed and approved by the national drug testing agency and its standard should not be lower than the quality standard of the product.

[0083] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0084] Unless otherwise specified, the "sample" in the present invention refers to a medicinal material that has been identified as a crude drug (e.g., identification of the original plant (animal) species, property identification, microscopic identification, physical and chemical identification, etc.) and used to establish a corresponding characteristic spectrum. For example, the "Purslane sample" in the present invention refers to a Purslane medicinal material that has been identified as a crude drug and used to establish a corresponding characteristic spectrum.

[0085] First aspect

[0086] The present invention provides a chromatographic method for identifying the authenticity of Scutellaria baicalensis, which comprises the following steps:

[0087] (1) Preparation of a Herba Crepisae reference substance solution: extracting the Herba Crepisae reference substance with an extraction solvent, collecting the extract, and obtaining a Herba Crepisae reference substance solution;

[0088] In some specific embodiments of the present invention, in step (1), the Herba Crescens reference substance is selected from one or more of Herba Crescens medicinal materials, Herba Crescens decoction pieces, Herba Crescens standard decoctions and Herba Crescens formula granules;

[0089] Among them, "medicinal materials" refer to raw materials of Chinese medicine that have not been processed or made into finished products; "decoction pieces" refer to Chinese medicines that are processed according to needs for prescription use, or Chinese medicines that can be directly used in clinical Chinese medicine; "standard decoctions" refer to single-ingredient Chinese medicine decoction 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-ingredient Chinese medicine decoction pieces processed according to traditional standards and then extracted and concentrated for use in clinical Chinese medicine prescriptions.

[0090] In addition, in some specific embodiments of the present invention, the extraction solvent is methanol, water or a mixture of the two; in some preferred embodiments, the extraction solvent is 30% to 70% v / v methanol aqueous solution. As for the amount of the extraction solvent, in some specific embodiments of the present invention, the mass volume ratio of the powder of the spatholobi reference substance to the extraction solvent is 1g: (5 to 50mL), specifically 1g: 5mL, 1g: 10mL, 1g: 12.5mL, 1g: 15mL, 1g: 25mL, 1g: 30mL, 1g: 40mL, 1g: 45mL, 1g: 50mL, etc.; in order to further optimize the extraction effect and material dosage, the mass volume ratio of the powder of the medicinal material, decoction piece, standard decoction and / or formula granule to the extraction solvent is preferably 1g: (5 to 15) mL; too small a concentration of the reference substance is not conducive to the detection of the chromatographic peak, and too large a concentration may cause concerns about poor chromatographic peak shape due to overloading of the chromatographic column.

[0091] Regarding the extraction method, in order to obtain more abundant chromatographic peak information, the extraction method can be carried out by ultrasonic treatment, shaking extraction or heating reflux extraction, preferably by ultrasonic treatment with simpler operation, the present invention has no special restrictions on the power, frequency and other conditions of ultrasonic treatment, in some preferred embodiments, the power of the ultrasonic treatment can be 200W to 300W, the frequency can be 35kHz to 45kH, and the further preferred power is 250W and the frequency is 40kHz. The extraction time is 30 to 120min, specifically 30min, 45min, 55min, 60min, 70min, 80min, 90min, 100min or 120min; in order to further optimize the extraction effect and treatment cycle, comprehensively consider the extraction efficiency of each chromatographic peak, the preferred extraction time is 35 to 60min, more preferably 45min.

[0092] For the Herba Coptidis, it is preferred to first sieve the powder of the medicinal material, which can be listed as No. 2 sieve (24 mesh), No. 3 sieve (50 mesh) and No. 4 sieve (65 mesh), and preferably No. 2 sieve can be used. For the Herba Coptidis standard decoction, the medicinal material is first subjected to pretreatment, decoction, low-temperature concentration, freeze-drying and other processes to obtain freeze-dried powder, and the freeze-dried powder is preferably ground; for the Herba Coptidis formula granules, the Herba Coptidis formula granules obtained by pretreatment, extraction, concentration, spray drying, granulation and packaging of the Herba Coptidis are extracted. In some specific embodiments of the present invention, the preparation method of the reference solution is as follows: take an appropriate amount of Herba Coptidis and Herba Coptidis decoction pieces, crush them, pass through a No. 2 sieve (24 mesh), accurately weigh 2 g, put them in a stoppered conical flask, accurately add 25 mL of a 30% methanol aqueous solution by volume, seal them, weigh them, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, cool them, weigh them again, make up the lost weight with a 30% methanol aqueous solution, shake them well, filter them, and take the filtrate to obtain;

[0093] Alternatively, in some specific embodiments of the present invention, take an appropriate amount of standard decoction of Herba Crescentellae Sinensis or Herba Crescentellae Sinensis formula granules, grind them into powder, take 0.8 g, put it in a stoppered conical flask, accurately add 25 ml of a 30% methanol aqueous solution by volume fraction, seal it, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0094] (2) Preparation of reference solution: comprising dissolving a reference substance of vanillic acid-4-β-D-glucoside and a reference substance of isorhamnetin-3,7-O-diglucoside in a solvent to obtain a reference solution;

[0095] The method of obtaining the reference solution comprises dissolving the reference substances in methanol or its aqueous solution respectively to prepare a vanillic acid-4-β-D-glucoside reference solution and an isorhamnetin-3,7-O-diglucoside reference solution with a concentration of 5 to 40 μg / mL; in some specific embodiments of the present invention, preferably, a vanillic acid-4-β-D-glucoside reference solution with a concentration of 20 μg / mL is prepared with a 50% v / v or higher methanol aqueous solution, and a isorhamnetin-3,7-O-diglucoside reference solution with a concentration of 15 μg / mL is prepared with methanol.

[0096] (3) Establishment of characteristic spectrum: using ultra-high performance liquid chromatography to detect the Herba Crepisae reference substance solution described in step (1) and the reference substance solution described in step (2), and after obtaining the Herba Crepisae reference substance detection spectrum and the reference substance detection spectrum respectively, establish the Herba Crepisae reference substance characteristic spectrum, and attribute the characteristic peaks in the characteristic spectrum according to the chromatographic peaks of the reference substance;

[0097] The present invention has determined, through in-depth research, the ultra-high performance liquid chromatography conditions that are applicable to Herba Crepisae herbs, Herba Crepisae decoction pieces, Herba Crepisae standard decoctions, and Herba Crepisae formula granules, which include:

[0098] Stationary phase: a chromatographic column with octadecylsilane bonded silica as a filler and a particle size (filler particle size) of 1.6 to 1.8 μm; further, the chromatographic column has a column length of 100 to 150 mm and an inner diameter of 2.1 mm; preferably, the column length is 100 mm, the inner diameter is 2.1 mm, and the filler particle size is 1.8 μm; in some specific embodiments of the present invention, the chromatographic column is selected from any one of Eclipse Plus C18 (Agilent, 100 mm × 2.1 mm × 1.8 μm), CORTECS UPLC T3 (Waters, 2.1 mm × 100 mm × 1.6 μm), and ACQUITY BEH C18 (Waters, 2.1 × 100 mm × 1.7 μm). Further, an Eclipse Plus C18 (Agilent, 100 mm × 2.1 mm × 1.8 μm) chromatographic column is used for separation, and the separation of characteristic peaks is better and the overall spectrum is more beautiful.

[0099] Mobile phase: acetonitrile is used as mobile phase A, and an aqueous solution of formic acid is used as mobile phase B. In some specific embodiments of the present invention, a 0.05% to 0.20% v / v formic acid aqueous solution is preferably used as mobile phase B, and a 0.1% v / v formic acid aqueous solution is more preferably used as mobile phase B.

[0100] The elution method of the present invention adopts gradient elution, and the elution procedure of gradient elution is:

[0101]

[0102] Considering the chromatographic peak separation effect, the preferred flow rate of the present invention is 0.28mL / min to 0.32mL / min; and / or, the column temperature is 15°C to 35°C. Within the range of column temperature or flow rate, the construction method of the present invention can meet the system suitability requirements with smaller column temperature changes or flow rate changes. In some preferred embodiments of the present invention, the flow rate can be 0.25mL / min to 0.30mL / min; and / or, the column temperature can be 20°C to 32°C; in some specific embodiments of the present invention, the flow rate is 0.3mL / min, the column temperature is 30°C, and the injection volume is 2μL.

[0103] The detector used in the present invention can be an ultraviolet detector, an evaporative light scattering detector or an charged aerosol detector; in some preferred embodiments of the present invention, an ultraviolet detector is preferably used, and the detection wavelength is 220nm to 300nm. In some preferred embodiments of the present invention, the detection wavelength can be 254nm. Under the detection wavelength in this range, the chromatographic peak information is relatively rich, the separation effect of each chromatographic peak is better, and the baseline is relatively stable.

[0104] The present invention establishes a characteristic spectrum of the creeping grass reference sample by testing at least 17 batches of creeping grass reference samples from different origins, and attributes characteristic peaks in the characteristic spectrum of the creeping grass reference sample according to the chromatographic peaks of the reference sample, thereby establishing an identification standard for creeping grass and its counterfeits.

[0105] The characteristic spectrum of the Herba Crepisae reference substance established by the above method includes 9 characteristic peaks, among which peak 3 is the peak of vanillic acid-4-β-D-glucoside, and peak 9 is the peak of isorhamnetin-3,7-O-diglucoside.

[0106] (4) Processing and identification of the test sample: taking the test sample, obtaining a test sample solution according to the method in step (1), and then establishing a test sample characteristic spectrum according to the method in step (3); comparing the test sample characteristic spectrum with the characteristic spectrum of the Herba Crepisae reference substance in step (3) to identify the test sample as Herba Crepisae or a counterfeit;

[0107] The identification criteria include: if the characteristic spectrum of the test product contains characteristic peak 9, the test product is Sedum sibiricum; otherwise, it is a fake product;

[0108] Furthermore, the identification criteria include:

[0109] If the characteristic spectrum of the test sample does not contain characteristic peak 1, the test sample is Portulaca oleracea or Eclipta prostrata;

[0110] If the characteristic spectrum of the test product contains characteristic peak 1 and characteristic peak 3, the test product is Sedum serrata;

[0111] If the characteristic spectrum of the test product contains characteristic peak 1 and characteristic peak 5, the test product is Sedum notoginseng;

[0112] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 9, the test sample is Sedum melongena.

[0113] The present invention improves the analysis efficiency by optimizing the preparation method of the reference substance of the creeping hyacinth, the preparation method of the reference substance, and the chromatographic analysis conditions, and establishes ultra-high performance liquid chromatography conditions that are simultaneously applicable to creeping hyacinth materials, creeping hyacinth decoction pieces, creeping hyacinth standard decoctions and / or creeping hyacinth formula granules and their counterfeits. The method can distinguish creeping hyacinth and its counterfeits including Portulaca oleracea, Eclipta prostrata, Sedum truncatum and / or Sedum notoginseng, and provides an analysis means and basis for quality control of creeping hyacinth and its counterfeits.

[0114] Second aspect

[0115] The present invention also provides the application of the method in identifying any two or more of Sedum spatholobi, Portulaca oleracea, Eclipta prostrata, Sedum concha and / or Sedum notoginseng, and provides the application of the method in quality detection of Sedum spatholobi and its counterfeits.

[0116] In a specific embodiment of the present invention, the method can identify Sedum sibiricum and Portulaca oleracea or Eclipta prostrata; in a specific embodiment of the present invention, the method can identify Sedum sibiricum and Sedum sibiricum; in a specific embodiment of the present invention, the method can identify Sedum sibiricum and Sedum notoginseng;

[0117] In addition, in a specific embodiment of the present invention, the method can identify Portulaca oleracea or Eclipta prostrata and Sedum multiflorum; in a specific embodiment of the present invention, the method can identify Portulaca oleracea or Eclipta prostrata and Sedum multiflorum; in a specific embodiment of the present invention, the method can identify Sedum multiflorum and Sedum multiflorum.

[0118] The method provided by the present invention is described in detail below in conjunction with examples. However, the specific experimental conditions and results described in the examples are only used to illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention.

[0119] Example

[0120] Instruments, reagents and samples

[0121] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters); Empower 3 workstation (Waters); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.).

[0122] Acetonitrile (chromatographic grade, Thermo Fisher); water is ultrapure water; formic acid (chromatographic grade, Aladdin); other reagents are of analytical grade.

[0123] Vanillic acid-4-β-D-glucoside (CAS NO.32142-31-7) was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. with the number 21Z017-Q1. Isorhamnetin-3,7-O-diglucoside reference substance (CAS NO.6758-51-6) was purchased from Shanghai Shidande Biotechnology Co., Ltd. with the number 10502. 17 batches of Herba Lycopodii, 3 batches of Ecliptae, 3 batches of Portulaca Oleracea, 3 batches of Sedum notoginseng, and 3 batches of Sedum serrata, a total of 29 batches of experimental samples came from major production areas and authentic production areas in China, all of which met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0124] Example 1: Establishment of a chromatographic method for identifying the authenticity of Herba Lycopodii

[0125] 1. Preparation of the reference solution of Scutellaria baicalensis

[0126] Take an appropriate amount of Herba Lycopodii, crush it, pass it through a No. 2 sieve (24 mesh), accurately weigh 2 g, put it in a stoppered conical flask, accurately add 25 mL of a 30% by volume methanol aqueous solution, stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 45 minutes, cool it, weigh it again, make up the lost weight with 30% v / v methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0127] 2. Preparation of reference solution

[0128] Take vanillic acid-4-β-D-glucoside reference substance and add 50% v / v methanol aqueous solution to make a solution containing 20 μg per 1 ml; then take isorhamnetin-3,7-O-diglucoside reference substance and add methanol to make a solution containing 15 μg per 1 ml as the reference substance solution.

[0129] 3. Determination of ultra-high performance liquid chromatography conditions

[0130] 3.1 Determination of detection wavelength

[0131] Accurately pipette 2 μL of the reference solution of the creeping sedge, inject it into the ultra-high performance liquid chromatograph, measure, and record the absorption spectrum in the range of 210-400 nm. Figure 1 . The chromatograms of Herba Lysimachiae at different wavelengths (240nm, 254nm, 280nm) are measured, such as Figure 2 According to the results, the chromatographic peak information at 254nm is relatively rich, the separation effect of each chromatographic peak is good, and the baseline is relatively stable.

[0132] 3.2 Optimization of chromatographic conditions

[0133] ACQUITY UPLC BEH C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used as the chromatographic column; acetonitrile was used as the mobile phase A, 0.1% v / v phosphoric acid solution was used as the mobile phase B, elution was performed according to the gradient 1 specified in Table 1, the flow rate was 0.3 ml / min, the column temperature was 30° C., and the detection wavelength was 254 nm.

[0134] Table 1:

[0135]

[0136] Get as Figure 3 The chromatogram of Herba Cibotii when eluted according to Gradient 1 is shown.

[0137] Next, elution was performed according to the gradient 2 specified in Table 2, with other conditions remaining unchanged.

[0138] Table 2:

[0139]

[0140] Get as Figure 4 The chromatogram of Herba Cibotii when eluted according to Gradient 2 is shown.

[0141] The chromatographic column was replaced with ACQUITY UPLC HSS T3 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) as the chromatographic column; acetonitrile was used as mobile phase A, 0.1% v / v phosphoric acid solution was used as mobile phase B, elution was performed according to the gradient 3 specified in Table 3, the flow rate was 0.3 ml / min, the column temperature was 30° C., and the detection wavelength was 254 nm.

[0142] Table 3:

[0143]

[0144] Get as Figure 5 The chromatogram of Herba Cibotii when eluted according to Gradient 3 is shown.

[0145] Next, elution was performed according to the gradient 4 specified in Table 4, with other conditions remaining unchanged.

[0146] Table 4:

[0147]

[0148] Get as Figure 6 The chromatogram of Herba Cibotii when eluted according to Gradient 4 is shown.

[0149] Continue to replace the chromatographic column, use ZORBAX Eclipse Plus C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) as the chromatographic column; use acetonitrile as mobile phase A, use 0.1% v / v phosphoric acid solution as mobile phase B, elute according to the gradient 5 specified in Table 5, the flow rate is 0.3 ml / min, the column temperature is 30° C., and the detection wavelength is 254 nm.

[0150] Table 5:

[0151]

[0152] Get as Figure 7 The chromatogram of Herba Cibotii when eluted according to Gradient 5 is shown.

[0153] The mobile phase was replaced, and ZORBAX Eclipse Plus C18 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used as the chromatographic column; acetonitrile was used as mobile phase A, 0.1% v / v formic acid solution was used as mobile phase B, and elution was performed according to the gradient 6 specified in Table 6, with a flow rate of 0.3 ml / min, a column temperature of 30° C., and a detection wavelength of 254 nm.

[0154] Table 6:

[0155]

[0156] Get as Figure 8 The chromatogram of Herba Cibotii when eluted according to Gradient 6 is shown.

[0157] The gradient was adjusted again, with octadecylsilane bonded silica gel as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile as mobile phase A, 0.1% v / v formic acid solution as mobile phase B, elution according to the gradient 7 specified in Table 7; the flow rate was 0.3 ml / min, the column temperature was 30°C, and the detection wavelength was 254 nm.

[0158] Table 7:

[0159]

[0160] Get as Fig. 9 The chromatogram of Herba Lysimachiae when eluted according to Gradient 7 is shown, and the final elution gradient is determined.

[0161] 3.3 Determination of mobile phase

[0162] Acetonitrile was selected as mobile phase A and 0.1% v / v formic acid solution was selected as mobile phase B.

[0163] 3.4 Investigation of chromatographic columns

[0164] Take the Herba Crepisae reference solution prepared in accordance with the above “1. Preparation of Herba Crepisae reference solution”, select commonly used chromatographic columns Eclipse Plus C18 (Agilent, 100mm×2.1mm×1.8μm), CORTECS UPLC T3 (Waters, 2.1mm×100mm×1.6μm), ACQUITY BEH C18 (Waters, 2.1×100mm×1.7μm), use acetonitrile as mobile phase A, use 0.1% v / v formic acid solution as mobile phase B, follow the elution gradient described in Table 7 above, perform injection measurement, and compare the separation effects of different chromatographic columns, as shown in FIG. Fig.10 shown.

[0165] The results showed that the Eclipse Plus C18 RRHD column (2.1 mm × 100 mm × 1.8 μm) had the best sample separation effect and a stable baseline.

[0166] 3.5 Investigation of column temperature

[0167] Take the Herba Crepisae reference solution prepared in the above “1. Preparation of Herba Crepisae reference solution”, use the same chromatographic column, follow the above chromatographic conditions, but inject at different column temperatures (25°C, 30°C, 35°C), and record the chromatograms, such as Fig.11 shown.

[0168] The results show that when the column temperature is in the range of 25℃~35℃, the chromatographic peaks are well separated and the durability is good. Combined with the actual analysis situation, the column temperature of 30℃ is selected for analysis.

[0169] 3.6 Investigation of flow rate

[0170] Take the Herba Crepisae reference substance solution prepared under the above “1. Preparation of Herba Crepisae reference substance solution”, use the same chromatographic column, according to the above chromatographic conditions, and measure at different flow rates of 0.25ml / min, 0.30ml / min, and 0.35ml / min, respectively, and record the chromatogram, such as Fig.12 shown.

[0171] The results show that within the flow rate range of 0.25ml / min to 0.30ml / min, small flow rate changes can meet the overall applicability requirements, and the fixed flow rate is 0.30ml / min.

[0172] 4. Establishment of characteristic profiles of the reference and counterfeit products of Sedum sibiricum

[0173] 17 batches of Herba Coptidis, 3 batches of Herba Ecliptae, 3 batches of Herba Portulacae, 3 batches of Herba Sedum, and 3 batches of Herba Sedum were taken, 2.0 g of each sample was prepared according to the above preparation method, 2 μL of sample solution and reference solution were accurately pipetted respectively, injected into ultra-high performance liquid chromatography, and the characteristic spectra of the samples were determined according to the above chromatographic conditions. The characteristic spectra of 17 batches of Herba Coptidis were superimposed using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" promulgated by the State Pharmacopoeia Committee. Fig.13 UPLC comparison charts of Sedum sutchuenensis, Eclipta prostrata, Portulaca oleracea, Sedum notoginseng, and Sedum sutchuenensis are shown in Fig.14 . Reference material identification diagram Fig.15 .

[0174] 5. Research on identification of Herba Lycopodii and its counterfeit medicinal materials

[0175] The characteristic spectra of common counterfeits of creeping grass, such as Eclipta prostrata, Portulaca oleracea, Sedum ternata, and Sedum serrata, are significantly different from those of creeping grass reference. The characteristic spectrum of creeping grass reference has 9 characteristic peaks, among which peak 3 is the peak of vanillic acid-4-β-D-glucoside, and peak 9 is the peak of isorhamnetin-3,7-O-diglucoside; taking characteristic peak 3 as the reference peak, the relative retention time of characteristic peak 1 and characteristic peak 2 is calculated, and the relative retention time should be within ±10% of the specified value, and the specified values ​​are 0.46 and 0.64 respectively; taking characteristic peak 9 as the reference peak, the relative retention time of characteristic peak 4, characteristic peak 5, characteristic peak 6, characteristic peak 7, and characteristic peak 8 is calculated, and the relative retention time should be within ±10% of the specified value, and the specified values ​​are 0.56, 0.71, 0.77, 0.82, and 0.97 respectively. Portulaca oleracea and Eclipta prostrata are plants of different families. Under the control characteristic spectrum of Sedum sphenanthera, there are almost no corresponding chromatographic peaks. Sedum sphenanthera has characteristic peaks 1 and 3, and other peaks are missing. Sedum sphenanthera has characteristic peaks 1 and 5, and other peaks are missing.

[0176] The characteristic spectra of the above-mentioned creeping sedge and its counterfeit are compared, and the differences in the characteristic peaks in each characteristic spectrum are compared. The differences are recorded to obtain the identification standard.

[0177] In some specific embodiments of the present invention, the differences include characteristic peak 1 which can distinguish Portulaca oleracea and Eclipta prostrata, characteristic peak 3 which can distinguish Sedum serrata, characteristic peak 5 which can distinguish Sedum notoginseng, and characteristic peak 9 which can distinguish Sedum sphenanthera.

[0178] Example 2: Chromatographic method for identifying the authenticity of Herba Crepisae Decoction Pieces, Herba Crepisae Standard Decoction and Herba Crepisae Formula Granules

[0179] Preparation of reference solution of Herba Crepisae

[0180] For the decoction pieces of Creeping Jasmine: take an appropriate amount of decoction pieces of Creeping Jasmine, crush them, pass through a No. 2 sieve (24 mesh), accurately weigh 2 g, put them in a stoppered conical flask, accurately add 25 mL of a 30% volume fraction methanol aqueous solution, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 45 minutes, cool it, weigh it again, make up the lost weight with 30% v / v methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain it.

[0181] For the standard decoction of Herba Crescens: take an appropriate amount of freeze-dried powder of the standard decoction of Herba Crescens prepared by pre-treatment, decoction, low-temperature concentration and freeze-drying of Herba Crescens, grind it into powder, take 0.8 g, put it in a stoppered conical flask, accurately add 25 ml of 30% methanol aqueous solution by volume fraction, seal it, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0182] For the formula granules of Creeping Jasmine: take an appropriate amount of the Chinese medicine formula granules obtained by pre-treatment, extraction, concentration, spray drying, granulation and packaging of Creeping Jasmine materials, grind them into powder, take 0.8g, put it in a stoppered conical bottle, accurately add 25ml of 30% methanol aqueous solution by volume fraction, seal it, ultrasonically treat (power 250W, frequency 40kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain.

[0183] In addition, the reference solution was prepared according to the method described under "2. Preparation of reference solution" in Example 1 above, using an Eclipse Plus C18 RRHD column as a chromatographic column, acetonitrile as mobile phase A, and 0.1% v / v formic acid aqueous solution as mobile phase B, eluting according to the gradient in Table 7 of Example 1 above, with a flow rate of 0.3 ml / min, a column temperature of 30° C., and a detection wavelength of 254 nm, to obtain characteristic spectra of Herba Ceratitise and its related preparations, as shown in FIG. Fig.16 Among them, after identification by the reference substance, peak 3 (S1) is the peak of vanillic acid-4-β-D-glucoside, and peak 9 (S2) is the peak of isorhamnetin-3,7-O-diglucoside.

[0184] Example 3: Application of chromatographic method for identifying the authenticity of Herba Lycopodii

[0185] The test solution was prepared according to the method in "1. Preparation of Scutellaria baicalensis reference solution" in Example 1, using an Eclipse Plus C18 RRHD column as the chromatographic column, acetonitrile as the mobile phase A, and 0.1% v / v formic acid aqueous solution as the mobile phase B. Elution was performed according to the gradient in Table 7 of the above Example 1, with a flow rate of 0.3 ml / min, a column temperature of 30°C, and a detection wavelength of 254 nm to obtain a characteristic spectrum of the test sample.

[0186] Next, the obtained characteristic spectrum of the test sample is compared with the characteristic spectrum of the Herba Crepisae reference substance established in Example 1 to identify whether the test sample is Herba Crepisae or a fake. The characteristic spectrum of the test sample is as follows: Fig.17 shown.

[0187] The identification standard is: if the characteristic spectrum of the test sample contains characteristic peak 9, then the test sample is Sedum sibiricum; otherwise it is a counterfeit.

[0188] Further identification is performed based on the characteristic peaks of the characteristic spectrum of the test sample.

[0189] If the characteristic spectrum of the test sample does not contain characteristic peak 1, the test sample is Portulaca oleracea or Eclipta prostrata;

[0190] If the characteristic spectrum of the test product contains characteristic peak 1 and characteristic peak 3, the test product is Sedum serrata;

[0191] If the characteristic spectrum of the test product contains characteristic peak 1 and characteristic peak 5, the test product is Sedum notoginseng;

[0192] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 9, the test sample is Sedum melongena.

[0193] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0194] The above-mentioned embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

[0195] Industrial Applicability

[0196] The method described in the present invention is suitable for industrial identification of creeping hyacinth and its counterfeits.

Claims

1. A chromatographic method for identifying the authenticity of Scutellaria baicalensis, characterized in that: The method comprises the following steps: (1) Preparation of a reference solution of the creeping hyacinth: extracting the creeping hyacinth reference substance with an extraction solvent, collecting the extract, and obtaining a reference solution of the creeping hyacinth; The Herba Crepisae reference substance is selected from one or more of Herba Crepisae medicinal materials, Herba Crepisae decoction pieces, Herba Crepisae standard decoctions and Herba Crepisae formula granules; (2) Preparation of reference solution: vanillic acid 4-β-D-glucoside and isorhamnetin-3,7-O-diglucoside, which are reference substances, are respectively dissolved in a solvent to obtain a reference solution; (3) Establishment of characteristic spectrum: using ultra-high performance liquid chromatography to detect the Herba Crepisae reference substance solution described in step (1) and the reference substance solution described in step (2), and after obtaining the Herba Crepisae reference substance detection spectrum and the reference substance detection spectrum respectively, establish the Herba Crepisae reference substance characteristic spectrum, and attribute the characteristic peaks in the characteristic spectrum according to the chromatographic peaks of the reference substance; (4) Processing and identification of the test sample: taking the test sample, obtaining a test sample solution according to the method in step (1), and then establishing a test sample characteristic spectrum according to the method in step (3); comparing the test sample characteristic spectrum with the characteristic spectrum of the Herba Crepisae reference substance in step (3) to identify the test sample as Herba Crepisae or a counterfeit; The test sample is selected from one or more of the tested medicinal materials, decoction pieces, standard decoctions and formula granules.

2. The method according to claim 1, characterized in that In step (1), the extraction solvent is methanol, water or a mixture of the two; preferably, the extraction solvent is a 30% to 70% v / v methanol aqueous solution; and / or, The extraction method is ultrasonic treatment, shaking extraction, heating reflux extraction; preferably, the extraction method is ultrasonic extraction, the power of the ultrasonic extraction is 200-300W, the frequency of the ultrasonic extraction is 35-45kHz, and the time of the ultrasonic extraction is 30-120min; and / or, The mass volume ratio of the Creeping Herb reference substance to the extraction solvent is 1 g: (5-50 mL).

3. The method according to claim 1 or 2, characterized in that: In step (2), the solvent is methanol or an aqueous solution thereof; preferably, the solvent is methanol or an aqueous solution of methanol at a concentration of 50% v / v or more. and / or, The concentration of the reference solution is 5-40 μg / mL.

4. The method according to any one of claims 1 to 3, characterized in that: In step (3), The chromatographic conditions of the ultra-high performance liquid chromatography include: Stationary phase: chromatographic column with octadecylsilane bonded silica gel as filler; Mobile phase: acetonitrile as mobile phase A, formic acid aqueous solution as mobile phase B; Elution mode: gradient elution.

5. The method according to claim 4, characterized in that In step (3), 0.05% to 0.20% v / v formic acid aqueous solution is used as mobile phase B; and / or, The elution procedure of the gradient elution is as follows:

6. The method according to claim 4 or 5, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography method also include: a column temperature of 20 to 35° C., and / or a flow rate of 0.25 to 0.30 mL / min, and / or a detection wavelength of 220 to 300 nm.

7. The method according to any one of claims 1 to 6, characterized in that: In step (3), The characteristic spectrum of the Herba Crepisae reference substance includes 9 characteristic peaks, wherein characteristic peak 3 corresponds to the chromatographic peak of the vanillic acid-4-β-D-glucoside reference substance, and characteristic peak 9 corresponds to the chromatographic peak of the isorhamnetin-3,7-O-diglucoside reference substance; Preferably, the relative retention time of characteristic peak 1 and characteristic peak 2 is calculated with characteristic peak 3 as the reference peak, and the relative retention time should be within ±10% of the specified value, and the specified values ​​are 0.46 and 0.64 respectively; Taking characteristic peak 9 as the reference peak, calculate the relative retention times of characteristic peak 4, characteristic peak 5, characteristic peak 6, characteristic peak 7 and characteristic peak 8. The relative retention times should be within ±10% of the specified values, and the specified values ​​are 0.56, 0.71, 0.77, 0.82 and 0.97 respectively.

8. The method according to any one of claims 1 to 7, characterized in that: In step (4), The identification criteria include: if the characteristic spectrum of the sample contains characteristic peak 9, the sample is Sedum serrata; otherwise, it is a fake; Preferably, the identification criteria also include: If the characteristic spectrum of the sample does not contain characteristic peak 1, the sample is Portulaca oleracea or Eclipta prostrata; If the characteristic spectrum of the test sample includes characteristic peak 1 and characteristic peak 3, the test sample is Sedum serrata; If the characteristic spectrum of the test sample includes characteristic peak 1 and characteristic peak 5, the test sample is Sedum notoginseng.

9. The method according to any one of claims 1 to 8, characterized in that: The counterfeits include one or more of Portulaca oleracea, Eclipta prostrata, Sedum truncatum and Sedum notoginseng.

10. Use of the method according to any one of claims 1 to 9 in quality detection of Creeping Jasmine and its counterfeits.

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