Chromatographic identification method for distinguishing zedoary and approximation product thereof

Abstract

The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a chromatographic identification method for distinguishing rhizoma wenyujin concisum and an approximate product thereof. Characteristic spectrums of rhizoma wenyujin concisum and similar products of rhizoma wenyujin concisum are established through a liquid chromatography-mass spectrometry technology, the characteristic spectrums are compared and researched, an identification standard is formulated, and then a test sample is detected and identified. According to the method, the characteristics of the rhizoma wenyujin concisum and the similar product thereof are comprehensively reflected, a new analysis means is provided for internal quality control of the rhizoma wenyujin concisum medicinal material, and the method is suitable for preparations such as standard decoction and / or formula granules of the rhizoma wenyujin concisum and the similar product thereof. 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 identification method for distinguishing turmeric slices and similar products thereof. Background Art

[0002] Turmeric is the dried rhizome of Curcuma wenyujin YHChen et C.Ling, a plant of the ginger family. It is a cold medicine and was first recorded in Renzhai Zhizhi Fanglun. It tastes pungent and bitter, is warm in nature, and enters the spleen and liver meridians. It has the effects of breaking blood, promoting qi, and relieving pain.

[0003] Modern research shows that turmeric contains volatile oil, alkaloids, polysaccharides and inorganic elements. The main active ingredient is sesquiterpenoids, which are the main components of volatile oil. Turmeric has significant effects in anti-tumor, anti-inflammatory and analgesic, anti-thrombotic and blood coagulation, and enhancing immunity. It is mainly used in clinical treatment of gynecological diseases, rheumatism, shoulder and arm pain and waist pain caused by cold.

[0004] The underground part of the same plant of Wenyujin can produce three kinds of medicinal materials, all of which are included in the 2020 "Chinese Pharmacopoeia", and their processing and uses are different. The tuber is boiled and dried to become the famous medicinal material "Wenyujin", which tastes slightly bitter and pungent, is slightly warm in nature, can relieve liver depression, promote qi and remove blood stasis, promote bile and relieve jaundice; the lateral rhizome is freshly cut into thick slices and dried to be called "sliced ​​turmeric", which tastes pungent and bitter, is warm in nature, can break blood and promote qi, relieve menstruation and relieve pain; the main rhizome is boiled and dried to be called "Wen Curcuma", which tastes bitter and pungent, is warm in nature, can break blood and disperse qi, eliminate symptoms and relieve pain.

[0005] In the 2020 edition of the Chinese Pharmacopoeia, only the dried rhizome of Curcuma wenyujin YHChenet C.Ling is included under the item of Curcuma as the legal origin of Curcuma, and only the dried rhizome of Curcuma longa L. of the Zingiberaceae family is included under the item of Curcuma as the legal origin of Curcuma; under the item of Curcuma, three types of dried rhizomes of Curcuma phaeocaulis Val., Curcuma kwangsiensis SGLee et CFLiang or Curcuma wenyujin YHChen et C.Ling are included as the legal origin of Curcuma; under the item of Curcuma, Curcuma xvenyujin YHChen et C.Ling, Curcuma longa L, Curcuma k-wangsiensis SGLee et CFLiang or Curcuma phaeocaulis are included The dried root tubers of Vai. are the legal origin of Curcuma.

[0006] Traditional Chinese medicines are mainly derived from plants and animals in nature, some of which have multiple origins. These traditional Chinese medicines often have confusing origins and are difficult to distinguish between true and false, which leads to unstable quality and greatly limits their clinical application effects. At present, there are many methods for comparative studies of turmeric slices and their similar products. The research on confusing products mainly focuses on the study of properties and chromatography. Zhang Xinhua et al. compared the sources, properties, microscopic, physical and chemical identification, ingredients and functional indications of turmeric and turmeric slices (Zhang Xinhua, Han Lijie. Discrimination of turmeric and turmeric slices in clinical application [J]. Guangming Chinese Medicine, 2011, 26 (9).). Li Dexun studied the thin layer chromatography identification of turmeric and turmeric slices (Li Dexun, Thin layer chromatography identification of turmeric and turmeric slices [J]. Medical and Health Science and Technology, 2001 (03).). Fang Hongzhuang et al. compared the chromatographic fingerprints of turmeric and turmeric slice volatile oils. The results showed that at least 10 chemical components were present in the chromatographic fingerprint of turmeric volatile oils but not detected in turmeric slices, at least 9 chemical components were present in turmeric slices but not in turmeric, and there were 9 chemical components in common in the fingerprints of turmeric and turmeric slice volatile oils (Fang Hongzhuang, Ren Shuqing. Comparison of chromatographic fingerprints of turmeric and turmeric slice volatile oils [J]. Chinese Medicinal Materials, 2006, (11).). The above-mentioned study based on the comparison of the properties of medicinal materials is not suitable for standard decoctions and formula granules of medicinal materials. The characteristic spectrum established by gas chromatography mainly analyzes the sesquiterpenoid components in volatile oils. The sample preparation is cumbersome and not suitable for standard decoctions.

[0007] Therefore, it is necessary to establish a comprehensive, accurate and rapid chromatographic identification method for turmeric slices and its similar products. This method can distinguish and identify turmeric slice standard decoctions or formula granules and its similar preparations, which is crucial for the quality detection of turmeric slices and its similar products including turmeric, Curcuma zedoaria of different origins, and Curcuma aromatica of different origins. Summary of the invention

[0008] Problem that the invention aims to solve

[0009] In order to solve the above-mentioned problems in the prior art, the purpose of the present invention is to provide a chromatographic identification method for distinguishing turmeric slice samples and their similar products. The method is applicable to standard decoctions or formula granules of turmeric slices and their similar products, and can distinguish turmeric slices from similar products including turmeric, zedoary, turmeric, etc., and provides a new analytical means for the intrinsic quality control of standard decoctions of turmeric slices, turmeric, zedoary, turmeric, etc., so as to achieve the purpose of simultaneously identifying the origin and authenticity of turmeric slices and their similar products.

[0010] Solutions for solving problems

[0011] [1] The present invention provides a chromatographic identification method for distinguishing a turmeric sample and its similar products, wherein the method comprises the following steps:

[0012] (1) Sample solution preparation: extracting the turmeric slice sample and the similar product sample with an extraction solvent, collecting the extracts, and preparing the turmeric slice sample solution and the similar product sample solution;

[0013] The turmeric slice sample is selected from the turmeric slice standard decoction or turmeric slice formula granules; the similar product sample is selected from the similar product standard decoction or similar product formula granules; the similar product is one or more of turmeric, zedoary turmeric, and curcuma;

[0014] (2) Preparation of reference substance solution: comprising the steps of taking one or more reference substances selected from curcumin, zedoarylol, zedoarylonediol, and zedoarylbicycloenone to prepare reference substance solution;

[0015] (3) Establishment of characteristic spectrum: using ultra-high performance liquid chromatography to detect the turmeric sample solution, the similar product sample solution and the reference solution in step (2), and obtaining a detection spectrum and a reference spectrum respectively, and using a Chinese medicine chromatographic fingerprint similarity evaluation system to analyze and establish a characteristic spectrum of the turmeric sample and a characteristic spectrum of the similar product sample;

[0016] (4) Establishment of identification standards: Compare the characteristic spectrum of the turmeric sample, the characteristic spectrum of the similar product sample and the reference spectrum in step (3) to establish identification standards;

[0017] (5) Sample processing: Take the sample to be tested, obtain the sample solution according to the method in step (1), and then generate the sample characteristic spectrum according to the method in step (3);

[0018] (6) Identification of the sample: According to the identification standard established in step (4), the characteristic spectrum of the sample generated in step (5) is compared to determine that the sample is turmeric or any one of the similar products.

[0019] [2] The chromatographic identification method according to [1], wherein in step (1), the medicinal material origin of the Curcuma zedoaria includes one or more of Curcuma wenyujin, Curcuma guangxiensis, and Curcuma pengshu; the medicinal material origin of the Curcuma zedoaria includes one or more of Curcuma wenyujin and Curcuma guangxiensis;

[0020] The extraction solvent is methanol or a methanol aqueous solution, preferably a methanol aqueous solution with a volume concentration of 30% to 70%; the extraction method is any one of ultrasonic treatment, shaking extraction or heating reflux extraction; preferably, the extraction method is ultrasonic extraction, with a power of 200W to 300W, a frequency of 35kHz to 45kHz, and an extraction time of 5min to 60min.

[0021] [3] The chromatographic identification method according to [1] or [2], wherein the preparation of the reference solution in step (2) comprises dissolving the reference solution in a methanol aqueous solution having a methanol volume percentage of not less than 50% or in methanol to prepare one or more of a curcumin reference solution, a zedoaryldiol reference solution, a zedoaryloxadiol reference solution, and a zedoarylbicycloalkenone reference solution having a concentration of 10 to 40 μg / mL.

[0022] [4] The chromatographic identification method according to any one of the technical solutions [1] to [3], wherein the chromatographic conditions of the ultra-high performance liquid chromatography in step (3) include:

[0023] Stationary phase: chromatographic column with octadecyl bonded silica gel as filler,

[0024] Mobile phase: acetonitrile is used as mobile phase A, and an aqueous solution of phosphoric acid is used as mobile phase B; preferably, a phosphoric acid solution with a volume fraction of 0.05% to 0.20% is used as mobile phase B;

[0025] Also included is a gradient elution program, wherein the gradient elution program is:

[0026] Time / minute) Mobile phase A / % Mobile phase B / % 0～4 20 80 4～8 20→30 80→70 8～12 30→32 70→68 12～20 32→43 68→57 20～25 43→75 57→25 25～30 75 25

[0027] ; Preferably also includes: a column temperature of 20 to 30°C, and / or a flow rate of 0.28 to 0.32 mL / min, and / or a detection wavelength of 220 to 270 nm.

[0028] [5]. The chromatographic identification method according to any one of the technical solutions [1] to [4], wherein the characteristic spectrum of the turmeric sample in step (3) includes 9 characteristic peaks, wherein peak 1 is the peak of curcuma oxadiol, peak 3 is the peak of curcuma diol, and peak 9 is the peak of curcuma bicycloenone.

[0029] [6] The chromatographic identification method according to any one of the technical solutions [1] to [5], wherein the formulation of the identification standard in step (4) further comprises:

[0030] Characteristic peaks are determined from the characteristic spectrum of the turmeric slice sample and the characteristic spectrum of the similar product sample respectively, and the characteristic peaks in the characteristic spectrum of the turmeric slice sample are attributed by liquid chromatography-mass spectrometry technology, and the distinguishing components are identified, and the differences of the characteristic peaks in each characteristic spectrum are compared, and the differences are recorded to obtain the identification standard.

[0031] [7]. The chromatographic identification method according to [6], wherein the differences include: the curcumin peak can distinguish turmeric, the relative peak area of ​​the curcuminone diol peak and the curcuma diol peak can distinguish turmeric and turmeric, and the relative peak area of ​​the curcuma bicycloenone peak and the curcuma diol peak can distinguish curcuma and turmeric.

[0032] [8] The chromatographic identification method according to any one of the technical solutions [1] to [7], wherein the step (6) of comparing the characteristic spectrum of the sample generated in step (5) comprises:

[0033] If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to that of the curcumin reference substance, the test sample is turmeric;

[0034] If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area of ​​the peak of curcuma oxadiol and the peak of curcuma diol is less than 2.4, then the test sample is curcuma;

[0035] If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area between the peak of curcuma bicycloenone and the peak of curcuma diol is higher than 6.2, then the test sample is Curcuma aromatica;

[0036] If the characteristic spectrum of the test sample has chromatographic peaks at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area of ​​the curcuma oxadiol peak to the curcuma diol peak is higher than 2.4; the relative peak area of ​​the curcuma bicycloenone peak to the curcuma diol peak is lower than 6.2, then the test sample is turmeric.

[0037] [9]. Use of the method according to any one of the technical solutions [1] to [8] in identifying two or more of turmeric, turmeric, zedoary zedoary and / or curcuma.

[0038]

[10] . Application of the method described in any one of the technical solutions [1] to [8] in the quality inspection of turmeric slices and its similar products.

[0039] Effects of the Invention

[0040] The present invention establishes a characteristic spectrum of turmeric and its similar products with different origins by liquid chromatography-mass spectrometry technology, and provides a chromatographic identification method for distinguishing turmeric and its similar products. Ultra-high performance liquid chromatography is used to reasonably control the chromatographic conditions, and 9 characteristic peaks of the turmeric sample are determined, forming a full picture of the characteristic spectrum of turmeric and its preparations. At the same time, a comparative study is also conducted on similar products of turmeric, turmeric, zedoary and turmeric preparations, which can more comprehensively reflect the characteristics of the standard decoction of turmeric and its similar products (zedoary, turmeric, turmeric), and achieve the purpose of distinguishing turmeric from standard decoctions or formula granules of turmeric, zedoary, turmeric and turmeric.

[0041] The present invention controls the intrinsic quality of the entire turmeric slice medicinal material, reflects the characteristics of the turmeric slice medicinal material and its similar products with different origins in a relatively comprehensive manner, achieves the purpose of simultaneously identifying the turmeric slice and its similar products, and provides a new analytical means for the intrinsic quality control of preparations such as turmeric slice standard decoctions or formula granules.

[0042] 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.

[0043] 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

[0044] Figure 1 The characteristic spectrum of the standard decoction of turmeric slices in Example 1

[0045] Figure 2 The characteristic spectrum of the standard decoction of turmeric in Example 1

[0046] Figure 3 Characteristic spectrum of the standard decoction of Curcuma zedoaria (Wen Yujin) in Example 1

[0047] Figure 4 Characteristic spectrum of the standard decoction of Curcuma zedoaria (Guangxi Curcuma zedoaria) in Example 1

[0048] Figure 5 The characteristic spectrum of the standard decoction of Curcuma zedoaria (Atractylodes lancea) in Example 1

[0049] Figure 6 The characteristic spectrum of the standard decoction of Curcuma (Wen Yujin) in Example 1

[0050] Figure 7 The characteristic spectrum of the standard decoction of Curcuma zedoariae (Guangxi Curcuma) in Example 1

[0051] Figure 8 Comparison of characteristic spectra of standard decoctions of turmeric, turmeric, zedoary and turmeric in Example 1

[0052] Fig. 9 The characteristic spectrum of the standard decoction of turmeric slices in Example 1 is compared with the characteristic spectrum of the standard decoction of turmeric

[0053] Fig.10 The characteristic spectrum of the standard decoction of turmeric slices in Example 1 is compared with the characteristic spectrum of the standard decoction of zedoaria from different origins.

[0054] Fig.11 The characteristic spectrum of the standard decoction of turmeric slices in Example 1 is compared with the characteristic spectrum of the standard decoction of different origins of turmeric.

[0055] Fig.12 Identification of reference substances in the characteristic spectrum of standard decoction of turmeric

[0056] Fig.13 Chromatograms of standard decoction of turmeric slices under different chromatographic column conditions

[0057] Fig.14 The chromatograms of the standard decoction of turmeric slices under different column temperature conditions are shown in Figure 2.

[0058] Fig.15 Chromatograms of standard decoction of turmeric slices under different flow rate conditions DETAILED DESCRIPTION

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Unless otherwise specified, the percentage "%" in this document referring to solution concentration refers to volume percentage.

[0073] 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.

[0074] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0075] 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, identification of properties, microscopic identification, physical and chemical identification, etc.) and used to establish a corresponding characteristic spectrum. For example, the "sliced ​​turmeric sample" in the present invention refers to a sliced ​​turmeric medicinal material that has been identified as a crude drug and used to establish a corresponding characteristic spectrum.

[0076] Unless otherwise specified, the Curcuma zedoaria (Wen Yu Jin) in the present invention refers to the dried rhizome of Wen Yu Jin of the Zingiberaceae plant, that is, the medicinal material base of Curcuma zedoaria is Wen Yu Jin.

[0077] Unless otherwise specified, the Curcuma zedoaria (Guangxi Curcuma zedoaria) in the present invention refers to the dried rhizome of the plant Curcuma zedoaria of the Zingiberaceae family, that is, the medicinal material base of Curcuma zedoaria is Guangxi Curcuma zedoaria.

[0078] Unless otherwise specified, the Curcuma zedoaria (Germanthus zedoaria) in the present invention refers to the dried rhizome of the plant Ginger family Ginger, that is, the medicinal material base of Curcuma zedoaria is Ginger.

[0079] Unless otherwise specified, the turmeric (Wen Yujin) in the present invention refers to the dried root of Wen Yujin of the ginger family, that is, the medicinal material base of turmeric is Wen Yujin.

[0080] Unless otherwise specified, the Curcuma aromatica (Guangxi Curcuma) in the present invention refers to the dried root tuber of the plant Curcuma aromatica of the Zingiberaceae family, that is, the medicinal material base of Curcuma aromatica is Guangxi Curcuma.

[0081] <First aspect>

[0082] The present invention provides a chromatographic identification method for distinguishing turmeric slices and similar products thereof, which comprises the following steps:

[0083] (1) Sample solution preparation: extracting the turmeric slice sample and the similar product sample with an extraction solvent, collecting the extracts, and preparing the turmeric slice sample solution and the similar product sample solution;

[0084] In some specific embodiments of the present invention, in step (1), the turmeric slice sample is selected from turmeric slice standard decoction or turmeric slice formula granules;

[0085] Among them, "standard decoction" refers to a single-ingredient Chinese medicine decoction prepared by standardized process under the guidance of TCM theory and based on clinical application, with reference to modern extraction methods. "Formulated granules" are granules made from single-ingredient Chinese medicine decoction pieces processed according to traditional standards, extracted and concentrated, for use in TCM clinical prescriptions.

[0086] The similar product samples are selected from similar product standard decoctions or similar product formula granules; the similar products are one or more of turmeric, curcuma, and turmeric; among them, the medicinal material origins of curcuma include one or more of warm curcuma, Guangxi curcuma, and curcuma; the medicinal material origins of turmeric include one or more of warm curcuma and Guangxi curcuma.

[0087] In addition, in some specific embodiments of the present invention, the extraction solvent is methanol or an aqueous solution of methanol; in some preferred embodiments, the extraction solvent is an aqueous solution of methanol with a volume concentration of 30% to 70%. For the amount of the extraction solvent, in some specific embodiments of the present invention, the mass volume ratio of the powder of the sliced ​​turmeric sample and its similar products to the extraction solvent is 1g: (30 to 100mL), specifically 1g: 30mL, 1g: 40mL, 1g: 50mL, 1g: 60mL, 1g: 70mL, 1g: 80mL, 1g: 90mL, 1g: 100mL, etc.; in order to further optimize the extraction effect and material dosage, the volume mass ratio of the powder of the standard decoction to the extraction solvent is preferably 1g: 100mL; the volume mass ratio of the powder of the formula granules to the extraction solvent is preferably 1g: 40mL; too small a sample concentration 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.

[0088] Regarding the extraction method, in order to obtain more abundant chromatographic peak information, the extraction can be carried out by ultrasound, shaking or heating reflux, preferably by ultrasonic treatment extraction, which is easier to operate. 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 5 to 60min, specifically 5min, 10min, 15min, 30min, 45min or 60min; in order to further optimize the extraction effect and treatment cycle, the extraction efficiency of each chromatographic peak is comprehensively considered, and the preferred extraction time is 10 to 45min, more preferably 15min.

[0089] For the standard decoction of turmeric and its similar products, the medicinal materials are first subjected to pre-treatment, decoction, low-temperature concentration, freeze-drying and other processes to obtain freeze-dried powder, and the freeze-dried powder is preferably ground into powder; the preparation method of the standard decoction is specifically as follows: soaking the medicinal materials in water, filtering while hot after decoction, decocting again after cooling and filtering while hot, combining the filtrate and concentrating under reduced pressure, freeze-drying, and obtaining the standard decoction.

[0090] In some specific embodiments of the present invention, the preparation method of the standard decoction of turmeric slices is as follows: take turmeric slices, weigh them, put them in a casserole, add water to soak them, use a mechanical split decoction pot, first boil them over high heat, then boil them over low heat, filter them while they are hot, and cool the filtrate in a cold water bath; add water for the second decoction, boil them over high heat, then boil them over low heat, filter them again while they are hot, and cool the filtrate in a cold water bath; combine the two filtrates, and then concentrate them under reduced pressure at 65°C, divide the concentrate into vials, place them in a freeze dryer, and freeze-dry them to obtain the standard decoction of turmeric slices.

[0091] For the formula granules of turmeric slices and its similar products, the Chinese medicine formula granules obtained by pre-processing, extracting, concentrating, spray drying, granulating and packaging the medicinal materials of turmeric slices and its similar products are extracted. In some specific embodiments of the present invention, the preparation method of the solution is as follows: take an appropriate amount of turmeric slice medicinal material, crush it, pass it through a No. 3 sieve (50 mesh), accurately weigh it, put it in a stoppered conical flask, accurately add a methanol aqueous solution with a volume fraction of 30% to 70%, seal it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 10min to 45min, let it cool, shake it well, filter it, and take the subsequent filtrate to obtain; or, take an appropriate amount of standard decoction of turmeric slices and its similar products or formula granules of turmeric slices and its similar products, grind it, accurately weigh it, put it in a stoppered conical flask, accurately add a methanol aqueous solution with a volume fraction of 30% to 70%, seal it, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 10min to 45min, let it cool, weigh it again, shake it well, filter it, take the subsequent filtrate, transfer it to a volumetric flask, add methanol to the scale, shake it well, filter it, and take the subsequent filtrate to obtain.

[0092] (2) Preparation of reference substance solution: comprising the steps of taking one or more reference substances selected from curcumin, zedoarylol, zedoarylonediol, and zedoarylbicycloenone to prepare reference substance solution;

[0093] Furthermore, the reference substance is dissolved in alcohol or alcohol-water solution, and the preparation of the reference substance solution includes dissolving the reference substance in a methanol-water solution or methanol with a methanol volume percentage of not less than 50%, respectively, to prepare one or more of a curcumin reference substance solution, a curcuma diol reference substance solution, a curcuma oxadiol reference substance solution, and a curcuma bicycloenone reference substance solution with a concentration of 10 to 40 μg / mL; in some specific embodiments of the present invention, it is preferred to prepare a curcumin reference substance solution with a concentration of 20 μg / mL, a curcuma diol reference substance solution with a concentration of 20 μg / mL, a curcuma oxadiol reference substance solution with a concentration of 20 μg / mL, a curcuma bicycloenone reference substance solution with a concentration of 20 μg / mL.

[0094] In some specific embodiments of the present invention, the preparation method of the above-mentioned reference solution is as follows: take appropriate amounts of curcumin, curcuma diol, curcuma oxadiol, and curcuma bicycloenone reference substances, accurately weigh them, add methanol to each to make a solution containing 20 μg per 1 ml, shake well, and use it as the reference solution. Further, the extraction solvent is an alcohol or an alcohol-water solution, such as methanol or a methanol-water solution. Further, the volume fraction of methanol in the methanol-water solution is not less than 50%, preferably the concentration of the extraction solvent is not less than 70% by volume fraction of methanol, and more preferably the extraction solvent can be methanol.

[0095] (3) Establishment of characteristic spectrum: using ultra-high performance liquid chromatography to detect the turmeric sample solution, the similar product sample solution and the reference solution in step (2), and obtaining a detection spectrum and a reference spectrum respectively, and using a Chinese medicine chromatographic fingerprint similarity evaluation system to analyze and establish a characteristic spectrum of the turmeric sample and a characteristic spectrum of the similar product sample;

[0096] The present invention has determined, through in-depth research, the ultra-high performance liquid chromatography conditions applicable to both the standard decoction of turmeric tablets and / or the turmeric tablets formula granules, which include:

[0097] Stationary phase: a chromatographic column with octadecyl bonded silica as 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, a chromatographic column with a column length of 150 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.6 μm; in some specific embodiments of the present invention, the chromatographic column is selected from any one of ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.8 μm), CORTECS UPLC T3 (150 mm×2.1 mm, 1.6 μm), and ZORBAX EclipsePlus C18 RRHD (100 mm×2.1 mm, 1.8 μm). Further, the chromatographic column preferably uses a CORTECS UPLC T3 (150 mm×2.1 mm, 1.6 μm) chromatographic column. When the chromatographic column is used for separation, the separation of characteristic peaks is better and the overall spectrum is more beautiful.

[0098] Mobile phase: acetonitrile is used as mobile phase A, and an aqueous solution of phosphoric acid is used as mobile phase B. In some specific embodiments of the present invention, a phosphoric acid solution with a volume fraction of 0.05% to 0.20% is preferred, and a phosphoric acid solution with a volume fraction of 0.1% is more preferred as mobile phase B.

[0099] The elution procedure of the present invention is:

[0100]

[0101] Considering the chromatographic peak separation effect, the preferred flow rate of the present invention is 0.2mL / min to 0.5mL / 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 applicability requirements with small changes in column temperature or flow rate. In some preferred embodiments of the present invention, the flow rate can be 0.28mL / min to 0.32mL / min; and / or, the column temperature can be 20°C to 30°C; in some specific embodiments of the present invention, the flow rate is 0.3mL / min and the column temperature is 25°C.

[0102] 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 270nm. 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 relatively good, and the baseline is relatively stable; in addition, the theoretical plate number calculated according to the peak of the curcuma diol reference substance should be not less than 5000.

[0103] Further, the present invention detects at least 18 batches of sliced ​​turmeric samples from different origins, 4 batches of turmeric samples, 4 batches of zedoaria (warm turmeric) samples, 3 batches of zedoaria (Guangxi zedoaria) samples, 8 batches of zedoaria (Peng zedoaria) samples, 3 batches of turmeric (warm turmeric) samples, 3 batches of zedoaria (Guangxi zedoaria) samples and reference solution, and analyzes and compares, selects common peaks to establish characteristic spectra, and identifies common peaks with reference to the positioning of reference chromatographic peaks. Finally, the obtained sample chromatograms are imported into the "Chinese medicine chromatographic fingerprint similarity evaluation system 2012 version", and the median method is used to generate respective superimposed characteristic spectra and reference characteristic spectra, so as to obtain the sliced ​​turmeric sample characteristic spectra and the similar product sample characteristic spectra.

[0104] The characteristic spectrum of the turmeric sample established by the above method contains at least 9 characteristic peaks, among which peak 1 is the peak of curcuma oxadiol, peak 3 is the peak of curcuma diol, and peak 9 is the peak of curcuma bicycloenone.

[0105] (4) Establishment of identification standards: After comparing the characteristic spectrum of the turmeric slice sample, the characteristic spectrum of the similar product sample and the reference spectrum in step (3), characteristic peaks are determined and identification standards are established; preferably, characteristic peaks are determined from the characteristic spectrum of the turmeric slice sample and the characteristic spectrum of the similar product sample, and the characteristic peaks in the characteristic spectrum of the turmeric slice sample are attributed by liquid chromatography-mass spectrometry technology, and the distinguishing components are identified, and the differences of the characteristic peaks in each characteristic spectrum are compared, and the differences are recorded to obtain the identification standards.

[0106] In some specific embodiments of the present invention, the components corresponding to the characteristic peaks in the characteristic spectrum of the turmeric slice sample include curcuma ketone diol, isocurcuma ketone diol, curcuma diol, aromatic turmeric ketone, dehydrocurcuma dione, protocurcumenol, geranone-4,5-epoxide, curcumenol and curcuma bicycloenone.

[0107] In some specific embodiments of the present invention, the differences include:

[0108] The characteristic components in the characteristic spectrum of turmeric samples include epicurenol, bis-demethoxybiredotin, demethoxybiredotin and curcumin;

[0109] The characteristic components in the characteristic spectra of Curcuma zedoaria (Guangxi Curcuma) and Curcuma zedoaria (Peng Curcuma) include geranone-1(10),4-diepoxide;

[0110] The curcumin peak can distinguish turmeric, the relative peak area of ​​the curcuminone diol peak and the curcuma diol peak can distinguish turmeric and turmeric, and the relative peak area of ​​the curcuminone peak and the curcuma diol peak can distinguish turmeric and turmeric.

[0111] (5) Handling of test samples:

[0112] Take the sample to be tested, obtain the test solution according to the method in step (1), and then generate the test sample characteristic spectrum according to the method in step (3);

[0113] In some specific embodiments of the present invention, the treatment of the test solution is specifically as follows: consistent with the above-mentioned method of "preparation of sample solution", take an appropriate amount of the sample to be tested, crush, sieve, accurately weigh, place in a stoppered conical flask, accurately add a methanol aqueous solution with a volume fraction of 30% to 70%, seal, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 10min to 45min, cool, shake well, filter, and take the filtrate to obtain.

[0114] In some specific embodiments of the present invention, the ultra-high performance liquid chromatography conditions used for detection are also consistent with the conditions described above.

[0115] (6) Identification of the sample: According to the identification standard established in step (4), the characteristic spectrum of the sample generated in step (5) is compared to determine that the sample is turmeric or any one of the similar products.

[0116] In some specific embodiments of the present invention, the step (6) of comparing the sample profiles generated in step (5) includes:

[0117] If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to that of the curcumin reference substance, the test sample is turmeric;

[0118] If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area of ​​the peak of curcuma oxadiol and the peak of curcuma diol is less than 2.4, then the test sample is curcuma;

[0119] If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area between the peak of curcuma bicycloenone and the peak of curcuma diol is higher than 6.2, then the test sample is Curcuma aromatica;

[0120] If the characteristic spectrum of the test sample has chromatographic peaks at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area of ​​the curcuma oxadiol peak to the curcuma diol peak is higher than 2.4; the relative peak area of ​​the curcuma bicycloenone peak to the curcuma diol peak is lower than 6.2, then the test sample is turmeric.

[0121] The invention shortens the analysis time and improves the analysis efficiency by optimizing sample preparation, reference substance preparation and chromatographic analysis conditions, establishes ultra-high performance liquid chromatography conditions applicable to standard decoction of turmeric slices and / or formula granules of turmeric slices and similar products thereof, truly reflects the original chemical composition of preparations such as standard decoction of turmeric slices and / or formula granules of turmeric slices and similar products thereof, and the method can distinguish turmeric slices and similar products thereof including turmeric, zedoary zedoary of different origins and curcuma of different origins, thereby providing an analysis means and basis for quality control of turmeric slices and similar products thereof.

[0122] <Second Aspect>

[0123] The present invention also provides the application of the chromatographic identification method in identifying two or more of turmeric, turmeric, zedoary turmeric and / or curcuma.

[0124] In addition, the invention also provides application of the chromatographic identification method in quality detection of turmeric slices and similar products thereof.

[0125] In a specific embodiment of the present invention, the identification method can identify turmeric flakes and turmeric.

[0126] In a specific embodiment of the present invention, the identification method can identify Curcuma longa and Curcuma zedoaria; wherein the medicinal material origin of Curcuma zedoaria includes one or more of Curcuma wenyujin, Curcuma guangxiensis, and Curcuma pengshuo.

[0127] In a specific embodiment of the present invention, the identification method can identify turmeric slices and curcuma; wherein the medicinal material origin of curcuma includes one or more of Wenyujin and Guangxi Curcuma.

[0128] In a specific embodiment of the present invention, the identification method can also identify turmeric and zedoary zedoary, turmeric and curcuma, and zedoary zedoary and curcuma.

[0129] 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.

[0130] Example

[0131] Instruments, reagents and samples

[0132] Waters-ACQUIYT-UPLC-H-Class, ultra-high performance liquid chromatography system; Waters-Quaternary-Solvent-Manager-quaternary pump; Sample-manager-FTN autosampler; Waters-UPLC-PDA detector; Empower-3 chromatography workstation. Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; 1290 DAD diode array detector; 1290 MCT column oven; 1290 Vial sampler autosampler; 1290 Fiexible pump quaternary pump; OpenLAB CDS 2.3 chromatography workstation. Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent)

[0133] METTLER TOLEDO XP6 one-millionth balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); ME204E / 02 electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); KQ-250E ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); HY-4 oscillator (Jintan Kexing Instrument Factory); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory).

[0134] Acetonitrile (chromatographic grade, Thermo Fisher); phosphoric acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); Milli-Q ultrapure water system (Millipore); other reagents were of analytical grade.

[0135] Curcumin reference substance was purchased from China Food and Drug Inspection Institute with the number 110823-202107; Curcuma zedoarylone reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd. with the number PS220826-01; Curcuma zedoarylol reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd. with the number PS200525-01; Curcuma bicycloenone reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd. with the number PS220805-02.

[0136] 18 batches of sliced ​​turmeric medicinal materials; 4 batches of turmeric medicinal materials, 4 batches of curcuma (Wen Yujin) medicinal materials, 3 batches of curcuma (Guangxi Curcuma) medicinal materials, 8 batches of curcuma (Peng Yujin), 3 batches of curcuma (Wen Yujin) medicinal materials, and 3 batches of curcuma (Guangxi Curcuma) medicinal materials. The above medicinal materials all comply with the provisions of the 2020 edition of the Chinese Pharmacopoeia under the items of sliced ​​turmeric, curcuma, curcuma, and turmeric.

[0137] Example 1: Establishment of characteristic spectrum of turmeric slices and its similar products

[0138] 1. Preparation of Sample Solution

[0139] 1.1 Preparation of turmeric sample solution

[0140] Preparation of standard decoction of turmeric slices: take 150g of turmeric slices, weigh it, put it in a casserole, add 1350ml of water and soak it for 30min, use a YMW mechanical split decoction pot, first boil it over high heat, then simmer it over low heat for 20min, select a 200-mesh standard sieve to filter it while hot, and cool the filtrate in a cold water bath; add 1050ml of water for the second decoction, boil it over high heat, then simmer it for 15min, filter it while hot through a 200-mesh standard sieve, and cool the filtrate in a cold water bath; combine the two filtrates and concentrate them under reduced pressure at 65°C; concentrate to a relative density of 1.01-1.03 (65°C), divide the concentrate into vials, place it in a freeze dryer, and freeze-dry it to obtain the standard decoction of turmeric slices.

[0141] Take about 0.2 g of the powder of this product, place it in a stoppered conical flask, add 20 ml of 70% methanol, stopper it tightly, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 15 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0142] 1.2 Preparation of similar sample solution

[0143] Take Curcuma zedoaria (Wen Yujin), Curcuma zedoaria (Guangxi Yujin), Curcuma zedoaria (Peng Yuzhu), Curcuma zedoaria (Wen Yujin) and Curcuma zedoaria (Guangxi Yuzhu) respectively, and prepare similar sample solutions according to the method of "1.1 Preparation of turmeric sample solution".

[0144] 2. Preparation of reference solution

[0145] Take appropriate amounts of curcumin reference substance, zedoaryl alcohol reference substance and zedoary bicycloenone reference substance, accurately weigh them, add methanol to make solutions containing 20 μg per 1 ml, shake well, and use them as reference substance solutions.

[0146] 3. Determination of liquid chromatography conditions

[0147] 3.1 Determination of mobile phase

[0148] Acetonitrile was selected as mobile phase A, and phosphoric acid solution with a volume fraction of 0.1% was selected as mobile phase B.

[0149] 3.2 Determination of elution gradient

[0150] Use CORTECS UPLC T3 (150mm×2.1mm, 1.6μm); perform gradient elution according to the regulations in Table 1; the flow rate is 0.3ml / min, the column temperature is 25℃, and the detection wavelength is 254nm. The theoretical plate number calculated based on the curcuma diol peak should not be less than 5000,

[0151] Table 1:

[0152]

[0153] 4. Establishment of characteristic spectra of turmeric slice samples and their similar samples

[0154] 1 μl of sample solution and reference solution were accurately drawn and injected into the ultra-high performance liquid chromatography instrument. The characteristic spectra of 18 batches of turmeric standard decoction samples were superimposed using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" issued by the Chinese Pharmacopoeia Committee to generate the characteristic spectrum of turmeric standard decoction. Figure 1 ; Four batches of characteristic spectra of turmeric standard decoction samples were superimposed to generate the characteristic spectra of turmeric standard decoction. Figure 2 , the characteristic spectra of 4 batches of samples of Curcuma zedoaria (Wen Yujin) standard decoction were superimposed to generate the characteristic spectrum of Curcuma zedoaria (Wen Yujin) standard decoction. Figure 3 , the characteristic spectra of three batches of standard decoction samples of Curcuma zedoaria (Guangxi Curcuma zedoaria) were superimposed to generate the characteristic spectrum of standard decoction of Curcuma zedoaria (Guangxi Curcuma zedoaria) Figure 4 , the characteristic spectra of 8 batches of standard decoction samples of Curcuma zedoaria (Peng Curcuma) were superimposed to generate the characteristic spectrum of standard decoction of Curcuma zedoaria (Peng Curcuma) Figure 5 The characteristic spectra of three batches of standard decoction samples of Curcuma (Wen Yujin) were superimposed to generate the characteristic spectrum of standard decoction of Curcuma (Wen Yujin). Figure 6 The characteristic spectrum of three batches of Curcuma (Guangxi Curcuma) standard decoction samples was superimposed to generate the characteristic spectrum of Curcuma (Guangxi Curcuma) standard decoction. Figure 7 The common peaks with relatively stable relative retention time and relative peak area RSD were selected as characteristic peaks.

[0155] 5. Comparison of turmeric and its similar products

[0156] The characteristic spectra of standard decoctions of turmeric slices and similar products were compared using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" issued by the Chinese Pharmacopoeia Committee. Figure 8 .

[0157] Compare the characteristic spectrum of the standard decoction of turmeric slices with that of the standard decoction of turmeric. Fig. 9 , the main differences between turmeric slices and turmeric are peaks 1 to 9, peaks a to d;

[0158] The characteristic spectrum of the standard decoction of turmeric slices was compared with the characteristic spectrum of the standard decoctions of Curcuma zedoaria from different origins (Curcuma zedoaria (Wenyujin), Curcuma zedoaria (Guangxi Curcuma), Curcuma zedoaria (Peng Curcuma)) Fig.10 , the main differences between turmeric slices and zedoaria are peak 1, peak 3, peak 9, and peak e;

[0159] The reference spectrum of the standard decoction of turmeric slices was compared with the characteristic spectrum of the standard decoctions of different origins of turmeric (curcuma (Wen Yujin), turmeric (Guangxi Curcuma)). Fig.11 The main differences between turmeric slices and turmeric are peak 1, peak 3, and peak 9.

[0160] 6. Research on the material basis of turmeric and identification of characteristic peaks

[0161] Liquid chromatography-mass spectrometry (LC-MS / MS) was used to collect high-resolution mass spectrometry data according to the liquid chromatography conditions of the characteristic spectrum of the standard decoction of Turmeric Pianhuang. The molecular formulas of the compounds in the standard decoction of Turmeric Pianhuang were inferred by combining isotope analysis. The common peaks were attributed by comparing with references and related reference substances.

[0162] The characteristic spectrum of turmeric slices is shown in Figure 1 The retention time of peak 1, peak 3, and peak 9 is consistent with the retention time of reference substances such as curcuma oxadiol, curcuma diol, and curcuma bicycloenone. Therefore, peak 1, peak 3, and peak 9 in the characteristic spectrum of the standard decoction of sliced ​​turmeric are identified as curcuma oxadiol, curcuma diol, and curcuma bicycloenone, respectively. Fig.12 In addition, 9 common peaks were identified by LC-MS, and the results are shown in Table 2 LC-MS analysis results.

[0163] Table 2: UPLC-Q-TOF / MS identification results of the characteristic spectrum of the standard decoction of turmeric

[0164]

[0165] 7. Study on the material basis of standard decoctions of turmeric, zedoary turmeric and zedoary curcuma and identification of distinguishing peaks

[0166] Liquid chromatography-mass spectrometry (LC-MS / MS) was used to collect high-resolution mass spectrometry data according to the liquid chromatography conditions of the characteristic spectrum of the standard decoction of turmeric slices. The molecular formulas of the compounds were inferred by combining isotope analysis. The common peaks were attributed by comparing with references and related reference substances.

[0167] The mass spectrometer was used to identify the peaks a to d of the distinguishing components in the standard decoction of turmeric, where peak d was the peak of the curcumin reference substance; the identification of the distinguishing peak e of the standard decoctions of Curcuma zedoaria (Guangxi Curcuma zedoaria) and Curcuma zedoaria (Pangguoshu) was shown in Table 2.

[0168] Table 2: UPLC-Q-TOF / MS identification results of characteristic spectra of standard decoctions of turmeric and zedoaria

[0169]

[0170] 8. Determine the standard for distinguishing turmeric from similar products such as turmeric, zedoary and curcuma

[0171] Preparation of test solution and reference solution

[0172] Take the sample to be tested and prepare the test solution according to the method of "1.1 Preparation of turmeric sample solution"; take the reference substance and prepare the reference substance solution according to the method of "2. Preparation of reference substance solution"; accurately pipette 1 μl of the test solution and the reference substance solution respectively, and inject them into the ultra high performance liquid chromatography; measure according to the above ultra high performance liquid chromatography conditions, generate the test sample spectrum, and compare it with the reference substance spectrum.

[0173] Comparison results:

[0174] Through mass spectrometry and reference substances, the differential components were determined, and the components that could be identified by the reference substances were screened to distinguish the standard decoctions of turmeric, turmeric, zedoary and turmeric. Curcuma can be distinguished by peak d; the minimum value (3.46) of the ratio of peak 1 to peak 3 in the turmeric decoction was increased by 30%, and the result was calculated to be 2.4, which was used to distinguish turmeric and zedoary; the maximum value (4.08) of the ratio of peak 9 to peak 3 in the turmeric decoction was increased by 50%, and the result was calculated to be 6.2, which was used to distinguish turmeric and turmeric. The specific relative peak area ranges are shown in the following table.

[0175] Table 3: Relative peak area ranges of peak 1 and peak 3, peak 9 and peak 3 of standard decoction of turmeric and turmeric

[0176]

[0177] Table 4: Relative peak area ranges of peak 1 and peak 3, peak 9 and peak 3 of standard decoction of turmeric and zedoaria

[0178]

[0179] Specifically:

[0180] (1) If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to that of the curcumin reference substance, the test sample is turmeric;

[0181] (2) If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to that of the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area between the peak of curcuma oxadiol and the peak of curcuma diol is less than 2.4, then the test sample is Curcuma;

[0182] (3) If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area between the peak of curcuma bicycloenone and the peak of curcuma diol is greater than 6.2, then the test sample is Curcuma aromatica;

[0183] (4) If the characteristic spectrum of the test sample has chromatographic peaks at the retention times corresponding to those of the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area between the peak of curcuma oxadiol and the peak of curcuma diol is greater than 2.4; the relative peak area between the peak of curcuma bicycloenone and the peak of curcuma diol is less than 6.2, then the test sample is turmeric.

[0184] Example 2: Methodological verification of the liquid chromatography method in Example 1

[0185] Durability test

[0186] 1) Chromatographic column inspection

[0187] In this study, the separation effects of chromatographic columns with three different fillers were investigated. Fig.13 The results showed that the use of CORTECS UPLC T3 (150 mm × 2.1 mm, 1.6 μm) chromatographic column can achieve better separation of each chromatographic peak.

[0188] 2) Column temperature investigation

[0189] Using CORTECS UPLC T3 (150 mm × 2.1 mm, 1.6 μm) chromatographic column, the chromatographic peak separation effect was investigated at column temperatures of 20°C, 25°C, and 30°C. The results are shown in Fig.14 .

[0190] 3) Flow rate investigation

[0191] The chromatographic peak separation effect was investigated using a CORTECS UPLC T3 (150 mm × 2.1 mm, 1.6 μm) column at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min. Fig.15 .

[0192] The results showed that small changes in column temperature and flow rate could meet the applicability requirements of the system and the analytical method had good durability.

[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.

Claims

1. A chromatographic identification method for distinguishing a turmeric sample and its similar products, characterized in that: The method comprises the following steps: (1) Sample solution preparation: extracting the turmeric flake sample and the similar product sample with an extraction solvent, collecting the extracts, and preparing the turmeric flake sample solution and the similar product sample solution; The turmeric slice sample is selected from the turmeric slice standard decoction or turmeric slice formula granules; the similar product sample is selected from the similar product standard decoction or similar product formula granules; the similar product is one or more of turmeric, zedoary turmeric, and curcuma; (2) Preparation of reference substance solution: comprising the steps of preparing reference substance solution by taking one or more reference substances selected from curcumin, curcumadiol, curcuma oxadiol, and curcuma bicycloenone; (3) Establishment of characteristic spectrum: using ultra-high performance liquid chromatography to detect the turmeric sample solution, the similar product sample solution and the reference solution in step (2), and obtaining a detection spectrum and a reference spectrum respectively, and using a Chinese medicine chromatographic fingerprint similarity evaluation system to analyze and establish a characteristic spectrum of the turmeric sample and a characteristic spectrum of the similar product sample; (4) Establishment of identification standards: Compare the characteristic spectrum of the turmeric sample, the characteristic spectrum of the similar product sample and the reference spectrum in step (3) to establish identification standards; (5) Sample processing: taking a sample to be tested, obtaining a sample solution according to the method in step (1), and then generating a sample characteristic spectrum according to the method in step (3); (6) Identification of the sample: According to the identification standard established in step (4), the characteristic spectrum of the sample generated in step (5) is compared to determine that the sample is turmeric or any one of the similar products.

2. The chromatographic identification method according to claim 1, characterized in that: In step (1), the medicinal material origin of the Curcuma zedoaria includes one or more of Curcuma wenyujin, Curcuma guangxiensis, and Curcuma pengshu; the medicinal material origin of the Curcuma zedoaria includes one or more of Curcuma wenyujin and Curcuma guangxiensis; The extraction solvent is methanol or a methanol aqueous solution, preferably a methanol aqueous solution with a volume concentration of 30% to 70%; the extraction method is any one of ultrasonic treatment, shaking extraction or heating reflux extraction; preferably, the extraction method is ultrasonic extraction, with a power of 200W to 300W, a frequency of 35kHz to 45kHz, and an extraction time of 5min to 60min.

3. The chromatographic identification method according to claim 1 or 2, characterized in that: The preparation of the reference solution in step (2) includes dissolving the reference substance in a methanol aqueous solution or methanol with a methanol volume percentage of not less than 50% to prepare one or more of a curcumin reference solution, a zedoaryldiol reference solution, a zedoaryl oxadiol reference solution, and a zedoaryl bicycloenone reference solution with a concentration of 10 to 40 μg / mL.

4. The chromatographic identification method according to any one of claims 1 to 3, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography in step (3) include: Stationary phase: chromatographic column with octadecyl bonded silica gel as filler, Mobile phase: acetonitrile is used as mobile phase A, and an aqueous solution of phosphoric acid is used as mobile phase B; preferably, a phosphoric acid solution with a volume fraction of 0.05% to 0.20% is used as mobile phase B; Also included is a gradient elution program, wherein the gradient elution program is: Preferably, the method further comprises: a column temperature of 20 to 30° C., and / or a flow rate of 0.28 to 0.32 mL / min, and / or a detection wavelength of 220 to 270 nm.

5. The chromatographic identification method according to any one of claims 1 to 4, characterized in that: The characteristic spectrum of the turmeric sample in step (3) includes 9 characteristic peaks, wherein peak 1 is the peak of curcuma oxadiol, peak 3 is the peak of curcuma diol, and peak 9 is the peak of curcuma bicycloenone.

6. The chromatographic identification method according to any one of claims 1 to 5, characterized in that: The formulation of the identification criteria in step (4) also includes: Characteristic peaks are determined from the characteristic spectrum of the turmeric slice sample and the characteristic spectrum of the similar product sample respectively, and the characteristic peaks in the characteristic spectrum of the turmeric slice sample are attributed by liquid chromatography-mass spectrometry technology, and the distinguishing components are identified, and the differences of the characteristic peaks in each characteristic spectrum are compared, and the differences are recorded to obtain the identification standard.

7. The chromatographic identification method according to claim 6, characterized in that: The differences include: the curcumin peak can distinguish turmeric, the relative peak area of ​​the curcuminone diol peak and the curcuma diol peak can distinguish turmeric and turmeric, and the relative peak area of ​​the curcuma bicycloenone peak and the curcuma diol peak can distinguish turmeric and turmeric.

8. The chromatographic identification method according to any one of claims 1 to 7, characterized in that: The step (6) of comparing the characteristic spectrum of the test sample generated in step (5) includes: If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to that of the curcumin reference substance, the test sample is turmeric; If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area of ​​the peak of curcuma oxadiol and the peak of curcuma diol is less than 2.4, then the test sample is curcuma; If the characteristic spectrum of the test sample has a chromatographic peak at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area between the peak of curcuma bicycloenone and the peak of curcuma diol is higher than 6.2, then the test sample is Curcuma aromatica; If the characteristic spectrum of the test sample has chromatographic peaks at the retention time corresponding to the reference substances of curcuma oxadiol, curcuma diol, and curcuma bicycloenone, and the relative peak area of ​​the curcuma oxadiol peak to the curcuma diol peak is higher than 2.4; the relative peak area of ​​the curcuma bicycloenone peak to the curcuma diol peak is lower than 6.2, then the test sample is turmeric.

9. Use of the method according to any one of claims 1 to 8 in identifying two or more of Curcuma longa, Curcuma longa, Curcuma zedoaria and / or Curcuma aromatica.

10. Use of the method according to any one of claims 1 to 8 in quality inspection of turmeric slices and its similar products.

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