Method for establishing fingerprint spectrum of tuckahoe and cinnamon mixed plant solid beverage and determining component content of tuckahoe and cinnamon mixed plant solid beverage

Abstract

The invention relates to a fingerprint establishment method, a detection method and a content determination method of a traditional Chinese medicine composition mild drink. The traditional Chinese medicine composition comprises the following raw materials: lily, pawpaw, Chinese yam, poria cocos, cinnamon, orange peel, rhizoma polygonati, radix polygonati officinalis and mulberry. The fingerprint establishment method comprises the following steps: preparing a reference solution and a test solution; detecting the reference substance solution and the test solution by high performance liquid chromatography; a mobile phase adopted by the high performance liquid chromatography is an acetonitrile and formic acid aqueous solution, and the elution mode is gradient elution. According to the fingerprint spectrum of the traditional Chinese medicine composition and the content determination method, the traditional Chinese medicine composition can be effectively represented, the content of various components can be simply, conveniently and rapidly determined, and richer chemical information and more comprehensive basis are provided for quality evaluation of the traditional Chinese medicine composition.

Description

Technical Field

[0001] The invention belongs to the technical field of quality analysis of traditional Chinese medicines, and particularly relates to the establishment of a high performance liquid chromatography (HPLC) fingerprint of a tuckahoe, cinnamon mixed plant solid beverage, a determination method and a component content determination method thereof. Background Art

[0002] Constitution in traditional Chinese medicine refers to the comprehensive and relatively stable inherent characteristics of the human body in terms of morphological structure, physiological function and psychological state formed on the basis of innate endowment and acquired acquisition. It is an individual characteristic of the human body that is formed in the process of growth and development and adapted to the natural and social environment. Studies on constitution in traditional Chinese medicine have found that constitution is correlated with disease, and constitutional factors affect the occurrence and syndrome type of disease, as well as the progression of syndromes and the prognosis of disease. Although constitution has its stability, it is also variable. Scientific intervention in constitution is conducive to the prevention and cure of diseases. According to the main clinical manifestations of overall characteristics, the current study divides constitution into nine types. Among them, people with a peaceful constitution have a harmonious balance of yin and yang, qi and blood, and are characterized by moderate body shape, ruddy complexion, and high energy. It is a relatively healthy constitution, and the focus of this constitution is maintenance.

[0003] The trade name of the mixed plant solid beverage of Poria cocos and cinnamon is "Pinghe Drink". It is a functional food specially developed by Yabao Jiuhe (Beijing) Health Management Co., Ltd. for people with a peaceful constitution. It is mainly composed of lily, papaya, yam, Poria cocos, cinnamon, tangerine peel, Polygonatum odoratum, Polygonatum odoratum, mulberry and other raw materials. In the formula, lily nourishes yin and moistens the lungs, clears the heart and calms the mind; papaya relaxes the muscles and activates the collaterals, harmonizes the stomach and eliminates dampness; yam nourishes the spleen and nourishes the stomach, promotes fluid and benefits the lungs, nourishes the kidney and astringes the essence; Poria cocos promotes diuresis and eliminates dampness, strengthens the spleen, and calms the heart; cinnamon replenishes fire and helps yang, guides fire back to the origin, dispels cold and relieves pain, warms and unblocks the meridians; tangerine peel regulates qi and strengthens the spleen, dries dampness and eliminates phlegm; Polygonatum odoratum replenishes qi and nourishes yin, strengthens the spleen, moistens the lungs, and benefits the kidneys; Polygonatum odoratum nourishes yin and moistens dryness, promotes fluid and quenches thirst; mulberry nourishes yin and replenishes blood, promotes fluid and moistens dryness. Each ingredient in the formula performs its own duties and can be used as an auxiliary diet therapy to maintain a peaceful constitution.

[0004] Pinghe Drink (Poria cocos, cinnamon mixed plant solid beverage) is a new product developed by the applicant. It is made of a variety of main raw materials, with complex ingredients and few quality inspection methods. There is currently a lack of very rigorous quality control standards, so production is only carried out according to actual conditions, resulting in different production processes, which will have a certain adverse impact on the standardized production of Pinghe Drink.

[0005] Traditional Chinese medicine compositions contain botanicals from a variety of sources and with complex ingredients. The efficacy of traditional Chinese medicine compositions does not come from a single active ingredient, but is the result of the combined action of the raw materials of the entire traditional Chinese medicine composition. Differences in the content and composition ratio of active ingredients in traditional Chinese medicine compositions will affect the efficacy. Due to the complexity of the ingredients of traditional Chinese medicine compositions, it is difficult to effectively evaluate the quality of traditional Chinese medicines with any single active ingredient or index ingredient. When evaluating the quality of traditional Chinese medicine compositions and their preparations, appropriate detection methods that can provide rich identification information should be used. Current methods such as microscopic identification, physical and chemical identification, and content determination are not sufficient to solve this problem.

[0006] At present, the quality control of Chinese medicine compositions such as plant solid beverages mostly uses a single ingredient or component as an evaluation indicator. There is a problem of a single evaluation indicator, which cannot reflect the overall quality level of plant solid beverages, and its quality stability is difficult to guarantee. Pinghe Drink contains a variety of Chinese medicinal ingredients, and the detectable active ingredients may reach dozens or even more. If the different active ingredients in Pinghe Drink are identified and determined one by one, a lot of screening work is required. There is currently no technical report on the establishment of a fingerprint spectrum of Pinghe Drink to detect the quality, which brings more technical obstacles to the establishment of a fingerprint spectrum detection method for Pinghe Drink.

[0007] The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine. It is mainly used to evaluate the authenticity, quality and stability of the quality of traditional Chinese medicine and semi-finished products of traditional Chinese medicine preparations. It specifically includes two aspects: (1) The authenticity of traditional Chinese medicine can be effectively identified through the characteristic characteristics of the fingerprint; (2) The quality of traditional Chinese medicine can be effectively controlled through the main characteristic peak area and ratio of the fingerprint. The establishment of the fingerprint of traditional Chinese medicine will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then make an overall description and evaluation of the quality of the medicine. This is also in line with the holistic theory of traditional Chinese medicine. On this basis, if further spectrum efficacy research is carried out, the quality of traditional Chinese medicine can be truly combined with its efficacy, which will help to clarify the mechanism of action of traditional Chinese medicine. The fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum that can indicate the chemical characteristics of traditional Chinese medicine or traditional Chinese medicine preparations after appropriate treatment using certain analytical methods. It is a key technology for realizing the overall related quality evaluation of multiple components.

[0008] At present, the fingerprint technology of traditional Chinese medicine has involved many methods, including chromatographic methods such as thin layer scanning (TLCS), high performance liquid chromatography (HPLC), gas chromatography (GC) and high performance capillary electrophoresis (HPCE), as well as spectroscopic methods such as ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR) and X-ray diffraction.

[0009] Therefore, there is a need for a method for quality monitoring of Pinghe Drink products through fingerprint analysis, as well as a standardized production method based on the quality monitoring method. Summary of the invention

[0010] In order to improve the above technical problems, the present invention provides a method for establishing a fingerprint of a mixed plant solid beverage of Poria cocos and cinnamon and determining the content of its components. The method is accurate, reliable, and simple to operate, and provides a basis for the quality control of the solid beverage. The mixed plant solid beverage of Poria cocos and cinnamon is a product with complex ingredients. Whether a high-quality fingerprint can be established and accurate content determination can be performed depends on whether the separation effect of the high-efficiency liquid phase is good. After exploration and verification, the liquid phase conditions provided by the present invention can achieve a very good separation effect, and a variety of components can be separated and peaks can be reflected. This is very important for traditional Chinese medicine products with complex ingredients.

[0011] The medicinal raw materials of the Poria cocos and cinnamon mixed plant solid beverage are lily, papaya, yam, Poria cocos, cinnamon, tangerine peel, Polygonatum sibiricum, Polygonatum odoratum and mulberry.

[0012] The fingerprint construction method or detection method of the mixed plant solid beverage of Poria cocos and cinnamon of the present invention comprises the steps of taking a test solution for high performance liquid phase detection to obtain a fingerprint, and the chromatographic conditions used are: C18 chromatographic column, 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, and the gradient elution program:

[0013]

[0014] In order to make the fingerprint more accurate, the reference solution can be detected under the same liquid phase conditions before and after the test sample is detected, and the chromatographic peak can be identified by the chromatogram of the reference. The reference used in the present invention is hesperidin, and the selection of the reference is based on the UPLC-Q-Exactive-Orbitrap-MS technical detection results of the mixed plant solid beverage of Poria cocos and cinnamon by the inventor in the early stage, and then the literature is consulted to screen the index components of each medicine in the mixed plant solid beverage of Poria cocos and cinnamon, and finally determined after experimental testing and other steps. The reference finally selected has a good peak shape in the chromatogram of the mixed plant solid beverage of Poria cocos and cinnamon. There is no interference from impurity peaks before and after, and the completely separated hesperidin peak is the reference peak of the fingerprint.

[0015] The preparation process of the reference solution is simple. The reference hesperidin and a solvent capable of dissolving hesperidin are taken and dissolved. Methanol and / or ethanol are preferably used in the present invention, and methanol is most preferred. There is no special requirement for the preparation concentration, as long as the upper and lower limit detection requirements of the instrument are met. The preparation concentration given in the embodiment of the present invention is 115 μg / mL to 120 μg / mL, specifically 117.5 μg / mL.

[0016] The test solution needs to be extracted from the mixed plant solid beverage of Poria cocos and cinnamon, and the extraction process is: take the mixed plant solid beverage of Poria cocos and cinnamon, add solvent for extraction. The solvent is a solvent commonly used for natural compound extraction in the art, including but not limited to alcohol or alcohol aqueous solution, such as methanol and ethanol. In some embodiments of the present invention, methanol and 60%-80% ethanol aqueous solution are used, specifically methanol.

[0017] Because the target components of the Poria cocos and cinnamon mixed plant solid beverage are all components with good solubility in alcohol and alcohol water, the conventional natural compound extraction method in the art can be used, such as ultrasonic extraction and reflux extraction. The embodiment of the present invention specifically adopts a water bath reflux process, and the test methanol is heated and refluxed in a water bath at 80 degrees Celsius for 1 hour. Comparing several different extraction methods, it is found that the chromatogram extracted by methanol water bath reflux has a relatively stable baseline, a higher chromatographic peak, and a better peak shape, which is the best way.

[0018] After the extraction is completed, the extract is filtered, the filtrate is evaporated, and the residue is dissolved with a re-dissolving solvent (for example, about 5 ml of methanol) and filtered to obtain a subsequent filtrate. The re-dissolving solvent is preferably the same as the extraction solvent, and the specific solution concentration after re-dissolving does not need to be forced to be within a certain range, as long as the equipment can detect the components. A more suitable range can be selected: Weight of Poria cocos and cinnamon mixed plant solid beverage: Volume of re-dissolving solvent = (0.5-1.5) g: 1 mL, for example 0.5 g, 1 g or 1.5 g of Poria cocos and cinnamon mixed plant solid beverage / mL.

[0019] The chromatogram obtained under the above conditions has 18 characteristic peaks, among which peak 16 is hesperidin. The fingerprint spectrum uses the chromatographic peak of peak 16 hesperidin as a reference (S). The relative retention times of the 17 common characteristic peaks are:

[0020]

[0021]

[0022] Usually, after the fingerprint spectrum determines the characteristic peaks in the traditional Chinese medicine product, the requirement can be met if the characteristic peaks appear at the characteristic peak positions. When there are higher requirements for the fingerprint spectrum, not only the characteristic peak positions are required to meet the requirements, but also the areas of each characteristic peak are required to meet certain requirements, so as to show that the proportion and content of each component in the composition meet certain requirements.

[0023] When the chromatographic peak of hesperidin peak 16 is used as the reference (S), the relative peak areas of the common characteristic peaks are:

[0024] Peak Relative peak area 1 0.11-0.17 2 0.22～0.33 3 0.07～0.09 4 0.02～0.03 5 0.03～0.04 6 0.02～0.03 7 0.05～0.06 8 0.03～0.04 9 0.05～0.06 10 0.14～0.16 11 0.05～0.06 12 0.09～0.10 13 0.06～0.08 14 0.03～0.04 15 0.36～0.37 16 1 17 0.04～0.05 18 0.03～0.04

[0025] When preparing the fingerprint, in order to prepare the fingerprint more accurately, the chromatogram peak can be identified by the chromatogram of the reference substance, and the fingerprint of the mixed plant solid beverage of Poria cocos and cinnamon can be prepared by the chromatogram of different batches of the mixed plant solid beverage of Poria cocos and cinnamon. The different batches are preferably at least 5 batches, preferably at least 10 batches. The fingerprint of the mixed plant solid beverage of Poria cocos and cinnamon to be tested is compared with the fingerprint of the mixed plant solid beverage of Poria cocos and cinnamon constructed by the present invention. The relative retention time should be within ±5% of the specified value for qualified products. The similarity evaluation is performed with the fingerprint, and the similarity is greater than 0.8, preferably greater than 0.90 for qualified products.

[0026] The present invention also provides a method for simultaneously determining the contents of multiple components in the Chinese medicine composition Pinghe Drink (Poria cocos, cinnamon mixed plant solid beverage), comprising the following steps:

[0027] (1) Preparation of reference solution:

[0028] (2) Preparation of test solution:

[0029] (3) HPLC detection of content.

[0030] The present invention adopts the same HPLC conditions as the aforementioned fingerprint detection, and can simultaneously detect the contents of protocatechuic acid, chlorogenic acid, ferulic acid and hesperidin in the Poria cocos and cinnamon mixed plant solid beverage under the same chromatographic conditions, with simple and quick operation, low reagent consumption and low cost; by selecting appropriate detection conditions, the method can determine the separation degree, accuracy, repeatability, stability and the like of the contents of each component in the Poria cocos and cinnamon mixed plant solid beverage, which meet the standard requirements.

[0031] The reference substance in the reference substance solution is selected from one or more of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin. The reference substance solution can be a solution of each of the four reference substances, or a solution of a mixture of any two or more of the four reference substances.

[0032] HPLC detection and analysis were performed on the reference solution and test solution of each concentration. The test sample concentration was calculated based on the determination results of the reference and test samples, and the content of the test sample components was calculated: the concentration-peak area standard curve of each reference sample component was drawn; the linear regression equation of each component was calculated with the chromatographic peak area Y of the reference solution component of each concentration as the ordinate and the mass concentration X of the reference solution component as the abscissa, and the test sample concentration was calculated by substituting the HPLC peak area of ​​the test sample into the linear regression equation.

[0033] In one embodiment of the present invention, HPLC detection and analysis are performed on reference solution and test solution of each concentration, and the chromatographic peak in the mixed standard (solution of reference mixture) is qualitatively characterized according to the chromatographic peak of each reference solution, and the concentration of the test sample is calculated according to the measurement results of the reference and the test sample, and the content of the test sample component is calculated: a concentration-peak area standard curve of each component in the mixed standard is drawn; the chromatographic peak area Y of the component of the reference solution of each concentration is used as the ordinate, and the mass concentration X of the component of the reference solution is used as the abscissa, and the linear regression equation of each component is calculated, and the concentration of the test sample is calculated according to the HPLC peak area of ​​the test sample.

[0034] For example, in the content determination process given in the embodiment of the present invention, the linear regression equation is first calculated:

[0035] The linear regression equation of protocatechuic acid is: y = 11480x + 335.7, with a linear range of 2.57 to 12.85 μg / mL; the linear regression equation of chlorogenic acid is: y = 13150x + 292, with a linear range of 1.54 to 7.7 μg / mL; the linear regression equation of ferulic acid is: y = 32176x–199.5, with a linear range of 1.24 to 6.2 μg / mL; the linear regression equation of hesperidin is: y = 17998x + 2934.8, with a linear range of 23.5 to 117.5 μg / mL. In the above linear regression equation, y is the peak area of ​​the corresponding component, and x is the mass concentration of the component in μg / mL. Then, the content of the corresponding substance in the test sample is calculated according to the linear regression equation and the peak area of ​​the test sample obtained by the determination.

[0036] When determining the content in this field, the area comparison method of the test sample and the standard sample can also be used for calculation, for example, the formula: S 供试品 / S 标准品 =Concentration 供试品 / concentration 标准品 The test sample concentration was calculated by .

[0037] For the content determination of the traditional Chinese medicine composition with complex ingredients of the present invention, it is more suitable to use the linear regression equation to calculate the content of each ingredient.

[0038] The reference substances are dissolved in organic solvents to obtain solutions of the reference substances; or multiple reference substances are dissolved in the same organic solvent to obtain solutions of the reference substances, and the organic solvent can be selected from methanol and / or ethanol, preferably methanol. When the reference substance solution is a series of concentration reference substance solutions, the concentration ranges of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin are 2 to 13 μg / mL, 1 to 8 μg / mL, 1 to 7 μg / mL, and 20 to 120 μg / mL, respectively; the series concentrations of each reference substance can be increased arithmetic or geometrically.

[0039] In one embodiment of the present invention, the method for preparing the series of concentrations of the reference substance solution comprises: preparing a solution of a mixture of four reference substances, wherein the concentrations of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin in the solution are 12.85 μg / mL, 7.7 μg / mL, 6.2 μg / mL, and 117.5 μg / mL, respectively; performing arithmetic dilution on the solution to obtain a series of protocatechuic acid concentrations of 2.57 μg / mL, 5.14 μg / mL, 7.71 μg / mL, 10.28 μg / mL, 12. 85μg / mL; the concentration series of chlorogenic acid were 1.54μg / mL, 3.08μg / mL, 4.62μg / mL, 6.16μg / mL, 7.7μg / mL; the concentration series of ferulic acid were 1.24μg / mL, 2.48μg / mL, 3.72μg / mL, 4.96μg / mL, 6.2μg / mL; the concentration series of hesperidin were 23.5μg / mL, 47μg / mL, 70.5μg / mL, 94μg / mL, 117.5μg / mL.

[0040] The method for preparing the test solution is the same as the method for preparing the fingerprint spectrum, but in order to accurately calculate the content, the tuckahoe cinnamon mixed plant solid beverage particles need to be accurately weighed. The ratio of the tuckahoe cinnamon mixed plant solid beverage particles to the re-dissolving solvent after extraction is controlled in the range of 1.0g: 0.5ml-1.5ml, preferably 1.0g: 0.9ml-1.1ml.

[0041] The chromatographic column used in the method for preparing fingerprint and content determination of the present invention is a commonly used C18 chromatographic column. Under more optimized conditions, the chromatographic column used is a 250mm×4.6mm, 3-5μm chromatographic column. According to the high performance liquid phase detection of the present invention, the C18 chromatographic column can be AQ-C18 or WELCH AQ-C18 chromatographic column, in one embodiment of the present invention, is WELCH AQ-C18 chromatographic column, for example, C 18 Column (4.6mm×250mm, 5μm). The number of theoretical plates calculated by hesperidin should not be less than 5000, and the chromatograms of different batches of tuckahoe cinnamon mixed plant solid beverage and the chromatograms of the reference substance were obtained. AQ-C18 column (4.6 mm × 250 mm, 5 μm) and WELCH AQ-C18 chromatographic column (4.6mm×250mm, 5μm), it was found that in WELCH The peak shapes of the components in the chromatogram separated by the AQ-C18 column (4.6 mm × 250 mm, 5 μm) are better and have the best separation degree, so it can be used as the preferred column for subsequent research.

[0042] The peaks of multiple components can basically be realized within the wavelength range of 280-286nm, which can meet the requirements of fingerprint preparation. Taking the four reference substances of the present invention as an example, the present invention uses a PDA ultraviolet detector to perform full-wavelength scanning on a mixed reference substance containing four components. Among them, the reference peak hesperidin has the best absorption effect at 283nm, and protocatechuic acid, chlorogenic acid and ferulic acid have maximum absorption wavelengths at 210nm, 219nm and 207nm, respectively. By comparison, it was found that there was no significant difference between the chromatographic peak response values ​​of protocatechuic acid, chlorogenic acid and ferulic acid at 283nm and the response values ​​at their respective corresponding maximum absorption wavelengths. Taking the above factors into consideration, the present invention selects 283nm or a wavelength near it as the detection wavelength, and the spectral information of each compound can be seen in Figure 3 and Figure 4 .

[0043] There is usually no fixed limit on the injection volume of HPLC, as long as the operation is convenient and the equipment meets the basic detection limit. When the peak area accuracy requirement is relatively high, the injection volume of each injection can be kept the same as much as possible during the content determination, especially the injection volume should be kept consistent each time during the content determination. The injection volume can be 5.0-20.0μL according to the conventional injection volume of HPLC method, for example, 10μL.

[0044] The flow rate of the mobile phase can be adjusted according to the equipment performance and work efficiency, provided that the peaks of each component are well separated. Too high a flow rate will result in large equipment losses, while too low a flow rate will take a long time. The embodiment of the present invention uses the most commonly used flow rate of 1 ml.min according to the conditions of the instrument and the work efficiency. -1 , and around the flow rate 0.8-1.3ml.min -1 range for detection.

[0045] The present invention does not have temperature-sensitive substances and operations, so there is no special restriction on the column temperature, and normal detection can be achieved under normal laboratory conditions. Therefore, it is recommended that the HPLC detection environment be 20-40°C, for example, 30°C or 25°C in the embodiment of the present invention.

[0046] The separation effect can be ensured within the range of gradient elution conditions given above in the present invention. The most preferred gradient conditions are:

[0047]

[0048]

[0049] In the present invention, "optional", "optionally" or "optionally present" means that the subsequently described event or circumstance can but does not have to occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0050] In the present invention, "include" and "comprise" are both open expressions, that is, including the contents specified in the present invention, but not excluding other aspects. It should be understood that "include" and "comprise" can include closed meanings, that is, "consisting of..."

[0051] Advantageous Effects of the Invention

[0052] (1) The present invention successfully established the fingerprint of the mixed plant solid beverage of Poria cocos and cinnamon by optimizing the HPLC chromatographic conditions, identified 18 common peaks in total, and simultaneously detected the contents of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin in the mixed plant solid beverage of Poria cocos and cinnamon, laying the foundation for the establishment of a comprehensive quality standard for the mixed plant solid beverage of Poria cocos and cinnamon.

[0053] (2) The HPLC fingerprint determination method of the mixed plant solid beverage of Poria cocos and cinnamon was proved to be reliable by analyzing 10 batches of the mixed plant solid beverage of Poria cocos and cinnamon. The quality was comprehensively evaluated, and the precision, repeatability and stability experiments verified that the method of the present invention has high stability and credibility, so it can be used as the determination method of the fingerprint of the mixed plant solid beverage of Poria cocos and cinnamon.

[0054] (3) The present invention adopts the same chromatographic conditions to simultaneously detect the contents of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin in the mixed plant solid beverage of Poria cocos and cinnamon, which is simple and quick to operate, with low reagent consumption and low cost. By selecting appropriate detection conditions, the method can determine the separation degree, accuracy, repeatability, stability, etc. of the content of each component in the mixed plant solid beverage of Poria cocos and cinnamon, which meet the standard requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 A in the middle is the chromatogram of hesperidin reference substance; Figure 1 B in the middle is the fingerprint of the mixed plant solid beverage of Poria cocos and cinnamon; Figure 1 Figure C is an overlay of HPLC fingerprints of 10 batches of Poria cocos and cinnamon mixed plant solid beverage.

[0056] Figure 2 A in the figure is the chromatogram of the upper limit of gradient elution; Fig. 9 B is the chromatogram of the lower limit of gradient elution.

[0057] Figure 3 A and B in the figure are the ultraviolet absorption diagrams of protocatechuic acid and chlorogenic acid in the wavelength range of 200-400nm, respectively.

[0058] Figure 4 C and D in the figure are the ultraviolet absorption diagrams of ferulic acid and hesperidin in the wavelength range of 200-400nm, respectively.

[0059] Figure 5 These are the fingerprints of the Poria cocos and cinnamon mixed plant solid beverage obtained at different detection wavelengths, where A, B, and C are the fingerprints of the Poria cocos and cinnamon mixed plant solid beverage at detection wavelengths of 283nm, 210nm, and 219nm, respectively.

[0060] Figure 6 These are the fingerprints of the Poria cocos and cinnamon mixed plant solid beverage obtained under different elution conditions, where A and B are the fingerprints of the Poria cocos and cinnamon mixed plant solid beverage under elution conditions one and two, respectively.

[0061] Figure 7 The fingerprints of the mixed plant solid beverage of Poria cocos and cinnamon obtained under different chromatographic columns, where A is Fingerprint of mixed plant solid beverage of Poria cocos and cinnamon under AQ-C18 column conditions; B is WELCH Fingerprint of Poria cocos and cinnamon mixed plant solid beverage under AQ-C18 chromatographic column conditions.

[0062] Figure 8 These are the fingerprints of the Poria cocos and cinnamon mixed plant solid beverage obtained under different sample extraction methods, where A, B, and C are the fingerprints of the Poria cocos and cinnamon mixed plant solid beverage under extraction methods A, B, and C, respectively.

[0063] Fig. 9 A in the middle is the chromatogram of the test solution of the mixed plant solid beverage of Poria cocos and cinnamon; Fig. 9 B is the chromatogram of the mixed reference solution (in the figure: 1-protocatechuic acid; 2-chlorogenic acid; 3-ferulic acid; 4-hesperidin). DETAILED DESCRIPTION

[0064] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0065] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0066] Experimental instruments and reagents:

[0067] 1. Instrument

[0068] High performance liquid chromatography (model: LC-20A, Shimadzu Corporation, Japan), magnetic stirring water bath (model: LC-WB-8+, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); XS105 electronic balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.); Milli-Q ultrapure water purification system (Beijing Wuzhou Oriental Technology Co., Ltd.).

[0069] 2. Drug reagents

[0070] Protocatechuic acid (purity ≥ 98%, batch number 110809-202207, China Food and Drug Inspection Institute), chlorogenic acid (purity ≥ 98%, batch number 110753-202119, China Food and Drug Inspection Institute), ferulic acid (purity ≥ 98%, batch number 110773-202316, China Food and Drug Inspection Institute), hesperidin (purity ≥ 98%, batch number 110721-202220, China Food and Drug Inspection Institute Research Institute); 10 batches of Poria cocos and cinnamon mixed plant solid beverage (S1-S10), with batch numbers 202204171, 202201221, 202110211, 202104161, 202101311, 2007091, 2005061, 2003141, 2001141, 1909181, source Shanxi Yabao Jiuhe Pharmaceutical Technology Co., Ltd.; chromatographic grade acetonitrile; chromatographic grade methanol; ultrapure water.

[0071] Example 1 Establishment of fingerprint of mixed plant solid beverage of Poria cocos and cinnamon

[0072] The fingerprint was prepared according to the following conditions and the results are shown in Figure 1 .

[0073] 1. Chromatographic conditions

[0074] HPLC fingerprint analysis conditions were adopted by WELCH AQ-C18 chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase A is water (0.1% formic acid in water), mobile phase B is acetonitrile; gradient elution; flow rate is 1.0 ml min -1 ; Column temperature 30℃; Injection volume 10μL, detection wavelength 283nm.

[0075] Table 1 Gradient elution method

[0076]

[0077]

[0078] 2. Preparation of reference solution

[0079] Take the hesperidin reference substance, weigh it accurately, and add methanol to prepare a hesperidin reference substance solution of 117.50 μg / ml.

[0080] 3. Preparation of test solution

[0081] Take about 5.0 g of the granules of this product, weigh accurately, place in a stoppered conical flask, add an appropriate amount of methanol, heat under reflux in a water bath at 80 degrees Celsius for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5 ml of methanol to the residue to dissolve, filter, and take the filtrate to obtain the product.

[0082] 4. HPLC fingerprint analysis of Poria cocos and cinnamon mixed plant solid beverage

[0083] 4.1 Precision test

[0084] Take the same batch (batch number: 202204171) of Poria cocos and cinnamon mixed plant solid beverage test sample, inject it 6 times continuously, record the retention time and peak area of ​​each common chromatographic peak, and use the retention time and peak area of ​​peak 16 hesperidin as a reference to convert the relative retention time and relative peak area of ​​each common peak to examine the consistency of the relative retention time and relative peak area ratio of the chromatographic peak. The results show that the relative retention time RSD of each common peak is less than 1.36%, and the relative peak area RSD is less than 3.82%, which shows that the determination method of the present invention is stable, the system tightness is good, and the method is stable and reliable.

[0085] 4.2 Stability test

[0086] Take the same batch (batch number: 202204171) of Poria cocos and cinnamon mixed plant solid beverage test sample, and analyze the sample at different time points (0h, 2h, 4h, 6h, 8h, 12h, 24h), record the retention time and peak area of ​​each common chromatographic peak, and use the retention time and peak area of ​​peak 16 hesperidin as a reference to convert the relative retention time and relative peak area of ​​each common peak to investigate the consistency of the relative retention time and relative peak area ratio of the chromatographic peak. The results show that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD is less than 3.15%. This shows that the Poria cocos and cinnamon mixed plant solid beverage sample of the present invention has good stability under experimental conditions, and the reliability of the analysis results can be guaranteed within 24h.

[0087] 4.3 Repeatability test

[0088] Take the same batch (batch number: 202204171) of Poria cocos and cinnamon mixed plant solid beverage test sample, prepare 6 test sample solutions in parallel according to the method under "3", inject and analyze them separately, and record the retention time and peak area of ​​each common chromatographic peak. Take the retention time and peak area of ​​peak 16 hesperidin as reference, and convert the relative retention time and relative peak area of ​​each common peak to examine the consistency of the relative retention time and relative peak area ratio of the chromatographic peak. It shows that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD is less than 3.76%, which shows that the method of the present invention is stable and reliable, and is conducive to promotion.

[0089] 4.4 Establishment and similarity evaluation of HPLC fingerprint of Poria cocos and cinnamon mixed plant solid beverage

[0090] Take 10 batches of Poria cocos and cinnamon mixed plant solid beverage (batch number: 202204171, 202201221, 202110211, 202104161, 202101311, 2007091, 2005061, 2003141, 2001141, 1909181), prepare the test solution according to the method under "3", and inject and analyze, and record the chromatograms respectively. Select a representative spectrum (Poria cocos and cinnamon mixed plant solid beverage with batch number 202204171) as the reference fingerprint, and use the "Chinese medicine chromatographic fingerprint similarity evaluation system" recommended by the Pharmacopoeia Commission for analysis, use chromatogram similarity as the fingerprint evaluation index, and use multi-point correction full spectrum matching to generate the common mode of the fingerprint. The similarities between each batch of samples and the control fingerprint were (0.999, 0.999, 0.997, 0.997, 0.996, 0.996, 0.996, 0.995, 0.996, 0.995), respectively.

[0091] 4.5 Identification of common peaks in fingerprints, calculation of relative peak areas and relative retention times

[0092] Using the similarity evaluation system software, 18 peaks were detected in 10 batches of fingerprints. Compared with the reference substance, peak 16 was calibrated as hesperidin, and its retention time was 84.700min. Peak 16 has a good peak shape, no impurity peak interference before and after, and complete separation, so it was selected as a reference peak to calculate the relative peak area and relative retention time of other common peaks. The results are shown in Tables 2 and 3 below.

[0093] Table 2 Relative peak areas of 18 common peaks in 10 batches of samples (S1-S10)

[0094]

[0095] Table 3 Relative retention times of 18 common peaks in 10 batches of samples (S1-S10)

[0096]

[0097]

[0098] 4.6 Range of gradient elution conditions

[0099] By adjusting the conditions of gradient elution, it can be found that under the currently determined elution conditions, the ratio of mobile phases A and B can be appropriately adjusted, and the upper and lower limits of the elution conditions can be determined through experiments.

[0100] Upper elution condition:

[0101]

[0102] Lower limit elution condition:

[0103]

[0104]

[0105] Results Figure 2 , Figure 2 A is the upper limit elution condition of the mobile phase. Figure 2 B is the lower limit elution condition of the mobile phase. Both conditions meet the effect of good separation and complete elution.

[0106] Example 2 Comparison of Fingerprint Preparation Methods of Poria Cocos and Cinnamon Mixed Plant Solid Beverage

[0107] 1. Determination of chromatographic conditions and exploration of fingerprint methods

[0108] The fingerprint results of Pinghe Drink prepared under different HPLC chromatographic conditions were investigated and screened.

[0109] (1) Detection wavelength

[0110] Prepare and test according to the method of Example 1. Take about 5.0g of the granules of this product, weigh accurately, put into a stoppered conical flask, add an appropriate amount of methanol, heat and reflux in a water bath at 80 degrees Celsius for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5ml of methanol to the residue to dissolve, filter, and take the filtrate.

[0111] HPLC fingerprint analysis conditions were adopted by WELCH AQ-C18 chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase A is water (0.1% formic acid in water), mobile phase B is acetonitrile; gradient elution; flow rate is 1.0 ml min -1; column temperature 30℃; injection volume 10μL, analysis was performed at detection wavelengths of 283nm, 210nm and 219nm, respectively. The results are shown in Figure 5 .

[0112] It can be seen that with 283nm as the detection wavelength, the chromatographic information is richer, the baseline is more stable, the response values ​​of each peak are more balanced, and it can relatively comprehensively reflect the component information in the sample. Therefore, 280-286nm was selected as the detection wavelength of the fingerprint spectrum of the mixed plant solid beverage sample of Poria cocos and cinnamon, and the optimal condition was 283nm.

[0113] (2) Elution conditions

[0114] The inventors compared several different elution conditions, and the results showed that the mobile phase elution conditions currently selected by the present invention are the best conditions. Due to the large number of elution conditions, the following is a brief example: Take the particles of this product, prepare them according to the method for preparing the test solution in Example 1, and compare the following two elution conditions according to the chromatographic conditions in (1) detection wavelength. The results are shown in Figure 6 It can be seen that in the chromatogram under elution condition 1, the peak shapes of each component are good and have good separation. Therefore, elution condition 1 is selected as the elution condition for the fingerprint of the mixed plant solid beverage sample of Poria cocos and cinnamon.

[0115] Elution condition 1:

[0116]

[0117] Elution condition 2:

[0118]

[0119] (3) Column type

[0120] Take the granules of this product and, according to the preparation method and chromatographic conditions of the test solution in Example 1, use chromatographic columns AQ-C18 (4.6mm×250mm, 5μm) and WELCH AQ-C18 (4.6mm×250mm, 5μm) was used for analysis. The results are shown in Figure 7 It can be seen that both can meet the test of fingerprint spectrum and can be used as chromatographic columns for preparing fingerprint spectrum. The peak shape of each component of the chromatogram separated by AQ-C18 column (4.6mm×250mm, 5μm) is good, and the separation degree is good. AQ-C18 chromatographic column (4.6mm×250mm, 5μm) was used as the chromatographic column for the fingerprint of the test sample of the mixed plant solid beverage of Poria cocos and cinnamon.

[0121] 3. Determination of sample extraction method

[0122] Pingheyin has complex ingredients, and different extraction solvents will affect the extraction effect of the active ingredients. Finding the most suitable extraction method is critical, and extracting as many ingredients as possible and showing peaks in the HPLC graph is one of the technical difficulties.

[0123] The samples were extracted using methods A, B, and C. The processes are as follows:

[0124] A: Take about 5.0g of the granules of this product, weigh accurately, place in a stoppered conical flask, add appropriate amount of methanol, heat under reflux in a water bath at 80 degrees Celsius for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5ml of methanol to the residue to dissolve, filter, and take the filtrate to obtain the product.

[0125] B: Take about 5.0g of the granules of this product, weigh accurately, place in a stoppered conical flask, add an appropriate amount of methanol, ultrasonically treat for 1h, cool, filter, evaporate the filtrate to dryness, add 5ml of methanol to the residue to dissolve, filter, and take the filtrate to obtain.

[0126] C: Take about 5.0g of the granules of this product, weigh accurately, place in a stoppered conical flask, add an appropriate amount of 70% ethanol, heat under reflux in a water bath at 80 degrees Celsius for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5ml of 70% ethanol to the residue to dissolve it, filter, and take the filtrate to obtain the product.

[0127] The test solutions prepared by the above three methods were tested according to the chromatographic conditions finally determined above. The results are as follows: Figure 8 As shown, from Figure 8 It can be seen that all three methods meet the requirements for preparing fingerprints and can be used as extraction methods for preparing fingerprints or content determination. However, after comparison, it was found that the methanol water bath reflux extraction method was the best. This method obtained the most stable chromatogram baseline, the best chromatographic peak separation effect, and the best peak shape.

[0128] Example 3 Method for determining the contents of four components in a mixed plant solid beverage containing tuckahoe, cinnamon and cinnamon

[0129] 1. Chromatographic conditions

[0130] Same as Example 1.

[0131] 2. Preparation of reference solution

[0132] Take appropriate amount of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin reference substances respectively, weigh accurately, and add methanol to prepare a mixed reference substance solution containing 12.85 μg / mL of protocatechuic acid, 7.70 μg / mL of chlorogenic acid, 6.20 μg / mL of ferulic acid, and 117.50 μg / mL of hesperidin.

[0133] At the same time, appropriate amounts of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin reference substances were taken and accurately weighed, and methanol was added to prepare single solutions of each reference substance with a concentration of 12.85 μg / mL for protocatechuic acid, 7.70 μg / mL for chlorogenic acid, 6.20 μg / mL for ferulic acid, and 117.50 μg / mL for hesperidin.

[0134] 3. Preparation of test solution

[0135] Take about 5.0 g of the granules of this product, weigh accurately, place in a stoppered conical flask, add an appropriate amount of methanol, heat under reflux in a water bath at 80 degrees Celsius for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5 ml of methanol to the residue to dissolve, filter, and take the filtrate to obtain the product.

[0136] 4. Content determination and methodological investigation of mixed plant solid beverage of Poria cocos and cinnamon

[0137] 4.1 Precision test

[0138] The same batch number (202204171) of Poria cocos and cinnamon mixed plant solid beverage was taken for testing, and the samples were injected 6 times continuously, and the peak areas were recorded respectively. After calculation, it was found that the RSD values ​​of the peak areas of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin were 0.63%, 1.7%, 0.68%, and 0.77%, respectively, which showed that the determination method of the present invention had good precision.

[0139] 4.2 Investigation of linear relationship

[0140] According to the chromatographic conditions under item "1" above, take a single solution of each reference substance for chromatographic detection, and then perform chromatographic detection on the solution of the reference substance mixture. Compare the chromatographic peaks of the single reference substance to confirm the ownership of the chromatographic peaks of the reference substance mixture, and then directly perform chromatographic analysis on the solution of the reference substance mixture to simultaneously determine the four components and obtain a linear equation.

[0141] Accurately measure 1mL, 2mL, 3mL, 4mL, and 5mL of the mixed reference solution, place them in 5mL volumetric flasks, dilute to the mark with methanol, and prepare mixed reference solutions of a series of concentrations. Analyze and measure according to the chromatographic conditions under the above "1", and record the liquid chromatogram. Use the chromatographic peak area (Y) of the mixed reference solution of each concentration as the ordinate and the concentration (X, μg / mL) of each mixed reference solution as the abscissa to draw the standard curve, and calculate the regression equation and correlation coefficient of each component, as shown in Table 4.

[0142] Table 4 Regression equations, correlation coefficients and linear ranges of the four chemical components

[0143]

[0144] 4.3 Stability test

[0145] Take the same batch number (202204171) of Poria cocos and cinnamon mixed plant solid beverage test sample, and according to the chromatographic conditions under item "1", sample analysis was performed when the test solution was placed for 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, respectively, and the peak areas were recorded respectively. The RSD values ​​of the peak areas of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin were calculated to be 1.12%, 0.58%, 0.79%, and 0.76%, respectively, indicating that the test solution of the present invention is stable within 24h.

[0146] 4.4 Repeatability test

[0147] Take the same batch number (202204171) of Poria cocos and cinnamon mixed plant solid beverage test sample, prepare 6 test sample solutions in parallel according to the method under "3", and analyze them according to the chromatographic conditions under "1", record the peak areas respectively, and calculate the contents of each component according to the linear regression equation of each component: protocatechuic acid 11.99 μg / mL, chlorogenic acid 7.38 μg / mL, ferulic acid 5.87 μg / mL, hesperidin 106.47 μg / mL, and the peak area RSD values ​​are 1.23%, 0.93%, 1.26%, and 0.76%, respectively, indicating that the method of the present invention has good repeatability.

[0148] 4.5 Sample recovery rate

[0149] Take a known content of Poria cocos and cinnamon mixed plant solid beverage (202204171), pour out the contents, accurately weigh 2.50g, and add 1mL of mixed reference solution containing 30μg / mL protocatechuic acid, 20μg / mL chlorogenic acid, 15μg / mL ferulic acid, and 265μg / mL hesperidin to each of the 6 parallel portions. Prepare the test solution according to the method under "3", and inject and analyze according to the chromatographic conditions under "1", record the peak area, and calculate the recovery rate of each component by the linear regression equation of each component. The results are shown in Table 5. The calculation method of "sample content" in Table 5 is: multiply the content of each component measured in item 4.4 by 5ml and divide by 2.

[0150] Table 5 The recovery results of the four components of the mixed plant solid beverage of Poria cocos and cinnamon (n=6)

[0151]

[0152]

[0153] 4.6 Sample determination

[0154] Take 10 batches of Poria cocos and cinnamon mixed plant solid beverage test solution, and analyze the sample according to the chromatographic conditions under item "1". Record the peak area of ​​each component respectively, and calculate the content of protocatechuic acid, chlorogenic acid, ferulic acid and hesperidin by the linear regression equation of each component. The results are shown in Table 6 below.

[0155] Table 6 Determination results of the contents of four components in 10 batches of Poria cocos and cinnamon mixed plant solid beverages (μg / mL, n=3)

[0156]

[0157] In summary, the present invention successfully established the fingerprint of the Poria cocos and cinnamon mixed plant solid beverage by optimizing the HPLC chromatographic conditions, determined a total of 18 common peaks, and could simultaneously detect the contents of protocatechuic acid, chlorogenic acid, ferulic acid and hesperidin in the Poria cocos and cinnamon mixed plant solid beverage; the methodological verification showed that all indicators of the detection method of the present invention were good, which improved the quality control standard of the Poria cocos and cinnamon mixed plant solid beverage and provided a reference basis for the establishment of its comprehensive quality standard.

[0158] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fingerprint construction or detection method for a Chinese medicine composition of a mixed plant solid beverage of Poria cocos and cinnamon, wherein the raw materials of the Chinese medicine composition are composed of: lily, papaya, yam, Poria cocos, cinnamon, tangerine peel, polygonatum, polygonatum odoratum, and mulberry, and the test solution is subjected to high performance liquid chromatography to obtain a fingerprint; The chromatographic conditions are as follows: the chromatographic column is a C18 chromatographic column, 0.1% formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed. The gradient elution program is:

2. The method according to claim 1, further comprising a high performance liquid phase detection step of a reference substance solution, wherein the reference substance is hesperidin.

3. The method according to claim 2, wherein the preparation method of the reference substance solution is as follows: take the reference substance hesperidin, add methanol or ethanol to dissolve it, preferably methanol.

4. The method according to claim 1, wherein the chromatogram has 18 characteristic peaks, wherein the 16th peak is hesperidin, the fingerprint spectrum uses the chromatographic peak of the 16th peak hesperidin as a reference (S), and the relative retention time of the 17 common characteristic peaks is:

5. The method according to claim 1, when the chromatographic peak of peak 16 hesperidin is used as a reference (S), the relative peak area of ​​each common characteristic peak is: 。 6. A method for simultaneously determining the contents of multiple components in a Chinese medicine composition of a mixed plant solid beverage of Poria cocos and cinnamon, wherein the raw materials of the Chinese medicine composition are composed of lily, papaya, yam, Poria cocos, cinnamon, tangerine peel, polygonatum, polygonatum odoratum, and mulberry, characterized in that: The reference solution and the test solution were analyzed by HPLC, and the concentration of the test sample and the content of the test sample components were calculated based on the determination results of the reference and test samples. The chromatographic conditions are as follows: the chromatographic column is a C18 chromatographic column, 0.1% formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed. The gradient elution program is: The reference substance is selected from one or more of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin, and the reference substance solution is a solution of each of the four reference substances, or a mixed solution of any two or more reference substances.

7. The method according to claim 6, wherein the reference substance solution is prepared by: adding an organic solvent to dissolve each reference substance to obtain a solution of each reference substance; or dissolving multiple reference substances in the same organic solvent to obtain a solution of a reference substance mixture, wherein the organic solvent is selected from methanol or ethanol, preferably methanol.

8. The method according to claim 7, wherein the concentration ranges of protocatechuic acid, chlorogenic acid, ferulic acid, and hesperidin in the reference solution are 2-13 μg / mL, 1-8 μg / mL, 1-7 μg / mL, and 20-120 μg / mL, respectively; and the concentration series of each reference substance increases arithmetic or geometrically. The method for calculating the content of the test sample is the linear regression equation method: draw a concentration-peak area standard curve for each reference sample component; use the chromatographic peak area Y of the reference sample solution component of each concentration as the ordinate and the mass concentration X of each reference sample solution component as the abscissa to calculate the linear regression equation for each component; according to the HPLC peak area of ​​the test sample, substitute it into the linear regression equation to calculate the test sample concentration.

9. The method according to claim 1 or 6, wherein the C18 chromatographic column is a chromatographic column of 250 mm×4.6 mm, 3-5 μm, preferably a chromatographic column of 5 μm.

10. The method according to claim 1 or 6, wherein the detection wavelength of the high performance liquid phase is 280-286 nm, preferably 283 nm.

11. The method according to claim 1 or 6, wherein the chromatographic conditions are: Chromatographic column: C18 column, 250mm×4.6mm, 5μm, detection wavelength 283nm; The gradient elution program was:

12. The method according to claim 1 or 6, wherein the preparation method of the test solution is: taking a mixed plant solid beverage of Poria cocos and cinnamon, adding an extraction solvent for extraction, filtering the extract after extraction, evaporating the filtrate to dryness, adding an extraction solvent for re-dissolving, filtering, and taking the subsequent filtrate as the test solution; when performing content determination, the ratio of the mixed plant solid beverage particles of Poria cocos and cinnamon to the re-dissolving solvent after extraction is 1.0g:0.9ml-1.1ml; Preferably, the extraction solvent is an alcohol or an aqueous solution of an alcohol, and the alcohol is selected from methanol and ethanol, and more preferably the extraction solvent is methanol; Preferably, the extraction process is water bath reflux or ultrasonic extraction; Preferably, the extraction method is to add methanol and heat under reflux in a water bath at 80 degrees Celsius for 0.5-2 hours, preferably 1 hour, filter the extract, evaporate the filtrate to dryness, add 5 ml of methanol to the residue to dissolve it, filter it, and take the filtrate to obtain the product.

13. Use of the method according to any one of claims 1 to 12 in the quality control of a mixed plant solid beverage of Poria cocos and cinnamon; preferably, the fingerprint of the tested mixed plant solid beverage of Poria cocos and cinnamon is compared with the fingerprint of a standard mixed plant solid beverage of Poria cocos and cinnamon, and the relative retention time should be within ±5% of the specified value to be a qualified product, and the similarity evaluation with the standard fingerprint is performed, and the similarity is greater than 0.8, preferably greater than 0.90 to be a qualified product.

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