Establishment of fingerprint of liquorice and bergamot mixed plant solid beverage and determination method of its components

Abstract

The present invention relates to a fingerprint establishment method, a detection method and a content determination method of a traditional Chinese medicine composition, Buddha's Hand Drink. The medicinal raw materials of the traditional Chinese medicine composition are composed of: Buddha's Hand, orange peel, malt, liquorice, jujube, perilla, citron, almond and donkey-hide gelatin. The fingerprint establishment method includes the preparation of a reference solution and a test solution; the reference solution and the test solution are detected by high performance liquid chromatography; the mobile phase used in the high performance liquid chromatography is acetonitrile and formic acid aqueous solution, and the elution method is gradient elution. The fingerprint and content determination method of the traditional Chinese medicine composition constructed by the above method can effectively characterize the traditional Chinese medicine composition, and simply and quickly determine the content of multiple components, providing richer chemical information and a more comprehensive basis for its quality evaluation.

Description

Technical Field

[0001] The invention belongs to the technical field of quality analysis of traditional Chinese medicines, and specifically relates to the establishment of a high performance liquid chromatography (HPLC) fingerprint spectrum of a liquorice and bergamot mixed plant solid beverage, a determination method thereof, and a method for determining the content of its components. 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 formed in the process of growth and development to adapt 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 outcome of syndrome 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 disease. The current study divides constitution into nine types according to the main clinical manifestations of overall characteristics. Among them, people with qi stagnation constitution are prone to chest and flank distension and discomfort, or wandering pain, sighing, or conscious foreign body sensation in the throat, poor sleep quality, forgetfulness, palpitations and easy fright, dry or loose stools, yellow urine, poor adaptability to the external environment, and are prone to depression, plum pit qi, goiter, lily disease and other diseases.

[0003] The trade name of the liquorice and bergamot mixed plant solid beverage is "bergamot drink". It is a functional food specially developed by Yabao Jiuhe (Beijing) Health Management Co., Ltd. for people with qi stagnation. It is mainly composed of bergamot, tangerine peel, malt, licorice, jujube, perilla, citron, almond, donkey-hide gelatin and other raw materials. In the formula, bergamot soothes the liver and regulates qi, harmonizes the stomach and relieves pain, and dries dampness and resolves phlegm; tangerine peel regulates qi and strengthens the spleen, dries dampness and resolves phlegm; malt promotes qi and digestion, strengthens the spleen and stimulates appetite; licorice nourishes the spleen and replenishes qi, clears away heat and detoxifies, eliminates phlegm and relieves cough, relieves acute pain, and harmonizes various medicines; jujube nourishes the middle and replenishes qi, nourishes blood and calms the mind; perilla relieves the cold, promotes qi and harmonizes the stomach; citron soothes the liver and regulates qi, relaxes the middle, and resolves phlegm; almonds lower qi, relieve cough and asthma, moisturize the intestines and relieve constipation; donkey-hide gelatin nourishes blood and nourishes yin, moisturizes dryness, and stops bleeding. Each ingredient in the formula performs its own duties and can be used as an auxiliary diet therapy for regulating qi stagnation.

[0004] Buddha's Hand Drink (Licorice and Buddha's Hand Mixed Plant Solid Drink) is a new product developed by the applicant. It is made of a variety of main raw materials, with a relatively complex composition, and there are few quality inspection methods. There is currently a lack of very rigorous quality control standards. Therefore, it is only produced according to actual conditions, resulting in different production processes, which will have a certain adverse impact on the standardized production of Buddha's Hand 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. Buddha's hand 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 Buddha's hand 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 Buddha's hand drink to detect quality, which brings more technical obstacles to the establishment of a fingerprint spectrum detection method for Buddha's hand 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 Buddha's hand drink product quality 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-mentioned technical problems, the present invention provides a method for establishing a fingerprint of a liquorice and bergamot mixed plant solid beverage 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. Licorice and bergamot mixed plant solid beverage 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 licorice and bergamot mixed plant solid beverage are bergamot, orange peel, malt, licorice, jujube, perilla, citron, almond and donkey-hide gelatin.

[0012] The fingerprint construction method or detection method of the liquorice and bergamot mixed plant solid beverage 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: a C18 chromatographic column, 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, and the gradient elution procedure:

[0013]

[0014]

[0015] 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 through the chromatogram of the reference. The reference used in the present invention is naringin, and the selection of the reference is based on the UPLC-Q-Exactive-Orbitrap-MS technology detection results of the licorice and bergamot mixed plant solid beverage by the inventor in the early stage, and then the literature is consulted to screen the index components of each medicine in the licorice and bergamot mixed plant solid beverage, and finally the chromatogram of the licorice and bergamot mixed plant solid beverage test solution is selected. The peak shape is better, there is no interference from impurity peaks before and after, and the completely separated naringin peak is used as the reference peak of the fingerprint.

[0016] The preparation process of the reference solution is simple. The reference naringin and a solvent capable of dissolving naringin 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 190 μg / mL to 195 μg / mL, specifically 191.25 μg / mL.

[0017] The test solution needs to be prepared by extracting the liquorice and bergamot mixed plant solid beverage, and the extraction process is: take the liquorice and bergamot mixed plant solid beverage, add a solvent for extraction. The solvent is a solvent commonly used in the art for extracting natural compounds, including but not limited to alcohol or an aqueous solution of alcohol, such as methanol and ethanol. In some embodiments of the present invention, methanol and a 60%-80% ethanol aqueous solution are used, specifically methanol.

[0018] Since the target components of the liquorice and bergamot mixed plant solid beverage are all components with good solubility in alcohol and alcohol water, the conventional natural compound extraction methods 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 uses methanol to heat reflux in a water bath at 80 degrees Celsius for 1 hour, 70% ethanol to heat reflux in a water bath at 80 degrees Celsius for 1 hour, and methanol to ultrasonic treatment for 1 hour. It is found that the chromatogram extracted by methanol water bath reflux has a more stable baseline, a higher chromatographic peak, and a better peak shape, which is the best way.

[0019] 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 consistent with the extraction solvent. The specific solution concentration after re-dissolution 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: Licorice and bergamot mixed plant solid beverage weight: re-dissolving solvent volume = (0.5~1.5) g: 1 mL, for example 0.5g, 1g or 1.5g licorice and bergamot mixed plant solid beverage / mL.

[0020] The chromatogram obtained under the above conditions has 18 characteristic peaks, among which peak 14 is naringin. Taking the chromatographic peak of peak 14 naringin as the reference (S), the relative retention time of the 17 common characteristic peaks of the fingerprint is:

[0021]

[0022]

[0023] 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.

[0024] When the chromatographic peak of naringin No. 14 is used as the reference (S), the relative peak areas of the common characteristic peaks are:

[0025] Peak Relative peak area 1 0.11～0.16 2 0.24～0.26 3 0.01～0.02 4 0.01～0.02 5 0.02～0.03 6 0.02～0.03 7 0.01～0.02 8 0.04～0.06 9 0.03～0.04 10 0.01～0.02 11 0.04～0.06 12 0.04～0.05 13 0.22～0.23 14 1 15 0.64～0.65 16 0.02～0.05 17 0.02～0.03 18 0.03～0.04

[0026] 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 liquorice and bergamot mixed plant solid beverage is prepared by the chromatogram of different batches of liquorice and bergamot mixed plant solid beverage, and the different batches are preferably at least 5 batches, preferably at least 10 batches. The fingerprint of the liquorice and bergamot mixed plant solid beverage to be tested is compared with the fingerprint of the liquorice and bergamot mixed plant solid beverage 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.

[0027] The present invention also provides a method for simultaneously determining the contents of multiple components in a traditional Chinese medicine composition, bergamot drink (licorice bergamot mixed plant solid drink), comprising the following steps:

[0028] (1) Preparation of reference solution:

[0029] (2) Preparation of test solution:

[0030] (3) HPLC detection of content.

[0031] The present invention adopts the same HPLC detection conditions as the aforementioned fingerprint detection, and can simultaneously detect the contents of caffeic acid, ferulic acid, naringin and hesperidin in a liquorice and bergamot mixed plant solid beverage under the same chromatographic conditions. The operation is simple and quick, and the reagent consumption and cost are low. 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 liquorice and bergamot mixed plant solid beverage, which meet the standard requirements.

[0032] The reference substance in the reference substance solution is selected from one or more of caffeic acid, ferulic acid, naringin, 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.

[0033] 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. 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: the linear regression equation of caffeic acid is: y = 14681x-119.6, and the linear range is 0.57-2.85 μg / mL; the linear regression equation of ferulic acid is: y = 20616x-189.4, and the linear range is 1.16-5.8 μg / mL; the linear regression equation of naringin is: y = 14760x-18679, and the linear range is 38.25-191.25 μg / mL; the linear regression equation of hesperidin is: y = 16246x-10401, and the linear range is 21.25-106.25 μg / mL. In the above linear regression equation, y is the peak area of ​​the corresponding component, and x is the mass concentration μg / mL of the component. Then, the content of the test sample is calculated according to the linear regression equation and the peak area of ​​the test sample obtained by the determination.

[0035] 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 .

[0036] 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.

[0037] 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 a solution of a reference substance mixture, wherein the organic solvent may 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 caffeic acid, ferulic acid, naringin, and hesperidin are 0.5-3 μg / mL, 1-6 μg / mL, 35-195 μg / mL, and 20-110 μg / mL, respectively; the series of concentrations of each reference substance may be increased arithmetic or geometrically.

[0038] 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 caffeic acid, ferulic acid, naringin, and hesperidin in the solution are 2.85 μg / mL, 5.8 μg / mL, 191.25 μg / mL, and 106.25 μg / mL, respectively; performing arithmetic dilution on the solution to obtain a series of concentrations of caffeic acid of 0.57 μg / mL, 1.14 μg / mL, 1.71 μg / mL, 2.28 μg / mL, 2.85 μg / mL, respectively. mL; the concentration series of ferulic acid were 1.16μg / mL, 2.32μg / mL, 3.48μg / mL, 4.64μg / mL, 5.8μg / mL; the concentration series of naringin were 38.25μg / mL, 76.5μg / mL, 114.75μg / mL, 153μg / mL, 191.25μg / mL; the concentration series of hesperidin were 21.25μg / mL, 42.5μg / mL, 63.75μg / mL, 85μg / mL, 106.25μg / mL.

[0039] The method for preparing the test solution is the same as the method for preparing the fingerprint. However, in order to accurately calculate the content, the licorice and bergamot mixed plant solid beverage particles need to be accurately weighed. The weight ratio of the licorice and bergamot mixed plant solid beverage particles to the volume of 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.

[0040] In the method for preparing fingerprint and determining the content of components of the present invention, the chromatographic column used is the 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 18Column (4.6mm×250mm, 5μm). The number of theoretical plates calculated by naringin should be no less than 5000, and the chromatograms of different batches of liquorice and bergamot mixed plant solid beverage and the chromatogram 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.

[0041] 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 naringin has the best absorption effect at 282nm, and caffeic acid, ferulic acid, and hesperidin have maximum absorption wavelengths at 322nm, 207nm, and 283nm. By comparison, it was found that there was no significant difference between the chromatographic peak response values ​​of naringin, caffeic 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 280-286nm as the detection wavelength range, preferably 283nm, and the spectral information of each compound is shown in Figure 3-4 .

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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:

[0046]

[0047]

[0048] 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.

[0049] 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..."

[0050] Advantageous Effects of the Invention

[0051] (1) The present invention successfully established the fingerprint of the liquorice and bergamot mixed plant solid beverage by optimizing the HPLC chromatographic conditions, identified 18 common peaks in total, and simultaneously detected the contents of caffeic acid, ferulic acid, naringin, and hesperidin in the liquorice and bergamot mixed plant solid beverage, laying the foundation for the establishment of a comprehensive quality standard for the liquorice and bergamot mixed plant solid beverage.

[0052] (2) The HPLC fingerprint determination method of licorice and bergamot mixed plant solid beverage was proved to be reliable by analyzing 10 batches of licorice and bergamot mixed plant solid beverage. 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 a method for determining the fingerprint of licorice and bergamot mixed plant solid beverage.

[0053] (3) The present invention uses the same chromatographic conditions to simultaneously detect the contents of caffeic acid, ferulic acid, naringin, and hesperidin in the liquorice and bergamot mixed plant solid beverage. The operation is simple and quick, and the reagent consumption is low and the cost is low. By selecting appropriate detection conditions, the method can determine the separation degree, accuracy, repeatability, stability, etc. of the content of each component in the liquorice and bergamot mixed plant solid beverage and meet the standard requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A in the middle is the chromatogram of naringin reference substance; Figure 1 Middle B is the fingerprint of liquorice and bergamot mixed plant solid beverage; Figure 1 Figure C is an overlay of HPLC fingerprints of 10 batches of licorice and bergamot mixed plant solid beverages.

[0055] Figure 2 A is the chromatogram of the upper limit of the HPLC gradient elution range, and B is the chromatogram of the lower limit of the HPLC gradient elution range.

[0056] Figure 3 A and B are the ultraviolet absorption diagrams of caffeic acid and ferulic acid in the wavelength range of 200-400nm, respectively.

[0057] Figure 4 C and D are the ultraviolet absorption graphs of naringin and hesperidin in the wavelength range of 200-400nm, respectively.

[0058] Figure 5 These are the spectra obtained under different detection wavelengths, where A, B, and C are the fingerprint spectra of the licorice and bergamot mixed plant solid beverage under detection wavelengths of 283nm, 322nm, and 207nm, respectively.

[0059] Figure 6 These are the spectra obtained under different elution conditions, where A and B are the fingerprint spectra of the liquorice and bergamot mixed plant solid beverage under elution conditions one and two in Example 2, respectively.

[0060] Figure 7 The spectra obtained under different chromatographic columns, A in the figure is Fingerprint of liquorice and bergamot mixed plant solid beverage under AQ-C18 column conditions; B is WELCH Fingerprint of liquorice and bergamot mixed plant solid beverage under AQ-C18 chromatographic column conditions.

[0061] Figure 8 These are the spectra obtained under different sample extraction methods, where A, B, and C are the fingerprint spectra of licorice and bergamot mixed plant solid beverage under extraction methods A, B, and C, respectively.

[0062] Fig. 9 A in the middle is the chromatogram of the liquorice and bergamot mixed plant solid beverage test solution; Fig. 9 B is the chromatogram of the solution of the reference substance mixture (in the figure: 1-caffeic acid; 2-ferulic acid; 3-naringin; 4-hesperidin). DETAILED DESCRIPTION

[0063] 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.

[0064] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0065] Experimental instruments and reagents:

[0066] 1. Instrument

[0067] 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.).

[0068] 2. Drug Reagents

[0069] Caffeic acid (purity ≥ 98%, batch number 110885-201703, China Food and Drug Inspection Institutes), ferulic acid (purity ≥ 98%, batch number 110773-202316, China Food and Drug Inspection Institutes), naringin (purity ≥ 98%, batch number 110722-202116, China Food and Drug Inspection Institutes), hesperidin (purity ≥ 98%, batch number 110721-202220, China Food and Drug Inspection Institutes); Ten batches of grass bergamot mixed plant solid beverages (S1-S10), with batch numbers 202204231, 202204221, 202204211, 202203291, 202203281, 202203271, 202203261, 202203041, 202203031, 202203021, source: Shanxi Yabao Jiuhe Pharmaceutical Technology Co., Ltd.; chromatographic grade acetonitrile; chromatographic grade methanol; ultrapure water.

[0070] Example 1 Establishment of fingerprint of liquorice and bergamot mixed plant solid beverage

[0071] The fingerprint was prepared according to the following conditions and the results are shown in Figure 1 .

[0072] 1. Chromatographic conditions

[0073] 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.

[0074] Table 1 Gradient elution method

[0075]

[0076]

[0077] 2. Preparation of reference solution

[0078] Take the naringin reference substance, weigh it accurately, and add methanol to prepare a naringin reference substance solution of 191.25 μg / ml.

[0079] 3. Preparation of test solution

[0080] 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.

[0081] 4. HPLC fingerprint analysis of liquorice and bergamot mixed plant solid beverage

[0082] 4.1 Precision test

[0083] The same batch (batch number: 202204231) of liquorice and bergamot mixed plant solid beverage was taken for testing, and the samples were injected 6 times continuously, and the retention time and peak area of ​​each common chromatographic peak were recorded. The retention time and peak area of ​​peak 14 naringin were used as references 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 1.14%, and the relative peak area RSD is less than 3.45%, which shows that the determination method of the present invention is stable, the system tightness is good, and the method is stable and reliable.

[0084] 4.2 Stability test

[0085] Take the same batch (batch number: 202204231) of liquorice and bergamot mixed plant solid beverage test sample, and analyze it 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 14 naringin 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 3.83%. This shows that the liquorice and bergamot 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.

[0086] 4.3 Repeatability test

[0087] Take the same batch (batch number: 202204231) of liquorice and bergamot mixed plant solid beverage test sample, prepare 6 test sample solutions in parallel according to the method under "3", 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 14 naringin 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 3.74%, which shows that the method of the present invention is stable and reliable, and is conducive to promotion.

[0088] 4.4 Establishment and similarity evaluation of HPLC fingerprint of liquorice and bergamot mixed plant solid beverage

[0089] Take 10 batches of liquorice and bergamot mixed plant solid beverage (batch number: 202204231, 202204221, 202204211, 202203291, 202203281, 202203271, 202203261, 202203041, 202203031, 202203021), prepare the test solution according to the method under "3", and inject and analyze, and record the chromatograms respectively. Select a representative spectrum (liquorice and bergamot mixed plant solid beverage with batch number 202204231) 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 fingerprint. The similarities between each batch of samples and the control fingerprint were (0.999, 0.999, 0.998, 0.999, 0.994, 0.996, 0.998, 0.999, 0.999, 0.996), respectively.

[0090] 4.5 Identification of common peaks in fingerprints, calculation of relative peak areas and relative retention times

[0091] Using the similarity evaluation system software, 18 peaks were detected in 10 batches of fingerprints. Compared with the reference substance, peak 14 was calibrated as naringin, and its retention time was 82.699min. Peak 14 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.

[0092] Table 2 Relative peak areas of 18 common peaks in 10 batches of samples (S1-S10)

[0093]

[0094] Table 3 Relative retention times of 18 common peaks in 10 batches of samples (S1-S10)

[0095]

[0096]

[0097] 4.6 Range of gradient elution conditions

[0098] 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.

[0099] Upper elution condition:

[0100]

[0101] Lower limit elution condition:

[0102]

[0103]

[0104] 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.

[0105] Example 2 Comparison of conditions for preparing fingerprint of liquorice and bergamot mixed plant solid beverage

[0106] 1. Determination of chromatographic conditions and exploration of fingerprint methods

[0107] The fingerprint results of Buddha's Hand Drink prepared under different HPLC chromatographic conditions were investigated and screened.

[0108] (1) Detection wavelength

[0109] Take the granules of this product, prepare them according to the method for preparing the test solution in Example 1, and analyze them according to the chromatographic conditions in Example 1 at detection wavelengths of 283nm, 322nm and 207nm, respectively. The results are shown in Figure 5 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, the range of 280-286nm is selected as the detection wavelength of the fingerprint spectrum of the licorice and bergamot mixed plant solid beverage test sample, and the optimal condition is 283nm

[0110] (2) Elution conditions

[0111] 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 examples, the following is a brief example: Take the particles of this product, prepare them according to the test solution preparation method of Example 1, and compare the following two elution conditions according to the chromatographic conditions of Example 1. The results are shown in Figure 6 It can be seen that in the chromatogram under elution condition 1, the peak shapes of the components are good and have good separation. Therefore, elution condition 1 is selected as the elution condition for the fingerprint of the licorice and bergamot mixed plant solid beverage test sample.

[0112] Elution condition 1:

[0113]

[0114] Elution condition 2:

[0115]

[0116]

[0117] (3) Column type

[0118] Take the granules of this product, prepare the test solution according to the method for preparing the test solution in Example 1, and use the chromatographic columns according to the chromatographic conditions in Example 1. 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 better and has better separation. Therefore, WELCH AQ-C18 chromatographic column (4.6mm×250mm, 5μm) was used as the chromatographic column for the fingerprint of the licorice and bergamot mixed plant solid beverage test sample in subsequent experiments.

[0119] 2. Determination of sample extraction method

[0120] Buddha's Hand Drink has complex ingredients, and different extraction solvents will affect the extraction effect of the effective ingredients. Finding the most suitable extraction method is very critical, and maximizing the extraction of more ingredients and showing peaks in the HPLC chart is one of the technical difficulties.

[0121] The samples were extracted using methods A, B, and C. The processes are as follows:

[0122] 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.

[0123] B: 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.

[0124] C: 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.

[0125] 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. The chromatogram obtained by this method had the most stable baseline, the highest chromatographic peak, and the best peak shape.

[0126] Example 3 Method for determining the contents of four components in liquorice and bergamot mixed plant solid beverage

[0127] 1. Chromatographic conditions

[0128] Same as Example 1.

[0129] 2. Preparation of reference solution

[0130] Take appropriate amount of caffeic acid, ferulic acid, naringin and hesperidin reference substances respectively, weigh accurately, and add methanol to prepare a mixed reference substance solution containing 2.85 μg / mL of caffeic acid, 5.8 μg / mL of ferulic acid, 191.25 μg / mL of naringin and 106.25 μg / mL of hesperidin.

[0131] At the same time, appropriate amounts of caffeic acid, ferulic acid, naringin, and hesperidin reference substances were taken and accurately weighed, and methanol was added to prepare single solutions of each reference substance at 2.85 μg / mL of caffeic acid, 5.8 μg / mL of ferulic acid, 191.25 μg / mL of naringin, and 106.25 μg / mL of hesperidin.

[0132] 3. Preparation of test solution

[0133] 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.

[0134] Chromatograms of control and test products are shown in Fig. 9 .

[0135] 4. Content determination and methodology investigation of liquorice and bergamot mixed plant solid beverage

[0136] 4.1 Precision test

[0137] The same batch number (202204231) of liquorice and bergamot 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 caffeic acid, ferulic acid, naringin, and hesperidin were 0.75%, 0.81%, 1.17%, and 1.05%, respectively, which showed that the determination method of the present invention had good precision.

[0138] 4.2 Investigation of linear relationship

[0139] 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.

[0140] 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.

[0141] Table 4 Regression equations, correlation coefficients and linear ranges of the four chemical components

[0142]

[0143] 4.3 Stability test

[0144] Take the licorice and bergamot mixed plant solid beverage test sample of the same batch number (202204231), 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 caffeic acid, ferulic acid, naringin, and hesperidin were calculated to be 1.5%, 0.86%, 2.18%, and 1.86%, respectively, indicating that the test solution of the present invention is stable within 24h.

[0145] 4.4 Repeatability test

[0146] Take the licorice and bergamot mixed plant solid beverage test sample of the same batch number (202204231), prepare 6 test sample solutions in parallel according to the method under item "3", and analyze them according to the chromatographic conditions under item "1". Record the peak areas respectively, and calculate the contents of each component by the linear regression equation: caffeic acid 1.98 μg / mL, ferulic acid 4.07 μg / mL, naringin 146.21 μg / mL, hesperidin 86.50 μg / mL, and the peak area RSD values ​​are 1.51%, 0.9%, 1.94%, and 1.64%, respectively, indicating that the method of the present invention has good repeatability.

[0147] 4.5 Sample recovery rate

[0148] Take a known content of liquorice and bergamot mixed plant solid beverage (202204231), pour out the contents, accurately weigh 2.50g, and add 1mL of mixed reference solution containing 5μg / mL of caffeic acid, 10μg / mL of ferulic acid, 365μg / mL of naringin, and 215μg / mL of hesperidin to 6 portions, respectively. 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.

[0149] Table 5 The recovery results of the four components of the liquorice and bergamot mixed plant solid beverage (n=6)

[0150]

[0151] 4.6 Sample determination

[0152] Take 10 batches of liquorice and bergamot mixed plant solid beverage test solution, and analyze them according to the chromatographic conditions under item "1". Record the peak area of ​​each component respectively, and calculate the content of caffeic acid, ferulic acid, naringin and hesperidin by the linear regression equation of each component. The results are shown in Table 6 below.

[0153] Table 6 Determination results of the contents of four components in 10 batches of liquorice and bergamot mixed plant solid beverages (μg / mL, n=3)

[0154]

[0155] In summary, the present invention successfully established the fingerprint of the liquorice and bergamot mixed plant solid beverage by optimizing the HPLC chromatographic conditions, determined 18 common peaks in total, and can simultaneously detect the contents of four components, namely, caffeic acid, ferulic acid, naringin and hesperidin, in the liquorice and bergamot 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 liquorice and bergamot mixed plant solid beverage and provided a reference basis for the establishment of its comprehensive quality standard.

[0156] 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 detection method for a Chinese medicine composition liquorice and bergamot mixed plant solid beverage, wherein the raw materials of the Chinese medicine composition are composed of: bergamot, orange peel, malt, liquorice, jujube, perilla, citron, almond, and donkey-hide gelatin, characterized in that: The test solution is subjected to high performance liquid chromatography (HPLC) detection to obtain a fingerprint spectrum; the detection wavelength of the HPLC is 280-286 nm; 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 solution, wherein the reference solution is naringin.

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

4. The method according to claim 1, wherein the fingerprint has 18 characteristic peaks, wherein the 14th peak is naringin, the fingerprint uses the chromatographic peak of the 14th peak naringin as a reference (S), and the relative retention time of the 18 common characteristic peaks is:

5. The method according to claim 1, wherein when the chromatographic peak of naringin No. 14 is used as a reference (S), the relative peak areas of the common characteristic peaks are:

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

7. The method according to claim 6, wherein the C18 chromatographic column is a 5 μm chromatographic column.

8. The method according to claim 1, wherein the detection wavelength of the high performance liquid phase is 283 nm.

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

10. The method according to claim 1, wherein the preparation method of the test solution is: taking a liquorice and bergamot mixed plant solid beverage, adding an extraction solvent for extraction, filtering the extract after extraction, evaporating the filtrate, adding an extraction solvent for re-dissolving, filtering, and taking the filtrate as the test solution; the extraction solvent is alcohol or an aqueous solution of alcohol, and the alcohol is selected from methanol and ethanol; the extraction process is water bath reflux or ultrasonic extraction.

11. The method according to claim 10, wherein the extraction solvent is methanol or 60% to 80% ethanol.

12. The method of claim 10, wherein the extraction method is to use methanol for reflux in a water bath at 80 degrees Celsius for 1 hour, 70% ethanol for reflux in a water bath at 80 degrees Celsius for 1 hour, or methanol for 1 hour under ultrasonic treatment.

13. The method according to any one of claims 1 to 12 is used for quality control of a liquorice and bergamot mixed plant solid beverage: the fingerprint of the liquorice and bergamot mixed plant solid beverage to be tested is compared with the fingerprint of a standard liquorice and bergamot mixed plant solid beverage. If the relative retention time is within ±5% of the specified value, the product is qualified. A similarity evaluation is performed with the fingerprint of the standard liquorice and bergamot mixed plant solid beverage. If the similarity is greater than 0.8, the product is qualified.

Images